Health promoting factors from milk of cows fed green...

B r i t a N g u m C h e P h D T h e s i s

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Health promoting factors from milk of cows fed green

plant material - The role of phytanic acid

Brita Ngum Che

MSc in Molecular Biology

Department of Food Science

Faculty of Science and Technology

Aarhus University

Denmark

A thesis submitted for the degree of Doctor of Philosophy at Aarhus University

September 2012


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Title of PhD project: Health promoting factors from milk of cows fed green plant

material - The role of phytanic acid

PhD student: Brita Ngum Che, MSc in Molecular Biology, Department of Food Science,

Faculty of Science and Technology, Aarhus University, Denmark

Supervisor: Associate professor Jette F. Young, Department of Food Science, Faculty of

Science and Technology, Aarhus University, Denmark

Co-supervisor: Assistant professor Mette K. Larsen, Department of Food Science,

Faculty of Science and Technology, Aarhus University, Denmark

Opponents:

1. Professor Karsten Kristiansen, Department of Biology, University of Copenhagen

2. Professor Ian Givens, Animal Science Research Group, University of Reding, United

Kingdom


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Preface

The work presented in the present PhD thesis titled “Health promoting factors from milk

of cows fed green plant material-the role of phytanic acid” centers around elucidating the

availability of phytanic acid in milk-fat and the relevance of this fatty acid on glucose and

fatty acid metabolism in humans. This work aimed at improving human health through the

differentiation of milk production enriched with phytanic acid.

This thesis is part of a “Green feed” project, where the metabolic impact of altered fatty

acid composition of milk-fat as affected by green feed was studied. I was specifically

interested in in vitro studies of glucose and fatty acid metabolism and the metabolic

impact of phytanic acid. The in vitro studies were conducted with a synthetic form of

phytanic acid, which is an isomeric mixture.

At the final stages of the project, the content of phytanic acid in milk-fat was accessed and

its diastereomers were observed to be differentially distribution under altered feeding

conditions. This was the reason behind the inclusion of the inclusion of studies on phytanic

acid diastereomers which was not part of the original setup of the project.

Regarding the setup of the thesis, an introduction to the thesis is presented, followed by a

literature review of factors that are relevant in understanding the work described in the

thesis. Results obtained from the study are discussed together, and some concluding

remarks are stated. Some future perspectives are included, and finally, an appendix of

manuscripts, which constitute work carried out in the project, is included.

This PhD project was based at the Department of Food Science at Aarhus University. Milk

analysis, in vitro cultures, and glucose metabolism studies were conducted at The

Department of Food Science, while the analyses of acylcarnitines were carried out at The


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Department of Forensic Science, in collaboration with The Research unit of Molecular

Medicine at Aarhus University.

The work presented in this PhD project was funded by The Danish Council for Strategic

Research and The Danish Cattle Federation, and was part of the scientific network called

“Tailored milk and human health”.


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Acknowledgements

I will commerce by expressing tremendous gratitude to my supervisor Jette F. Young,

Department of Food Science, for excellent supervision and guidance all through my PhD

studies. Thanks for the extreme understanding and support from you, especially during the

tough times. I am grateful for all the support right to the very last minute of completing my

thesis. To Lars I. Hellgren of The Technical University of Denmark, I am very grateful for

the fact that you conceptualized this project, thus making it at all possible. Your inputs at

every level have been indispensable for the realization of this thesis. Special thanks go to

my co-supervisor Mette K. Larsen, Department of Food Science, for her support both in

the laboratory and during the writing of manuscripts. I am grateful for the enthusiastic

discussion we have had concerning the milk-fat studies.

Special thanks to Niels Oksbjerg, Department of Food Science, for his contribution in the

isolation and culturing of porcine myotubes and the many discussions on glucose uptake

experiments and statistics. I will like to thank Jacob Holm Nielsen of Arla Foods, Braband,

Århus, for his fruitful input especially at the early stages of the project.

Special thanks to Mogens Johannsen and Rune Isaac Dupont Birkler at The Department of

Forensic Science at Aarhus University, for your help and support in the analysis of

acylcarnitines, and for you contribution in writing of manuscript. My gratitude also goes to

Niels Gregersen at The Research unit of Molecular Medicine at Aarhus University, thanks

for your inputs concerning the conception of acylcarnitine studies, but also for the warm

atmosphere and time spent at your laboratory.

Bente Andersen, Camilla Bjerg Kristensen, Caroline Nebel and Anne-Grethe Pedersen are

heartily thanked for contributing with excellent technical support.


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Aase Sørensen and Anne Hjorth Balling, special thank you for your patience and excellent

help in proof-reading my thesis and manuscripts

My office mates Sumangala Bhattacharya and Bjørn Nielsen, thanks for the many

interesting discussions, and a good working atmosphere. My colleagues in Foulum, thank

you for creating an interesting working environment and for your support in one way or

the other.

To my friend Ngonidzashe Chirinda, I very much appreciate the interesting discussions all

through the years in Foulum, and your suggestions regarding the structure of my thesis. To

my friend Steen Pedersen, thank you so much for excellent support in preparing some of

the figures for my thesis.

To my friend Bülent Kocaman, thank you for giving me the courage and support eight

years ago to quit my cleaning job and continue on my academic career. I am grateful to my

friend Marceline Pirkaniemi for her moral support. To Ina Lindgård and family, thank you

so much for being such a caring and supporting family. Your unlimited help with my kids

Nicole and Victoria has been invaluable and I will always be grateful for that.

Special thanks to my family for their moral support and up-backing, and to you Dad

thanks for implanting the will and zeal in me; you knew I would make it, and I am almost

there…..

Last but not the least, I am grateful for the tolerance, understand and love of my two

daughters Nicole and Victoria. It is incredibly cool of you two. You do not stop to amaze

me; first, you came into my life, then you gave me the chance to accomplish what I have

always dreamt of. For that, and all the unsaid, this work is dedicated to you Nicole and

Victoria.


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Abstract

Phytanic acid (PA) is a fatty acid (FA) that is present in ruminant products, and a natural

agonist of the peroxisome proliferator-activated receptors (PPAR). Because the PPARs are

pivotal in the regulation of glucose and FA homeostasis, it has been suggested that PA

could positively regulate these processes, in which case, it could be considered as a

bioactive compound with health-improving properties that can be used to fight against the

metabolic syndrome (MS). The objectives of this PhD study were to determine the impact

of total PA on glucose uptake and FA β-oxidation in skeletal muscles, and to elucidate the

total content of PA and the distribution of its diastereomers in milk as affected by feed

composition.

In this project, we established primary porcine myotubes as an efficient skeletal muscle

model for metabolic studies. Satellite cells (SC) derived from porcine muscles were

cultured to generate differentiated primary porcine myotubes. Viability studies were

performed to determine which concentrations or length of treatments could be tolerated

by the myotubes under glucose uptake, glycogen synthesis, and FA oxidation (FAO)

experiments. Optimization of glucose uptake assay using cytochalasin B revealed that both

the insulin-mediated and non-insulin mediated mechanisms of glucose uptake were

functioning in the myotubes. Exposures to myotubes of excess glucose during the analysis

of glucose uptake, and palmitate during the analysis of acylcarnitine, rendered the

myotubes insulin resistant and inhibited the oxidation of palmitoylcarnitine (C16),

phenomena of which can be expressed by skeletal muscles.

During the elucidation of the metabolic impact of PA, glucose uptake, glycogen synthesis

and FAO were analyzed by the use of tritiated 2-deoxyglucose, 14C- glucose and 13C-

palmitate, respectively. It was shown that physiological amounts of PA, which included 10


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µM, enhanced glucose uptake, especially at low concentrations of insulin, but PA could not

activate the incorporation of glucose into glycogen, except in the presence of insulin. Thus,

PA seemed to regulate glucose uptake in both an insulin-dependent and insulin-

independent fashion. When the myotubes were rendered insulin resistant by exposure to

excess glucose, it was neither possible for PA nor insulin to stimulate glucose uptake or

glycogen synthesis. During the analysis of β-oxidation using acylcarnitine profiling, we

could show that PA enhanced the β-oxidation flux in the myotubes, as it caused an increase

in the content of acetylcarnitine (C2) and a decrease in the C16/C2 ratio. Regarding

stimulation of β-oxidation also 10 µM PA was an effective dose. However, we could not

conclude if the induction in FAO by PA was through the induction of PPARα, since a

PPARα agonist was necessary as a control to validate the changes observed.

It has previously been shown that cow breed and feed composition affect milk-output and

FA composition, respectively, and that green feed increases the content of PA in milk-fat.

In this project, milk was sampled from grazing Danish Holstein and Danish Jersey cows

May and September periods, and the total content of PA and its diastereomers (RRR PA

and SRR PA) in the milk-fat of the cows were studied by gas chromatography-mass-

spectrometry analysis. The milk yield was higher in the Danish Holstein than the Danish

Jersey cows, but the breed did not affect the total content of PA in milk-fat. The total

content of PA was higher during the grazing period of September than in May. However,

differences were small and the intake of green feed could not be related positively to the

total content of PA. The distribution of the diastereomers was affected by feeding, as the

content of the RRR PA was positively related to the intake of grazed legumes. This finding

indicates that it is possible to manipulate the PA isomer distribution through strategic

feeding.


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In conclusion, a concentration of 10 µM PA achievable through dietary intake, is an

acceptable amount in in vitro studies, and can stimulate both glucose uptake and FAO.

The mechanisms behind these inductions need subsequent elucidations.


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Dansk Sammendrag

Fytansyre (PA) er en fedtsyre (FA) der findes i produkter fra drøvtyggere og er en naturlig

agonist for peroxisomere proliferator-aktiverede receptorer (PPAR). Fordi PPAR’erne er

centrale for reguleringen af glukose og FA homeostase, er det blevet foreslået, at PA kan

regulere disse processer positivt, og i så fald betragtes som en bioaktiv komponent med

sundhedsfremmende egenskaber, der vil kunne bruges i forbindelse med forebyggelse af

metabolisk syndrom (MS). Formålene med dette PhD-projekt har været at fastslå

betydningen af det totale PA på glukoseoptaget og FA β-oxidation i skeletmuskulatur, og

undersøge det totale indhold af PA og distributionen af dets diastereomerer i mælk

afhængig af fodersammensætning.

I dette projekt har vi etableret primære svine myotubes som en effektiv model for

skeletmuskulatur til metaboliske studier. Satellitceller (SC) fra muskelceller fra grise er

blevet dyrket for at etablere differentierede primære svine myotubes. Levedygtigheden af

myotubes blev undersøgt, for at bestemme hvilke koncentrationer og hvilke varigheder af

behandlinger som kunne tolereres af myotubes i forbindelse med glukoseoptag,

glykogensyntese og FA oxidations (FAO) eksperimenter. Optimering af glukoseoptag ved

brug af cytochalasin B viste, at både det insulinmedierede og ikke-insulinmedierede

glukoseoptag fungerede i myotubes. Overeksponering af glukose til Myotubes i forbindelse

med analysen af glukoseoptag, og palmitat i forbindelse med analyser af acylcarnitin,

gjorde myotubes insulinresistente og hæmmede oxidationen af palmitoylcarnitin (C16),

fænomener som er velkendte i skeletmuskulatur.

I forbindelse med undersøgelsen af PA’s metaboliske betydning, blev glukoseoptag,

glycogensyntese og FAO analyseret ved hjælp af henholdsvis tritieret 2-deoxyglukose, 14C-

glukose og 13C-palmitat. Det blev vist, at den fysiologiske mængde af PA, inklusiv 10 µM,


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forbedrede glukoseoptaget, især ved lave koncentrationer af insulin, men PA kunne ikke

aktivere dannelsen af glykogen fra glukose, medmindre der var insulin til stede. Dette

indikerer at PA regulerer glukoseoptag via både en insulin-afhængig og insulin-uafhængig

mekanisme. Når myotubes blev gjort insulinresistente ved overeksponering af glukose, var

det hverken muligt for PA eller insulin at stimulere glukoseoptag eller glykogensyntese. I

forbindelse med analysen af β-oxidation ved acylcarnitine profilering, kunne vi se, at PA

forbedrede β-oxidationen i myotubes, fordi det øgede indholdet af acetylcarnitin (C2) og

mindskede C16/C2 ratioen. Også i stimuleringen af β-oxidationen viste 10 µM PA sig at

være en effektiv dosis. Vi kunne dog ikke konkludere om PA-induceret FAO sket via

PPARα, fordi en veletableret PPARα agonisten ville være nødvendig som kontrol for at

kunne validere de ændringer, der blev observeret.

Det er tidligere vist, at kvægrace og fodersammensætning har betydning for såvel

mælkeudbytte som mælkens FA sammensætning, og at grønne fodermidler øger indholdet

af PA i mælkefedtet. I dette projekt blev der taget mælkeprøver fra græssende Holstein og

Jersey køer i maj og september, og det totale indhold af PA og dennes diastereomere (RRR

PA og SRR PA) i mælkefedtet fra køerne blev analyseret ved brug af gaschromatografi og

massespektrometri. Mælkeudbyttet var højere for Holstein end for Jersey køer, mens

racen ikke havde nogen betydning for det totale indhold af PA. Det totale indhold af PA var

højere i mælk fra september sammenlignet med maj. Forskellene var dog små, og det var

ikke muligt at relatere indtaget af grønne fodermidler positivt til mælkens PA indhold.

Fordelingen af diastereomererne var afhængig af fodringen, hvor indholdet af RRR PA var

positivt relateret til indtaget af bælgplanter i afgræsningen. Dette resultat indikerer, at det

er muligt at styrefordelingen af PA diastereomerer gennem strategisk fodring.


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Det kan konkluderes, at koncentrationen på 10 µM PA, der kan opnås via kosten, er en

acceptabel mængde i in vitro studier, og at dette kan stimulere både glukoseoptag og FAO.

Mekanismerne bag disse aktiveringer kræver dog nærmere undersøgelser.


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Key words

Primary porcine myotubes, phytanic acid, phytanic acid diastereomers, insulin, palmitic

acid, glucose uptake, fatty acid β-oxidation, acylcarnitine, pasture, legumes


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Lists of disseminations:

A: Abstracts and posters

1. Separate and combined effects of phytanic acid and insulin on glucose uptake in

primary porcine myotubes; 2009.Poster presented at International Food Research Day,

Aarhus University, Aarhus, Denmark

2. Production of phytanic acid-rich milk by feeding cows green-plant material; 2009.

Poster presented at LMC PhD congress, Copenhagen, Denmark

3. Giver grønt foder sundere mælk? Forbedres de ernæringsmæssige egenskaber af

mælkefedt, når køerne fodres med grønt plantemateriale? Mælkeritidende, Nr.

16,2009, s. 344-345

4. Phytanic acid enhances glucose uptake in primary porcine myotubes; 2010. Abstract

for The 9th Conference of the International Society for the Study of Fatty Acids and

Lipids, Maastricht, Holland

5. Phytanic acid-induced glucose uptake in primary porcine myotubes may be mediated

by glucose transporter 4; 2011. Abstract presented at LMC Food Congress, Odense,

Denmark

6. The distribution of phytanic acid diastereomers in food; 2011. Poster presented at 3rd

COST Feed for Health Conference, Copenhagen Denmark


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B: Manuscripts

1. Manuscript I; MI:

Phytanic acid stimulates glucose uptake in a model of skeletal muscles, the primary

porcine myotubes

2. Manuscript II; MII:

Effects of phytanic acid on beta-oxidation flux in a primary porcine myotube model as

detected by an UPLC-MS/MS acylcarnitine assay

3. Manuscript III; MIII:

Content and distribution of phytanic acid diastereomers in organic milk as affected by

feed composition


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List of abbreviations

ACAD acyl-CoA dehydrogenase

Acyl-CoA acyl coenzyme A

AMCAR α-methylacyl-CoA racemase

AMPK adenosine monophosphate-activated protein kinase

AS160 Akt substrate 160

BMI body mass index

C16 palmitoylcarnitine

C2 acetylcarnitine

CACT carnitine-acetylcarnitine translocase

CAT carnitine acyltransferase

CLA conjugated linoleic acid

COT carnitine O-octanoyltransferase

CPT carnitine palmitoyl transferase

DBD DNA binding domain

EH enoyl-CoA hydratase

FA fatty acid

FAO fatty acid oxidation

GLUT glucose transporters

GS glycogen synthase

GSK glycogen synthase kinase

HAD 3-hydroxyacyl-CoA dehydrogenase

IGF insulin growth factor

IRAP insulin-responsive aminopeptidase

IRS insulin receptor substrate

KAT 3-ketoacyl-CoA thiolase


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LBD ligand binding domain

LCEH long-chain enoyl-CoA hydratase

LCHAD long-chain hydroxyacyl-CoA dehydrogenase

LCKAT long-chain ketoacyl-CoA thiolase

MCAD medium-chain acyl-CoA dehydrogenase

MCKAT medium-chain ketoacyl-CoA thiolase

MS metabolic syndrome

mTOR rapamycin complexed with rictor

MTP mitochondrial trifunctional protein

MyoD myogenic differentiation

OCTN2 organic cation transporter number 2

PA phytanic acid

PAX paired box

PDK1 phosphoinositide dependent protein kinase-1

PI3K phosphatidyl inositol 3-kinase

PIP phosphatidyl inositol 3, 4, 5- triphosphate

PKB protein kinase B

PKC protein kinase C

PPAR peroxisome proliferator-activated receptor

PPRE peroxisome proliferator response element

RXR retinoid x receptor

SC satellite cells

SCAD short-chain acyl-CoA dehydrogenase

SCEH short-chain enoyl-CoA hydratase

SCHAD short-chain hydroxyacyl-CoA dehydrogenase

T2D type 2 diabetes


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TAG triacylglyceride

UDP uridine diphosphate

VI vastus intermedius

VLCAD very long-chain acyl-CoA dehydrogenase


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Table of contents

Title page………………………………………………………………………………………………………. 1

Authors addresses, supervisors, reviewers, opponents………………………………………. 2

Preface………………………………………………………………………………………………………….. 3

Acknowledgements………………………………………………………………………………………… 5

Abstract………………………………………………………………………………………………………… 7

Dansk Sammendrag……………………………………………………………………………………….. 10

Key words…………………………………………………………………………………………………….. 13

Lists of disseminations…………………………………………………………………………………… 14

Abbreviations………………………………………………………………………………………………… 16

Table of content……………………………………………………………………………………………… 19

1. INTRODUCTION………………………………………………………………………………………. 21

1.1 Problem description……………………………………………………………………….. 21

1.2 Hypotheses……………………………………………………………………………………. 23

1.3 Aims……………………………………………………………………………………………… 24

1.4 The metabolic syndrome………………………………………………………………… 25

1.4.1 Insulin resistance in skeletal muscles………………………………………………. 26

1.4.2 Obesity………………………………………………………………………………………….. 29

1.4.3 Type 2 diabetes (T2D)……………………………………………………………………… 30

1.5 The Satellite cell (SC)………………………………………………………………………. 31

1.5.1 From SCs to skeletal muscles…………………………………………………………… 33

1.6 Insulin signaling in skeletal muscles………………………………………………… 34

1.6.1 Glucose uptake……………………………………………………………………………… 36

1.6.2 Glycogen synthesis………………………………………………………………………… 37

1.7 The β- oxidation……………………………………………………………………………. 38


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1.7.1 Acylcarnitine shuttling ……………………………………………………….………… 39

1.7.2 Β- oxidation enzymes……………………………………………………………………. 41

1.8 Peroxisome proliferator activated-receptors (PPAR)………………………… 43

1.8.1 PPARα…………………………………………………………………………………………. 44

1.8.2 PPARβ………………………………………………………………………………………….. 45

1.8.3 PPARγ………………………………………………………………………………………….. 45

1.9 Phytanic acid (PA)…………………………………………………………………………. 46

1.9.1 Origin of PA and its diastereomers………………………………………………….. 46

1.9.2 Metabolism of PA in humans………………………………………………………….. 48

1.9.3 Milk fat composition of PA as affected by cow feed…………………………… 51

2 RESULTS AND DISCUSSION……………………………………………………………………. 52

2.1 Primary porcine myotubes; a model of skeletal muscles, for studying glucose

and FA metabolism………………………………………………………………………… 52

2.2 The metabolic impact of PA……………………………………………………………. 55

2.3 Concerns regarding excess content of total PA in humans…………………. 58

2.4 PA in milk-fat as affected by cow feed……………………………………………… 58

2.5 Concerns regarding the distribution of PA diastereomers in humans…. 61

3 CONCLUDING REMARKS………………………………………………………………………... 63

4 PERSPECTIVES………………………………………………………………………………………… 64

5 REFERENCES…………………………………………………………………………………………… 66

6 APPENDIX………………………………………………………………………………………………. 83

6.1 Manuscript I; MI…………………………………………………………………………… 83

6.2 Manuscript II; MII………………………………………………………………………… 117

6.3 Manuscript III; MIII……………………………………………………………………… 152


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1 INTRODUCTION

1.1 Problem description

Small changes in food components that can improve pathological conditions and leave the

population healthier are in focus. Milk is a global commodity and harbors numerous

bioactive compounds that have been suggested to improve diseases such as type 2 diabetes

(T2D). Besides proteins, minerals and vitamins, many fatty acids (FA) are constituents of

milk and share nutritional, functional and physiological importance in humans.

Accumulating data indicates that the intake of milk alleviates symptoms of the metabolic

syndrome (MS); an observation that is contrary to the fact that milk fat is highly saturated

and, thus, believed to be unhealthy. A milk-fat paradox has originated from these findings

and prompts the elucidation of potential bioactives responsible for the protective effect of

milk.

Phytanic acid (PA); 3, 7, 11, 15 tetramethylhexadecanoic acid, is a milk-fat- derived FA,

which is a natural ligand of the peroxisome proliferator-activated receptors (PPAR) and

the retinoid X receptor (RXR). The PPARs are known for their crucial role in the regulation

of glucose homeostasis and FA metabolism. Being natural agonists of these receptors, PA

has been suggested to be a bioactive compound with potential health-improving abilities

and might to some extent alleviate MS and T2D. Reviews on the metabolism and

physiological potentials of PA have thrown more light and focus on PA as an important

bioactive compound just like conjugated linoleic acid (CLA).

So far, one study in rat hepatocytes has shown that PA can improve glucose uptake, even

though very high concentrations of the FAs were utilized. Also, not much has been done

regarding the regulation of FA metabolism by PA. Thus, very little is known of the effect of

PA on glucose homeostasis and FA oxidation (FAO). Furthermore, works in this thesis


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show that the content of the two naturally occurring stereomers of PA (RRR PA and SRR

PA) in milk fat is differentially affected by the feed composition of the cow. Not much is

known about the physiological activities of the diastereomers of PA. More so, in light of the

fact that enzymes involved in the metabolism of FA are stereo-specific, the distribution of

the PA diastereomers in milk fat becomes relevant.

T2D is a global epidemic that is fast-growing. People with diabetes live 12 years less than

the rest of the population, and the mortality rate in Denmark is 3-6 times higher amongst

people with diabetes ≤ 65 years than the rest of the population. Early and coordinated

treatment can reduce diabetic-related mortality by 50 %. To reduce the growth of the

diabetic epidemic and the use of medication, which is extremely expensive, the search for

solutions in food products with better health quality is of interest.


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1.2 Hypotheses

Dairy fat is rich in PA, which exists naturally as SRR PA and RRR PA. PA is a natural

agonist of the peroxisome proliferator-activated receptors (PPAR). The PPARs are

regulators of glucose homeostasis and lipid metabolism, and have been documented to

improve insulin sensitivity and FAO. There is, thus, an incitement that PA can improve

these processes. Measurements in glucose uptake and acyl carnitines have been used to

follow changes in glucose homeostasis and FA metabolism, as they are indispensable

metabolites required for these processes. Skeletal muscles account for the bulk of insulin-

mediated glucose uptake and therefore chosen as the model for exploring the impact of PA

on glucose and FA metabolism. As PA diastereomers are derived from chlorophyll, their

content in milk fat is dependent on the feed composition, and increased amounts of green

feed are expected to affect the content of PA diastereomers. From these bases, four

hypotheses arose:

1. Primary porcine myotubes can be an effective model of skeletal muscles for studying

glucose and FA metabolism.

2. Glucose uptake in skeletal muscles can be improved by PA.

3. The exposure of skeletal muscles to PA will induce β-oxidation.

4. The total content of PA and its diastereomers is differentially affected by specific

feeding patterns.


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1.3 Aims

The aims of this PhD project were to study the impact of total PA on glucose uptake and FA

β-oxidation in primary porcine myotubes, and to determine the total content of PA and the

distribution of its diastereomers in milk as affected by feed composition. The overall aim

was to produce differentiated milk of improved bioactive capacity.


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1.4 The metabolic syndrome(MS)

The metabolic syndrome (MS) is a disease state consisting of abdominal obesity,

dyslipidemia, hypertension, pro-inflammatory and pro-thrombotic states, insulin

resistance and glucose intolerance (Table 1). These factors usually occur in clusters and

promote the prevalence of T2D and cardiovascular diseases directly [1]. Due to the inter-

mix of the various components constituting MS, it is very difficult to pin-point and treat.

Populations with MS are insulin resistant and at higher risk of developing cardiovascular

diseases than people without MS [2]. The prevalence of MS is about 20-30 % in normal

subjects and 70-80 % in diabetic subjects [2, 3]. In Denmark, the frequency of MS is 38 %

and 22 % amongst men and women ≥ 60 years of age, respectively [4]. Low physical

activity, excess dietary intake, maternal obesity and ethnicity are some of the main factors

that promote the prevalence of MS [5-10].

Table 1: Risk factors related to the metabolic syndrome (Adopted from [11])

Waist circumference* Men: ≥ 94 cm

Women: ≥ 80 cm

Triglyceride (TAG) levels >1.7 mM (150 mg/dL)

HDL-cholesterol Men: < 1.03 mM (40 mg/dL)

Women: < 1.29 mM (50 mg/dL)

Blood pressure systolic: ≥ 130 mm Hg

Diastolic: ≥ 85 mm Hg

Fasting plasma glucose ≥ 5.6 mM (100 mg/dL)

*The waist circumference is ethnic-specific and the values stated are for europids. It is also valid

for sub-Saharan Africans and eastern Mediterranean and Middle East populations until useful data

are available.


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1.4.1 Insulin resistance in skeletal muscle

Insulin resistance (IR) in skeletal muscle is one of the early signs and a core phenomenon

in the pathophysiology of T2D [12, 13]. It occurs when normal concentrations of insulin are

not enough to execute effective uptake and disposal of glucose. The increase in blood

insulin levels as well as glucose levels, thus, characterize IR, and in muscles glucose uptake

is impaired with the down regulation of the glucose receptor transporters (GLUT) [14, 15].

Figure 1 illustrates the dysfunction in GLUT-4, which results in diminished glucose uptake

and IR. Since IR is not completely abolished by GLUT-1 and GLUT-4, it denotes that

players other than the glucose transporters are involved in the generation of IR [16, 17].

Decrease in the protein content of insulin receptor substrate-I (IRS-I) and impairment of

its phosphorylation, reduced activity of phosphatidyl inositol 3-kinase (PI3K) and

downstream enzymes are partly responsible for the defects of the insulin-signaling

pathway [18]. IR also results from a defect in glycogen synthesis, due to a reduced activity

of glycogen synthase (GS) originating from a drop in insulin-stimulated GS [19]. Defects in

the adenosine monophosphate-activated protein kinase (AMPK) pathway, has also been

shown to cause IR, in a manner that does not involve insulin [20].

Excess food intake overloads the muscles with glucose and lipids, which are known to

generate IR. High glucose levels have been shown to generate insulin resistance by

activation of the glucosamine pathway [21, 22], while accumulation of fats in the muscles

has also been linked to IR [23]. Lipid accumulation is thought to cause IR by inhibiting

mitochondrial function [24], and possibly the rate of FAO. Shulman et al proposed that

the mechanism by which FAs causes IR in the muscle lies in the presence of their

metabolites, generated through excess feeding or through defects in FAO, which cause the


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phosphorylation of IRS at its serine residue, rendering it inactive [25]. When this occurs,

the translocation of GLUT 4 is obstructed and the result is reduced glucose uptake.

Figure 1: IR in muscles results in defect in GLUT 4 signaling (Adopted from [26])

Irresponsiveness of GLUT-4 to insulin hinders its expression and translocation to the plasma

membrane, thus, reducing the ability of glucose uptake in the muscles.

It was suggested that exercise and muscle contraction in the absence of insulin can

stimulate glucose uptake [27] and even restore insulin sensitivity in obese mice [28]. It has

actually been proven that glucose uptake in muscles can also occur in a non-insulin

dependent manner, and that IR could actually occur in muscles with an intact insulin-

signaling system [17]. The activation of AMPK activity has been suggested to be

responsible for this non-insulin control of glucose [17]. Moreover, lipids accumulating in


28

muscles generate ceramides and other intermediates, which have been linked to the

development of IR [29], not only through the inhibition of protein kinase B (PKB) or IRS-1

but also by the generation of oxidative stress and apoptosis [25, 30, 31]. Even so, defects in

fat tissue and liver are extended to skeletal muscles [32], and because of this cross-talk

between tissues, other pathways and organs are involved in the generation of IR in the

muscles. An illustration of the link between adipose tissues, liver and muscles in the

generation of IR is shown on Figure 2.

Figure 2: Defects in other organs contributes to IR in the muscles (Adapted from [25])

Dysfunction due to for example changes in the partitioning and storage of lipids in the liver adipose

tissue or muscle results in IR, through decreased oxidation capacity of the muscles


29

1.4.2 Obesity

Obesity correlates strongly with T2D and arises as a result of excess body fat [33, 34].

Excess food intake and inactivity are known to be principal causes of obesity. An extensive

review on the many other causes of obesity, which includes smoking, medication,

pollutants and genetic factors, have been done by Keith et al [35]. The body mass index

(BMI) is used as a generic measure of obesity as it is closely related to the total body fat

[36]. A person with a BMI ≥ 30 kg/m2 is considered obese, and once this is the case, their

waist circumference is not considered during diagnosis [11]; see Table 2. Obesity increases

the prevalence of many pathological factors that constitute MS, including diabetes [34]. In

Europe, it is responsible for 80 % of type 2 diabetic incidents and 55 % of diseases related

to high blood sugar [37]. As much as 1 million deaths in Europe each year are related to

excess body weight [37].

Table 2: Classification of obesity using BMI (adopted from [38])

BMI range – kg/m2 Health Risk

≥ 30 Obese (high risk of developing MS)

25-29.9 Overweight (moderate risk of developing MS)

18.5 to 24.9 Healthy range (low risk)

< 18.5 Underweight (risk of developing problems such as nutritional

deficiency and osteoporosis)


30

1.4.3 Type 2 diabetes (T2D)

Contrary to type 1 diabetes, which is insulin-dependent and affects 10 % of patients

suffering from diabetes [39], T2D is a non-insulin dependent disease that affects 90 % of

diabetic patients and is common in old age but is now spread to the entire population [40].

The incidence of cardiovascular diseases is elevated four-folds when T2D is involved [41],

and it has been estimated that the living costs of obese men and women are 15 % and 21 %

higher, respectively, when compared to healthy individuals, regardless of their shorter

lifespans [38].

In Europe, over 50 million people are affected by T2D, and in Denmark, every twelfth

person is diabetic, but only half of them are aware of it [42]. Furthermore, it is estimated

that about 0.5 million people in Denmark are pre-diabetic [42]. A central player and

predictor of T2D is IR [43]. Other disorders such as oxidative stress, hyperglycemia and

dyslipidemia and β-cell malfunction also result in T2D [21, 39, 44, 45]. Figure 3 illustrates

the metabolic progress of T2D.

Figure 3: The metabolic progress of T2D (adopted from [13] )

The onset of T2D starts with the presence of excess fat, gene defects and/or aging. These factors

cause the muscle to become intolerant to insulin. As a result, glucose levels become elevated and

the β-cells of the pancreas produce more insulin to cope with the high glucose level. Eventually,

other tissues such as adipose tissue and liver become defective due to either a change in their fat

content or rate of glucose production, respectively. They become IR and contribute to increase

plasma glucose. The stress on the β-cells of the pancreas is even more, which with time, becomes

ineffective in its insulin production. Eventually, the whole body becomes intolerant to glucose.


31

1.5 The Satellite cell (SC)

Skeletal muscle is one of the few adult tissues that possess the ability to regenerate itself,

and credit for this ability is given to a population of dormant cells residing within the

muscles and known as satellite cells (SC). SC were first described by Mauro A. in 1961 as

mono-nucleated cells situated between the basal membrane and cell membrane of fibers,

and coexisting alongside multinucleated muscle fibers, as the name implies [46]. They are

quiescent by nature and located between the sarcolemma and basement membrane of

terminally- differentiated muscle fibers [47, 48]. The SC provide nuclei to the growing

muscle fibers during postnatal development [49]. More so, it has been revealed that SC are

crucial in the repair of damaged fibers by facilitating their re-innervation and


32

vascularization [50]. Although SC retain the ability to grow and differentiate during adult

life, they however, acquire a quiescent nature and become active only upon response to

need [51]. Figure 4 is a sketch of the different functions of SC.

Figure 4: Function of SC (adopted from [52])

When activated, dormant SCs re-new themselves (A), while the majority proliferates and take part

in either the formation of new muscle fibers (B) or fuse to injured muscle fibers to repair them (C).

The SCs constitute only 3-6 % of fiber nuclei, but perform a strikingly fast and efficient

task in the regeneration of damaged skeletal muscle, given that it takes approximately 3-5

days to completely generate a large portion of new myotubes after acute damage [53, 54].

Studies revealing the existence of a mixture of fast cycling cells and a small portion of slow

cycling cells in the SC population have led to the belief that SCs are a heterogenic pool [55,

56]. However, the molecular identification of any subpopulation of SC still remains to be


33

achieved, and even though cells of non-muscular origin can contribute to muscle

production, it has not been possible to achieve efficient muscle regeneration using these

cells [57]. Much evidence points instead to a more homogeneous nature of SCs. It is

believed that following activation, SCs adopt a bifacial state, whereby the maintenance of a

satellite pool as well as proliferation to generate new nuclei is established [58]. This dual

fate is necessary for the SC to maintain a self-renewal pool of muscle progenitor cells [58].

1.5.1 From SCs to skeletal muscles

The transition of SCs to muscles occurs through the processes of activation, proliferation

and differentiation of the quiescent SCs (see Figure 4) and is effected by various

transcription and growth factors including insulin growth factor-1 (IGF-1) [59, 60]. While

a small portion of the activated SCs down-regulate the myogenic differentiation gene

(MyoD) but maintain the paired box (PAX)-7 gene to re-attain a quiescent state of renewed

SCs [58], the majority of the activated SCs loose PAX-7 and, thus, proliferate into

myoblasts [61]. Proliferating myoblasts are therefore characterized by a high expression of

MyoD, and in its absence, muscle regeneration is delayed [62]. The up- and down-

regulation of myogenin and myostatin, respectively, allows for the differentiation of

proliferating myoblasts [61, 63-66], while insulin growth factor-I (IGF-I) not only activates

proliferation, but is also involved in the innervation of the differentiated myoblasts [59, 60,

67]. The differentiation of myoblasts is also marked by the upregulation of IGF-II and IGF-

I and II receptors.[68], when compared to the proliferation process [68]. Skeletal muscle

actin and myosin heavy chain are typical markers of mature muscles, which are present at

the late stages of differentiation [54, 69]. Figure 5 shows the morphology of muscles cells

during myogenesis.


34

A B

C D

Figure 5: Transition from SCs to myotubes (adopted with modifications from Figure 1, MI)

Isolated porcine SCs cultured for 24 h on a matrigel matrix (A) proliferate into 50 % confluent

myoblasts after 3 days (B) and by day 5, myoblasts are 100 % confluent (C). The closely packed

myoblasts start to differentiate, and serum reduction enhances the differentiation process to

generate multinucleated myotubes (D).

1.6 Insulin signaling in skeletal muscles

Skeletal muscle is the main site of insulin action and accounts for over 70 % of glucose

disposal after a meal [70]. Insulin is a hormone that has a crucial role in the regulation of

whole body metabolism, and defects in its signaling generate IR, as discussed earlier. The

binding of insulin to its receptors initiates an auto-phosphorylation of the receptor and

sets off a signaling cascade, which eventually causes the translocation of GLUT 4 receptors


35

to the plasma membrane to ease the transportation of glucose [71, 72]. The

phosphorylated insulin receptor activates IRS-I and II by phosphorylating them at their

tyrosine residues [73]. Many signaling routes are affected by the phosphorylated IRS, but

the important pathway in the regulation of glucose is the phosphatidyl inositol 3-kinase

(PI3K) route [74]. PI3K, once activated catalyzes the formation of phosphatidylinositol 3,

4, 5- triphosphate (PIP) and the subsequent phosphorylation of the phosphoinositide

dependent protein kinase-1 (PDK1) enzyme [75]. It is the activated PDK1 that

phosphorylates protein kinase B (PKB) and a subtype of protein kinase C (PKCλ), and this

action effectuates the translocation of vesicles harboring GLUT4 [76, 77], via the action of

the enzyme Akt substrate 160 (AS160) [77]. PI3K has also been shown to activate PKB

through the mammalian target of rapamycin complexed with Rictor (mTORC2) [75]. The

action of insulin also causes PKB to catalyze the phosphorylation of Glycogen synthase

kinase (GSK3), which renders it inactive and allows for multiple dephosphorylation and,

hence, activation of GS [78]. Figure 6 is a sketch showing some of the enzymes involved in

insulin signaling.

Figure 6: Insulin signaling and glucose uptake in skeletal muscles (adopted from [77]).

The presence of glucose in plasma initiates the production of insulin by the beta cells in the islets of

the pancreas. The released insulin binds to its receptor on the plasma membrane and activates a

cascade of enzymes through phosphorylation. At the end of the reaction cascade is PKB, whose

activation leads to the transcription of GLUT 4 and translocation of GLUT 4 vesicles to the plasma

membrane, where they facilitate the transport of glucose into the cells.


36

1.6.1 Glucose uptake

Glucose uptake has been defined as the rate limiting step in the metabolism of glucose in

the muscles [79]. The regulation of glucose uptake in the muscles is accomplished by at

least GLUT 1 and GLUT 4 [80]. It has been established that GLUT 4 is the protein

responsible for the insulin-mediated glucose uptake in skeletal muscles while GLUT 1 is

responsible for basal glucose disposal, i.e. not dependent on insulin action [16, 81].

In the presence of insulin, the GLUT 4 vesicles, which are normally situated around the

nuclear space and in the cytosol, are activated to translocate and merge to the PM. The

insulin-responsive aminopeptidase (IRAP), which co-locates with insulin in the vesicles,


37

has been documented to assist insulin in the translocation process of GLUT 4 [82, 83].

GLUT 1 is found in the cytosol but also in the PM at basal conditions. It has been reported

that non-insulin-dependent mechanisms such as hypoxia and contraction also cause an

increase in the uptake of glucose, via the activation of AMPK [84, 85]. It has also been

revealed that hypoxia not only stimulates the expression and translocation of GLUT 1, but

also reduces the degradation of GLUT 1 [86, 87]. Therefore, one can conclude that it is

partly GLUT 1 that is responsible for the activation of glucose uptake in these instances.

1.6.2 Glycogen synthesis

The glucose taken up by the muscles can be used in many metabolic processes. About

50,000 glucose moieties make a single glycogen molecule, and 80 % of the glycogen in the

body is stored in skeletal muscles [88]. The principal role of glycogen in the muscle is to

produce energy during high level activity [89], whereas glycogen in the liver is utilized for

gluconeogenesis during fasting [77]. During glycogen synthesis, glucose taken up by the

cells is converted to glucose-6-phodphste, which is further converted into uridine

diphosphate (UDP)-glucose and finally to glycogen [25]. The enzymes hexokinase II and

GS are responsible for these processes, which are rate limiting in the synthesis of glycogen

[25]. Figure 7 shows the enhancing action of insulin on glycogen synthesis in primary

porcine cultures.

Other factors involved in the synthesis and regulation of glycogen in the muscles include

the auto-glucosylating protein; glycogenin, which is responsible for forming

oligosaccharide necessary for the further build-up of glycogen [90]. The glycogen

degrading enzymes; glycogen debranching enzyme and glycogen phosphorylase, which are


38

highly expressed in fast-twitched glycolytic fibers also regulate the synthesis of glycogen

[91].

b

*

Insulin concentration (nM)

0 1 10 100Fo

ld c

han

ge i

n g

lyco

gen

syn

thesis

(d

pm

/mg

pro

tein

)

0.9

1.0

1.1

1.2

1.3

1.4

1.5

a

ab

bc

cd

Figure 7: Glycogen synthesis is regulated by insulin (Adopted with modifications from M I,

Figure 4b)

Glycogen synthesis in primary porcine myotubes is enhanced by increasing amount of insulin. Data

points with different letters (a-d) denote significant difference.

1.7 β-oxidation

As skeletal muscles are not tuned for storing fats, they need a constant supply of energy,

which is provided by FAs generated from lipolysis in adipocytes. β-oxidation is the prime

route for the degradation of FAs [92] and is a core energy producing process of the cell,

which occurs in both the peroxisomes and mitochondria [92, 93]. Long-chain FAs once


39

transported into the cell for oxidation are directed towards the mitochondria, and these

processes are aided by transport and binding proteins [94-96]. The enzyme acyl-Coenzyme

A (acyl-CoA) synthase converts the FAs into acyl-CoAs, which are active intermediates of

FAs that are recognizable by enzymes involved in FA oxidation (FAO).

1.7.1 Acylcarnitine shuttling

The acyl-CoAs have to be shuttled into the mitochondria or the peroxisomes, where the

enzymes responsible for β-oxidation are situated [97]. L-carnitines (referred to hereafter

as carnitines) and their esters; acylcarnitines, are indispensable compounds in the β-

oxidation of FAs, with high levels present in the kidney, heart, and skeletal muscles [98,

99]. External sources of carnitines include dairy products and fish, but they can also be

produced endogenously by the kidney, heart and brain [99]. Carnitines are transported

into the cells by the aid of the sodium-dependent carnitine transporter; organic cation

transporter number 2 (OCTN2), and assist in the transportation of FAs by forming esters

of the FAs (acylcarnitines) that can easily be transported across the mitochondrial matrix

to undergo FAO [100]. See Figure 8 for a sketch describing acylcarnitine shuttling.

The esterification process is catalyzed by the enzyme carnitine-palmitoyl transferase I

(CPT I), using free Coenzyme A (CoASH), and occurs at the hydroxyl group of L-carnitine.

Unlike acetyl- and palmitoyl-transferases, the enzyme carnitine-acylcarnitine translocase

(CACT), has a wide specificity, ranging from free L-carnitine to acylcarnitine of all chain

lengths [101]. CACT assists in the transportation of acylcarnitines across the inner

mitochondrial matrix [101].


40

Once the acylcarnitine is inside the mitochondria, the enzyme carnitine-palmitoyl

transferase II (CPT II) helps in the release of L-carnitine and regeneration of acyl-CoA. The

reversible nature of the esterification of FAs with carnitines, not only enables

transportation of the FAs across the mitochondrial matrix, but also holds the content of

acyl-CoA in the cell at a non-toxic level [102, 103]. With the aid of short chain- acyl-

transferases, the carnitine system also assists in the transportation of short-chain FAs

from the peroxisome into the mitochondria [104, 105].

Figure 8: Acylcarnitine shuttling and FAO in the mitochondria (adopted with

modifications from [97])


41

Activated FAs in the form of acyl-CoAs are shuttled into the mitochondria in the form of

acylcarnitine and by the help of the enzymes CPT I, CACT and CPT II. Acyl-CoA dehydrogenases

and the trifunctional proteins execute a chronological degradation of the FAs to release acetyl-CoA

as the end-product of β-oxidation, which is shuttled into the kreb’s cycle

.

1.7.2 The β-oxidation enzymes

Many enzymes with specificity to either the long, medium, or short acyl-CoAs are involved

in the process of β-oxidation [106]; see Figure 9. Four main enzyme groups namely acyl-

CoA dehydrogenase (ACAD), enoyl-CoA hydratase (EH), 3-hydroxyacyl-CoA

dehydrogenase (HAD) and 3-ketoacyl-CoA thiolase (KAT), are responsible for the cyclic

degradation of acyl-CoAs by reducing a two carbon atom during each cycle [97, 107]. A

thorough review of ACADs has been achieved [108]. EHs, HADs and KATs are members of

the mitochondria trifunctional protein (MTP), which is a hetero-octomer consisting of four

α- and β- units, each [109]. The activities of EHs and HADs are situated within the α- unit,

while that of KAT is found in the β-unit [97, 110].

ACADs are known to exist in many forms in humans and depending on their chain-length

specificity to acyl-CoA, they are called short-chain acyl-CoA dehydrogenase (SCAD),

medium-chain acyl-CoA dehydrogenase (MCAD) or very long-chain acyl-CoA

dehydrogenase (VLCAD)[97]. ACAD are responsible for the first step of β-oxidation, where

FAD is necessary to dehydrogenate the long-chain acyl-CoA conjugate, producing a trans-

2-enoyl-CoA as intermediate [97] see Equation 1.

EHs exist in many forms in the mitochondria and include long-chain enoyl-CoA hydratase

(LCEH) and short-chain enoyl-CoA hydratase (SCEH), and their activities are different

from those in the peroxisomes [111]. LCEH and SCEH catalyze the addition of water to the


42

long-chain trans-2-enoyl-CoA and short-chain trans-2-enoyl-CoA substrates, respectively,

to generate L-3-hydroxyacyl-CoA [97, 112]; see Equation 2.

With regards to HAD, long-chain hydroxyacyl-CoA dehydrogenase (LCHAD) and short-

chain hydroxyacyl-CoA dehydrogenase (SCHAD) are two of the many forms that exist [97].

While LCHAD is specific only for long-chain L-3- hydroxyacyl-CoA, SCHAD has a broader

specificity and accepts substrates in the range of short chain (C4) to long chain (C16),

emphasizing on its importance in β-oxidation. HAD catalyzes the formation of 3-ketoacyl-

CoA from L-3-hydroxyacyl-CoA, using the cofactor NAD+ [113] see Equation 3.

Three types of KATs have been detected in the mitochondria and are responsible for the

conversion of 3-ketoacyl-CoA to an acyl-CoA that is short of two carbon atoms [114, 115];

see Equation 4. Medium-chain ketoacyl-CoA thiolase (MCKAT) effectively degrade

medium-chain FAs, as defects in this enzyme lead to fatal oxidation disorders [116]. Long-

chain ketoacyl-CoA thiolase (LCKAT) is crucial for the efficient degradation of LCFAs

[117], and utilizes substrates ranging from 3-ketohexanoyl-CoA to 3-ketopalmitoyl-CoA

[97]. The end-product of β-oxidation; acetyl-CoA, is either shuttled into the Krebs cycle to

produce ATP, or converted into acetylcarnitine and exported out of the mitochondria [97].


43

1.8 Peroxisome proliferator-activated receptors (PPAR)

The peroxisome proliferator-activated receptors (PPAR ) are members of the nuclear

receptor hormone family and consist of three isoforms (PPARα, PPARβ and PPARγ),

which control the expression of a well of genes by binding to peroxisome proliferator

response elements (PPRE) on the DNA strand [118]. Just like all nuclear receptors, the

PPARs consist of a DNA-binding domain (DBD) and a ligand-binding domain (LBD).

When activated by the binding of FAs or synthetic ligands to the LBD, PPAR forms

heterodimers with the retinoid X receptors (RXR), and this action causes their release

from the co-repressor [119]. The PPAR: RXR complex binds to PPRE via the DBD, and

initiates the expression of genes involved in lipid and glucose metabolism [120-122]. See

Figure 9 for a sketch of the mode of action of PPARs. Several unsaturated FAs, long-chain

FAs and branched-chain FAs are natural ligands of PPARs. As discussed earlier, PA is also

a natural ligand of the PPARs, especially PPARα [123-125].


44

Figure 9: Sketch of mode of action of PPARs (Adopted from [126] )

1.8.1 PPARα

PPARα is highly expressed in many organs, including skeletal muscle, liver, heart brown

adipose tissue and kidney [127]. PPARα activates FA uptake, transport and oxidation, as

well as regulates lipolysis and fat storage [128]. Attempts to use PPARα as targets of

treating insulin resistance and T2D have been made [129, 130]. Synthetic ligands of

PPARα include fibrates and the drug WY-14643 [131, 132], and they have been proven

effective to mend metabolic malfunction related to MS [133]. One possible mechanism of

action of PPARα is by a stimulation of glucose utilization in the muscles and adipose

tissue, resulting in a reduction in the glucose and insulin plasma levels [134, 135].

http://www.sciencedirect.com.ez.statsbiblioteket.dk:2048/science/article/pii/S104366180400194X


45

1.8.2 PPARβ

PPARβ is also involved in FAO [136]. Recent findings about PPARβ show that it helps

against heat injury in fibroblasts by increasing cell proliferation [137]. It has also been

shown to improve myogenesis through the down-regulation of myostatin expression [138],

and it is involved in the prostaglandin-induced activation of blastocyst development in

mice [139]. Synthetic agonists of PPARβ include GW501516, which has been reported to

inhibit dyslipidemia, by slowing down the activity of cholesterol ester transfer protein, and

reducing the content of very low-density lipoprotein [140].

1.8.3 PPARγ

PPARγ is mainly expressed in adipose tissues but it is also found in other tissues such as

the liver [127]. The isoform exists in two forms (PPARγ1 and PPARγ2), and it is PPARγ2

that predominates in adipocytes [141]. It activates protein synthesis and adiponectin

secretion in adipocytes, and it is crucial for efficient white and brown adipocyte

differentiation [142]. PPARγ also regulates lipogenesis in the liver [143] and increases

insulin sensitivity in adipocytes, thus, ameliorating the dysfunction of fat cells in T2D

[144]. In muscles, PPARγ also increases insulin sensitivity and improves FAO [121, 145].

Targeting PPARγ for the modulation of IR is common [145, 146]. The thiazolidinedione

(TZDs) are well known synthetic ligands of PPARγ, and they are important in the

treatment of T2D [147]. Some mechanisms employed by TZDs in improving IR are by

stimulating the production of adiponectin, which suppresses gluconeogenesis, and

reducing the level of circulating free FAs through their esterification to glycerol [148, 149].

Nevertheless, PPARγ has shown to induce the production of adiposites [150, 151].


46

1.9 Phytanic acid (PA)

1.9.1 Origin of PA and its diastereomers

The alcohol unit of chlorophyll; phytol, is the substrate for PA production (Figure 10). In

ruminants and in marine environments, oxidative processes of bacteria lead to the first of

the four steps in the degradation of chlorophyll; cleavage and oxidation of phytol from the

porphyrin ring of chlorophyll [155-157]. This step is the rate limiting step in chlorophyll

catabolism of chlorophyll and is controlled by the enzyme chlorophyllase [157]. The

generated phytol exists in two forms; E-phytol and Z-phytol, and when ingested by animals

or humans, can undergo a series of enzymatic reactions, including biohydrogenation, to

generate phytanic acid and afterwards, phytanoyl-CoA [158, 159], which can be

metabolized further. It was observed that E-phytol is the preferable substrate for the

PPAR-related metabolism of phytol [159, 160]. This conclusion was drawn from the fact

that E-phytol accumulates in the liver of PPARα-knockout mice fed phytol-rich diets [159].

More so, the enzymes responsible for phytol conversion to phytanoyl-CoA turned out to be

stereospecific [159]. It has also been shown that the aerobic and anaerobic activity of

bacteria on phytol generate both E- and Z-phytenic acid [161]. Thus, the role of bacteria in

the degradation of phytol is crucial, as these bacteria possess enzymes that are required for

the decarboxymethylation and oxidation of methyl-branched metabolites, without the

need of oxygen or the enzymes normally involved in methyl-branched FAs [162].


47

Figure 10: Production of PA from phytol (Adopted with modifications from [158])

The generation of Z- and E-phytol from chlorophyll requires bacterial action. Both forms of phytol

can be differentially processed to phytenic acid, which in turn, is converted to either RRR PA or

SRR PA by an enoyl-CoA reductase. The esterification of both forms of PA generates RRR- and

SRR-phytanoyl-CoA.

PA is 16 C- long and has four methyl groups positioned at carbons 3, 7, 11 and 15, with the

carbon 3, 7 and 11 being chiral centers. Carbons 7 and 11 have R configurations, just like in

phytol, while carbon 3 is either R- or S- configured owing to the biohydrogenation of


48

oxidized phytol. For this reason, PA occurs naturally as two diastereomers; SRR PA and

RRR PA [163]; see Figure 10. The SRR form has been shown to predominate in marine

animals while some terrestrial mammals have more of the RRR form [163-165]. Little is

known about the production and distribution of the two forms of PA in ruminal products

or about their physiological role. PA can be stored in the phospholipid and neutral

fractions of lipids in tissues and in milk [166]. Since humans lack the ability to cleave

phytol from chlorophyll [167], PA can only be obtained through the consumption of

ruminant or marine products [168, 169]. Differential deposition in lipid fractions of tissues

and metabolic rates of the diastereomers insinuates possible differences in the biological

activities of the two natural diastereomers of PA in humans [166, 170, 171].

1.9.2 Metabolism of PA in humans

After a phytol-rich meal, phytol is usually transported to the liver for catabolism, where

phytenic, phytanic and pristanic acids are formed [160]. Free phytol or PA taken up from

the diet can be converted into phytanoyl-CoA by the enzyme phytanoyl-CoA hydroxylase,

and further esterified and stored in lipids, hydrolyzed back to PA or used as a substrate for

alpha (α)- oxidation [172, 173]. Phytanoyl-CoA can also come directly from the diet, since it

is produced in ruminants. β-oxidation of PA is not directly possible due to the presence of

a methyl group on carbon 3 of PA ( see structure of PA in Figure 10).

The α- oxidation of PA occurs in the peroxisome [174] and the enzymes involved in the

process are illustrated in Figure 11. The actions of the hydroxylase and lyase enzymes on

phytanoyl-CoA and 2-hydroxylphytanoyl-CoA, respectively, are involved to produce

pristanic acid, with the release of formic acid as the primary product of α- oxidation [173,


49

175, 176]. Pristanic acid is further converted by a synthase reaction to pristanoyl-CoA, and

is used for β- oxidation. Phytanoyl-CoA and pristanoyl-CoA, thus, are the substrates for α-

and β- oxidations, respectively [173].

Another bottle neck in the degradation of PA is the presence of an R-form [177]. The

enzymes responsible for β- oxidation are stereospecific [178], and as such, an α-

methylacyl-CoA racemase (AMCAR) is required to convert 2R-pristanoyl-CoA to its 2S

isomer before it can be oxidized [93]; see Figure 11. At first, two cycles of β-oxidation, give

rise to 2, 6, 10 trimethylundecanoyl-CoA, which is R-configured. A racemase action

converts this product into its S-isomer, and a third β-oxidation is performed to generate 4

,8 dimethylnonanoyl CoA and short-chain acyl-CoAs [179]. These products of peroxisomal

β-oxidation are either hydrolyzed to acids (route 1) or esterified to carnitine esters by the

enzymes carnitine O-octanoyltransferase (COT) and carnitine acyltransferase (CAT), and

eventually transported to the mitochondria for further β-oxidation or processing (route 2)

[159, 173]. Besides acetate, propionate has been identified as the main degradation product

of PA oxidation [173, 180].

PA can also be degraded via omega (ω)-oxidation. For more information on ω-oxidation of

PA, see the reviews by Komen et al. (2004) [181] and Wanders et al. (2011) [182].


50

Figure 11: Peroxisomal α- oxidation and subsequent β-oxidation of PA (Adopted with

modifications from [173])

Phytanoyl-CoA and pristanoyl-CoA are the substrates for α- and β- oxidation, respectively.

Hydrolysis or esterification of the final peroxisomal oxidation products are transport to the

mitochondria for further processing.


51

1.9.3 Milk fat composition of PA as affected by cow feed

Organic farming practices and consequently milk fat composition vary with climate and

country [183]. More so, differential feeding strategies have been shown to improve the

quality of milk [184, 185]. Grazing or green grass feeding is an obligatory part of organic

farming in Denmark but other components of feed include maize and concentrates. Being

a by-product of chlorophyll, the content of PA in milk fat ought to correlate with greed feed

intake and has been reported so in various studies [169, 186]. As such, the content of PA in

organic milk has been proposed as a marker for organic dairy products [169]. In addition,

the distribution of PA diastereomers can be used for a better authentication of organic milk

[187]. It was recently shown that the distribution of PA diastereomers changes from more

to less RRR PA, when feeding shifts from green feed (organic) to concentrates

(conventional) [165, 188]. However, very little is known of the physiological roles of the

diastereomers of PA.


52

2 RESULTS AND DISCUSSION

In this section, the results regarding the use of primary porcine myotubes as a model for

skeletal muscles to study glucose and FA metabolism will be discussed. Furthermore, the

results of the effects of PA on the metabolic parameters, glucose uptake, glycogen

synthesis, and FA β-oxidation, shall be discussed in relation to glucose and FA

homeostasis. The effects of excess PA in humans shall be adressed and the impact of cow

feed on the content of PA in milk-fat will be elaborated. Finally, the metabolic implications

of the distribution of PA diastereomers in humans shall be discussed.

2.1 Primary porcine myotubes; a model of skeletal muscles, for studying

glucose and FA metabolism

The muscle is the major site for insulin-dependent glucose uptake [70, 189] and the initial

site in the generation of IR observed in T2D [189]. Therefore, the muscle represents an

ideal model to study the metabolic effects on glucose and FA homeostasis. The porcine

primary myotube model is similar to human myocytes in many ways [190], making it an

excellent choice. More so, because they are primary cells, they have not been modified in

any way other than enzymatic or physical dissociation from tissues, during purification.

Using well-established methods [191], SCs obtained from the semi-membranosus of

piglets were successfully proliferated and differentiated into multinucleated myotubes in

culture for use in this project (Figure 1, M I). As hypothesized, the porcine myotubes were

efficient for use in the analyses of glucose and FA metabolism.

In the analysis of glucose uptake, the myotubes were observed to harbor functional

insulin- mediated and non-mediated pathways, as revealed by their response to insulin


53

and by the inhibiting action of cytochalasin B in both pathways (Figures 2, M I). GLUT-1

and 4 were responsible for the basal and insulin-stimulated glucose uptake, respectively,

an observation that is common in human muscles [192], but the fact that 20 % of the

glucose taken up by the myotubes could not be accounted for by neither GLUT-1 nor

GLUT-4 denotes that there might be other transporters in the pig model that aid in the

uptake of glucose. Actually, GLUT-10, 11, and 12 are, for example, known to be expressed

in mammalian skeletal muscles, and may be involved in glucose uptake, as they bind to

glucose with different affinities [193-195]. So there is a possibility that these GLUTs are

also present in pigs. It should be noted that cytochalasin B independent glucose uptake of

about the same magnitude (20 %) has been observed before [196].

GLUT-1 and 4 each activated glucose uptake in the porcine myotubes by about 4o %

(Figure 2b, M I).The GLUT-4-mediated uptake seem low when compared to a study by

Nedachi et al. (2006), where 70 % of glucose uptake was insulin-mediated [70]. This

disparity could be due to the fact that Nedachi et al used a murine cell line for the glucose

uptake analysis, and furthermore, cell lines are known to produce more proteins and

GLUT-4 than primary cells, probably because cell lines do not age or become senescent as

normal cells [197].

Another explanation of the low level of insulin-mediated uptake registered could lie in the

fact that the myotubes generated for this study were isolated from the semi-membranosus

muscles (SM) of piglets, which consists predominantly of white-type fibers. White-type

fibers are known to be low in their contents of GLUT 4 and adiponectin [198, 199], both

factors of which are necessary for the insulin-mediation of glucose uptake in the muscles.

Actually, we have seen in our lab that red-type vastus intermedius muscles (VI) show 30 %

increased insulin-mediated glucose uptake, when compared to SM (unpublished data).


54

In muscles, up to 80 % of the insulin-activated glucose uptake is converted into glycogen

[200], underlining the importance of glycogen synthesis in the regulation of glucose

metabolism. In this study, the induction of glycogen synthesis in the porcine myotubes by

insulin was also effective (Figure 4b, M I), and hinted the up-regulation and activation of

GS [78]. In a previous study on insulin signaling in primary porcine myotubes, insulin

administration did not activate GS [201]. This controversial finding probably lies in

experimental methodology, rather than in the pig culture as a model, since cultures in the

latter example were exposed to supra-physiological concentrations of insulin for several

days before the analysis was performed [201]. The long-term exposure to insulin is the

likely cause of insulin-insensitivity, as hyperinsulinemia has been shown to cause IR [202].

More so, it is possible, that the chosen pigs for SC isolation was generally insulin

insensitive, especially as glycogenin which is also a rate limiting factor in glycogen

synthesis [203], was unaffected in the study [201]. We have seen in our lab that myotubes

isolated from different pigs vary in their insulin-regulated glucose uptake, with some

myotubes being completely insulin-resistant (unpublished data).

The porcine myotubes were also able to metabolize FAs. Their regulation of FA metabolism

seemed to lie in both the areas of uptake and oxidation of FAs. This is because the same

conditions that caused the generation of acetylcarnitine (C2); the end product of β-

oxidation, did not change or even reduced the degradation of palmitoylcarnitine (C16)

(Figures 4a and b, M II).

The myotubes could also be rendered resistant in both the uptake of glucose (Figure 5a, M

I) and the oxidation of FAs (Figure 3c, M II) using high concentration of glucose and

palmitate, respectively; a feature that is characteristic of human muscle cultures.


55

2.2 The metabolic impact of PA

During glucose uptake analysis, it was shown that physiological amounts of PA enhanced

glucose uptake in the primary porcine myotubes, especially at low concentrations of

insulin (Figure 4a, MI), while further increments in the concentration of PA did not further

improve glucose uptake (Table 2, MI). This finding revealed that PA and insulin may have

similar mode of action in activating glucose uptake, as a combination of both compounds

did not have any synergistic effect on the activation of glucose uptake. In a human

intervention study, where PA-rich dairy fat was administered, there were no significant

changes in the risk markers of MS (Cholesterol, triglycerides, C-reactive protein, serum

insulin and serum glucose) [204]. Especially the observation of an unchanged serum

glucose level after the intake of PA-rich fat [204], was controversial to the results in our

project, and could be attributed to other involved factors that are present at the organism

level. As shown by the involvement of many tissues in IR [25, 32], the regulation of glucose

involves many organs, and studies of the metabolic effect of PA in an organism, as a whole,

presents an intermix of many pathways whose results can be different from those obtained

from studies in single tissue (myotubes).

It was observed that the time-dependent activation of glucose uptake by PA was biphasic,

as incubation of myotubes with PA for 4-8 h turned to increase glucose uptake, while

incubation for 12-16 h reduced glucose uptake and by 24-h incubation, there was an

increase again in glucose uptake (Figure 3a, MI). It was suggested that depletion of GLUTs

was the reason for the drop in glucose uptake, but given that the preceding rise in glucose

uptake was mild, other factors must have been involved in the biphasic effect.

PA-induced glucose uptake was significant after 60 min of incubation with glucose,

although at this time, the glucose uptake process seemed saturated (Figure 3b, MI);


56

glucose uptake during the first 15 min was steepest when compared to the last 15 min. This

observation showed that other factors are involved in the regulation of glucose uptake,

when PA is involved.

We showed that the glucose taken up by the myotubes in the presence of PA could not be

incorporated into glycogen, unless insulin was available (Figure 4b, MI). This observation

insinuates that the mechanism, of which PA activates glucose uptake, is different from that

which regulates glycogen synthesis. Thus, PA regulates glucose homeostasis in both an

insulin-dependent and insulin-independent manner. Our results also show that pathways,

other than glycogen synthesis are involved in the metabolism of the PA-incorporated

glucose.

When the myotubes were rendered insulin resistant by exposure to excess glucose (Figure

5b, MI), it was not possible for PA or insulin to stimulate glucose uptake or glycogen

synthesis (Figures 5c and d). One could, therefore, suggest PA to be beneficial in the

regulation of glucose in normal individuals, who generally have very low or insignificant

content of PA in their serum, or in persons that have inadequate amounts of insulin in

their serum.

During the analysis of β-oxidation in the myotubes using acylcarnitine profiling, PA

enhanced the content of C2 generated in the myotubes, as well as reduced the content of

C16, as shown by a reduced C16/C2 level (Figures 5a and b, MII). These findings denote

that PA, possibly in a PPAR-manner [133, 205, 206] improves β-oxidation by both

enhancing the degradation of C16 and increasing the content of C2.

The effect of PA on β-oxidation was not dose-dependent but longer incubation periods

with PA increased β-oxidation. During 4 h incubation of myotubes with PA, no change was


57

observed in the content of C16 (shown as C16/C2) and C2, but after 24 h incubation, the

content of C2 increased as the level of C16 fell and the tendency continued even when the

exposure to PA was increased to 48 h (Figure 5a and b, MII). PA has been shown to

improve FA uptake [207], which could be a reason for the unchanged level of C16, during 4

h incubation. To concretize the time-dependent effect, a lower concentration of PA (5 µM ),

which could not reduce the content of C16 or increase the level of C2 during exposure for 4

h, actually reduced and increased the levels of C16 and C2, respectively, after exposure for

48 h. These findings insinuate that PA needs time (possibly to generate more potent

metabolites [208]) to exert its effect on β-oxidation.

The fact that 5 µM PA exposed to the myotubes for 24 h caused a reduction in the content

of C16, but not an elevation of C2, suggests that PA might affects β-oxidation at different

levels and different magnitudes. Experiments with oleic acid (OLA); a saturated FA that

has been shown to have PPAR characteristics [209], revealed that it also improves β-

oxidation, at least at two levels, just like PA; at the level of uptake of FAs into the cell

and/or mitochondria, and at the level of β-oxidation of FAs. The regulation by PA, of

intermediate carnitines at the level of acyl-dehydeogenases, carnitine acyl-transferases and

MTPs cannot be excluded as a means of regulating FAO. In fact, the metabolism of PA is

known to up-regulate octanoyl-transferase [210], and possibly propionyl-transferase, given

that propionate is one of the main products of PA degradation [104, 173]. Octonoyl-

transferases have been implicated in the induction of β-oxidation of VLCFA and MCFA

[211].

Despite the observations regarding the induction of β-oxidation by PA, we could not

unequivocally conclude if it was via a PPAR-manner, since a PPAR control agonist was not

included in the study. Thus, the use of a positive control, for example a PPARα agonist


58

such as bezafibrates or the well-known synthetic agonist; WY-14643 [133, 205, 212], is

necessary to validate the results.

2.3 Concerns regarding excess content of total PA in humans

It is a desire to increase the total content of PA in milk fat, to achieve levels that are

physiologically advantageous. The PA content in normal human subjects that consume

ruminant products has been documented to range from 0.04-11.5 µM, and consumers of

non-ruminant products have negligible amounts of PA in their plasma, compared to

consumers of ruminant products, whose total content of PA is in the upper range [204,

213, 214]. In our studies, 10 µM PA was utilized as standard concentration as it lies within

the physiological range. More so, the viability of the myotubes were negatively affected

when the concentration of PA was over 10 µM (table 1, M I), and results from FAO

experiments with 20 µM PA where not generally better, when compared to results

achieved with 10 µM (Figure 5 b and c, M II).

2.4 PA content in milk-fat as affected by cow feed

As shown in Figure 1, M III, two naturally-occurring stereomers of PA were identified in

milk-fat, with the SRR form dominating, just as in previous findings [165]. Milk-fat

produced organically or even by conventional feeding has been shown to have higher

contents of PA than in our study [187, 188]. This discrepancy in the content of PA lies in

different farming practices between farms or regions/countries.


59

Surprisingly, we found that an increase in the total content of PA was attributed to feed

concentrates, while the reverse was attributed to the content of pasture in feed (Table 2, M

III). These correlations were, however not very strong and could be attributed to random

variation. Nevertheless, our finding could actually explain the difference in PA contents

between the grazing seasons. The total content of PA was higher in September than in

May, and this is probably because the use of concentrates was generally augmented in

September to compromise for the weather-mediated low dry matter intake (DMI) from

grazing in September, and not because of the content of legumes that constituted a larger

part of pastures in September. Nevertheless, it could be that the availability of light during

the May season (there is generally more light in the May than September season), also

affected the results. The degradation of chlorophyll in leaves have been shown to be

enhanced by photo-oxidation [215]. In this case, the production of phytol should be

enhanced and, thereby generating more PA during the May season. But given our findings,

it is obvious that other factors (e.g. the developmental state of the plants) affect the

production and content of PA in milk-fat more.

We suggested that the use of grass pellets in concentrates was the cause for the elevated

content of total PA, since grass feed has been shown to generate higher contents of PA

[186]. Actually, dried grass has been shown to increase the degradation of chlorophyll in

sheep rumen [216]. As PA can only be produced from free phytol, chlorophyll degradation,

therefore, will increase the production of PA.

The inverse correlation of pasture to the total content of PA observed in our studies

contradicts previous conclusions that green feeding increase the content of PA [169, 217].

In our experiments, pasture was dominated mostly by legumes (ryegrass and white clover).

Howard et al. (1993) found that legumes like soya beans present a “stay green”


60

phenomenon, whereby chlorophyll degradation is resisted [218]; maybe this phenotype is

common for the legumes in the pasture used in our study, and would thereby explain the

decrease in the total content of PA recorded. Nevertheless, as we found no correlation

between the total content of PA and legumes, it is obvious that other unknown factors are

involved in the production of PA. A study conducted by Lee et al. (2006) showed that the

total content of PA in duodenal flow was reduced in line with a reduction in

biohydrogenation [186]. Although this trend was affected by the intake of red clover, it is

likely that, partly the influence of legumes on biohydrogenation is causing the reduction in

the total content of PA after pasture intake, observed in our study.

Many factors other than proteolytic activities in the pasture might alter ruminal micro

flora [219], and, thus, responsible for driving the mechanisms behind our observations of

altered PA content with feed. The rate limiting step in the catabolism of chlorophyll, which

generates free phytol, is controlled by the chlorophyllase and has been showed to be post-

translationally regulated [157], possibly by a well of factors, some of which can originate

from the rumen of the cow. In that case, the feed can have an influence on chlorophyllase.

Phytol exists in two forms (E- and Z- form) and the degradation and production of each

form from chlorophyll depend on the bacteria community [161, 220], whose population

and action can be affected differently by the feed of the cow. Moreover, the enzymes

involved in the degradation of phytol are stereospecific, favoring the degradation of E-

phytol, and these enzymes can be altered by the feed [159].

The values of the total content of PA obtained in our study do not necessarily indicate the

lack or limited use of green feed in organic farming, but merely indicate that the type of

(green) feed can alter the acquired content of total PA in milk-fat. It is apparent, therefore,


61

to suggest that it is the feed composition that in many levels and ways controls the total

content of PA.

To support this suggestion, we also showed that although pasture did not affect the total

content of PA, the increasing content of legumes in the pasture caused an elevation in the

content of RRR PA (Table 2, M III). Schröder et al. (2011 and 2012) also reported that

organic feeding, which is dominated by green feed (grass silage) or conventional feeding

rich in concentrates, increased the content of SRR PA in milk-fat, while organic feeding

dominated by hay feeding increased the content of RRR PA instead [187, 188]. Likely

reasons for the altered content of PA diastereomers are similar to some of those that

caused an alteration in the total content of PA; namely, the altered distribution or action of

ruminal micro flora bacteria in response to feed, and also the stereospecificity of

degradation enzymes. As mentioned earlier, β-oxidation enzymes prefer SRR-pristanic

acid (generated from SRR PA) as substrate, and in that case, one would expect to have

different contents of the diastereomers in milk. More so, effects on AMCAR (enzyme

necessary to convert RRR PA to SRR PA) by feed may be possible.

2.5 Concerns regarding the distribution of PA diastereomers in

humans

The notion to elevate the total PA content of milk fat was borne with little knowledge of the

distribution of the individual stereomers of PA and what impact the diastereomers might

have on the physiology. Studies carried out by Tsai et al. (1973) showed that SRRR PA

instead of RRR PA was preferred for oxidation [170], but this conclusion was drawn from

experiments with very high concentrations of PA, like those found in a diseased state, and


62

revealed nothing about the PPAR-effects of PA. Nevertheless, Heim et al. (2002) later

showed that both forms of PA affected PPARs equally [124], but this study was performed

just in one tissue; the liver. Results from other tissues or organs would give more

convincing observations and information on the effects of PA diastereomers.

The knowledge that β-oxidation of FAs is stereospecific [178] with preference towards the

SRR form generates interests in the diastereomatic distribution of PA in milk fat. Because

of a methyl side chain on position C3, the metabolism of PA occurs through α- oxidation in

the peroxisome, resulting in a more potent PPAR-inducer; pristanic acid [208]. Pristanic

acid existing in the RRR-form has to be converted by AMCAR to the SRR form, for it to be

β-oxidized. These bottle-necks in the metabolism of PA create a scenario with regards to

what feeding strategy to embark on. On the one hand, feed composition that will give a

higher ratio of SRR/RRR PA could be thought desirable, as its metabolism would be

preferably faster and less dependent on AMCAR. On the other hand, it is hard to conclude

that milk-fat with higher RRR/SRR PA ratio is undesirable; one can speculate that a slower

degradation of RRR PA may cause an increase in the level of pristanic acid, and since

pristanic acid is a more potent PPARα agonist than PA, the expression of PPARα would be

up-regulated. However, it is important that the elevated level of PA or pristanic acid is

within a physiological range.


63

3 CONCLUDING REMARKS

As illustrated in Figure 12, we show in this study that manipulating the feeding strategy of

the cow can alter the total content and distribution of PA diastereomers in the milk-fat of

the cow. More so, we could show that the exposure of a physiologically relevant

concentrations of a mixture of PA isomers induced glucose uptake and β-oxidation in

primary porcine myotubes, which we established as an efficient skeletal muscle model.

However, the mechanisms behind the control of glucose and FA metabolism by PA remain

to be investigated.

Figure 12: An illustration of the effects of cow feed on the total content of PA and its

diastereomers, and the metabolic impact of PA on skeletal muscles


64

4 PERSPECTIVES

During glucose uptake analysis, we found that PA at physiological concentrations

enhanced glucose uptake but not glycogen synthesis. It would, thus, be relevant to

investigate the fate of the glucose after uptake and whether other metabolic pathways of

glucose, than glycogen synthesis are affected by PA. While PA, in the presence of insulin,

generated a synergistic effect on the activation of glycogen synthesis, no such effect was

observed during glucose uptake. These observations insinuate that more than one

mechanism is involved in the PA-mediated metabolism of glucose, and needs to be

analyzed further.

PA was observed to enhance the degradation of C16 and increase the production of C2.

However, a positive PPARα control, such as bezafibrates, needs to be investigated in

parallel with PA, before we can conclude on what mechanism is responsible for this effect.

More so, the effect of PA on intermediate acylcarnitines is relevant to get an overview of

the general impact of PA on FA metabolism.

In our studies, synthetic PA was used. Given that this product is a mixture of up to eight

isomers of unknown distribution, it is hard to tell whether our in vitro finding concerning

the metabolic impact of PA could have been better, if a pure PA product consisting only of

the two naturally known isomers had been used. Thus, studies with the purified naturally

occurring forms of PA are of interest. The finding that RRR PA can be enhanced by cow

feed makes it even more relevant to elucidate the metabolic effects of this form. Also,

analysis of other tissues than muscles would give more information about the distribution

and effects of PA diastereomers.


65

Gene expression experiments and protein determination of specific markers of glucose

uptake and FAO are necessary to fully concretize the findings in our studies. In this regard,

human muscle cell lines could be used as a supplement to the primary porcine model,

since cell lines generate enough proteins for biochemical and histological studies. More so,

the human muscles express uncoupling proteins that regulate thermogenesis, and this

process has been shown to be regulated by PA. Even more interesting is the fact that the

use of cell line would exclude constrains of dealing with variation between pigs, although

this factor is a natural aspect that needs to be kept in mind when dealing with cell lines.

Which of the natural forms of PA, that influence this metabolic process could be of

relevance.

In our analysis with Jersey and Holstein cows, we could not link breed to the total content

of PA or distribution of its diastereomers. As the composition of feed intake varied between

breeds, one can speculate whether the conclusion would have been the same, had it been

the same variation in feed composition applied to both breeds. Such an experiment could

be of relevance.

Drawing from the observations in this project, the desired content of PA and distribution

of its diastereomers in milk-fat can be achieved by manipulating the composition of the

cow feed. Therefore, it will be interesting to analyze the impact of the legumes and other

feed components commonly used in Denmark, on the total content PA and its

diastereomatic distribution. Analysis from the level of chlorophyll degradation, through

the breakdown of the two forms of phytol, micro flora involvement, and ruminal

production and degradation of the two forms of PA would be relevant.


66

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213. Al-Dirbashi OY, Santa T, Rashed MS, et al. Rapid UPLC-MS/MS method for routine analysis of plasma pristanic, phytanic, and very long chain fatty acid markers of peroxisomal disorders. Journal of Lipid Research 2008; 49: 1855-62.

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83

6. APPENDIX

6.1 Manuscript I; MI


84

Phytanic acid stimulates glucose uptake in a model of skeletal

muscles, the primary porcine myotubes

Brita N. Che1, Niels Oksbjerg1, Lars I. Hellgren2, Jacob H. Nielsen3, Jette F. Young1*

1: Department of Food Science, Aarhus University, Blichers Allé 20, 8830 Tjele, Denmark

2: Department of System Biology, Technical University of Denmark, 2800 Kgs. Lyngby,

Denmark

3: Arla Foods, Sønderhøj 14, DK-8260 Viby J, Denmark

*Corresponding author: Jette Feveile Young

[email protected]

Tel: +45 87 15 80 51

Fax: +45 87 15 48 91

mailto:[email protected]


85

Abstract 1

Objectives: Phytanic acid (PA) is a chlorophyll metabolite with potentials in regulating 2

glucose metabolism, as it is a natural ligand of the peroxisome proliferator-activated 3

receptor (PPAR) that is known to regulate hepatic glucose homeostasis. This study aimed 4

to establish primary porcine myotubes as a model for measuring glucose uptake and 5

glycogen synthesis, and to examine the impact of physiological amounts of PA on glucose 6

uptake and glycogen synthesis either alone or in combination with insulin. 7

Methods: Porcine satellite cells were cultured into differentiated myotubes and tritiated 8

2-deoxyglucose (2-DOG) was used to measure glucose uptake, in relation to PA and 2-DOG 9

exposure times and also in relation to PA and insulin. The MIXED procedure model of SAS 10

was used for statistical analysis of data. 11

Results: PA increased glucose uptake by approximately 35%, and the presence of insulin 12

further increased the uptake, but this further increase in uptake was non- additive and less 13

pronounced at high insulin concentrations. There was no effect of PA alone on glycogen 14

synthesis, while the insulin stimulation of glycogen was increased by 20% in the presence 15

of PA. PA neither stimulated glucose uptake nor glycogen synthesis in insulin-resistant 16

myotubes generated by excess glucose exposure. 17

Conclusions: Primary porcine myotubes were established as a model of skeletal muscles 18

for measuring glucose uptake and glycogen synthesis, and we showed that PA can play a 19

role in stimulating glucose uptake at no or inadequate insulin concentrations. 20

Keywords: Phytanic acid, palmitic acid, insulin, glucose uptake, glycogen synthesis, 21

viability, primary porcine myotubes, excess glucose, free fatty acids 22

23


86

Background 24

Due to its high concentration of saturated fatty acids, dairy fat intake has been associated 25

with increased risk of cardiovascular diseases and type 2 diabetes in humans [1,2]. 26

However, recent meta-analysis of data from prospective cohort-studies shows an inverse 27

association between intake of dairy products and both cardiovascular diseases and 28

incidence of type 2 diabetes [3], and available data do not indicate any increased risk of 29

high-fat dairy products compared to low-fat dairy products [4]. Similarly, studies using the 30

odd-chained fatty acids C15:0 and C17:0 as validated biomarkers of dairy fat intake showed 31

that a higher intake of dairy fat was associated with a decreased risk of myocardial-32

infarction and development of the metabolic syndrome [5-8]. These findings suggest the 33

existence of a milk-fat paradox; despite the saturated profile of milk-fat, it does not seem 34

to increase metabolic risks, but might actually be protective. Hence, further research is 35

necessary to elucidate this controversy and to identify potential components in dairy fat 36

that might be responsible for the protective effect. 37

Milk is a very rich source of potentially bioactive lipids such as short-chained fatty acids 38

and conjugated linoleic acid (CLA) [9,10]. Another fatty acid of interest in milk is phytanic 39

acid (PA); a C20 saturated fatty acid with four methyl-branches, which is known to be a 40

natural ligand of the nuclear receptors peroxisome proliferator- activated receptors 41

(PPAR) and retinoid-X- receptors (RXR) [11-13]. The PPARs are lipid sensors, which act by 42

regulating body glucose and lipid homeostasis [14], and it has been shown that PA 43

enhances glucose uptake in rat hepatocytes, possibly in a PPAR-dependent manner, at 44

least at high concentrations [15]. Based on these observations, it was suggested that PA 45

might improve glucose- homeostasis, and thereby protect against the development of the 46

metabolic syndrome [16]. Dairy products or meat from ruminants are the major sources of 47


87

PA in the human diet [17]. The concentration of PA in plasma from healthy humans vary 48

between 0.04 and 11.5 µM [18], and is strongly dependent on the intake of ruminant 49

products [19]. 50

Skeletal muscle fibers are the major site for insulin-regulated glucose uptake, accounting 51

for over 70% of glucose disposal after a meal [20]. Furthermore, excess glucose and free 52

fatty acid exposure lead to insulin resistance in these muscle fibers [21,22]. Hence, effects 53

of PA on skeletal muscle glucose metabolism are expected to have an impact on total 54

glucose homeostasis at the organism level. The main aim of this study was therefore to 55

establish primary porcine myotubes as a model for glucose uptake and glycogen synthesis, 56

and to examine whether physiological concentrations of PA alter glucose uptake and the 57

rate of glycogen synthesis in primary porcine myotube cultures, either alone or in 58

combination with insulin. 59

60

Results 61

Viability assay 62

Primary porcine myotubes were incubated with doses of PA ranging from 1-500 µM (table 63

1). The viability of the myotubes was not affected by 1-10 µM PA. Exposure of the myotubes 64

to 20 µM PA markedly reduced their viability by about 20%. This viability dropped by 36% 65

after exposure to 500 µM PA. For comparative reasons, the effect of palmitic acid (PAM) 66

on viability was also assayed (table 1). Exposure of myotubes to 1 or 10 µM PAM had no 67

effect on viability while 50 µM PAM reduced their viability by about 15% and by 500 µM 68

PAM the viability of the myotubes were reduced to only 54 %. 69


88

Optimization in relation to glucose uptake assay 70

Glucose uptake in primary porcine myotubes was insulin-dose dependent (fig 2a). At 0.1 71

nM insulin, a significant increase in glucose uptake was recorded and at a 72

supraphysiological concentration of 500 nM insulin, a 70% increase in uptake was 73

obtained. To verify the presence of carrier-mediated glucose uptake in the primary porcine 74

myotube model, different doses of cytochalasin B (cyto B) (0-50 µM) were added to the 75

differentiated myotubes (fig. 2b). At 10 µM cyto B, the glucose uptake in myotubes with 76

and without insulin was reduced to 54 and 40%, respectively, when compared to that of 77

control cells without insulin or cyto B addition. In the presence of 20-50 µM cyto B, the 78

glucose uptake was reduced to approximately 70-90% of controls without cyto B, 79

irrespective of insulin addition. 80

Glucose uptake as a function of PA incubation time and 2-DOG incubation 81

time 82

Based on the results from the viability studies and the concentration of PA in human 83

plasma, glucose uptake assays were performed in myotubes that were incubated with 10 84

µM PA over a period of 24 h to define an effective incubation time with PA (fig. 3a). No 85

significant increase in uptake was observed after 1 h incubation with PA. There was, 86

however, a tendency of elevated glucose uptake between 4-8 h of incubation with PA 87

followed by a temporary marked decrease between 12-16 h of incubation, when compared 88

to 1 h of incubation. 89

To estimate an optimal incubation time for 2-DOG to achieve effective uptake, myotubes 90

were exposed to 2-DOG for 5-60 min with or without 10 µM PA (fig. 3b). There was a 91

general, steady increase in glucose uptake with increasing time of incubation with 2-DOG. 92


89

The steepest increase in 2-DOG uptake was observed during the first 15 min, while the 93

process was slowest during the last 10 min of incubation. In the presence of PA, glucose 94

uptake tended to be higher at all the different incubation times, but this difference was 95

significant only after 60 min of incubation. 96

Glucose uptake in response to PA and PAM 97

When testing the effect of PA on glucose uptake (table 2), a 23% increase in uptake was 98

noted even at the sub-physiological concentration of 0.5 µM PA. Increasing the PA 99

concentration further did not significantly increase the uptake, although average uptake 100

increased to between 30-35%. The effect of 1-10 µM PA could not be mimicked by same 101

concentrations of PAM (table 2). However, when myotubes were exposed to higher doses 102

of PAM, there were marked changes in glucose uptake. Glucose uptake dropped to 75% 103

when the myotubes were exposed to 100 µM PAM and at 200 µM and 400 µM PAM, the 104

glucose uptake ability of the myotubes was only 50 and 30%, respectively, when compared 105

to controls 106

Glucose uptake and glycogen synthesis with or without PA as a function of 107

insulin 108

The impact of PA on glucose uptake and glycogen synthesis was also studied in relation to 109

various insulin concentrations. Addition of 10 µM PA caused a further increase in glucose 110

uptake at all levels of insulin(fig.4a), but a significant improvement of glucose uptake by 111

addition of PA was only achieved at lower insulin concentrations (0 and 0.1nM). The 112

incorporation of glucose into glycogen increased in response to insulin treatment from 0-113

100 nM, with a significant increase at 10 and 100 nM insulin (fig.4b). Overall, glycogen 114

synthesis was not significantly affected by PA alone, regardless of the increase tendency. 115


90

However, in the presence of 10 nM insulin, PA caused an additional increase in glycogen 116

synthesis of about 20%. 117

Exposure of myotubes to extracellular glucose 118

The viability of the myotubes after exposure to excess glucose (12mM) was not significantly 119

affected (fig. 5a). However, addition of 7, 10 and 15 mM of extracellular glucose reduced 120

glucose uptake to about 80, 65 and 23%, respectively, when compared to myotubes 121

exposed to 4 mM of extracellular glucose (fig. 5b). Higher amounts of glucose did not cause 122

further drops in glucose uptake. While 10 µM PA and10 nM insulin caused an approximate 123

40 and 75% increase, respectively, in glucose uptake in myotubes exposed to 6 mM 124

extracellular glucose, no effect of neither PA nor insulin was observed when myotubes 125

were exposed to 14 mM extracellular glucose (fig. 5c). Glycogen synthesis was reduced to 126

25% when myotubes were treated with 14 mM glucose, compared to myotubes treated with 127

6 mM glucose (fig. 5d). The marked effect of 10 nM insulin on glycogen synthesis in the 128

presence of 6 mM glucose was abolished by exposing the myotubes to 14 mM glucose (fig. 129

5d). 130

131

Discussion 132

Phytanic acid (PA) is a natural ligand and activator of the PPARs, specifically PPAR-α and 133

γ [23]. The PPARs have been implicated not only in the regulation of adipose tissue 134

development and insulin signaling [24] but also in modulating insulin sensitivity of 135

muscles [25,26] and increasing exercise endurance [27]. It has been hypothesized that PA 136

can function as a natural PPAR agonist and be useful in the prevention and treatment of 137

diabetes [28]. Hence, PA may have a positive influence on the regulation of glucose 138


91

metabolism. As skeletal muscle is the major site of insulin-dependent glucose uptake [20], 139

we established a primary myotube model to study the effect of PA on glucose-homeostasis. 140

The porcine primary myotube model shares many similarities to the human myocytes and 141

has fewer artifacts when compared to immortal cell line systems [29]. Satellite cells were 142

grown (fig.1 a and c) and successfully differentiated into characteristic multinucleated 143

myotubes (fig.1 b and d), just as in previous findings [30]. 144

Initially, the suitability of the primary porcine myotube model was tested, and assay 145

conditions optimized in relation to both glucose (2-DOG) and PA exposure. Viability 146

assays were performed to determine an appropriate concentration of PA that did not 147

compromise the cells metabolic activities. The viability of the myotubes was noticeably 148

reduced when exposed to concentrations of PA equal to or above 20 µM (table 1). The toxic 149

effect of PA at 20 µM or above may be attributed to apoptosis of the cells, as shown 150

previously by studies in human skin fibroblasts and rat liver [31,32]. The average 151

concentration of PA in human plasma has been reported to be between 0.04-11.5 µM 152

[18,19,33], with higher values found predominantly in meat eaters and dairy product 153

consumers [18,19]. Based on this, and the results from our viability studies, the working 154

concentration of PA was chosen as 10 µM. Palmitic acid (PAM); a fatty acid of same chain 155

length as PA but without the branching methyl-groups was used as a control, and we 156

showed that 1 and 10 µM PAM had no effect on myotube viability. At concentrations of 100 157

µM PAM and above, the viability of the myotubes were reduced, possibly in a manner 158

similar to that of higher amounts of PA [31,32]. 159

The non-metabolizable analogue of glucose; 2-deoxyglucose (2-DOG), is a suitable marker 160

for the measurement of muscle glucose transport [34], and it was used in this study to 161

measure glucose uptake. Glucose transport in the muscles is insulin-dependent and 162


92

mediated by GLUT-4 transporters, as well as insulin-independent, mediated by GLUT-1 163

[35]. Insulin treatment of varying concentrations caused a dose-dependent increase in 164

glucose uptake in the differentiated myotubes cultures (fig. 2a), proving that the primary 165

porcine myotubes have a functional insulin-stimulated GLUT-4 translocation to the 166

plasma membrane [36]. The action of both GLUT-1 and GLUT-4 in the carrier-mediated 167

glucose uptake was checked by treatment of the myotubes with cytochalasin B (cyto B); a 168

drug known to inhibit glucose transport by binding to glucose transporters with a higher 169

affinity than glucose [37,38]. From our observation, cyto B inhibited both the insulin- and 170

non-insulin-mediated glucose uptake to a similar magnitude in primary porcine myotubes 171

(fig. 2b). The reduction in glucose uptake by cyto B confirms that both the insulin- and the 172

non-insulin-mediated transport systems are functional in our primary porcine myotube 173

model. Our observation with cyto B also showed that about 20% of glucose uptake in the 174

myotubes was not affected by cyto B. One can speculate that a minimal steady state of non-175

specific glucose uptake is maintained in the porcine cultures. It is also likely that other 176

GLUT receptors than GLUT 1 and 4 are present in the myotubes [39], and cyto B cannot 177

bind to them. In that case glucose transport is possible even in the presence of cyto B. 178

A biphasic effect on glucose uptake was observed during the 24 h incubation of myotubes 179

with 10 µM PA (fig. 3a). Since only a subtle increase in glucose uptake was noticed after 8 h 180

exposure to PA, it is less likely that the subsequent fall in glucose uptake is due to depletion 181

of GLUT depots. Other unknown factors could, thus, be responsible for this drop in glucose 182

uptake. A rise in glucose uptake at 24 h could be linked to consequent up-regulation of 183

transcripts responsible for the translation of proteins that skew up glucose uptake. 184

Glucose uptake has been shown to be linear for over 20 min in a human skeletal muscle 185

cell line study [40], and in the present study, the steepest increase in glucose uptake was 186


93

observed during the first 15 min of incubation with 2-DOG and by 45 min, the curve 187

started to level off (fig. 3b). It is possible that after 45 min incubation with 2-DOG, a 188

partial saturation of the normal glucose pathway is attained. Following these observations, 189

the incubation time with 2-DOG was set at 30 min. 190

Considering the effects of PA on glucose uptake, it is noteworthy that a concentration as 191

low as 1 µM caused a 25% increase in glucose uptake. Increasing the PA concentration to 192

10 µM slightly increased glucose uptake further (table 2), while equivalent amounts of 193

PAM had no effect on glucose uptake. The effect of PA was, thus, a specific effect, which is 194

in agreement with a similar study in rat hepatocytes, in which 100 µM PA markedly 195

increased glucose uptake following incubation with 2-DOG, when compared to 196

docosahexaenoic acid [15]. The reduction in glucose uptake observed at PAM 197

concentration of over 100 µM (table 2) was partly due to inhibition of glucose transport 198

activity [41], but also most likely a consequence of stress [31]. 199

The effect of PA diminished with increasing insulin stimulation of glucose uptake (fig. 4a). 200

This could indicate a similar mode of action between insulin and PA. Our results also 201

indicate that the PA concentrations normally found in plasma (> 1 µM), are sufficient to 202

enhance non-insulin dependent glucose uptake and that alteration of plasma PA 203

concentrations within normal physiological range, only have minor effects. Hence, we 204

suggest that normal levels of PA is a relevant factor in controlling fasting glucose 205

homeostasis in vivo and that increased intake of PA only will affect glucose uptake in 206

subjects with extremely low PA-levels (for example strict vegetarians with no intake of 207

dairy products or ruminant meat). 208

Despite the fact that 10 µM PA induced an increased glucose uptake in the absence of 209

insulin (fig 4a), it did not enhance the rate of glycogen synthesis (fig. 4b). Thus, PA at 210


94

physiological concentrations increases glucose uptake in the absence of insulin, without 211

causing a concomitant increase in glycogen synthesis, why the absorbed glucose must be 212

shunted into other metabolic pathways. However, the combination of 10 µM PA and 10 nM 213

insulin more than doubled the rate of glycogen synthesis, compared to the effect of insulin 214

alone (45% vs. 20%). This creates a somewhat paradoxical situation that increasing insulin 215

concentrations attenuate the effect of PA on glucose uptake, but enhance the effect on 216

glycogen synthesis. Hence, the two effects must be explained by different mechanisms of 217

action of PA. It should be noted, that the effect of PA on both glucose uptake and glycogen 218

synthesis is not a general effect of fatty acids, as the same concentration of PAM did not 219

cause these effects, neither with nor without insulin (data not shown). 220

Previous studies have shown that excess glucose causes glucose toxicity that is manifested 221

by the inhibition of glucose uptake and induction of insulin resistance in the muscles [42]. 222

The toxicity of excess glucose is due to the activation of the hexamine pathway and 223

production of glucosamine, which inhibits insulin-stimulated glucose uptake and 224

subsequently glycogen synthesis [43]. Our observations that increasing amounts of glucose 225

inhibit glucose uptake (fig. 5b and c) and glycogen synthesis (fig. 5d) are in agreement with 226

these findings. Excess glucose did not affect the viability of the myotubes (fig 5a), ruling 227

out the possibility that the fall in glucose uptake was due to decreased viability of the 228

myotubes. These findings stress the role of excess glucose in generating insulin resistance; 229

it is therefore also noteworthy that neither PA nor insulin had any effect on glucose uptake 230

or glycogen synthesis in myotubes that had been exposed to excess glucose. 231

232

Conclusions 233


95

In the present study, we have been able to generate a suitable primary porcine cell model 234

for studying both insulin and non- insulin-stimulated glucose uptake at physiological 235

levels of insulin, as 0.1 nM insulin induces a significant increase in glucose uptake. We 236

have shown that PA alone can improve glucose uptake but not glycogen synthesis and that 237

PA probably competes with insulin in insulin-signaling. PA can neither stimulate glucose 238

uptake nor glycogen synthesis in insulin-resistant cells generated by exposure to excess 239

glucose. A potential role for PA may, thus, be stimulating glucose uptake in muscle cells at 240

inadequate insulin concentrations, although the consequences of increasing glucose uptake 241

without a concomitant increase in glycogen synthesis need to be studied further. 242

243

Methods 244

Materials and reagents 245

Palmitic acid (PAM), PA, human insulin, dimethyl sulfoxide (DMSO), cytochalasin B (cyto 246

B), essentially fatty acid-free bovine serum albumin (defatted BSA), glycogen, 247

haematoxylin, chloral hydrate, cytosine arabinoside and antibiotics were all purchased 248

from Sigma. Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS), 249

horse serum (HS) and phosphate buffered saline (PBS) were obtained from Life 250

Technology. The WST-1 reagent was obtained from Boehringer Mannheim. The matrigel 251

reagent was from Becton Dickinson, and the BCA kit was from Pierce Rockford. [1-3H] - 2-252

deoxyglucose (2-DOG) was purchased from GE Healthcare and [1-14C]-D-glucose from 253

PerkinElmer. 254

Buffers and media 255


96

The proliferation growth media (PGM) consisted of DMEM containing 22 mM D-glucose, 256

10% FBS, 10% HS, 1% penicillin-streptomycin, 1.2% amphotericin B and 0.2% gentamycin. 257

The first differentiation media (DM1) was as PGM but without HS and D-glucose was 258

reduced to 6 mM. The second differentiation media (DM2) was as DM1, except for a 259

reduction of FBS to 5% and addition of cytosine arabinoside to stop proliferation. The 260

third differentiation media (DM3) was as DM2 but lacked serum. Hepes-buffered saline 261

(HBS) pH 7.4 consisted of 20mM Hepes, 140 mM NaCl, 5 mM KCl, 2.5 mM MgSO4 and 1 262

mM CaCl2. The storage medium was made up of PGM and 10% DMSO. To haematoxylin 263

solution (5 g of hydrated potassium sulphate, 0.01 g sodium iodate and 0.1 g haematoxylin 264

dye in 100 mL of distilled water) were added 5 g chloral hydrate and 0.1 g acetic acid to 265

improve shelf-life of the solution. 266

The experimental media normally consisted of the stock media (2.5 mM D-glucose and 267

defatted BSA) but where indicated, included different doses of insulin and/or glucose and 268

fatty acids. Fatty acids were solubilized either in DMSO or ethanol. Ethanol was 269

evaporated under a stream of liquid nitrogen while the final amount of DMSO in the 270

experimental media was less than 0.1%. The experimental media with fatty acid was 271

sterilized using a sterile filter and left overnight at room temperature to ensure efficient 272

binding of defatted BSA to free fatty acid. In this study, the fatty acid:BSA ratio was limited 273

to 5:1. The amount of glucose, DMSO and defatted BSA in control samples was equivalent 274

to that of treated samples, unless stated otherwise. 275

Media for glycogen analysis consisted of 5 mM D-glucose, 0.2 µCi [1-14C]-D-glucose and 276

0.1% defatted BSA in HBS. Glycogen carrier stock solution consisted of 0.5 g glycogen in 277

50 ml 30% KOH solution. 278

Isolation and culture of primary porcine satellite cells 279


97

Primary satellite cells were obtained from the left semi-membranosus muscle of six-week 280

old female pigs, weighing between 12-13.5 kg following a method described by Theil et al 281

[44]. Isolated cells were placed in storage medium and stored in liquid nitrogen until use. 282

Cells were thawed in a 37oC water bath and seeded on 4% matrigel-coated plates 283

containing PGM. After 2 days, cells were washed with 1xPBS, pH 7.4 (with Ca2+, Mg2+) and 284

allowed to proliferate for 3-4 days to 80% confluence (fig. 1 a and c). Cells were given DM1 285

until 100% confluent, and thereafter DM2 was given to stop proliferation and initiate 286

differentiation. At this stage, the cells were denoted to be at their first day of 287

differentiation. Cells were given DM2 until day 2 of differentiation, where fully developed 288

myotubes were observed microscopically (fig. 1 b and d). Myotubes were given DM3 16 h 289

prior to experimental treatment. When the experimental treatment was administered for 290

more than 4 h, DM3 was omitted. Cell culturing was performed at 37°C with 5% CO2 and at 291

100% humidity. 292

Haematoxylin staining 293

Cells were rinsed twice with 1xPBS (without Ca2+, Mg2+) and fixed with ice-cold methanol 294

for 5 min. The methanol was replaced by haematoxylin solution for 10 min, after which the 295

dye was aspirated and cells were rinsed several times to discard of unwanted stain. Cells 296

were viewed for appropriate staining on a microscope at 100 x magnification. 297

Viability test 298

After cell growth, differentiation and incubation with experimental media for 24 h, 299

myotubes were rinsed with 1xPBS (with Ca2+, Mg2+) and WST-1 reagent was used as 300

described by Okura et al [45]. The generation of a formazan dye by cleavage of a 301

tetrazolium salt present in the reagent reflects the amount of active cells present in culture. 302


98

The absorbance of the culture media, which was corrected for blank readings in the 303

presence of medium only, was measured at A450-630 using the Envision Multilabel Plate 304

Reader model 2104 PerkinElmer. 305

Protein determination 306

The protein concentration of myotube samples was determined using the bicinchroninic 307

acid (BCA) assay as described earlier by Smith et al [46]. 308

2-deoxyglucose uptake 309

Cells seeded in 48-well plates were cultured as described earlier until the 2nd or 3rd day of 310

differentiation. They were rinsed once with 1xPBS, pH 7.4 (with Ca2+, Mg2+) and incubated 311

at given time points with experimental media (see figure legend). Cells were washed 3 x 312

with HBS solution, and glucose uptake was carried out using [1-3H] 2-DOG as described by 313

Klip et al [47]. Unless stated otherwise, cells were incubated with glucose for 30 min. 314

Carrier-dependent glucose uptake was checked using [1-3H]2-DOG solution containing 0-315

50 µM of cyto B [40]. Samples were assayed for [1-3H] 2-DOG-uptake as disintegrations 316

per min and normalized to the basal level of glucose uptake in control samples. 317

Glycogen synthesis 318

Glycogen analysis was carried out on cells grown in 48-well plates as described by Al-319

Khalili et al [36] with some modifications. On day 2 or 3 of differentiation, DM3 media was 320

replaced with experimental media. Cells were rinsed afterwards with HBS solution and 321

then incubated with fresh HBS solution containing 0.1 mM defatted BSA (and insulin 322

where indicated) for 2h. Media for glycogen analysis was added to the cells during the last 323

1.5 h of incubation. Cells were washed 3 times with ice-cold 1xPBS, pH 7.4 (without Ca2+, 324


99

Mg2+) and lysed with 200 µL 30% KOH per well for at least 30 min. The cell lysate was 325

transferred into eppendorf tubes, heated for 10 min at 96oC and 3 mg/ml of carrier 326

glycogen was added to the cell lysate. A portion of the cell lysate was used for protein 327

determination. Glycogen was precipitated by overnight incubation with 5 volumes of 328

ethanol and 5% sodium sulfate at -20oC. The precipitated glycogen was collected by 329

centrifugation at 20.800 x g for 5 min, and re-dissolved in distilled water. Radioactivity 330

was measured in a scintillation mix as disintegrations per min and the values were 331

corrected to the protein content of the cells. 332

Statistical analysis 333

Statistical analyses were performed using the MIXED procedure in SAS version 9.2 (SAS 334

Institute Inc., Cary, NC, USA). The results are presented as least square mean estimates 335

(LSmeans) and the standard error of mean (SEM). When the main effects were significant, 336

the least square mean estimates were separated by least significant difference (p < 0.05). 337

All data are expressed as fold changes normalized to control samples. 338

339

List of abbreviations 340

2-DOG: 2-deoxyglucose, BCA: bicinchroninic acid, CLA: conjugated linoleic acid, cyto B: 341

cytochalasin B, defatted BSA: fatty acid-free bovine serum albumin, DM1:first 342

differentiation media, DM2: second differentiation media, DM3: third differentiation 343

media, DMEM: Dulbecco's Modified Eagle's Medium, DMSO: dimethyl sulfoxide, FBS: 344

fetal bovine serum, GLUT: glucose transporter, HBS: Hepes-buffered saline, HS: horse 345

serum, LSmeans: least square mean estimates, PA: phytanic acid, PAM: palmitic acid, 346


100

PBS: phosphate buffered saline, PGM: proliferation growth media, PPAR: peroxisome 347

proliferator-activated receptor, RXR: retinoid-X-receptor, SEM: standard error of mean 348

349

Competing interests 350

The authors declare that they have no competing interests. 351

352

Authors’ contributions 353

B.N.C designed and performed experiments, analyzed data and wrote the manuscript. N.O. 354

contributed to statistical analysis of data, discussion, review and editing of manuscript. 355

L.I.H contributed to the conception of the project, discussion, interpretation, review and 356

editing of the manuscript. J.H.N. contributed to the conception of the project. J.F.Y 357

contributed to the experimental design, discussion, review and editing of manuscript. 358

359

Acknowledgements 360

This study is part of the project; Tailored milk for human health and was supported by the 361

Danish Council for Strategic Research and The Danish Cattle Federation. The authors 362

thank Aase Sorensen for proof reading of the manuscript and Inge-Lise Sørensen and 363

Anne-Grete Dyrvig Pedersen for technical support. 364

365

366


101

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337. 495

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Hexose Uptake in Muscle-Cells in Culture. Biochem J 1987, 242:131-136. 503

504

505

506

507

508

509


106

Table legends 510

Table 1: Effect of fatty acids on the viability of primary porcine myotubes 511

Fatty acid concentration (µM)

Fold change in viability

PA PAM

Control (0) 1a ± 0.023 1a ± 0.036

1 1.01a ± 0.030

10 0.97a ± 0.022 0.95a ± 0.031

20 0.81b ± 0.036

50 0.74bc ± 0.042 0.85b ± 0.032

100 0.70c ± 0.046 0.82b ± 0.035

200 0.74c ± 0.029

300 0.61d ± 0.051

500 0.64c ± 0.025 0.54d ± 0.030

512

Differentiated porcine myotubes were incubated with different concentrations of fatty 513

acids (PA or PAM) for 24 h, and then treated with WST-1 reagent as mentioned in the 514

methods. Control samples contained experimental media without fatty acids. Data are 515

expressed as LSmean values from five-eight replicate wells, carried out in three separate 516

experiments with cells isolated from a different pig each time. LSmeans with different 517

letters (a-d) within each row denote significantly different responses related to fatty acid 518

treatments; (P < 0.05). 519


107

520

Table 2: Glucose uptake in response to PA and PAM 521

Fatty acid concentration (µM) Glucose uptake

PA PAM

Control (0) 1.00a ± 0.045 1.00a ± 0.067

1 1.23b ± 0.105 1.09a ±.051

5 1.29b ± 0.071

10 1.32b ± 0.075 1.14a ± 0.061

20 1.34b ± 0.166

40 1.37b ± 0.076

50 0.92a ± 0.032

60 1.37b ± 0.079

100 0.75b ± 0.032

200 0.48c ± 0.04

400 0.32d ± 0.028

522

Glucose uptake was carried out on differentiated myotubes that had been incubated with 1-523

60 µM PA or 1and 10 µM PAM for 4 h. PAM from 50-400 µM was administered for 24 h. 524

Control samples lacked fatty acids. Data are expressed as LSmean values of triplicate wells, 525

carried out in three separate experiments with cells isolated from a different pig each time. 526


108

LSmeans with different letters (a-d) within each row denote significantly different 527

responses related to fatty acid treatments; (P < 0.05). 528

529

Figure legends 530

Fig. 1: Microscopic view of proliferating and differentiating porcine satellite 531

cells 532

Proliferating myoblasts at 80 % confluence (a) or myotubes at day 2 of differentiation (b) 533

were stained as described in methods to reveal the single nuclei cells of myoblasts (c) and 534

multi-nuclei tubular structure of the myotubes (d). Picture magnification was 100 x. 535

536

Fig. 2: Glucose uptake in porcine myotubes in response to insulin and cyto B 537

Glucose uptake was performed on differentiated porcine myotubes in the presence of 538

varying concentrations of insulin (a), or varying concentrations of cyto B with (open 539

circles) or without (full circles) 500 µM insulin (b). Control samples lacked cyto B. Data 540

are expressed as LSmean values of four replicate wells, carried out in two separate 541


letters (a-f) denote significantly different responses related to the different treatments; (P 543

< 0.05). 544

545

546


109

Fig. 3: Glucose uptake with or without PA as a function of 2-DOG incubation 547

time 548

Glucose uptake was performed on differentiated myotubes that had been incubated with 549

(a) 10 µM PA for different times and then treated with 2-DOG for 30 min, or (b) with 550

(dashed line) or without (full line) 10 µM PA for 4 h and afterwards treated with 2-DOG for 551

5-60 min. Data are expressed as LSmean values of triplicate wells carried out in three 552

separate experiments with cells isolated from a different pig each time. LSmeans with 553

different letters (a-e) denote significantly different responses related to incubation times, 554

while * denotes LSmeans with significantly different responses with or without PA; (P < 555

0.05). 556

557

Fig. 4: Glucose uptake and glycogen synthesis with or without PA as a function 558

of insulin 559

Glucose uptake (a) was carried out on differentiated myotubes that had been incubated 560

with no (full line) or 10 µM (dashed line) PA for 4 h, followed by different concentrations 561

of insulin during the last 1 h. Glycogen synthesis (b) were performed on myotubes treated 562

with no (full line) or 10 µM PA (dashed line) for 4 h, followed by 0-100 nM insulin for 2 h. 563

Control samples lacked PA and insulin. Data are expressed as LSmean values collected 564

from three-four wells, carried out in three separate experiments with cells isolated from a 565

different pig each time. LSmeans with different letters (a-d) denote significantly different 566

responses related to the insulin concentration, while * denotes LSmeans with significantly 567

different responses with or without PA; (P < 0.05). 568

569


110

Fig. 5: Exposure of myotubes to extracellular glucose 570

Differentiated myotubes treated with two different concentrations of glucose for 24 h were 571

subjected to viability assay (5a).Glucose uptake was also performed on myotubes exposed 572

to various gluose concentrations (5b). Myotubes exposed to 6mM glucose (controls) or 14 573

mM glucose for 24 h in the absence (open bars) or presence of PA (black bars) or insulin 574

(grey bars) were subjected either to (5c) glucose uptake or (5d) glycogen synthesis 575

measurements. PA was administered for 24 h while insulin was given during the last 1 h of 576

incubation for glucose uptake measurements and 2 h for glycogen synthesis. Data are 577

expressed as LSmean values of four replicate wells, carried out in three separate 578


letters (a and b) denote significantly different responses related to PA or insulin 580

treatments, while statistical difference in LSmeans between controls and myotubes given 581

14 mM glucose is denoted with *; (P < 0.05). 582

583

584

585

586

587

588

589

590


111

Figures 591

a b 592

c d 593

Fig. 1 594

595


112


0 0.01 0.1 0.5 1 10 100 500

Fold

change in g

lucose u

pta

ke (

dpm

)

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

a

ab

bcbc

cdcd

cd

d

596

Fig. 2a 597

cyto B concentration (µM)

control 10 20 30 40 50

Fold

change in g

lucose u

pta

ke (

dpm

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

a

c

b

dde

defdef

efef

fef

f

598

Fig. 2b 599


113

PA incubation time (h)

1 2 4 8 12 16 24

Fo

ld c

ha

ng

e in

glu

co

se

up

take

(d

pm

)

0.6

0.8

1.0

1.2

1.4

aa

a

a

bb

a

600

Fig. 3a 601

2-DOG incubation time (min)

0 10 20 30 40 50 60 70

Fo

ld c

ha

nge

in

glu

co

se

up

take

(d

pm

)

0

2

4

6

8

10

12

14

e

d

d

d

c

bab

a

a

ab

b

c

*

602

Fig. 3b 603


114

Insulin concentration(nM)0 0.1 1 10

Fo

ld c

ha

nge

in

glu

co

se

up

take

(dp

m)

1.0

1.2

1.4

1.6

1.8

2.0

bc

c

b

a

b

a

a

a

*

*

604

Fig. 4a 605

b

*


0 1 10 100

Fold

change in g

lycogen s

ynth

esis

(dpm

/mg p

rote

in)

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

1.7

a

a

ab

a

bc

b

cd

b

*

*

606

Fig. 4b 607


115

Glucose concenteation (mM)5.6 12

Fo

ld c

ha

nge

in v

iab

ility

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

a

a

608

Fig. 5a 609

Glucose concentration (mM)

4 7 10 15 25

Fold

change in g

lucose u

pta

ke (

dpm

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

a

b

c

d

d

610

Fig. 5b 611


116


6 14

Fo

ld c

hang

e in

glu

cose u

pta

ke (

dp

m)

0.0

0.5

1.0

1.5

2.0

2.5

control

10 µM PA

10 nM Ins

*a

b

b

612

Fig. 5c 613


6 14

Fold

change in g

lycogen s

ynth

esis

(d

pm

/mg p

rote

in)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

control

10 µM PA

10 nM Ins

b

aa

*

614

Fig. 5d 615


117

6.2 Manuscript II; MII


118

Effects of phytanic acid on beta-oxidation flux in a primary porcine

myotube model as detected by an UPLC-MS/MS acylcarnitine assay

Brita Ngum Chea, Rune Isak Dupont Birklerc, Mogens Johannsenc, Niels Gregersenb, Lars

I. Hellgrend, Jette Feveile Younga*

a) Department of Food Science, Aarhus University, Blichers Allé 20, 8830 Tjele, Denmark

b) Department of Clinical Medicine, Research Unit for Molecular Medicine, Aarhus University,

Brendstrupgårdsvej 100, 8200 Aarhus N Denmark

c) Department of Forensic Medicine, Aarhus University, Brendstrupgårdsvej 100, 8200 Aarhus N

Denmark

d) Department of System Biology, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark

*Corresponding author: Jette Feveile Young

[email protected]

Tel: +45 87 15 80 51

Fax: +45 87 15 48 91



119

Abstract 1

Primary porcine myotubes were successfully established as an efficient model for the 2

determination of β- oxidation flux, as a simple underivatized and sensitive UPLC-MS/MS 3

method was utilized. It was revealed that the myotubes could easily take up and oxidize 4

labeled palmitate, since they generated palmitoylcarnitine (C16) and acetylcarnitine (C2). 5

Phytanic acid (PA) is a natural ligand of the peroxisome proliferator-activated receptors 6

(PPAR) and has been suggested to have health-improving properties. If PA can improve 7

FAO just like PPARα ligands do, then it will have a positive implications in relation to e.g. a 8

fight against the metabolic syndrome (MS). PA was shown to improve β-oxidation in the 9

myotubes, as it significantly reduced the content of C16 and increased the content of C2 in 10

a time-dependent manner. However, the impact of PA on the myotubes can only be 11

confirmed if the findings are validated with a known PPARα agonist. 12

Keywords: fatty acid β-oxidation, phytanic acid, oleic acid, acylcarnitine, primary porcine 13

myotubes, viability, palmitic acid 14

15

16

17

18

19

20

21

22

23

24


120

1. Introduction 25

One of the most important metabolic functions of fatty acids (FAs) is to supply energy to 26

the body; FAs account for up to 90% of the cellular energy requirement during fasting 1. 27

Lipid oxidation and utilization of FAs during fasting are reduced in obese and diabetic 28

patients2;3 and this reduced ability of muscles to oxidize fats has been linked to the 29

accumulation of intramuscular triacylglycerides (TAG) in the muscles 4;5. The rate of FAO 30

is one of the many factors that influence the content of TAG. These observations direct 31

focus towards the search of components and mechanisms that might improve FAO. 32

The peroxisome proliferator-activated receptors (PPARs) are nuclear receptors, their 33

activation of which initiate the transcriptional regulation of genes that improve FAO, 34

insulin signaling and glucose metabolism 6;7. Three forms of PPAR exists (PPAR α, β, and 35

γ), and especially synthetic ligands of PPAR α such as benzofibrates have been 36

documented to improve FAO 8-10. More so, some unsaturated FAs such as oleic acid (OLA), 37

conjugated linoleic acid (CLA) and branched-chain FAs like phytanic acid (PA) have been 38

established as natural ligands of PPARs11-13. OLA and CLA have already been shown to 39

improve FOA or have therapeutic potentials, respectively11;14. 40

PA, which is of ruminant origin and present in dairy products15 has in our lab recently been 41

shown to improve glucose uptake in primary porcine myotubes at physiological 42

concentrations (unpublished data). Phytol-rich diets have been shown to increase 43

peroxisomal and mitochondrial oxidation enzymes in the liver of mice, in both a PPAR α-44

dependent and independent fashion16. To our knowledge, it is unknown whether PA can 45

improve FAO in the muscles, but given that it is a natural PPAR ligand, we hypothesize 46

that it is capable of mediating the metabolism of FAs, by improving β-oxidation in the 47

muscles. 48


121

In order to undergo FAO, long-chain FAs have to be taken up into the cells and shuttled 49

through the carnitine system17-19. FAs taken up into the cells are converted into acyl-50

coenzyme A (acyl-CoA) by the enzyme acyl-CoA synthetase, which is located on the outer 51

membrane of the mitochondria. By using free carnitine taken up into the cells through 52

carnitine transporters, carnitine palmitoyl transferase (CPT I) catalyzes the esterification 53

of acyl-CoA into acylcarnitines. Acylcarnitines are transported through the inner 54

mitochondria membrane by carnitine-acylcarnitine translocase (CACT), and inside the 55

mitochondria CPT II catalyzes the reformation of acyl-CoA, which is the substrate for β-56

oxidation. The β-oxidation of acyl-CoA to acetyl-CoA requires the action of acyl-57

dehydrogenases and the mitochondria trifunctional proteins (MTP) 19. 58

Assays using labeled palmitate to track changes in FAO in vitro is common 20;21. In line 59

with such assays, in vitro acylcarnitine profiling is performed in cells that are incubated in 60

a medium supplemented with labeled palmitate and excess of free L-carnitine, resulting in 61

an elevation and extracellular accumulation of the acylcarnitines derived from FAO. The 62

analyzed content of the produced acylcarnitines reflects the content of acyl-CoA in the 63

mitochondria. 64

In this study, primary porcine myotubes with similar characteristics to human muscle 65

fibers 22, were established as a model for analyses of β-oxidation flux in the mitochondria, 66

using the acylcarnitine assay. The time- and dose-dependent impact of PA on FAO was 67

tested by using a simple underivatized and sensitive UPLC-MS/MS method that measured 68

palmitoylcarnitine and acetylcarnitine pools in cell media. 69

70

71


122

72

2. Methods 73

2.1. Materials and reagents 74

Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS), horse serum 75

(HS) and 1x phosphate buffered saline (Ca 2+ and Mg 2+ (PBS)) were obtained from Life 76

Technology. Essentially fatty acid-free bovine serum albumin, Cytosine arabinoside and 77

antibiotics were purchased from Sigma Aldrich, Denmark. PA, OLA and L-Carnitine were 78

also from Sigma Aldrich, Denmark, while [U-13 C]-Potassium palmitate (13C-PAM) was 79

from Cambridge Isotopes. WST-1 reagent, matrigel and BCA kit were bought from 80

Boehringer Mannheim, Becton Dickinson and Pierce Rockford, respectively. HPLC-grade 81

methanol, HPLC-grade acetonitrile, and formic acid 98-100% were purchased from Sigma 82

Aldrich, Denmark. Acetyl- and palmitoyl-L-carnitine.HCl were purchased from 83

Departamento de Química Orgánica, Universidad Autónoma de Madrid, Cantoblanco, 84

Madrid, España. Acylcarnitine Internal standard (IS) solution was purchased from 85

Cambridge Isotope Laboratories, Inc., Andover, MA, USA. The IS stock solution contained 86

L-carnitine-d9, L-acetylcarnitine-d3 and L-palmitoylcarnitine-d3; deuterium labelled 87

acylcarnitine analogues. 88

2.2. Media and buffers for cell culture 89

The proliferation growth media (PGM) was made up of DMEM containing 22mM D-90

glucose, 1% penicillin-stretomyocin, 1.2% Amphotericin B, 0.2% Gentamycin, 10% FBS 91

and 10% HS. The storage medium (SM) contained PGM and 10 % DMSO. The first fusion 92

media (FM1) was as PGM, but lacked HS and D-glucose was reduced to 6mM. The second 93

fusion media (FM2) was as FM1, however, it contained 10 µM Cytosine arabinoside and 94


123

FBS was only 5%. Hepes buffered saline (HBS) pH 7.4 consisted of 20 mM Hepes, 140 mM 95

NaCl, 5 mM KCl, 2.5 mM MgSO4 and 1 mM CaCl2. 96

2.3. Preparation of experimental media 97

A stock of 10% defatted BSA in PBS, 400 mM L-Carnitine in H2O and DMEM (25 mM 98

glucose) was used to prepare a stock medium containing 4 or 60 µM BSA, 400 µM L-99

carnitine and 5mM D-glucose. Experimental medium I (EM I) consisted of the stock 100

medium (with 60 µM BSA) and varying concentrations of 13C-PAM, while experimental 101

medium II (EM II) consisted of stock medium (with 4 µM BSA) and different 102

concentrations of FAs. When EM I was used in the second experimental set up, the 103

concentration of 13C-PAM was kept constant at 50 µM (see Figure 1b). To prepare the 104

experimental media, 13C-PAM, FAs were dissolved in ethanol and dried under a stream of 105

liquid nitrogen. Stock medium was then added and vigorously mixed for 5 min. The 106

experimental medium was left overnight at room temperature with gentle shaking and was 107

afterwards sterilized using a sterile filter. Experimental medium with lower concentrations 108

of 13C-PAM or PA were made by titration of higher concentrations with the stock media. 109

The minimum ratio of FAs to defatted BSA was 5:1 in EM I, EM II and stock media. 110

2.4. Isolation and culture of primary porcine satellite cells 111

Cell culturing was carried out at 37° C with 5% CO2. Satellite cells obtained from the semi-112

membranous muscle of young pigs 23 were placed in storage medium and stored in liquid 113

nitrogen until use. Cells were cultured as described earlier in Che et al (unpublished data). 114

Briefly, cells were thawed at 37o C and seeded on 4% matrigel coated plates containing 115

PGM. The cells were rinsed with PBS after two days, and allowed to proliferate into 116

myoblast. PGM were given to myoblasts every second day until they were 80 % confluent. 117


124

Then, FM1 was given to myoblasts until they were 100% confluent. At this point, FM2 was 118

given to the confluent cells to stop proliferation and initiate differentiation. When the cells 119

in FM2 were fully differentiated, as confirmed by microscopic observations, they were 120

rinsed with PBS and ready for experimental treatments. 121

2.5. Exposure of experimental media to myotubes 122

Two experimental setups (Figures 1a and b) were established to study the acylcarnitine 123

profiles in the primary porcine myotubes. On day 3 of differentiation, FM3 was discarded 124

and the myotubes were rinsed and incubated with stock media (white bars). Stock media 125

was replaced with EM I (black bars) for up to 4 days or EM II (grey bars) for up to 2 days 126

followed by a short rinse and then EM I for 1 day. EM I collected from the myotubes was 127

stored at -20 oC until use for acyl carnitine analysis, and the myotubes were used for 128

protein determination using the BCA method 24. 129

2.6. Preparation of collected media for acylcarnitine analysis 130

Cell growth media (50 µL) was mixed with 10 µL internal standard solution. 440 µL cold (-131

18° C) ACN was afterwards added and the solution was subsequently centrifuged at 20.800 132

g for 1 minute to separate the precipitate from the solution. 200 µL of the supernatant was 133

transferred to HPLC vials for further acylcarnitine analysis. 134

2.7. Acylcarnitine analysis 135

Acylcarnitines were analysed without derivatization prior to analysis using a Waters 136

ACQUITY UPLC coupled to a Waters Xevo TQS tandem mass spectrometer. Acylcarnitines 137

were separated using gradient elution on a HILIC (hydrophilic Interaction 138

Chromatography) column (ACQUITY BEH Amide column, 2.1 mm i.d. x 100 mm, particle 139


125

size 1.7 µm) equipped with an ACQUITY UPLC column inline filter (0.2 µm). 140

Acylcarnitines were analysed in positive ionization mode using the common acylcarnitine 141

fragments m/z 85 and [MH-59]+. The product ion m/z 85 were used as the quantifier ion 142

and the [MH-59]+ ion as qualifier when quantifying and verifying detected peaks of the 143

acylcarnitines respectively. Quantification of acylcarnitines in the cell growth media was 144

calculated using external calibration samples. Linear regression analysis of the peak area 145

ratio analyse/IS (weighed 1/X) versus concentration was used for each of the 146

acylcarnitines. 147

2.8. Viability test 148

Cells grown in 96-well plates were used for the viability test. After incubation of 149

differentiated myotubes with EM I, they were rinsed with PBS and exposed to the WST-1 150

reagent following a method described by Okura et al 25, with a few modifications. The cells 151

were incubated with WST-1 media for 4 h with a short shaking every hour. The action of 152

mitochondria on tetrazolium salts present in the WST-1 reagent generates a formazan dye, 153

the concentration of which reflects the number of active cells available. The absorbance at 154

A450-630 of the WST-1 media was recorded in the absence of cells, immediately after WST-1 155

addition and after 4 h of incubation, using the Envision Multi label Plate Reader model 156

2104 PerkinElmer. 157

158

3. Results 159

3.1. Viability assay 160

The viability of the primary porcine myotubes was affected by both time and dose of 13C-161

PAM (Figure 2). With doses of 13C-PAM ranging from 0-100 µM, there was a general 162


126

increase in the viability of the myotubes as the time of incubation with 13C-PAM increased 163

(from 1 day - 4 days). Incubation of myotubes with 25 µM or more of 13C-PAM for up to 2 164

days reduced the viability of the myotubes relative to controls without 13C-PAM. At 3 days 165

of incubation, the viability of the cells decreased only when 13C-PAM ≥ 50 µM was used. 166

Cell viability was significantly reduced by 200 and 300 µM 13C-PAM during all incubation 167

periods. Based on these results, subsequent experiments on determining the flux of β-168

oxidation in the myotubes were performed with concentrations of 13C-PAM ranging from 169

0-100 µM. 170

3.2. Acylcarnitine quantification using UPLC-MS/MS 171

Acylcarnitine concentrations in the cell growth media were measured using UPLC-MS/MS. 172

The concentrations were afterwards correlated to the protein content of the cells. An 173

example of 13C-C16 is shown in a chromatogram in Figure 3. Together with the two 174

transitions shown in the chromatogram, the retention time of 0.84 minutes is used for the 175

identification of 13C-palmitoylcarnitine. The first entry in the chromatogram shows the 176

transition 416.34 > 85.05, which is used as quantifier. The transition 416.34 > 357.34 177

(common loss of 59) is used as qualifier. For both C2 and C16 analogues, retention time, 178

quantifier, and qualifier transitions were used for exact identification. 179

3.3. Acylcarnitine profile as affected by incubation period and dose of 180

13C-PAM 181

182

3.3.1. Acetylcarnitine (C2) 183

Generally, the contents of C2 increased with increasing dose of 13C-PAM, regardless of the 184

duration of exposure of the myotubes to 13C-PAM (Figure 4a). At doses of 13C-PAM ≤ 25 185

µM, treatment for 4 days caused the highest increase in the content of C2, while treatments 186


127

for 1-3 days with 13C-PAM ≤ 10 µM resulted in amounts that were not significantly 187

different from each other. However, incubation for 2 or 3 days, with 25 µM 13C-PAM 188

resulted in similar C2 contents. When 50 µM 13C-PAM was administered to the myotubes 189

for 4 days, the content of C2 was the highest, although not significantly different from that 190

obtained after 3 days of incubation. More so, the level of C2 after 1 day of treatment with 191

50 µM 13C-PAM was lowest, even though it was not significantly different from that 192

obtained after 2 days of treatment. Exposure of the myotubes to 100 µM 13C-PAM for 1 day 193

resulted in a lowest C2-content, even though it was not significantly different from that 194

generated after 4 days of incubation. Nevertheless, treatments with 100 µM 13C-PAM for 195

over 1 day resulted in contents of C2 that were not significantly different from each other 196

(Figure 4a). 197

3.3.2. Palmitoylcarnitine (C16) 198

The exposure of 13C-PAM ≥ 25 µM generated C16-levels, which were bi-symmetrically 199

grouped, with respect to the duration of exposures; incubation for 1 and 2 days generated 200

higher levels, when compared to 3 and 4 days of incubation (Figures 4b). C16 generally 201

increased with increasing dose of 13C-PAM, even though no clear increase was noticed after 202

2 days of incubation with 0- 10 µM 13C-PAM, and unlike with C2, the content of C16 was 203

highest after 1 day of incubation with 13C-PAM, although they were not significantly 204

different from levels generated after 2 days of incubation (Figure 4b). 205

3.3.3. C16/C2 206

The ratio of C16/C2 was calculated to get a clear picture of inhibition or activation of β-207

oxidation during varying exposure of myotubes to different doses of 13C-PAM. Increasing 208

the incubation time of myotubes with 13C-PAM clearly increased the content of C16, as the 209


128

C16/C2 ratio dropped from 1 - 3 days of incubation (Figure 4c). Incubation for more than 3 210

days did not significantly increase the content of C16 except when a dose of 5 µM 13C-PAM 211

was used. With regards to the effect of 13C-PAM concentration, treatment for just 1 day 212

had no overall impact on the generation of C16, as the C16/C2 content was unchanged 213

when myotubes were exposed to all doses of 13C-PAM (Figure 3c). Treatments for 2 days 214

with U-13 C-PAM tended to increase the content of C16/C2 and by 50 and 100 µM 13C-PAM, 215

the content of C16/C2 was significantly increased. When myotubes were treated for over 2 216

days with 13C-PAM, the content of C16/C2 also tended to increase, but only at 100 µM was 217

the increase significant (Figure 4c). 218

As the content of C16/C2 showed that treatments of myotubes for 1 day with 50 µM 13C-219

PAM did not have any overall effect on the content of C16, this concentration and time was 220

used for further analysis of the effects of treatment of PA on β-oxidation. 221

3.4. PA effects on acylcarnitine contents 222

As shown in Figure 5a, the incubation of myotubes with PA enhanced the production of 223

C2, which was dependent on the amount of PA and the duration of exposure. No effect of 224

PA was noticed during an exposure of 4 h, but by 24 h, PA ≥ 10 µM increased the content 225

of C2 while at 48 h exposure of the myotubes also 5 µM PA caused an increase in the 226

content of C2. For comparison, OLA caused a significant increase in the content of C2 as 227

from 4 h of incubation. 228

The content of C16/C2 dropped after myotubes were exposed to PA ≥ 5 µM or OLA for 24 229

h, and 48 h exposure resulted in a further reduction of C16/C2 (figure 5b). 230

231


129

4. Discussion 232

4.1 Optimization of myotubes for acylcarnitine analysis 233

Optimization experiments based on viability and acylcarnitine measurements, after 234

exposure of myotubes to different concentrations of 13C-PAM at varying time points, were 235

performed to establish the myotubes as an efficient model for the analysis of β-oxidation. 236

To determine an appropriate concentration range of 13C-PAM and duration of 13C-PAM 237

exposure to the myotubes, viability studies were performed. Doses of 13C-PAM above 10 238

µM and administered for just 1 day significantly reduced the viability of the cells, and 200 239

and 300 µM 13C-PAM reduced the viability of myotubes, regardless of the exposure 240

periods (Figure 2). The reduction in viability by palmate has been observed before, and 241

suggested to be caused by stress-induced apoptosis, or through the accumulation of 242

ceramides, which are intermediates produced by palmitate metabolism 26. The reduction 243

of viability of the myotubes by 200 and 300 µM of 13C-PAM was extreme, and therefore, 244

these concentrations were excluded during further analysis. 245

During longer exposures of myotubes to 0-100 µM 13C-PAM, we observed that the viability 246

of the cells was increased (Figure 2). This could be attributed to the fact that myotubes 247

adapted to 13C-PAM as an extra energy source; FAs are predominant primary sources of 248

energy 1. 249

A concentration range of 0-100 µM 13C-PAM was exposed to the myotubes for up to 4 250

days, and the flux in β- oxidation was followed by analyzing the contents of C2 and C16 251

(discussed later). In the analysis of C2 and C16, the retention times, quantifications and 252

qualifications of their analogues were used in combination, to exactly identify each 253

acetylcarnitine. 254


130

Following the observations, a concentration of 50 µM 13C-PAM administered for just 1 day 255

was chosen for further analysis of the effects of PA on β- oxidation. The chosen 256

concentration and duration of incubation, regardless of a 10 % drop in viability, was based 257

predominantly on the fact that the content of C16 was unchanged under these conditions 258

(discussed later). More so, long-term incubation with palmitate could affect the general 259

metabolism of the myotubes as palmitate has been implicated in the inhibition of glucose 260

uptake and FAO 27-29. Thus, it was of interest not to expose the myotubes to palmitate for a 261

longer period. Furthermore, muscle cells can undergo senescence when cultured for 262

longer periods and this may also dysregulate their metabolism 30. Actually, myotubes 263

cultured for longer periods are known to exhibit physiological, morphological and 264

mechanical changes31-34. 265

266

267

4.2 Dose and time-dependent generation of acylcarnitines by 13C- PAM 268

The dose of 13C-PAM and duration of incubation with myotubes affected the content of C2, 269

possibly through the extent to which it was generated (Figures 4a). The content of C2 270

increased with increasing dose of 13C-PAM and this indicated that FAO was enhanced with 271

increased 13C-PAM (Figure 4a). More so, incubation of the myotubes for 4 days with 13C-272

PAM < 25 µM, resulted in the highest level of FAO, as the content of C2 was highest. 273

Studies on the effect of palmitate in rat muscles have shown that increasing the 274

concentration of palmitate and the duration of exposure to the muscles, led to an increase 275

in FAO 35. As the same palmitate has also been shown to inhibit FAO, as previously 276


131

mentioned, it seems apparent that a certain amount of palmitate available in a specific 277

duration is beneficial to the cells. 278

Although in the work of Rasmussen et al 35, the duration of exposure of muscles to 279

palmitate was in the range of minutes, and CO2 was measured as the oxidation product, 280

one can suggest the extrapolation of the physiological changes necessary to cause these 281

effects, e.g. changes in malonyl-CoA35, to also affect the content of C16. Analysis of the 282

content of C16 showed that it was increased during the first two days of incubation with 283

13C-PAM > 10 µM, when compared to the third and fourth day of incubation (Figure 4b), 284

but as observed in the level of C16/C2, the actual content of C16 was markedly reduced 285

when the duration of exposure of 13C-PAM ≤ 50 µM was more than 1 day (Figure 4c). This 286

disparity in the effect of dose and duration could lie in the fact that the contents of C16 287

and C2 generated in the myotubes are controlled possibly by same mechanisms in varying 288

magnitudes, or by different mechanisms. 289

The reduction in the content of C16 denotes part of the increase in the β-oxidation flux, 290

and reasons for this could be the same that caused an increase in the content of C2. The 291

activation of PPARα and γ, as well as the up-regulation of the CPT-1 have been shown to 292

enhance β-oxidation36-38. The fact that exposures to 100 µM 13C-PAM for more than 2 days 293

did not significantly change the level of C2 generated in the myotubes, denotes that during 294

these conditions, FAO is limited or even inhibited. This suggestion was confirmed by the 295

observation of an increased content of C16/C2 at 100 µM 13C-PAM, during 2-4 days of 296

incubation (Figure 4c). FAO is known to be inhibited by high doses of palmitate 39. Thus, 297

as the content of 13C-PAM in the myotubes is elevated by increasing the dose from 5-100 298

µM, there is a gradual tendency of reduction in the flux of C16, which eventually becomes 299

significant, regardless of the length of incubation (Figure 4c) 300


132

Many factors can account for the reduction in β-oxidation flux in the myotubes at high 301

palmitate concentrations. It is likely, that at higher concentrations of 13C-PAM, its uptake 302

into cells is either increased, or unaffected, but its oxidation is reduced through 303

increments and drops in the activities of malonyl CoA and CPT-1, respectively40. High 304

amounts of C2 generated in the presence of high doses of 13C-PAM can also be converted 305

to malonyl-CoA, which will, in turn suppress the generation of C16 by suppressing the 306

action of CPT-1 307

4.3 Impact of PA on β- oxidation 308

Myotubes were treated with different doses of the potential PPAR agonist PA together with 309

20 µM of the well-known PPAR agonists OLA 11 for 4h, 24 h or 48 h. Afterwards, the 310

myotubes were analyzed for β- oxidation capacity by exposure to 50 µM 13C-PAM for 24 h, 311

and analysis of the C2 and C16/C2 contents of the cell culture media, reflecting the 312

myotube content (Figures 5a and b). By relating the contents of C16 to that of C2, it was 313

possible to observe induced inhibition (higher C16/C2 ratios, when compared to controls) 314

or activation (lower C16/C2 ratios, when compared to controls) of β- oxidation. 315

The effect of PA on the content of C16 or C2 was not dose-dependent, and when there was 316

a significant effect of treatment, 10 µM PA activated β-oxidation either equally or even 317

more than 20 µM (Figures 5a and b). The used of PA over 10 µM compromises the viability 318

of the myotubes (unpublished data) and could be a reason for these observations. 319

After 24 h of incubation with 10 µM PA or OLA, FAO in the myotubes was increased, and 320

during further incubation, even 5 µM PA enhanced FAO, as observed in the increase in the 321

content of C2 (Figure 5a) and in the reduction in the content of C16/C2 (Figure 5b). Both 322

OLA and PA are PPAR agonists, and thus have FAO-inducing potentials11;41. The activation 323


133

of PPAR α and γ, as well as the up-regulation of the CPT-1 are possible causes to this 324

increase in C16 oxidation 36-38. More so, being PPARα agonists, one can anticipate that PA 325

and OLA, just like bezafibrates activate very long chain acyl coenzyme A dehydrogenases 326

42, thus, enhancing the oxidation of long-chain FAs. 327

Exposure time also affected FAO, as longer incubation periods with PA or OLA increased 328

the content of C2 (Figure 4a), as well as reduced the content of C16/C2 (Figure 5b). 329

Already after 4h of incubation with OLA, the content of C2 was elevated, showing that OLA 330

is possibly a more potent enhancer of FAO, being an unsaturated FA 43. The metabolism of 331

PA involves peroxisomal α- oxidation and racemase activity, in order to generate active 332

metabolites that are involved in FA metabolism, through the activation of PPARs 44;45. This 333

might account for a delay in its activation of FAO, as seen by the increase in the content of 334

C2, or reduction of C16/C2 only after 24h of incubation (Figures 5a and b). 335

As shown by the contents of C16/C2, no changes in the degradation of C16 by the 336

treatments were recorded during the first 4 h of incubation (Figure 5b). This could be 337

attributed to minimal oxidation rates during this time, but possibly because the treatments 338

simultaneously enhanced the uptake of U-13 C-PAM and eventually its activated form 339

(palmitoyl-CoA) into the cell and mitochondria, respectively. Both PA and OLA have been 340

reported to enhance FA binding proteins46;47, and as such, FA uptake. One could therefore 341

suggest that both PA and OLA affect β-oxidation by affecting both the uptake and the 342

degradation of 13C-PAM. More so, during 4 h treatment with OLA, the content of C2 was 343

increased but that of C16/C2 was unchanged, insinuating that OLA affects the rate of FAO 344

and that of FA uptake into the cell and/or mitochondria, differently. 345

5. Conclusions 346


134

In this study, primary porcine myotubes were established as an effective model for 347

determining the flux of β- oxidation, as they could successfully take up 13C-PAM into the 348

cell, generate and incorporate C16 into the mitochondria, and eventually degrade the 349

incorporated C16 into C2. Furthermore, the time and dose-dependent effect of palmitate 350

on FAO revealed that, longer exposures of myotubes to palmitate induced FAO by 351

enhancing the generation of both C2 and C16, while only the generation of C2 was induced 352

with different doses of palmitate. Thus, the FA metabolism of the myotubes was regulated 353

at both the uptake and oxidation levels, as same experimental conditions enhanced the 354

generation of C2 but not of C16. The treatments of myotubes with PA or OLA enhanced 355

FAO in a time-dependent fashion, and possibly in a PPARα- dependent manner. However, 356

concrete conclusions on our observations of PA on β-oxidation, awaits further analysis 357

with the inclusion of a PPARα control, e.g. bezafibrates, which will give a more convincing 358

understanding of the impact of PA on β- oxidation in primary porcine myotubes. 359

Abbreviations 360

Acyl-CoA, acylcoenzyme A; 13C-PAM, [U-13 C]-Potassium palmitate;C2, acetylcarnitine; 361

C16, palmitoylcarnitine; CACT, carnitine-acylcarnitine translocase; CLA, conjugated 362

linoleic acid; CPT, carnitine palmitoyl transferase; DMEM, Dulbecco's Modified Eagle's 363

Medium; EM, experimental media; FA, fatty acid; FAO, fatty acid oxidation; FBS, fetal 364

bovine serum; HS, horse serum; IS, internal standard; MS, metabolic syndrome; MTP, 365

mitochondria trifunctional protein; OLA, oleic acid; PA, phytanic acid; PGM, 366

proliferation/growth medium; PPAR, peroxisome proliferator-activated receptor; 367

368

369


135

Acknowledgments 370

The authors thank Aase Sørensen and Anne Hjorth Bailing for proof reading of the 371

manuscript, and Niels Oksbjerg for his help in statistical analysis. 372

Funding 373

This study is part of the project; Tailored milk for human health, and was supported by 374

the Danish Council for Strategic Research and The Danish Cattle Federation. 375

Disclosure of statement 376

The authors declare no competition in interests 377

Authors’ contribution 378

B.N.C. designed and performed experiments, analyzed data and wrote the manuscript. 379

R.I.D.R. contributed in the analysis of acylcarnitine data. M.J. contributed in providing 380

instrumentation for acylcarnitine measurements, discussion and interpretation of 381

manuscript. N.G. contributed in the conception, discussion, interpretation, review and 382

editing of manuscript. L.I.H. contributed to the conception of the project, discussion, 383

interpretation, review and editing of the manuscript. J.F.Y. contributed to the 384

experimental design, discussion, review and editing of manuscript. 385

386


136

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of Physiology - Endocrinology and Metabolism 289:E151-E159, 2005 470

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non-diabetic insulin resistant subjects retain defects in insulin action. Journal 472

of Clinical Investigation 98:2346-2350, 1996 473

31. Lieth E, Cardasis CA, Fallon JR: Muscle-derived agrin in cultured myotubes: 474

Expression in the basal lamina and at induced acetylcholine receptor clusters. 475

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32. Gopinath SD, Rando TA: Stem Cell Review Series: Aging of the skeletal muscle stem 477

cell niche. Aging Cell 7:590-598, 2008 478

33. Oksbjerg N, Gondret F, Vestergaard M: Basic principles of muscle development and 479

growth in meat-producing mammals as affected by the insulin-like growth 480

factor (IGF) system. Domestic Animal Endocrinology 27:219-240, 2004 481

34. Mann C, Leckband D: Measuring traction forces in long-term cell cultures. Cellular 482

and Molecular Bioengineering 3:40-49, 2010 483

35. Merrill GF, Kurth EJ, Rasmussen BB, et al: Influence of malonyl-CoA and palmitate 484

concentration on rate of palmitate oxidation in rat muscle. Journal of Applied 485

Physiology 85:1909-1914, 1998 486

36. Guan Y, Zhang Y, Breyer MD: The role of PPARs in the transcriptional control of 487

cellular processes. Drug News and Perspectives 15:147-154, 2002 488


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syndrome and type 2 diabetes mellitus. Current Diabetes Reviews 3:33-39, 490

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38. Ciaraldi, T. P., Cha, B. S., Park, K. S., et al: Free fatty acid metabolism in human 492

skeletal muscle is regulated by PPAR gamma and RXR agonists. Annals of the 493

New York Academy of Sciences 967, 66-70. 2002. 494

39. Molé PA, Oscai LB, Holloszy JO: Adaptation of muscle to exercise. Increase in levels 495

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dehydrogenase, and in the capacity to oxidize fatty acids. Journal of Clinical 497

Investigation 50:2323-2330, 1971 498

40. Rasmussen BB, Holmbäck UC, Volpi E, et al: Malonyl coenzyme A and the regulation 499

of functional carnitine palmitoyltransferase-1 activity and fat oxidation in 500

human skeletal muscle. Journal of Clinical Investigation 110:1687-1693, 2002 501

41. Gloerich J, van den Brink DM, Ruiter JPN, et al: Metabolism of phytol to phytanic 502

acid in the mouse, and the role of PPAR alpha in its regulation. Journal of 503

Lipid Research 48:77-85, 2007 504

42. Djouadi F, Aubey F, Schlemmer D, et al: Bezafibrate increases very-long-chain acyl-505

CoA dehydrogenase protein and mRNA expression in deficient fibroblasts and 506

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Genetics 14:2695-2703, 2005 508

43. Rivellese AA, Maffettone A, Vessby B, et al: Effects of dietary saturated, 509

monounsaturated and n-3 fatty acids on fasting lipoproteins, LDL size and 510

post-prandial lipid metabolism in healthy subjects. Atherosclerosis 167:149-511

158, 2003 512

44. Jansen GA, Wanders RJA: Alpha-Oxidation. Biochimica et Biophysica Acta - 513

Molecular Cell Research 1763:1403-1412, 2006 514

45. Wanders RJA, Jansen GA, Lloyd MD: Phytanic acid alpha-oxidation, new insights 515

into an old problem: A review. Biochimica et Biophysica Acta - Molecular and 516

Cell Biology of Lipids 1631:119-135, 2003 517

46. Wolfrum C, Ellinghaus P, Fobker M, et al: Phytanic acid is ligand and transcriptional 518

activator of murine liver fatty acid binding protein. Journal of Lipid Research 519

40:708-714, 1999 520

47. Meunier-Durmort C, Poirier H, Niot I, et al: Up-regulation of the expression of the 521

gene for liver fatty acid-binding protein by long-chain fatty acids. Biochemical 522

Journal 319:483-487, 1996 523


140

524

525

526


141

Figure legends 527

Figure 1: Experimental setups 528

Two experimental setups were established to study the effects of time and dose of 13C-PAM 529

(1a), and the effects of time-dependent treatments (1b) on the palmitoyl-and 530

acetylcarnitine profiles in primary porcine myotubes. Myotubes in stock media (white 531

bars) were given either EMI (black bars) on a daily basis for 1 - 4 days (Figure 1a) or EM II 532

(grey bars) for 4 h, 24 h or 48 h, followed by a quick rinse and afterwards EM I for 24 h 533

(Figure 1b). In both setups, the EM I collected from the cells was stored at -20oC until use 534

for acyl carnitine analysis. 535

536

Figure 2: Viability of porcine primary myotubes 537

Differentiated porcine myotubes were exposed to varying doses of 13C-PAM (0-300 µM) for 538

up to 4 days and afterwards subjected to viability assay as described in the methods. 539

Control samples (0) had the same media (EM I) as other samples, but lacked 13C- PAM. 540

Data are expressed as mean values with SEM from triplicate wells, carried out in three 541

separate experiments with cells isolated from a different pig for every experiment. * denote 542

significant difference between doses of U-13 C-PAM used within a time period, with regards 543

to their respective controls (P < 0.05). 544

545

Figure 3: Extracted UPLC-MS/MS chromatogram of 13C-palmitoylcarnitine 546 (C16). 547

A peak specific to C16 is detected by use of the retention time, a quantifier and a qualifier. 548


142

549

Figure 4: Acylcarnitine contents of C2 and C16 as affected by incubation time 550

and dose of 13C- PAM 551

Differentiated porcine myotubes were exposed to varying doses of 13C- PAM (0-100 µM) 552

for 1 day (closed circles), 2 days (open circles), 3 days (closed triangles) or 4 days (open 553

triangles) and the media collected for acyl carnitine analysis. The contents of C2 (a) and 554

C16 (b) were measured using a fast and simple extraction procedure followed by UPLC-555

MS/MS analysis.The ratio of C16/C2 (c) was calculated in relation to the content generated 556

after 1-day incubation with 5 µM 13 C-PAM. Data are expressed as mean values with SEM, 557

and are generated from samples collected from triplicate wells, carried out in three 558

separate experiments, with cells isolated from a different pig each time. * applies only to 559

figure 4c and denotes significant difference in relation to treatments with 5 µM 13C-PAM 560

during each incubation period. 561

Figure 5: Contents of palmitoylcarnitine and acetylcarnitine as affected by 562

time-dependent treatments of myotubes 563

Differentiated porcine myotubes were exposed to varying doses of PA (0-20 µM) or 20 µM 564

OLA for 4 h, 24 h or 48 h and afterwards to EM II containing 50 µM U-13 C-PAM for 24 h. 565

Media was collected and analyzed for their C2 (a) and C16/C2 contents (b). Control 566

samples were only exposed to stock media before EM II. Data are expressed as mean 567

values with SEM and are related to control samples. The data are generated from samples 568

collected from 6-9 different wells, carried out in two separate experiments, with cells 569

isolated from a different pig for every experiment. Significant differences with regards to 570

controls for each time period (4h, 24 h or 48 h) are denoted with *; P < 0.05. 571


143

572

573

574


144

Figures 575

576

577

Figure 1a 578

579

Figure 1b 580


145

Incubation time with varying doses of µM 13

C-PAM (days)

1 2 3 4

Fo

ld c

ha

ng

e in

via

bil

ity

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0

5

10

25

50

100

200

300

***

*

*

* *

*

*

*

*

*

*

*

*

*

581 582

Figure 2 583

584


146

585

Figure 3 586

min 0.620 0.640 0.660 0.680 0.700 0.720 0.740 0.760 0.780 0.800 0.820 0.840 0.860 0.880 0.900 0.920 0.940

%

0

100 F11:MRM of 5 channels,ES+

416.34 > 357.34

13C-Palmitoylcarnitine (C16)

0.84 min.

min

%

0

100 F11:MRM of 5 channels,ES+

416.34 > 85.05

13C-Palmitoylcarnitine (C16)

0.84 min.


147

C2

13C-PAM concentration (µM)

control 5 10 25 50 100

Ace

tylc

arn

itin

e co

nte

nt

x10

(µ

M/m

g p

rote

in)

0

2

4

6

C2

587

Figure 4a 588


148

13C-PAMconcentration (µM)

control 5 10 25 50 100

Pa

lmit

oy

lca

rnit

ine

con

ten

t x

10 (

µM

/mg

pro

tein

)

0

1

2

3

4

5

6

C16

589

Figure 4b 590


149

C16/C2

13C PAM concentration (µM)

5 10 25 50 100

Fo

ld c

han

ge

in C

16/C

2 c

on

ten

t (µ

M/m

g p

rote

in)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

*

*

*

*

591 592

Figure 4c 593


150

594

C2

Incubation time time (h)

4 24 48

Fo

ld c

han

ge i

n C

2 c

on

ten

t (µ

M/m

g p

rote

in)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

*

* **

*

*

*

*

595

Figure 5a 596

597


151

C16/C2

Incubation time (h)

4 24 48

Fo

ld c

ha

ng

e in

C16

/C2

co

nte

nt

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

**

*

* **

**

598

Control 1 µM PA 5 µM PA 10 µM PA 20 µM PA 20 µM OLA 599

Figure 5b 600


152

6.3 Manuscript III; MIII

Content and distribution of phytanic acid diastereomers in organic

milk as affected by feed composition

Brita N. Che†, Troels Kristensen‡, Caroline Nebel†, Trine K. Dalsgaard†, Lars I.

Hellgren§, Jacob H. Nielsen#, Jette F. Young† Mette K. Larsen†*

† Department of Food Science, ‡ Department of Agroecology, Aarhus University, AU

Foulum, Blichers Allé 20, DK-8830 Tjele, Denmark

§ Department of System Biology, Center for Biological Sequence Analysis, Technical

University of Denmark, Kemitorvet, Building 208, DK-2800 Kgs. Lyngby, Denmark

# Arla Foods, Sønderhøj 14, DK-8260 Viby J, Denmark

* Corresponding author: Mette K. Larsen

[email protected]

Tel: +45 87 15 80 62

Fax: +45 87 15 48 91



153

ABSTRACT: Phytanic acid (PA) is a bioactive compound of milk, which is derived from 1

the phytol chain of chlorophyll, and the content of PA in milk-fat depends on the 2

availability of phytol from feed. In this study, the content of PA diastereomers was 3

analyzed in milk sampled from five organic herds, twice during the grazing season (May 4

and September). The total content of PA was higher in September, when compared to May, 5

but was not affected by breed (Danish Holstein or Danish Jersey). Total PA could not be 6

directly related to intake of green feed items. The distribution between PA diastereomers 7

was closely related to the amount of grazed legumes, where a higher intake resulted in a 8

higher share of the RRR isomer. 9

10

KEYWORDS: Organic milk, phytanic acid diastereomers, pasture, legumes, silage 11


154

INTRODUCTION 12

Recent findings point to an inverse correlation between increased dairy consumption 13

and the metabolic syndrome (MS), 1, 2 even though dairy products are a rich source of 14

saturated fatty acids. This controversy has directed the focus towards the search for 15

components in milk that are responsible for neutralizing the adverse effects of the 16

metabolically detrimental saturated fatty acids. One such component is 3, 7, 11, 15 17

tetramethylhexadecanoic acid (phytanic acid (PA)). In the human food chain, PA is 18

primarily found in dairy products, ruminant meat and some marine fats. PA is formed 19

from phytol, which is cleaved from chlorophyll and subsequently oxidized by the rumenal 20

microbiota and certain marine organisms.3, 4 21

As PA is derived from chlorophyll, the content in milk-fat is dependent on the feed 22

composition, where increased amounts of green feed increase the content of PA. Due to the 23

higher use of grass-based products, PA content is higher in organic milk, when compared 24

to conventional milk, and a threshold value of the PA content in milk-fat of at least 200 25

mg/g has been suggested as a marker of organic milk products.5 26

PA, as well as its primary metabolite pristanic acid, are natural agonists of the 27

peroxisome proliferator activated receptor-α (PPARα) and the retinoid X receptor (RXR).6 28

As RXRs and the PPARα are known to control fatty acid and glucose metabolism,7 PA has 29

been suggested to have health-improving properties and a protective effect on MS.6, 8 30

Recently, we have shown that PA can increase glucose uptake in primary porcine myotubes 31

(unpublished data), although there was no concomitant increase in glycogen synthesis. 32

PA has 3 chiral centers positioned on carbons 3, 7 and 11. Carbons 7 and 11 are R- 33

configured as in phytol, whereas carbon 3 is either R- or S- configured as a result of the 34


155

biohydrogenation of oxidized phytol. As such, PA occurs naturally as two diastereomers; 35

SRR PA and RRR PA.3 The distribution profile of the PA diastereomers in lipid fractions in 36

serum varies, with higher SRR/RRR ratio in phospholipids than in other lipid fractions.9 37

More so, the biological activity of the diastereomers of PA in humans differ. At higher PA 38

concentrations, the oxidation of SRR PA is preferred by up to 50%, compared to the RRR 39

form,10 whereas at lower PA concentrations, both forms exhibit similar oxidation rates.10, 11 40

The initial step in PA oxidation is peroxisomal α-oxidation to pristanic acid (2,6,10,14 41

tetramethylpentadecanoic acid) which is further degraded by β-oxidation.12 The β-42

oxidation is stereospecific and requires α-methylacyl-CoA racemase (AMCAR) to convert 43

2R-pristanoic acid to its 2S isomer.12 These differences in the oxidation and distribution 44

profiles of PA diastereomers hint on potential variations in their physiological functions 45

and warrant an elucidation of their origin in ruminants. 46

The distribution of the two diastereomers of PA varies in food, with the SRR form 47

predominant in marine animals and some terrestrial mammals having more of the RRR 48

form.3, 13 The distribution between the two isomers varies between bovine milks of 49

different origin where organic farming gives higher shares of the RRR isomer.13, 14 Based on 50

these results it has been suggested that the threshold value of 200 mg/g PA in organic milk 51

fat5 should be replaced by a combination of total PA content and the ratio between the 52

diastereomers for authentication of organic milk.14 53

Organic dairy management varies between countries due to tradition as well as climatic 54

differences.15 In Danish organic dairy farming there is a compulsory use of grazing when 55

climatic conditions allow, which is in accordance with the definition in the European 56

Council Regulations,16 however, the extent of grazing varies considerably between farms. 57

Other main feed items include grass and maize silage, and concentrates.15 The feed 58


156

composition differs significantly from the ratios of pure hay or pure grass silage 59

investigated by Schröder et al (2012),14 and PA concentrations in milk fat may be lower as 60

the content of grass based feed is lower. 61

The present study was carried out as a survey of five Danish commercial organic herds 62

in the grazing season, and the purpose was to test whether the suggested target value of 63

200 mg/g PA in milk fat5 could be met, to investigate how the content of the two 64

diastereomers of PA varied in bulk milk, and to test the hypothesis that these differences 65

could be related to specific feeding patterns. 66

67

MATERIALS AND METHODS 68

The experiment was conducted at five Danish commercial organic dairy herds. 69

Registration of feed consumption and milk production was made during two periods of 14 70

days within the grazing season, where the first period started in spring about two weeks 71

after start of grazing, and the second period started in late summer at the end of August. 72

Milk was sampled from the bulk tank milk on two days at the end of each period. 73

Herds and feeding. Three herds (DH1, DH2 and DH3), were Danish Holstein cows 74

(DH), while the two other herds (DJ1 and DJ2) were Danish Jersey (DJ). All farms 75

practiced calving all year round, and all lactating cows participated in the experiment, as 76

the herd was the experimental unit. Feeding of supplemental feed as roughage (grass 77

silage, maize silage, hay or straw), or a mix of roughage and concentrate were applied 78

indoors at the feeding lane with one feeding space to each cow, whereas concentrate 79

(commercial mix and cereals) in all herds was applied in the milking parlour, divided in 80

equal amounts and given twice daily. 81


157

Pastures. At all farms, pastures dominated by perennial ryegrass (Lolium perenne) 82

and white clover (Trifolium repens) were part of a cropping rotation with arable crops. 83

Rotations were typically based on 3-year leys and grassland was established by seeding 84

cereals with grass clover mixtures. 85

Registration and calculation of feed intake. Milk yield was recorded once in 86

each period for each cow and the contents of fat, protein and urea were analysed 87

(Milkoscan 4000, Foss Electric A/S, Hillerød, Denmark). Milk yield as energy-corrected 88

milk (ECM) was calculated according to Sjaunja et al. (1990)17, and average contents of fat, 89

protein and urea at herd level were calculated based on the milk recordings from each 90

lactating cow. 91

Registration of the pasture was made three times during each period. Samples were 92

collected in 25 places at each farm by hand-plucking the pasture at the height at which 93

cows were observed to graze. The botanical composition of the dry matter (DM) was 94

determined after drying at 60°C to constant weight. The relative amounts of the individual 95

species were recorded, and the relative amount of legumes calculated as the sum of white 96

clover and red clover. 97

Three times during each period, intake of supplemental feed was determined by weight 98

over one day at herd level. Feed samples of concentrates and roughage were taken once 99

during each period. Each sample of roughage and supplement feed was analysed by near 100

infrared reflectance spectroscopy. Net energy value of lactation (NE) was estimated based 101

on Hvelplund et al (2007).18 102

The energy requirements for maintenance, grazing activity, lactation and live weight 103

change was calculated as described by Macoon et al. (2003)19 based on animal 104


158

performance data at group level, as the intake of the indoor feed was registered at group 105

level. Pasture DM intake (DMI) was estimated as the difference between the requirement 106

and NE in supplemental feed at group level divided by the NE concentration of the hand-107

plucked samples. 108

The DMI of the different species in the pasture was estimated proportionally to the 109

botanical analysis of the hand-plucked samples assuming no selectivity. DMI of individual 110

feed items was expressed as average relative distribution (kg DM/kg total DMI) at herd 111

level for each period. 112

Fatty acid extraction from milk. Milk (10 mL) was centrifuged at 1700 g at 4oC for 113

20 min and the cream phase transferred to a new tube. The cream was centrifuged for 10 114

min at 13000 g at 20°C and afterwards placed on a 60oC heater for 10 min. The heated 115

cream was then centrifuged for 10 min at 13000 g at 40oC and fat was sampled from the 116

liquid fat layer. A volume of 15 mg of fat was weighed out and mixed with 1 ml heptane 117

containing 0.4 mg/mL of C12:1 cis 11 triglyceride (Nu-Chek-Prep Inc, Elysian, MN, USA) 118

as an internal standard. For methylation 10 µl of a 2.2M sodium methylate was added. The 119

mixture was vortexed for 1 min and allowed to stand for 10 min at room temperature. The 120

methylated mixture was centrifuged for 5 min at 13000 g at 4oC and the heptane phase 121

collected for GC-MS analysis. 122

GC-MS analysis of methyl esters of PA diastereomers. Methylated milk fat 123

extracts were analyzed in details for PA isomers on a GC-MS (GC 6890N from Agilent 124

Technologies (Waldbronn, Germany)) equipped with a Restek 2560 column (100 m x 0.25 125

mm x 0.20 µm, Supelco, Bellafonte, PA, USA). A 1 μL aliquot of the extracts was injected in 126

split mode (20:1) into an inlet with a temperature of 250° C. The programmed column 127

temperature was isothermal at 100° C for 5 min followed by an increase to 140° C with a 128


159

rate of 3°C/min, a hold time of 5 min, followed by a raise to 160° C with a rate of 5° C/min 129

and a hold time for 20 min. Subsequently, the temperature was raised to 220° C with a rate 130

of 12° C/min and held there for 8 min. Finally, an increase to 240 ° C was performed with a 131

rate of 25° C/min and held there for 10 min before the next injection. Mass spectral 132

analysis was performed on a quadrupole MSD 5975 (Agilent Technologies, Germany) using 133

an external standard curve in selected ion monitoring using the ion 101 m/z as target and 134

the ions 57, 74, and 171 m/z as qualifier ions for the quantification of the PA isomers (SRR 135

and RRR). For the internal standard (C12:1) the ion 96 m/z was used as target ion, and 136

110, 123, 138 m/z were used as qualifier ions. The analysis was performed keeping the 137

quadrupole temperature of 150° C and a fragmentation voltage of 70 eV. The ion source 138

temperature was 230° C, and the interface was 280° C. The flow rate for helium carrier gas 139

was set at 1.0 mL/min. 140

Content of PA in the milk samples was calculated based on a standard curve made from 141

synthetic PA methyl esters (Sigma-Aldrich A/S, Brøndby, Denmark) and variation in 142

response between different samples was corrected using the internal standard. 143

Calculations and statistical treatment. The total amount of PA in milk fat was 144

calculated as the sum of the two isomers and the share of RRR isomer in total PA was 145

calculated as a measure of the distribution of isomers. Data were treated statistically by use 146

of SAS, version 9.2 for Windows (SAS Institute Inc, Cary, NC). The first model investigated 147

the main effects of breed and period, the second model was a one-way ANOVA of the effect 148

of combinations of herd and period. Pearson correlation coefficients between PA results 149

and share of individual feed items were calculated to determine relations between feed 150

components and PA. 151

152


160

RESULTS 153

Identification of PA diastereomers in milk fat. GC-MS analysis showed two PA 154

peaks of milk samples and three peaks of the synthetic standard (see Figure 1). A constant 155

ratio between the target (m/z101) and the qualifier ions (m/z 57, m/z 74, m/z 171) over the 156

whole peak was used for peak authentication and the first peak of the milk samples was 157

assigned as the SRR isomer of PA whereas the second peak of the milk samples was 158

assigned as the RRR isomer of PA according to literature.3, 13 159

Effect of breed and period. The concentration of total PA in milk fat was generally 160

higher in September compared to May (1.21 and 0.96 mg/g fat, respectively, p < 0.001), 161

and this difference could be ascribed to results for September of one DH herd and one DJ 162

herd (DH3 and DJ1) (see Figure 2). No overall breed difference was detected. There were 163

no significant effects of period or breed on the share of RRR isomer in total PA, despite 164

large variations (0.24 to 0.49) between different herds at different periods as shown in 165

Figure 3. 166

Effect of feed composition. The milk yield and feed consumption data are 167

presented in Table 1. Concentrations of fat and protein were higher and milk yields lower 168

for DJ compared to DH as normally observed for these breeds. There was a large variation 169

in feed composition between herds and the share of pasture ranged from 0.21 to 0.76, the 170

share of maize silage ranged from 0 to 0.48, the share of grass silage including hay and 171

straw ranged from 0 to 0.31, whereas the share of concentrate ranged from 0.10 to 0.34 172

corresponding to a total share of roughage from 0.66 to 0.90. Due to weather conditions 173

DMI from grazing was lower in September which was accompanied by a higher DMI of 174

silage as well as concentrate. Legumes constituted a larger part of pastures in September. 175


161

Pearson correlation coefficients of total PA as well as share of RRR isomer in total PA, 176

on individual feed groups are given in Table 2. Total PA was negatively correlated to intake 177

of pasture and positively correlated to intake of concentrate, whereas the share of RRR 178

isomer was positively correlated to the grazed amount of legumes. This latter correlation 179

coefficient was most significant and the share of RRR isomer in milk fat from different 180

herds at different periods related to the grazed amount of legumes is presented in Figure 4. 181

182

DISCUSSION 183

The concentrations of total PA in milk fat were only about half of the suggested 184

threshold value of 200 mg PA per100 g milk fat for organic milk.5 This could be due to 185

differences in Danish and German organic feeding practice; however, differences in 186

analytical procedures may have also affected results. The impact of different feeding 187

regimes on the content of PA in milk fat has been demonstrated by Schröder et al (2012)14 188

where cows have been switched from a conventional total mixed ratio to a ratio consisting 189

entirely of hay and afterwards back to the mixed ratio or to a ratio of only grass silage. 190

Average PA content in milk fat have been reported as 98-116 mg/100g after feeding the 191

mixed ratio, 153 mg/100 g at hay feeding, and 259 mg/100 g at grass silage feeding.14 192

These results demonstrate a positive relationship between the consumption of green 193

fodder and the PA content in milk fat; such relationship has also been documented by 194

Schröder et al (2011)20 where 50% or 87.5% green fodder has resulted in PA contents in 195

milk fat of 146 mg/100 g and 314 mg/100 g, respectively. Leiber et al (2005)21 have shown 196

a three-fold increase in PA content of milk-fat (from 0.15 to 0.45 g/100 g), when cows were 197

shifted from a mixed ratio based on hay, grass silage, maize silage and concentrate to a 198

ratio based entirely on fresh grass (either barn fed or pasture). Contrarily, in our present 199


162

results, grazing was negatively correlated and concentrates positively correlated to the 200

concentration of PA in milk fat. The negative correlation with grazing may be ascribed to 201

fatty acid oxidation products formed during the destruction of fresh plant material, which 202

alter the activity and composition of rumen microorganisms.22 The positive effect of 203

concentrate could be due to the use of grass pellets in concentrate mixtures which is 204

common in Danish organic farming. However, as differences in PA content in milk samples 205

were small and most samples did not differ significantly, the observed correlations may to 206

some extent reflect random variation. 207

The distribution between the diastereomers of PA depends on dairy management and 208

the share of SRR isomer has been reported as 39-71% in organic cheeses and 51-84% in 209

conventional cheeses.13 In milk from one organically and one conventionally fed cow the 210

share of SRR isomer has been reported as 47% and 85% , respectively.13 In the experiment 211

where feed ratios was shifted between a mixed ratio, pure hay and pure grass silage (see 212

above)14 the share of SRR isomer was reported as 50% during hay feeding and 70-80% 213

during feeding the mixed ratio as well as during grass silage feeding. These results 214

demonstrate that feed composition regulates the distribution of isomers, and hay favors 215

the formation of the RRR isomer. Our present results showed a high positive correlation 216

between the amount of grazed legumes and the share of RRR isomer. This relation 217

between legumes and isomer distribution could be an explanation of the higher share of 218

RRR isomer in organic products as the use of legumes is increased in organic agriculture to 219

ensure nitrogen fertilization. 220

The mechanisms involved in the formation of either SRR or RRR isomers have to our 221

knowledge not been previously reported. The R or S configuration of carbon 3 is formed 222

during the biohydrogenation of oxidized phytol, and possibly different microbial strains 223


163

form either predominantly SRR or RRR isomers. The biohydrogenation of most 224

unsaturated fatty acids takes place at carbon 9 or higher and different microorganisms are 225

involved in the hydrogenation step depending on the position and number of double 226

bonds.23 Similarly, the final hydrogenation of phytenic acid may involve other groups of 227

microorganisms. The hydrogenation of unsaturated fatty acids from feed has been 228

reported to be reduced by secondary plant metabolites from legumes.21 Likewise, these 229

secondary plant metabolites may favor strains forming predominantly RRR isomer or 230

suppress strains forming predominantly SRR isomer. 231

Very little is known on whether the intake of PA with different isomeric composition 232

has any physiological relevance. There is no difference in the agonist activity on PPARα, 233

between the isomeric forms of PA.11 However, pristanic acid, which is produced by α-234

oxidation of PA , is a considerable more potent PPARα agonist than PA,24 hence would a 235

shift towards more pristanic acid be expected to enhance PPARα activation. 236

It has been shown that the SRR-form is more rapidly oxidized in mitochondrial 237

preparations at high PA concentrations.10 However, since peroxisomal α-oxidation is 238

required for efficient β-oxidation of PA, the in vivo relevance of this is uncertain. But, as 239

the RRR-form requires isomerization by the AMCAR prior to β-oxidation,12 it is reasonable 240

to assume that the SRR-form of pristanic acid is more rapidly oxidized in the β-oxidation, 241

than the RRR-form, hereby decreasing the PA/pristanic acid ratio. 242

Thus, the intake of PA with a greater portion of SRR might be desirable as its oxidation 243

would be faster and less dependent on AMCAR, but on the other hand, a greater portion of 244

RRR might lead to higher concentrations pristanic acid, and hence more efficient 245

activation of PPARα. Which of these situations that is most advantageous for the 246

consumer cannot be concluded at present. In either case, feeding strategies can be 247


164

manipulated, for example by changing the content of legumes in the feed, to achieve an 248

altered content of RRR share of PA in dairy products. 249

In conclusion this study shows that total PA content in milk fat cannot be directly 250

related to the intake of green fodder items and other elements in the feed composition may 251

affect PA synthesis as well. The distribution of isomers was highly dependent on the 252

amount of legumes in feed and the results suggest that it should be possible to increase the 253

share of RRR isomer of PA by increasing the amount of legumes in feed. However, results 254

do not show whether this effect only relates to grazed legumes or similar effects could be 255

obtained by preserved legumes. The physiological relevance of the distribution of PA 256

isomers remain to be investigated. 257

258

ABBREVIATIONS USED 259

PA, phytanic acid; PPARα, peroxisome proliferator activated receptor-α; RXR, retinoid 260

X receptor; AMCAR, α-methylacyl-CoA racemase; DH, Danish Holstein; DJ, Danish 261

Jersey; ECM, energy corrected milk; DM, dry matter; metabolic syndrome, MS; NE, net 262

energy value of lactation; DMI, dry matter intake; GC-MS, gas chromatography mass 263

spectroscopy. 264

265

266

267

268

269


165

REFERENCES 270

(1) Azadbakht, L.; Mirmiran, P.; Esmaillzadeh, A.; Azizi, F. Dairy consumption is 271

inversely associated with the prevalence of the metabolic syndrome in Tehranian adults. 272

Am. J. Clin. Nutr. 2005, 82, 523-530. 273

(2) Pereira, M. A.; Jacobs, D. R.; Van Horn, L.; Slattery, M. L.; Kartashov, A. I.; Ludwig, 274

D. S. Dairy consumption, obesity, and the insulin resistance syndrome in young adults - 275

The CARDIA study. J. Am. Med. Ass. 2002, 287, 2081-2089. 276

(3) Ackman, R. G.; Hansen, R. P. Occurrence of Diastereomers of Phytanic and Pristanic 277

Acids and Their Determination by Gas-Liquid Chromatography. Lipids 1967, 2, 357-362. 278

(4) Patton, S.; Benson, A. A. Phytol Metabolism in Bovine. Biochim. Biophys. Acta 1966, 279

125 (1), 22-32. 280

(5) Vetter, W.; Schroder, M. Concentrations of phytanic acid and pristanic acid are higher 281

in organic than in conventional dairy products from the German market. Food Chem. 282

2010, 119, 746-752. 283

(6) Zomer, A. W. M.; van der Burg, B.; Jansen, G. A.; Wanders, R. J. A.; Poll-The, B.; van 284

der Saag, P. T. Pristanic acid and phytanic acid: naturally occurring ligands for the nuclear 285

receptor peroxisome proliferater-activated receptor alpha. J. Lipid Res. 2000, 41, 1801-286

1807. 287

(7) Grimaldi, P. A. Peroxisome Proliferator-Activated Receptors as sensors of fatty acids 288

and derivatives. Cell. Mol.Life Sci. 2007, 64, 2459-2464. 289

(8) Hellgren, L. I. Phytanic acid - an overlooked bioactive fatty acid in dairy fat? Ann. 290

N.Y. Acad. Sci. 2010, 1190 (1), 42-49. 291

(9) Eldjarn, L.; Try, K. Different Ratios of Ldd and Ddd Diastereoisomers of Phytanic 292

Acid in Patients with Refsums Disease. Biochim. Biophys. Acta 1968, 164 (1), 94-100. 293

(10) Tsai, S. C.; Steinber, D.; Avigan, J.; Fales, H. M. Studies on Stereospecificity of 294

Mitochondrial Oxidation of Phytanic Acid and of Alpha-Hydroxyphytanic Acid. J. Biol. 295

Chem. 1973, 248, 1091-1097. 296


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(11) Heim, M.; Johnson, J.; Boess, F.; Bendik, I.; Weber, P.; Hunziker, W.; Fluhmann, B. 297

Phytanic acid, a natural peroxisome proliferator-activated receptor agonist, regulates 298

glucose metabolism in rat primary hepatocytes. FASEB J. 2002, 16, 718-720 299

(12) Ferdinandusse, S.; Rusch, H.; van Lint, A. E. M.; Dacremont, G.; Wanders, R. J. A.; 300

Vreken, P. Stereochemistry of the peroxisomal branched-chain fatty acid +¦- and +¦-301

oxidation systems in patients suffering from different peroxisomal disorders. J. Lipid Res. 302

2002, 43, 438-444. 303

(13) Schroder, M.; Vetter, W. GC/EI-MS determination of the diastereomer distribution of 304

phytanic acid in food samples. J. Am. Oil Chem. Soc. 2011, 88, 341-349. 305

(14) Schroder, M.; Lutz, N. L.; Tangwan, E. C.; Hajazimi, E.; Vetter, W. Phytanic acid 306

concentrations and diastereomer ratios in milk fat during changes in the cow's feed from 307

concentrate to hay and back. Eur. Food Res. Technol. 2012, 234, 955-962. 308

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Leifert, C. The effects of dairy management and processing on quality characteristics of 310

milk and dairy products. NJAS - Wageningen J. Life Sci. 2011, 58 (3-4), 97-102. 311

(16) Council Regulation (EC) No 834/2007 of 28 June 2007 on organic production and 312

labelling of organic products and repealing Regulation (EEC) No 2092/91. http://eur-lex. 313

europa. eu/LexUriServ/LexUriServ. do?uri=OJ:L:2007:189:0001:0023:EN:PDF 2007. 314

(17) Sjaunja, L. O.; Baevre, L.; Junkkarinen, L.; Pedersen, J.; Setala, J. A Nordic Proposal 315

for An Energy Corrected Milk (Ecm) Formula. EAAP European Association for Animal 316

Production Publication 1991, 50, 156-157. 317

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used for dairy cows in Denmark. Proc. Soc. Nutr. Physiol. 2007, 16, 129-131. 319

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Comparison of three techniques for estimating the forage intake of lactating dairy cows on 321

pasture. J. Anim. Sci. 2003, 81, 2357-2366. 322


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potential marker fatty acids for organic milk in milk from conventionally and organically 324

raised cows. Eur. Food Res. Technol. 2011, 232, 167-174. 325

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causes for the elevated n-3 fatty acids in cows' milk of alpine origin. Lipids 2005, 40, 191-327

202. 328

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107, 716-729 337

338


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Table 1: Milk production, milk content of fat and protein, and feed consumption and composition of five herds in May and

September.

Period May September

Herd DH1 DH2 DH3 DJ1 DJ2 DH1 DH2 DH3 DJ1 DJ2

ECM kg cow-1day-1 29.4 29.8 27.1 22.3 26.5 30.9 26.7 25.6 21.6 23.1

Fat g kg milk-1 39.0 38.4 38.5 58.0 55.5 42.1 35.1 40.2 56.8 51.7

Protein g kg milk-1 32.7 32.1 33.0 41.0 40.0 34.7 32.6 36.1 41.1 38.1

Feed dry matter intake (DMI) kg

day-1

20.0 19.2 19.6 15.7 17.7 20.5 20.2 19.8 15.8 16.8

Feed composition kg kg DMI-1

Total pasture 0.47 0.76 0.52 0.66 0.33 0.42 0.21 0.28 0.43 0.29

Grazed grass 0.33 0.50 0.29 0.46 0.20 0.17 0.09 0.13 0.25 0.17

Grazed legumes 0.11 0.20 0.18 0.14 0.01 0.23 0.11 0.13 0.16 0.09

Grazed other species 0.03 0.06 0.05 0.06 0.12 0.02 0.01 0.02 0.02 0.03


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Maize silage 0.23 0.06 0.13 0.00 0.15 0.17 0.48 0.20 0.00 0.11

Grass silage, hay and straw 0.04 0.07 0.06 0.14 0.30 0.17 0.00 0.19 0.23 0.31

Total concentrates 0.25 0.10 0.30 0.19 0.22 0.24 0.31 0.32 0.34 0.29

Brita Ngum Che PhD Thesis 2012

170

Table 2: Pearson correlation coefficients and corresponding p-values for the relations

between total relative amount of DMI of individual feed groups, and content of total PA in

milk-fat and share of RRR isomer of PA, respectively.

Total PA in milk fat Share of RRR isomer in

total PA

Correlation

coefficient p-value

Correlation

coefficient p-value

Feed

Total pasture -0.41 0.019 0.33 0.060

Grazed grass -0.42 0.014 0.11 0.526

Grazed legumes 0.07 0.697 0.85 <0.001

Grazed other species -0.52 0.002 -0.32 0.070

Maize silage 0.08 0.650 -0.28 0.110

Grass silage and hay

and straw

0.15 0.401 -0.02 0.898

Total concentrates 0.58 <0.001 -0.11 0.529


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172

Figure 1: GC-MS-SIM chromatograms (m/z 101) of methyl esters of a sythetic phytanic

acid standard and a milk sample.

Elution time [min]

46.1 46.2 46.3 46.4 46.5 46.6 46.7

Ab

un

da

nc

e m

/z1

01

0

1e+5

2e+5

3e+5

4e+5

5e+5

standard

milk

SRR

RRR


173

Herd

DH1 DH2 DH3 DJ1 DJ2

To

tal

ph

yta

nic

ac

id i

n m

ilk

fa

t [m

g/g

]

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

May

September

bc bc

bc

b

bc

a

a

c

bc bc

Figure 2: Total content of phytanic acid in milk fat of samples from five herds in May and

September. Error bars denote standard deviations and different letters above columns

indicate significant differences (p < 0.05).


174

Herd

DH1 DH2 DH3 DJ1 DJ2

RR

R p

hyta

nic

ac

id i

n t

ota

l p

hyta

nic

ac

id [

mg

/mg

]

0.0

0.1

0.2

0.3

0.4

0.5

0.6

May

September

a

b

f g

c

d

h

e

f f

Figure 3: Share of RRR phytanic acid in total phytanic acid in milk fat of samples from

five herds in May and September. Error bars denote standard deviations and different

letters above columns indicate significant differneces (p < 0.05).


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Share of grazed legumes in dry matter intake

0.00 0.05 0.10 0.15 0.20 0.25

Sh

are

of

RR

R i

so

me

r in

to

tal

ph

yta

nic

ac

id

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

Figure 4: Relations between share of grazed legumes in dry matter intake and share of

RRR phytanic acid in total phytanic acid in milk fat.

Health promoting factors from milk of cows fed green...

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