Retrospective Theses and Dissertations Iowa State University Capstones, Theses and Dissertations

1-1-2004

Prevention of fatty liver in transition dairy cows by subcutaneous Prevention of fatty liver in transition dairy cows by subcutaneous

glucagon injections glucagon injections

Rafael Albertovich Nafikov Iowa State University

Follow this and additional works at: https://lib.dr.iastate.edu/rtd

Recommended Citation Recommended Citation Nafikov, Rafael Albertovich, "Prevention of fatty liver in transition dairy cows by subcutaneous glucagon injections" (2004). Retrospective Theses and Dissertations. 20207. https://lib.dr.iastate.edu/rtd/20207

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Prevention of fatty liver in transition dairy cows by subcutaneous glucagon

injections

by

Rafael Albertovich Nafikov

A thesis submitted to the graduate faculty

in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

Major: Animal Nutrition

Program of Study Committee: Donald C. Beitz, Major Professor

Ronald L. Horst Kenneth J. Koehler

James M. Reecy

Iowa State University Ames, Iowa

2004

Copyright© Rafael Albertovich Nafikov, 2004. All rights reserved.

ii

Graduate College Iowa State University

This is to certify that the master's thesis of

Rafael Albertovich Nafikov

has met the thesis requirements of Iowa State University

Signatures have been redacted for privacy

iii

TABLE OF CONTENTS

LIST OF FIGURES iv

GENERAL INTRODUCTION Thesis Organization 1 Literature Review 1

Introduction 1 Etiology of Fatty Liver Disease 2 Glucagon and Its Involvement in Regulation of Glucose Homeostasis 8 Gluconeogenesis 15 Preventatives and Treatments of Fatty Liver Disease 20

PREVENTION OF FATTY LIVER IN TRANSITION DAIRY COWS BY 14-DAY SUBCUTANEOUSE GLUCAGON INJECTIONS Abstract Introduction Materials and Methods

Experimental Design Sampling and Analysis Statistical Analysis

Results Discussion Conclusions Acknowledgments References

GENERAL CONCLUSIONS General Discussion Recommendations for Future Research

REFERENCES

ACKNOWLEDGMENTS

24 25 28 28 29 32 33 44 51 52 52

58 60

63

72

iv

LIST OF FIGURES

GENERAL INTRODUCTION

Figure 1. Mechanism of fatty liver development in transition dairy cows 4

Figure 2. The "two hits" hypothesis of nonalcoholic steatohepatitis 7

Figure 3. Pancreatic processing of preproglucagon 9

Figure 4. Intestinal cell processing of preproglucagon 10

Figure 5. Signal transduction pathway for glucagon 12

Figure 6. Regulatory signal transduction pathway of hepatic gluconeogenesis 14 and fatty acid oxidation

Figure 7. Glucose biosynthesis from different gluconeogenic precursors in 17 ruminant animals

PREVENTION OF FATTY LIVER IN TRANSITION DAIRY COWS BY 14-DAY SUBCUTANEOUSE GLUCAGON INJECTIONS

Figure 1. Liver lipid composition of cows treated with saline, 7.5, and 15 mg/d 34 of glucagon

Figure 2. Liver glycogen concentration of cows treated with saline, 7 .5, 35 and 15 mg/d of glucagon

Figure 3. Plasma metabolite concentrations of cows treated with saline, 7.5, 37 and 15 mg/d of glucagon

Figure 4. Plasma hormone concentrations of cows treated with saline, 7 .5, 39 and 15 mg/d of glucagon

Figure 5. Milk production of cows treated with saline, 7.5, and 15 mg/d 40 of glucagon

Figure 6. Milk composition of cows treated with saline, 7.5, and 15 mg/d 41 of glucagon

v

Figure 7. Feed intake of cows treated with saline, 7.5, and 15 mg/d of glucagon

Figure 8. Body condition scores of cows from saline, 7.5, and 15 mg/d of glucagon treatment groups

43

45

1

GENERAL INTRODUCTION

Thesis Organization

This thesis is presented as one paper prepared for the submission to the Journal of

Dairy Science from research performed to partly fulfill the requirements for a Master of

Science degree. The paper is complete in itself including an abstract, introduction, materials

and methods, results, discussion, conclusions, and references. A title of the paper is

"Prevention of fatty liver in transition dairy cows by 14-day subcutaneous glucagon

injections". The data from the paper indicate the potential use of a peptide hormone glucagon

for fatty liver prevention in transition dairy cows. The paper is preceded by a literature

review and followed by general conclusions containing a general discussion and

recommendations for future research sections. References conclude the thesis.

Literature Review

Introduction.

Fatty liver (hepatic lipidosis) is a metabolic disorder of dairy cows that occurs

generally during the first two weeks of lactation and is characterized by excessive lipid

accumulation in the liver. However, cows can develop fatty liver at any other time during

2

lactation because of many different reasons. The prevalence of fatty liver can be up to 54%

of a dairy herd (Jorritsma et al., 2000a), and the morbidity and mortality from this syndrome

may reach 90 and 25%, respectively (Raoofi et al., 2001). The consequences of fatty liver

development in dairy cows can be attributed to their reduced productivity, reproductive

performance, suppressed immunity, and increased susceptibility to different infectious and

metabolic diseases (Bremmer et al., 2000; Jorritsma et al., 2000a; Raoofi et al., 2001). In

particular, cows prone to fatty liver can develop ketosis, milk fever, udder edema, displaced

abomasum, retained fetal membranes, metritis, and mastitis (Drackley, 1999; Katoh, 2002;

Morrow, 1976; Van Winden et al., 2003). Metabolic disorders such as fatty liver, milk fever,

and ketosis tend to occur in complexes and probably could have some general cause.

Accumulation of high amounts of triacylglycerols (TAG) by liver of lactating dairy cows is

highly correlated with their decreased fertility and an increase in the interval from parturition

to first ovulation (Jorritsma et al., 2000b; Rukkwamsuk et al., 1999b). Fatty liver

development during the first two weeks postpartum is associated with decreased functional

capacities of polymorphonuclear neutrophilic granulocytes derived from blood and uterus,

which may be why cows with fatty liver have a higher incidence of infectious diseases such

as endometritis (Zerbe et al., 2000).

Etiology of Fatty Liver Disease.

Dairy cows after parturition are in a negative energy balance (NEB) state. This means

that their feed intake and body reserves cannot supply enough energy and nutrients for

maintenance and milk production. With the onset of lactation, cows have increased

requirements for glucose production, which is used as a fuel and for lactose biosynthesis.

3

High demands for glucose in dairy cows at the beginning of lactation exacerbate the energy

deficit. One of the reasons for cows not being able to meet their energy needs is a decline in

their feed intake a few days before calving. After parturition, feed intake of dairy cows rises

much slower than their energy needs.

To meet their energy and glucose production needs, cows mobilize TAG from

adipose tissue and use nonesterified fatty acids (NEFA) as a primary energy source. Thereby,

they can repartition glucose towards lactose biosynthesis. For some reasons, cows in a NEB

mobilize more TAG than they can utilize, which leads to an increased blood NEF A

concentration. A high blood NEF A concentration is considered to be one of the major risk

factors for the development of fatty liver disease (Herdt, 1988).

In liver, NEFA can undergo mitochondrial (Figure 1) or peroxisomal ~-oxidation to

acetyl-CoA, which is oxidized to carbon dioxide in the citric acid cycle with concomitant

ATP production or uncoupling of oxidative phosphorylation and production of reactive

oxygen species (ROS). Alternatively, NEFA can undergo incomplete oxidation with

production of ketone bodies. In addition, NEFA can be esterified into TAG and stored in the

liver. Triacylglycerols can be used for production of very low density lipoproteins (VLDL),

which are secreted into the blood stream (Gruffat et al., 1996). An imbalance between

complete and incomplete NEF A oxidation, production and secretion of VLDL, and TAG

formation and storage in liver can lead to the development of hepatic steatosis.

High blood NEFA concentration in dairy cows is highly correlated with fatty liver

development postpartum. The hepatic uptake of NEF A depends on its concentration in blood

and the rate of blood flow to the liver (Gruffat et al., 1996). Therefore, higher blood NEFA

concentration results in more fatty acid being taken up by liver. Ruminant animals are known

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to have a relatively low capacity for production and secretion of VLDL from the liver

(Hocquette and Bauchart, 1999). So, the inability of liver to oxidize all NEPA that enters it

and impaired production and/or secretion of VLDL can lead to TAG accumulation and fatty

liver development.

Impairment in production and secretion of VLDL by the liver is considered as one of

the factors that contributs to the development of fatty Ii ver disease postpartum in dairy cows

(Bremmer et al., 2000; Katoh, 2002). The rate of liver TAG synthesis in ruminants and

nonrurninants is similar, but the rate of their secretion via VLDL is much slower in ruminants

than in nonruminants (Bremmer et al., 2000; Pullen et al., 1990). A possible explanation for

this could be the fact that ruminants, unlike nonruminants, use adipose tissue as a major site

for fatty acid synthesis, and as such, they do not need to have as good a mechanism for TAG

secretion from the liver (Katoh, 2002).

Cows with fatty liver disease have reduced VLDL secretion (Rayssiguier et al., 1988)

by the liver. Reduction in the secretion of VLDL from cows with fatty liver was attributed to

lower concentrations of apolipoprotein B-100 (apoB-100), which is a TAG-binding protein

that required for assembly of VLDL particles (Pullen et al., 1990; Yamamoto et al., 1995).

The apoB-100 rnRNA concentration in liver does not decrease substantially during earlier

stage of lactation, which indicates that apoB-100 concentration may be regulated

posttranslationally by proteolytic degradation of apoB-100 protein (Gruffat et al., 1997). The

reasons for increased proteolytic degradation of apoB-100 postpartum are not known.

Because of the high correlation between elevated blood NEPA concentration and

fatty liver development in transition dairy cows (Herdt et al., 1988), there have been several

attempts to relate the fatty acid composition of liver, blood NEPA, and adipose tissue to the

6

etiology of fatty liver development (Rukkwamsuk et al., 2000; Sato et al., 2004). Cows with

fatty liver disease had higher concentrations of palmitic, oleic, linoleic, but not stearic acid,

in liver. However, the composition of fatty acids in adipose tissue was the same in healthy

and fatty liver cows before and after parturition (Rukkwamsuk et al., 2000). Palmitic, stearic,

and oleic acids were the major fatty acids found in adipose tissue and blood. Different

feeding strategies (feed restriction or overfeeding) did not affect fatty acid composition in

adipose tissue and plasma.

Hepatic steatosis occurs in two forms: microvesicular and macrovesicular (Haque and

Sanyal, 2002). Microvesicular steatosis is characterized by cytoplasmic accumulation of

numerous small, fat-filled, globules that do not cause displacement of the nucleus. This

condition is associated with disorders of the urea cycle, which results in mitochondrial

abnormalities and hyperammonemia. Macrovesicular steatosis is characterized by

cytoplasmic accumulation of one or two large globules of fat, which causes displacement of

the nucleus to the periphery of the hepatocyte.

Even though there is a lack of the information about histopathological studies of fatty

liver cases in dairy cows, at least mild fatty liver in dairy cows could be characterized as a

microvesicular steatosis. This assumption is supported by data indicating that cows with fatty

liver could have mitochondrial dysfunction and suppressed ureagenesis (Hocquette and

Bauchart, 1999; Reid and Collins, 1980; Strang et al., 1998). However, severe fatty liver in

dairy cows could be an example of macrovesicular steatosis, which can develop into

steatohepatitis and/or fibrosis with the possible progression of fibrosis into cirrhosis.

It is obvious that fatty liver itself is not just an innocent lipid accumulation. Two main

pathways of liver injury are considered to be responsible for the pathogenesis of fatty liver-

7

oxidative stress-induced lipid peroxidation and cytokine-mediated injury (Day, 2002).

According to the "two-hit" hypothesis (Figure 2), high blood NEPA concentration can cause

steatosis (lipid accumulation by liver), which is "the first hit". Then, steatosis itself makes

liver vulnerable for "the second hit"-oxidative stress, endotoxin, and ATP depletion (Day,

2002).

The second 'hits'

Normal fa'./ content

The first 'hit'

t Peroxidation t Sensitivity to

TNF-a

Figure 2. The 'two hits' hypothesis of nonalcoholic steatohepatitis. ATP, adenosine triphosphate; MMPT, mitochondrial membrane permeability transition; UCP-2, uncoupling protein 2; TNF-a, tumor necrosis factor-a. Adopted from Day, 2002.

8

Glucagon and Its Involvement in Regulation of Glucose Homeostasis.

Glucagon is a 29-amino acid peptide hormone that is produced and secreted by the a-

cells of the islets of Langerhans in the pancreas (Drucker, 1990). In addition to the pancreas,

the glucagon gene expression also is observed in the small and large intestine, stomach, and

brain. However, instead of producing glucagon these other tissues produce different bioactive

peptides. The proglucagon cDNAs isolated from all these tissues are identical, which

indicates that the glucagon gene produces a single and unique mRNA transcript, despite

tissue specific glucagon gene localization.

Translation of glucagon mRNA results in a 180-amino acid peptide called

preproglucagon, which contains 20-amino acid signal peptide. After elimination of the signal

peptide, the ' 160-amino acid preglucagon peptide undergoes endoproteolytic processing in a-

cells of the pancreas (Figure 3) and intestinal L-cells (Figure 4), which leads to the

production of tissue specific bioactive peptides. Prohormone convertases 1 and 2 (PCl and

PC2) are responsible for tissue-specific posttranslational processing of the preglucagon

molecule (Tucker et al., 1996). The prohormone convertase 1 is localized in intestinal L­

cells, and PC2 is localized in pancreatic a-cells. Posttranslational processing of preglucagon

molecule in pancreatic a-cells by PC2 leads to the production of glucagon, a 29-amino acid

peptide. However, preglucagon processing in intestinal L-cells leads to the production of

glicentin, oxyntomodulin, glucagon-like peptide 1 (GLP-1), and small amounts of glucagon­

like peptide 2 (GLP-2) and intervening peptide 2 (IP-2).

Presence of the glucagon sequence in glicentin and oxyntomodulin makes these

peptides reactive with antisera against glucagon (Holst, 1997), which sometimes makes

determination of glucagon concentration by radioimmunoassays a bit challenging .

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Pancreatic glucagon and intestinal GLP-1 have attracted the most attention because of

their biological activities. Glucagon-like peptide 1 can stimulate insulin secretion even in

patients with severe type-2 diabetes mellitus and can inhibit glucagon secretion (Drucker et

al., 2000; Holst, 1997). In addition, GLP-1 may increase glucose uptake and insulin

sensitivity. The unique feature of GLP-1 is its very short half-life, which makes difficult to

study or to use that peptide for therapeutic purposes.

Glucagon is secreted from the pancreas in a pulsatile fashion, and such a delivery of

glucagon is more effective in stimulating hepatic glucose output in vivo (Jiang and Zhang,

2003). Glucagon signals via its receptor, which is localized to the liver, adipose, kidney,

heart, spleen, pancreatic islets, ovary, and thymus (Unson, 2002). At least two classes of G­

protein coupled receptors, namely Gha and Gq, are involved in signal transduction pathways

associated with glucagon signalling (Jiang and Zhang, 2003). The activation of the Gha class

of glucagon receptors leads to the activation of adenylate cyclase (Figure 5), increase in

intracellular cAMP concentration, and activation of protein kinase A (PKA). Glucagon

signalling through this pathway is involved in the regulation of hepatic glucose output by

activating PKA, which results in a stimulation of glycogenolysis and gluconeogenesis and

inhibition of glycogenesis and glycolysis. The activation of Gq class of glucagon receptors

leads to the activation of phospholipase C, production of inositol 1,4,5-triphosphate, and

subsequent release of intracellular calcium. Diacylglycerol, the product of

phosphatidylinositol 4,5-bisphosphate hydtrolysis by phospholipase C, binds in concert with

calcium ions to protein kinase C and thereby activates PKA. Activated protein kinase C and

increased intracellular calcium concentration can have an effect on different metabolic

pathways regulated by glucagon.

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Regulation of hepatic gluconeogenesis is achieved by the combined action of

glucagon and insulin. Glucagon activates hepatic gluconeogenesis. It binds to a glucagon

receptor and stimulates production of the cyclic AMP ( cAMP), a cell second messenger

molecule. Cyclic AMP activates PKA, which phosphorylates cAMP response element

binding (CREB) protein (Figure 6). Phosphorylated CREB protein activates peroxisome

proliferative activated receptor-y co-activator 1 (PGC-1) gene expression (Herzig et al.,

2001). Recently, it was discovered that PGC-1, found before in skeletal muscle and brown

fat, is also involved in the regulation of hepatic gluconeogenesis (Yoon et al., 2001).

Peroxisome proliferative activated receptor-y co-activator 1 binds to hepatic nuclear factor-4a

(HNF-4a) and forkhead transcription factor (FOXOl). This binding of PGC-1 can co-activate

the expression of gluconeogenic enzymes such as phosphoenolpyruvate carboxykinase

(PEPCK) and glucose-6-phosphatase (Figure 6).

Interaction between transcription factor FOXOl and co-activator PGC-1 is required

for robust activation of gluconeogenesis (Puigserver et al., 2003). Insulin suppresses hepatic

gluconeogenesis by disrupting the interaction of FOXO 1 and PGC-1. This suppression is

achieved by insulin-stimulated phosphorylation of FOXOI through protein kinase B (PKB)

and Akt protein kinase (Akt) pathway (Figure 6). FOXOl phosphorylation does not only

disrupt the interaction of FOXO 1 and PGC-1, but also excludes FOXO 1 from the nucleus

(Puigserver et al., 2003).

Glucagon is not only involved in the regulation of hepatic gluconeogenesis, but it also

activates fatty acid oxidation by up-regulating gene expression of liver carnitine

palmitoyltransferase 1 (L-CPT 1) enzyme (Louet et al., 2002). Liver camitine

palmitoyltransferase 1 gene has to bind both phosphorylated CREB protein and HNF-4a

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15

transcription factor to become active (Figure 6). Binding of PGC-1 co-activator to HNF-4a

transcription factor can strongly up-regulate L-CPT I gene expression. So, glucagon can

regulate L-CPT I gene expression through abundance of PGC-1 co-activator in the nucleus

(Louet et al., 2002). Adequate regulation of fatty acid oxidation is required for the prevention

of fatty liver development in dairy cows, because L-CPT I enzyme activity is lower in cows

with fatty liver (Mizutani et al., 1999).

Gluconeogenesis and fatty acid oxidation can also be regulated by nutritional means.

For example, HNF-4a transcription factor is involved in the regulation of both

gluconeogenesis and fatty acid oxidation (Hertz et al., 1998; Louet et al., 2002). Fatty acids

in the form of CoA thioesters can modulate the transcriptional activity of HNF-4a gene and

affinity of HNF-4a transcription factor for enhancers and/or promoter region on target genes

(Figure 6). Agonistic ligands of HNF-4a include fatty acyl-CoAs of C14:0 and C16:0 fatty

acids, whereas antagonistic ligands include w-3, w-6, and C18:0 fatty acyl-CoAs (Hertz et

al., 1998).

Gluconeogenesis.

Control of glucose metabolism postpartum is one of the major metabolic challenges

with which a dairy cow must cope. At the beginning of lactation, dairy cows have a great

demand for glucose, which is used primarily for lactose production in milk synthesis

(Overton et al., 1998).

The formation of glucose from nonhexose precursors is called gluconeogenesis.

Gluconeogenesis occurs primarily in liver and to a lesser extend in the kidney. The main

precursors for gluconeogenesis in dairy cows are propionate, some amino acids (mainly

16

alanine and glutamate), lactate, and glycerol (Greenfield et al., 2000a). The importance of

gluconeogenesis for dairy cows can be explained by the fact that only small amounts of

carbohydrates can escape microbial fermentation in the rumen and subsequently be available

for hydrolysis to glucose in the small intestine. The vast majority of the glucose used by a

dairy cow is endogenously produced via gluconeogenesis.

Propionate is converted first to succinyl-CoA in a series of reactions in which

propionyl-CoA carboxylase catalyzes the rate-limiting step (Figure 7). Glutamate is

converted to a-ketoglutarate. Then, both succinyl-CoA and a-ketoglutarate is converted to

oxaloacetate via TCA cycle. Oxaloacetate is exported from the mitochondrion into the

cytosol, where it is used for phosphoenolpyruvate production by PEPCK. Two molecules of

phosphoenolpyruvate are converted to one molecule of glucose via gluconeogenesis, and

glucose is released into a blood stream from the liver.

Alanine and lactate have to be converted to pyruvate before they can enter

gluconeogenesis (Figure 7). Pyruvate is then converted to oxaloacetate by pyruvate

carboxylase, which after conversion to phosphoenolpyruvate can undergo reverse glycolysis.

Glycerol undergoes changes to fructose 1,6-bisphosphate, which enters gluconeogenesis

(Figure 7). The rate-limiting step for the conversion of glycerol to glucose is a reaction

catalyzed by fructose 1,6-bisphosphatase.

In ruminants, PEPCK and glucose-6-phosphatase are the principal enzymes

responsible for the regulation of glucose production. The explanation for this phenomenon is

illustrated by the fact that the majority of the glucose in ruminants is produced from

propionate. Consequently, PEPCK is a good regulatory point. The appropriateness of using

glucose-6-phosphatase to regulate blood glucose concentration comes from the fact that this

Phosphoenol pyruvate

carboxykinase

Oxaloacetate ..

Pyruvate carboxylase

Pyruvate

/\

17

Glucose

Glucose-6-phosphatase

Fructose 1, 6-bisphosphatase

.ill ~ Glycerol

Phosphoenol

·. ·.

pyruvate

···· ... .

TCA cycle

Alanine L<lctate

ProJJionyl-C:ofi carboxylase

Propionate Glutamate

Figure 7. Glucose biosynthesis from different gluconeogenic precursors in ruminant animals. TCA cycle, tricarboxylic acid cycle.

18

enzyme can catalyze the conversion of glucose-6-phosphate, produced by glycogen

degradation, to glucose independent of gluconeogenesis.

Amino acids such as alanine and glutamate are considered to be important

gluconeogenic precursors in addition to propionate during the first two or three weeks of

lactation. However, it is very important not to overestimate the significance of amino acids as

gluconeogenic precursors at the beginning of lactation. The concentration of ammonia and

glutamine, and blood ammonia to urea ratio increases right after parturition (Zhu et al.,

2000). This indicates dysfunction of ureagenesis, which is accentuated by liver TAG

accumulation right after calving. Furthermore, amino acids have to be deaminated before

they can enter gluconeogenesis. The extra ammonia released as a result of amino acid

deamination leads to an increased blood ammonia concentration and possible overloading of

the urea cycle.

The feeding of extra rumen-undegradable protein (RUP) to ruminant animals before

and after parturition has been proposed to create a pool of extra amino acids that could be

used for glucose biosynthesis. However, feeding high amounts of crude protein (CP) and

RUP (CP=16 and 18%, RUP=33 and 40% on dry matter basis) to dairy cows during 28 days

before parturition resulted in decreased feed intake and milk production postpartum

(Greenfield et al., 2000b ). This result can be explained by the fact that excess of protein in a

diet could overwhelm the ability of liver to detoxify ammonia to urea at calving and right

after it. Consequently, the current NRC guidelines for protein content in diets of dry cows

(12% CP and 26% RUP on dry matter basis) seems adequate (Greenfield et al., 2000b).

Feeding extra RUP to dairy cows with fatty liver postpartum led to further TAG

accumulation by the liver and did not have an effect on gluconeogenesis (Murondoti et al.,

19

2002). In summary, even though alanine and glutamate are good substrates for glucose

biosynthesis, it is not reasonable to focus on them as a potential solution to the problem of

glucose production during the first weeks of lactation.

During lactation, hepatic gluconeogenesis is the major source of glucose production

in ruminants (Young, 1977). Therefore, there was an attempt to evaluate the effects of

overfeeding during a dry period together with fatty liver development on the function of key

gluconeogenic enzymes around parturition (Rukkwamsuk et al., 1999a). The activity of

PEPCK in the liver of cows overfed during a dry period was significantly lower than that in

the liver of control cows. The possible explanation underlying this finding is that overfed

cows were able to get most of their glucose from the ration, and, because of that, the rates of

their hepatic gluconeogenesis were low. The activity of PEPCK continued to be low after

parturition in the liver of overfed cows because of low adaptability of their livers to

gluconeogenesis during the dry period. Overfed cows had higher rates of lipolysis and,

consequently, higher glycerol concentration in blood. However, the increase in blood

glycerol concentration did not change the activity of liver fructose 1,6-bisphosphatase in

overfed cows. Possibly, blood glycerol concentration was not high enough to cause an

increase in the function of the enzyme. Surprisingly, the activity of glucose-6-phosphatase

was higher postpartum in the liver of overfed cows. Those cows possibly had higher rates of

glycogen degradation than did control cows because of limited availability of glucose from

gluconeogenesis in overfed cows.

The decreased rates of hepatic gluconeogenesis in fatty liver cows could be explained

not only by feeding high energy diet during dry period (Rukkwamsuk et al., 1999a), but also

by a fat infiltration of liver tissue itself (Cadomiga-Valino et al., 1997). Interestingly,

20

glucagon was able to increase the rates of hepatic gluconeogenesis regardless of whether

liver tissue had high TAG concentration or not (Cadomiga-Valino et al., 1997).

Preventatives and Treatments of Fatty Liver Disease.

Fatty liver, ketosis, and other peripaturient diseases associated with them decrease

productivity, reproductive performance, and well being of dairy cows. These all together

contribute to economic losses of dairy producers. Development of a preventive and/or

treatment for pathological fatty liver would place a tool in the hands of veterinarians and

dairy farmers that would improve the productivity, health, and well being of many cows thus

resulting in major savings for dairy producers.

Many research groups are trying to use different strategies to prevent or treat fatty

liver disease. There was an attempt to decrease blood NEFA and liver TAG concentrations

without decreasing blood glucose concentration and feed intake in healthy dairy cows during

the first week of lactation by a single intramuscular injection of several doses of slow-release

insulin (Hayirli et al., 2002). The results of this study showed that, even though there was

some decrease in blood NEF A and liver TAG concentrations and increase in dry matter

intake and milk production after insulin administration, high blood insulin concentration was

not able to suppress lipolysis from adipose tissue sufficiently because of existence of some

degree of insulin resistance in dairy cows right after calving.

Another attempt to decrease blood NEFA concentration and prevent liver TAG

accumulation in dairy cows postpartum was done by using a 3-day drenching with 500 ml of

propylene glycol, 454 g of fat, or the combination of both beginning at day 0 postpartum

(Pickett et al., 2003). Even though drenching with propylene glycol decreased blood NEFA

21

concentration in dairy cows during the first 21 days postpartum, it did not have any effects on

liver TAG concentration. The possible explanation to the inability of propylene glycol to

decrease liver TAG concentration lies in the magnitude of a difference in blood NEFA

concentration between control and the treatment group. The difference was not big enough to

cause any changes in liver TAG concentration.

Dairy cows have a low capacity for synthesis and secretion of VLDL from liver,

which is considered to be a contributing factor for the development of fatty liver disease

(Grummer, 1993). Increasing VLDL synthesis and secretion could prevent and/or treat fatty

liver. There was an attempt to feed extra rumen-protected choline (RPC) during

periparturient period to increase phosphatidylcholine synthesis and VLDL production and

secretion in dairy cows (Piepenbrink and Overton, 2003). There were no changes in plasma

BHBA, NEFA and liver TAG concentrations after feeding extra RPC. However, the hepatic

capacity for storage of palmitate in the form of TAG within liver slices tended to decrease as

the amount of RPC consumed by cows increased. The hepatic fatty acid oxidation to C02

was not affected by dietary choline.

The idea of using glucagon for fatty liver treatment and/or prevention originated from

investigators who studied secretion and clearance rates of glucagon in dairy cows under

different physiological conditions and changes in metabolite and hormone concentrations in

response to glucagon administration (De Boer et al., 1986). A single intravenous glucagon

injection increased blood insulin and glucose concentrations, thus creating a condition that

could lead to inhibition of lipolysis from adipose. Then, metabolic responses to 14-d

intravenous infusions of glucagon in lactating dairy cows were studied (She et al., 1999a).

The continuous glucagon infusion increased plasma glucose and insulin concentrations and

22

did not change the concentrations of plasma NEFA, BHBA, and urea. Liver glycogen

concentration was not significantly affected by glucagon but increased dramatically after the

cessation of glucagon administration. The continuous intravenous glucagon administration

decreased PEPCK mRNA concentration in normal cows but did not have any effects on

PEPCK mRNA in cows with fatty liver (She et al., 1999b). However, the concentration of

PEPCK was higher in fatty liver cows. There was a decrease in PEPCK mRNA in normal

midlactation cows after glucagon infusion. The data indicate that the expression of PEPCK

gene in normal cows is decreased after glucagon administration because of inhibitory effect

of insulin, which is secreted to balance elevated carbohydrate status after glucagon infusion.

The inability of glucagon to decrease PEPCK mRNA in fatty liver cows may be related to

some degree of insulin resistance existing in those cows.

Intravenous infusions of glucagon for 14 d alleviated fatty liver and ketosis in

lactating dairy cows (Hippen et al., 1999a). Feeding extra com during the dry period combine

with feed restriction and diet supplementation with ketogenic precursor postpartum were

used to induce fatty liver in those cows. In this study, glucagon did not increase plasma

insulin concentration but decreased plasma BHBA and NEFA and liver TAG concentrations,

and increased plasma glucose concentration. Liver glycogen concentration was not affected

by glucagon but increased after the end of glucagon administration.

To make a glucagon therapy easily applicable to field conditions in the dairy industry,

subcutaneous glucagon injections were used to test the treatment of fatty liver in transition

dairy cows (Bobe et al., 2003b). Glucagon was able to increase plasma glucose and insulin

concentrations and decrease plasma NEF A concentration. However, the treatment effect of

glucagon, as based on the changes in liver TAG concentration, was observed only in older

23

cows. Subcutaneous glucagon injections did not have any effects on the concentration of

different fractions of lipoproteins in plasma (Bobe et al., 2003a).

24

PREVENTION OF FATTY LIVER IN TRANSITION DAIRY

COWS BY 14-DAY SUBCUTANEOUS GLUCAGON

INJECTIONS

A paper to be submitted to the Journal of Dairy Science

Rafael A. Nafikov, Burim N. Ametaj, Kenneth J. Koehler, J. W. Young,

and Donald C. Beitz

Abstract

The main objective of this study was to test the extent to which subcutaneous

glucagon injections for 14 days beginning at d 2 postpartum would prevent fatty liver

development in transition dairy cows. Twenty-four multiparous Holstein cows were fed 6 kg

of cracked com in addition to dry cow ration during last 30 d of a dry period to induce fatty

liver development postpartum. Subcutaneous glucagon injections at either 7 .5 or 15 mg/d

dosages or saline as a control were given 3 times per day for 14 d beginning at d 2

postpartum. Glucagon administration at a dosage of 15 mg/d prevented liver triacylglycerol

accumulation and, consequently, fatty liver development in transition dairy cows postpartum.

Glucagon administration at 7.5 mg/d dosage showed a potential for fatty liver prevention.

25

Glucagon administration increased plasma glucose and insulin concentrations and decreased

plasma nonesterified fatty acid concentration. There were no significant effects of glucagon

administration on plasma B-hydroxybutyrate (BHBA) concentration. Cows treated with the

15 mg/d dosage of glucagon, however, tended to show an increase in plasma BHBA

concentration around d 9 postpartum. Glucagon had no effects on feed intake and milk

production of dairy cows. Moreover, milk composition was not altered by subcutaneous

glucagon injections. Milk urea concentration decreased, and plasma urea concentration

tended to decrease during glucagon administration, which indicates that treatment with

glucagon may improve protein utilization. Liver glycogen concentration was not affected by

glucagon administration. There were no significant differences in body condition scores

between cows from different treatment groups throughout the study. Our results show that

subcutaneous glucagon injections can be used for fatty liver prevention in transition dairy

cows.

Introduction

Transition dairy cows defined as 3 wk prior to and 3 wk after parturition, are at high

risk for the development of different metabolic diseases (Drackley, 1999). Fatty liver is one

of them. The prevalence of fatty liver in dairy herds could reach up to 50% (Jorritsma et al.,

2000a). The consequences of fatty liver development are decreased reproductive success and

suppressed immune function (Jorritsma et al., 2000b; Wentink et al., 1999). Increased liver

triacylglycerol (TAG) concentration increases the interval from parturition to first ovulation

and decreases functional capacities of polymorphonuclear neutrophilic granulocytes derived

26

from blood and uterus (Rukkwamsuk et al., 1999b; Zerbe et al., 2000). Fatty liver does not

develop alone and could be associated with other periparturient diseases such as ketosis,

retained fetal membranes, endometritis, displaced abomasum, mastitis, and milk fever

(Katoh, 2002; Van Winden et al., 2003). Ketosis is believed to be associated with fatty liver,

which is commonly the prerequisite for the development of ketosis (Katoh, 2002). Being able

to prevent and/or treat ketosis and other metabolic diseases is very important, because they

cause substantial economical losses to dairy farmers (Fourichon et al., 1999).

Around parturition dairy cows are in a state of negative energy balance (NEB),

because they cannot meet their nutrient requirements for maintenance, fetus growth and milk

production from feed consumption (Jorritsma et al., 2003). At the onset of lactation, the

mammary gland has increased demands for glucose, used primarily for lactose biosynthesis,

which exacerbates NEB, because glucose is in short supply. To meet energy requirements,

cows mobilize fatty acids from adipose tissue, which results in an elevated blood

nonesterified fatty acid (NEFA) concentration (Drackley, 1999; Grummer, 1993). Excess

blood NEFA is taken up by the liver and metabolized in different ways. Nonesterified fatty

acids can be oxidized completely to carbon dioxide or incompletely to ketone bodies,

esterified into TAG for storage in the liver, or secreted into blood as a part of very low

density lipoproteins (VLDL). When the rates of NEFA esterification into TAG for storage

exceed the rates of NEFA disposal, fatty liver can develop. The exact causes of fatty liver are

unknown. However, any factor that causes increased lipid mobilization could be responsible

for disease development. Periparturient ketosis is associated with fatty liver and developed

because of the condition of increased liver lipid infiltration and decreased liver glycogen

(Drackley et al., 1992).

27

Hepatic gluconeogenesis is impaired in cows fed high concentrate diets during the dry

period because of low phosphoenol-pyruvate carboxykinase (PEPCK) activity 1 wk before

and 2 wk after parturition (Rukkwamsuk et al., 1999a). As a result, inadequate glucose

production further increases the ongoing high rates of lipolysis and promotes fatty liver

development. The fatty liver condition itself, however, can inhibit ureagenesis, leading to

increased blood ammonia concentration (Zhu et al., 2000), which can decrease the rates of

hepatic gluconeogenesis from propionate (Overton et al., 1999). Thus, it was demonstrated in

vitro that hepatic TAG accumulation can decrease the rates of gluconeogenesis (Cadomiga­

Valino et al., 1997).

Glucagon is a 29-amino acid peptide hormone produced and secreted by the a-cells in

the islets of Langerhans in the pancreas. The secretion of glucagon occurs in a pulsatile

fashion, which generates a maximal effect on hepatic glucose output. Glucagon is involved in

the regulation of glucose homeostasis by stimulating gluconeogenesis and glycogenolysis

and by inhibiting glycogenesis and glycolysis. Half-life of a glucagon molecule in blood of

dairy cows is about 5 min. (De Boer et al., 1986). Based on in vitro studies glucagon has

been considered for a long time to have a lipolytic effect in humans (Richter et al., 1989).

However, recent data indicate that glucagon does not have a direct effect on the rates of

lipolysis in human adipose tissue (Bertin et al., 2001). In rats, the glucagon to insulin molar

ratio is responsible for the control of ketogenesis, but it has no effect on the rates of lipolysis

(Ubukata et al., 1996). Thus, a high glucagon to insulin ratio increases the concentration of

ketone bodies in blood and a low glucagon to insulin ratio has an opposite effect.

Previously, we demonstrated that 14-d intravenous infusion of glucagon could be

used to treat fatty liver and, associated with it, ketosis in early lactation cows (Hippen et al.,

28

1999a). In this study, feeding extra com grain to dry cows together with energy restriction

and diet supplementation with 1,3-butanediol postpartum were used to induce fatty liver and

ketosis in dairy cows. Intravenous infusions of glucagon, however, are not practical for use in

the dairy industry, and the conditions associated with naturally occurring fatty liver and

ketosis could be different from those artificially created in the Hippen et al. study. The

objectives of this study were to determine whether subcutaneous glucagon injections for 14 d

beginning at d 2 postpartum would prevent development of fatty liver in transition dairy

cows and to evaluate the effects of glucagon treatment on feed intake, milk production, and

blood composition of dairy cows.

Materials and Methods

Experimental Design

In this study, 24 multiparous Holstein cows were randomly assigned to saline

(control), 7.5 mg/d, or 15 mg/d glucagon treatment groups with 8 cows per group. One cow

from the 7.5 mg/d of glucagon treatment group was dropped from the study, because she

developed a fatty liver disease prepartum before we started the treatment administration for

preventive purposes. During wk 4 of gestation, all cows were supplemented with 6 kg of

cracked com in addition to their regular dry cow diet (Bobe et al., 2003b), which was fed

according to NRC requirements (National Research Council, 2001), to stimulate the

development of fatty liver disease postpartum (Hippen et al., 1999a). Cows were housed in a

straw-bedded free-stall with free access to water and hay. After parturition, cows were

29

housed in a tie-stall barn and fed ad libitum a lactation diet (Bobe et al., 2003b) that was

formulated to meet NRC requirements (National Research Council, 2001).

Beginning at d 2 postpartum, cows were injected subcutaneously with saline (0.15 M

NaCl), 7.5 mg/d, or 15 mg/d of glucagon for 14 d. Injections were given in the area between

the fifth and seventh intercostal space three times per day in 8-h intervals. The volume of a

single injection was 60 ml. Lyophilized glucagon (donated by Eli Lilly and Co.,

Indianapolis, IN) was dissolved in 0.15 M NaCl (pH 10.25) at 41.67 mg/Land 83.33 mg/L to

create the dosage of 7.5 and 15 mg/d of glucagon, respectively. Glucagon solution were

prepared every other day, stored at 4 °c and used within 48 h when needed. To prevent

adherence of glucagon, all glassware and utensils were rinsed with 1 % bovine serum albumin

in 0.15 M NaCl before use. All animals were treated in accordance with guidelines

established by the Iowa State University Committee on Animal Care.

Sampling and Analysis

Liver. Liver samples were obtained by puncture biopsies at d -4, 2, 6, 9, 16, 20, 27,

34, and 42 postpartum and stored at -80 c0 for later analysis for concentration of total lipids,

TAG, phospholipids, total cholesterol, and glycogen. Total lipid concentration of liver was

determined gravimetrically by using the Folch method (Folch et al., 1957) with the following

modifications. Liver tissue with wet weight between 300-400 mg was homogenized in 8 ml

of chloroform-methanol (2:1 vol/vol) in a screw-top 25 x 150 extraction tube and sonicated

for 30 s (model 350 sonifier, Bronson Sonic Power Corp., Danbury, CT). The samples were

shaken for 1 h (model 75 wrist-action shaker, Burrel Scientific, Pittsburg, PA), and 2 ml of

0.15 M NaCl was added. After vortexing for a few seconds, the mixture was centrifuged at

30

500 x g for 20 min. (model K centrifuge, International Equipment Co., Needham Heights,

MA), and the methanol-water layer was aspirated.

The chloroform-containing fraction was filtered through a Buchner funnel and a 42.5

mm glass microfiber filter (Whatman International Ltd., Maidstone, England) and washed

three times with 2, 4, and 4 ml of chloroform, respectively. Liver total lipid extract was dried

under constant air flow at 50 c0 for 2.5 h (SC/48R sampler concentrator, Brinkmann

Instruments, Inc., Westbury, NY). Liver total lipid extraction was performed twice using the

same set of liver samples. One of the liver total lipid extracts was used for liver phospholipid

and total cholesterol analysis, and the other extract was used for total lipid and TAG

determination. For phospholipid and total cholesterol determination, dried liver lipid extracts

were reconstituted with 5 ml of 1 % Triton X-405 (Amresco Inc., Solon, OH) in 0.15 M NaCl

at 37 c0, and liver phospholipid (Phospholipids B kits number 991-38492, Wako, Richmond,

VA) and total cholesterol (Cholesterol kit number C7510, Pointe Scientific, Lincoln Park,

Ml) concentrations were determined on a microplate spectrophotometer (SPECTRA max

Plus, Sunnyvale, CA). For liver TAG determination, dried liver lipids were reconstituted

with chloroform, and a small aliquot of the liver lipid in chloroform solution was used for

analysis. After chloroform evaporation, lipids were subjected to base hydrolysis in a solution

containing 20% of 2 N KOH and 80% of 96% ethanol for 1hat80 c0.

The pH of the mixture was neutralized by 12.1 N hydrochloric acid, and the

concentration of a glycerol in the mixture was determined enzymatically (TAG kit number

T7532, Pointe Scientific, Lincoln Park, Ml) and expressed as a TAG concentration. Liver

glycogen determination was done as described earlier (Bobe et al., 2003b ).

31

Plasma. Blood samples were collected in the morning from the coccygeal vein at d 1

through 17, 20, 27, 34, and 41 postpartum. During the treatment period, blood samples were

taken at 1 h after the morning injection of glucagon or saline. We used 10-ml Vacutainer

tubes (Beckton Dickinson and Co., Rutherford, NJ) containing Na3-EDTA for blood

sampling. After centrifugation, blood plasma samples were stored at -20 c0 until further

analysis for the concentration of glucose (glucose kit number 315 Sigma), NEPA (NEFA-C

kit number 994-75409; Wako, Richmond, VA), BHBA (BHBA; Pointe Scientific, Lincoln

Park, MI), PUN (BUN kit number B 7552, Pointe Scientific; Lincoln Park, Ml), and

glucagon (glucagon kit number GL-32K; LINCO Research, Inc. St Charles, MO). Aprotinin

(Boehringer -Mannheim, Indianapolis, IN), a protease inhibitor, was added at 500 KIU to the

1 ml plasma to be analyzed for glucagon.

Plasma insulin concentration was determined by RIA using antisera for bovine insulin

(Bovine Insulin Antisera catalog number 1010, LINCO Research, Inc.; St. Charles, MO). The

rest of the reagents for RIA were from the rat insulin kit (rat insulin RIA kit number RI-13K).

Bovine insulin (bovine insulin catalog number I 0516; Sigma) standards were prepared in the

prescribed assay buffer.

Other measurements. Feed intake was measured twice daily at d 1 through 17

postpartum. Cows were milked at 6.30 and 18.30 h, and production was recorded daily at d 1

through 41 postpartum. Milk samples were collected from two adjacent milking at d 2, 6, 9,

16, 20, 27, 34, and 41 postpartum and analyzed for fat, protein, lactose, and urea N by mid­

infrared spectrophotometry (Milk-0-Scan 203, Foss Food Technology, Eden Prairie, MN).

Body condition scores (BCS) were evaluated by three individuals at d -22, -9, 5, 19, and 32

32

postpartum. Cows were tested for urinary ketones (i.e., acetone plus acetoacetate, Ketostix®;

Miles Inc. Diagnostics Division, Elkhart, IN).

Statistical Analysis

Data were analyzed as repeated measures using the MIXED procedure of SAS,

Version 8.2 (2001). Effects of 7.5 and 15 mg/d of glucagon administration on response

variables were evaluated by comparing each glucagon-treated group with the saline (control)

group. Separate comparisons were made for data obtained during the treatment period and

the post treatment period.

The statistical models contained response variables as the dependent variables. The

fixed effects were treatment (saline, 7.5, and 15 mg/d of glucagon), time, and treatment by

time interaction. A different set of random effects was used for the set of measurements taken

on each cow. The Toeplitz covariance structure was selected based the Schwarz's Bayesian

Information Criterion (BIC, smaller is better), the design of the study, and the biology of the

experiment. The Toeplitz covariance structure assumes equal variances on the main-diagonal,

equal correlations for any pair of measurements equally distant in time, but different

correlation for pairs of observations with separation in time. The logarithmic transformation

of some data was used to meet the requirements for normality. The overall effects of the

different treatment administration were evaluated by comparing changes in response

variables from the glucagon-treated groups with corresponding changes in response variables

from saline (control) group by using a t-test in the LSMEAN statement. The

KENW ARDROGER option was used to obtain the correct degrees of freedom. Means and

33

SEM shown in the figures were calculated from the original, non-transformed data.

Significance was declared at P:S0.05.

Results

Liver lipid composition and glycogen. Glucagon administration tended to prevent

lipid accumulation by liver of lactating dairy cows (Figure lA), although the effects of 7.5

and 15 mg/d of glucagon treatments were not significant (P:S0.096 and P:S0.057,

respectively). A postpartal increase in liver TAG concentration was prevented by glucagon

administration at 15 mg/d dosage (P:S0.028), (Figure lB). Liver TAG concentration in cows

from 7 .5 mg/d of glucagon treatment group, however, was not as effectively suppressed

(P:S0.20). Concentrations of liver phospholipids and total cholesterol (Figure 1 C and 1 D,

respectively) in the glucagon-treated groups were not significantly different from those

concentrations in the control group (liver phospholipids: P:S0.12 and P:S0.31 for 7.5 and 15

mg/d of glucagon treatment groups, respectively; liver total cholesterol: P:S0.41 and P:S0.32

for 7.5 and 15 mg/d of glucagon treatment groups, respectively). Nevertheless, the

concentrations of liver phospholipids and total cholesterol tended to be higher in the control

group than in the glucagon-treated groups. There were significant time effects on liver TAG,

phospholipids, and total cholesterol concentrations (P:S0.024, P:S0.038, and P:S0.024,

respectively) for the posttreatment period (d 17 through 41 postpartum).

Liver glycogen concentration declined after parturition through d 6 postpartum in all

treatment groups (Figure 2). Then, it started to rise with different paces for the different

treatment groups, reaching prepartal values around d 34 postpartum. There were no

9

~8 Ill

:E 7 c.

!6 0 ... ; 5 > :J 4

l 1.2

... Q) ~ 1.1 ~ 0

~ 1.0 ·c.. ~ 0.9 c. t/I _g 0.8 c. a..

1 Treatment 1 A 1 l\l --o-Soll•• 1 1 _._7.5 mg glucagon y I -+-15 mggl""'°" ,-

I

~· jj( I

~~ -4 0 4 8 12 16 20 24 28 32 36 40

Time postpartum, d

: Treat\ment : C 1 I --o- Saline

I / I -+-7.5 mggl"'"°" I I ......._ 15 mg glucagon I

I I ~~ ........... I I e

~ 0.1-----------........ :J -4 0 4 8 12 16 20 24 28 32 36 40

Time postpartum, d

34

7

~ 6 ... ~ 5 ~ 0

~ 4 (!) <C I- 3 a.. Q)

> :J 2

l ... 0.45

; ~ 0.40

e o.35 ! ~ 0.30 0 ..c: (.,) 0.25

J!! .s 0.20 a..

~ :J

1 Treatment 1 B

: l\--o-Soll•o I 1 _._ 7.5 mg glucagon

~ I -+-15 mggl""O°" ,'a""

~ -~ +~ I I

-4 0 4 8 12 16 20 24 28 32 36 40

Time postpartum, d

I Treatment 1 D

1 1 ~7.5 mg glucagon

I I I I

/

1 a\ : --o-Saline

I ........_s......._15 mg glucagon

,- ~[J

l:t~Y~~ It: : ~ ~

-4 0 4 8 12 16 20 24 28 32 36 40

Time postpartum, d

Figure 1. Liver lipid composition of cows treated with saline, 7.5, and 15 mg/d of glucagon. A. Liver total lipids. Effects of 7.5 and 15 mg/d of glucagon administration during treatment period are PS0.096 and PS0.057, respectively; and during post treatment period are PS0.21 and PS0.23, respectively. SEM= 0.36 to 0.78. B. Liver TAG. Effects of 7.5 and 15 mg/d of glucagon administration during treatment period are PS0.20 and PS0.028, respectively; and during post treatment period are PS0.52 and PS0.24, respectively. Time effect for post treatment period is PS0.024. SEM= 0.38 to 0.84. C. Liver phospholipids. Effects of 7.5 and 15 mg/d of glucagon administration during treatment period are PS0.12 and PS0.31, respectively; and during post treatment period are PS0.73 and PS0.87, respectively. Time effect for post treatment period is PS0.038. SEM= 0.07 to 0.11. D. Liver total cholesterol. Effects of 7.5 and 15 mg/d of glucagon administration during treatment period are PS0.41 and PS0.13, respectively; and during post treatment period are PS0.91 and PS0.32, respectively. Time effect for post treatment period is PS0.024. SEM= 0.02 to 0.05.

l 3.0

..... ; 2.5

';!. c: 2.0 Cl> C> 8 1.5 ~ -C> ..... 1.0 g? ·-

I Treatment I

35

..J 0.5 ~-_.....,__ ................ ....-i_ ..... ,...... ..........................

-4 0 4 8 12 16 20 24 28 32 36 40

Time postpartum, d

Figure 2. Liver glycogen concentration of cows treated with saline, 7.5, and 15 mg/d of glucagon. Effects of 7.5 and 15 mg/d of glucagon administration during treatment period are P::;0.11 and P::;0.36, respectively; and during post treatment period are P::;0.094 and P::;0.045, respectively. Time effect for treatment period is P<0.0001. Treatment by time interaction for post treatment period is P::;0.0025. SEM= 0.17 to 0.20.

36

significant effects of glucagon administration on liver glycogen concentration during the

treatment period (PS0.11 and PS0.36 for 7.5 and 15 mg/d of glucagon treatment groups,

respectively). However, glucagon administration at 15 mg/d dosage caused an increase in

liver glycogen concentration during the posttreatment period (PS0.045). Liver glycogen

concentration also tended to be higher in the 7.5 mg/d of glucagon treatment group during the

posttreatment period as compared with that of the control (PS0.094). There were a significant

time effect on liver glycogen concentration for treatment period (P<0.0001) and a significant

time by treatment interaction for posttreatment period (PS0.0025).

Plasma metabolites. Plasma glucose concentration increased during the treatment

period in 7.5 and 15 mg/d of glucagon treatment groups (P<0.0001 and P<0.0001,

respectively) in a dosage-dependent manner (PS0.017, Figure 3A). There were no significant

differences in plasma glucose concentrations after the end of treatment period. Plasma NEF A

concentration was very high in cows from all treatment groups around d 2 postpartum

(Figure 3B). After glucagon administration, plasma NEFA concentration declined rapidly as

compared with that of the control group. As a result, plasma NEF A concentration was lower

in glucagon-treated groups (PS0.046 and PS0.007 for 7.5 and 15 mg/d of glucagon treatment

groups, respectively) during the treatment period. There were significant time effects on

plasma NEFA concentration for treatment (PS0.0089) and posttreatment (PS0.0013) periods.

Plasma BHBA concentration tended to be lower in the 7 .5 mg/d of glucagon treatment group

(P:s;0.35, Figure 3C) during treatment period. Glucagon administration at 15 mg/d dosage did

not have a significant effect on plasma BHBA concentration (P:s;0.77). Moreover, the pattern

of changes in BHBA concentration during the treatment period was the same for both

90

'C -en 80 E CIJ Ill

8 70 ::I

en

!¥ 60 Ill ~ a..

:0

12.0

Ci 10.5 E <(,~

9.0 al :::c al ns 7.5 E Ill ~ a.. 6.0

I Treatment 1

:. ll~lft 1\ ~. • l /'.· ,v.. ...

A

~Saline

~7.5 mg glucagon ......._15 mg glucagon

0 4 8 12 16 20 24 28 32 36 40

Time postpartum, d

1 Treatment 1 C I a~. I I I

1 I ~Saline I a I ~7.5 mg glucagon

:~1M\ ~ : \J~~"/~ I I

0 4 8 12 16 20 24 28 32 36 40

Time postpartum, d

37

700

:E 600 ::1.

<i 500 LL. w z 400 ns E Ill 300

.!:!! a..

200

20

32 en E 18 ns e ::s 16 ns E Ill ns a: 14

B I Treatment I cl I

Pl\ ~Saline CcC\i

1 \ 1 ~7.5 mg glucagon

11 1 ......._15 mg glucagon

I 1,.\ \a& : ~·11.'f•\ a ~~ , \~i9r-aA -~ ~ / : ~ /~ I I

0 4 8 12 16 20 24 28 32 36 40

Time postpartum, d

I Treatment I

-o-Saline _.,_7,5 mg glucagon --.t.-15 mg glucagon

0 4 8 12 16 20 24 28 32 36 40

Time oostoartum. d

Figure 3. Plasma metabolite concentrations of cows treated with saline, 7.5, and 15 mg/d of glucagon. A. Plasma glucose. Effects of 7.5 and 15 mg/d of glucagon administration during treatment period are P<0.0001 and P<0.0001, respectively; and during post treatment period are P:S0.96 and P:S0.33, respectively. Dosage effect for treatment period is P:S0.017. SEM=l.31to1.74. B. Plasma NEFA. Effects of 7.5 and 15 mg/d of glucagon administration during treatment period are P:S0.046 and P:S0.007, respectively; and during post treatment period are P:S0.99 and P:S0.48, respectively. Time effect for treatment period is P:S0.0089; and for post treatment period is P:S0.0013. SEM=26.52 to 64.80. C. Plasma BHBA. Effects of 7.5 and 15 mg/d of glucagon administration during treatment period are P:S0.35 and P:S0.77, respectively; and during post treatment period are P:S0.69 and P:S0.43, respectively. SEM=0.49 to 1.37. D. Plasma urea. Effects of 7.5 and 15 mg/d of glucagon administration during treatment period are P:S0.24 and P:S0.17, respectively; and during post treatment period are P:S0.65 and P:S0.76, respectively. Time effect for treatment period is P:S0.0008; and for post treatment period is P:S0.034. SEM=0.76 to 0.89.

38

15 mg/d of glucagon and saline treatment groups. Plasma urea concentration tended to be

lower in glucagon-treated groups (P:S0.24 and P:S0.17 for 7.5 and 15 mg/d of glucagon

treatment groups, respectively, Figure 3D) during the treatment period. There were

significant time effects on plasma BHBA concentration during the treatment (P:S0.0008) and

posttreatment (P:S0.034) periods.

Plasma insulin concentration rose steadily after the beginning of glucagon

administration (Figure 4A). Cows from the glucagon treatment groups had higher plasma

insulin concentration as compared with that of the control group (P:S0.012 and P:S0.0011 for

7.5 and 15 mg/d of glucagon treatment groups, respectively) during the treatment period.

There were no significant differences in plasma insulin concentration between the glucagon

treatment groups and the control group during the posttreatment period.

Milk production and composition. Glucagon administration did not have a

significant effect on milk production during the treatment (P:S0.78 and P:S0.51 for 7.5 and 15

mg/d of glucagon treatment groups, respectively) and posttreatment (P:S0.46 and P:S0.50 for

7.5 and 15 mg/d of glucagon treatment groups, respectively) periods (Figure 5). There were

significant time effects on milk production during the treatment (P<0.0001) and

posttreatment (P:S0.049) periods.

Although milk lactose concentration tended to be slightly higher in 7 .5 mg/d of

glucagon treatment group it was not a statistically significant difference either during the

treatment (P:S0.19) or posttreatment (P:S0.11) periods as compared with that of the control

group (Figure 6A). Glucagon administration at 15 mg/d dosage did not have a significant

effect on milk lactose concentration (P:S0.96 and P:S0.71) for the treatment and posttreatment

1.5

E c, 1.2 c ~

c ·-:; 0.9 en c

E 5 -C> c r! 4 0 C> CG (.)

~3 C> ns E en 2 ns -D.

•

39

A

~Saline

----7.5 mg glucagon __..._ 15 mg glucagon

0 4 8 12 16 20 24 28 32 36 40

Time postpartum, d

Treatment I e e I

:~ .. jA f"f~

B

~Saline

1 ----7.5 mg glucagon __..._ 15 mg glucagon

0 4 8 12 16 20 24 28 32 36 40

Time postpartum, d

Figure 4. Plasma hormone concentrations of cows treated with saline, 7.5, and 15 mg/d of glucagon. A. Plasma insulin. Effects of 7.5 and 15 mg/d of glucagon administration during treatment period are P:S0.012 and P:S0.0011, respectively; and during post treatment period are P:S0.33 and P:S0.62, respectively. SEM=0.05 to 0.08. B. Plasma glucagon. Effects of 7.5 and 15 mg/d of glucagon administration during treatment period are P:S0.47 and P:S0.86, respectively; and during post treatment period are P:S0.80 and P:S0.82, respectively. SEM=0.12 to 0.40.

42

en 36 ~

"' s:: 30 0 ·-... (.)

::J 24 "t:J 0 "'" Q.

~ 18 -::?:

12

40

I Treatment I 0 °b I I ~ l \

D D d D I • orfoDD o lid o NA. • 1 rY1~ .. -.. ... I 'f..aD -.~ ~ 1;• 'A,t..&AA~ I

~Saline

• 7.5 mg glucagon .. 15 mg glucagon

•

0 4 8 12 16 20 24 28 32 36 40

Time postpartum, d

Figure 5. Milk production of cows treated with saline, 7.5, and 15 mg/d of glucagon. Effects of 7.5 and 15 mg/d of glucagon administration during treatment period are P:'.S0.78 and P:S0.51, respectively; and during post treatment period are P:'.S0.46 and P:'.S0.50, respectively. Time effect for treatment period is P<0.0001; and for post treatment period is P:'.S0.049. SEM=l.32 to 2.79.

5· 1 1 Treatment I

4.8

~ ai 4.5 I Ill .s (.)

j 4.2

3.9

: ~Saline 1 -+-7.5 mg glucagon 1 -6- 15 mg glucagon

A

0 4 8 12 16 20 24 28 32 36 40

Time postpartum, d

c

6 I Treatment I

• 5

c

,Sl 4 I

e

'~saline :-+-7.5 mg glucagon

1 -6-15 mg glucagon

Q.

.ll::

~ 3

2 ........................................................................... _... 0 4 8 12 16 20 24 28 32 36 40

Time postparum, d

41

4.8

~ 4.4

..J

.f!

B

~Saline

-+-7.5 mg glucagon -6- 15 mg glucagon

.ll:: 4.0 I

:!!:

3.6

0 4 8 12 16 20 24 28 32 36 40

Time postpartum, d

Treatment 1 I

D 18 I

Nlr ·i' "C -~ 15

C'O ~ :::s 12

.ll::

I

~Saline L ' --+-7.5 mggl""'"" I -6-15 mg glucagon I

•

0 4 8 12 16 20 24 28 32 36 40

Time postpartum, d

Figure 6. Milk composition of cows treated with saline, 7.5, and 15 mg/d of glucagon. A. Milk lactose. Effects of 7.5 and 15 mg/d of glucagon administration during treatment period are P:S0.19 and P:S0.96, respectively; and during post treatment period are P:S0.11 and P:S0.71, respectively. Time effect for treatment period is P<0.0001. SEM=0.078 to 0.083. B. Milk fat. Effects of 7.5 and 15 mg/d of glucagon administration during treatment period are P:S0.71 and P:S0.66, respectively; and during post treatment period are P:S0.63 and P:S0.83, respectively. Time effect for treatment period is P:S0.0031. SEM=0.11 to 0.22. C. Milk protein. Effects of 7.5 and 15 mg/d of glucagon administration during treatment period are P:S0.13 and P:S0.20, respectively; and during post treatment period are P:S0.64 and P:S0.52, respectively. Time effect for treatment period is P<0.0001; and for post treatment period is P:S0.028. SEM=0.052 to 0.098. D. Milk urea. Effects of 7.5 and 15 mg/d of glucagon administration during treatment period are P:S0.0208 and P:S0.0018, respectively; and during post treatment period are P:S0.50 and P:S0.49, respectively. SEM= 0.55 to 1.34.

42

periods, respectively). There was a time effect on milk lactose concentration during the

treatment period (P<0.0001). Milk fat concentration was not affected by glucagon treatments

during both the treatment (P:S:0.71 and P:::;0.66 for 7.5 and 15 mg/d of glucagon treatment

groups, respectively) and posttreatment (P:S:0.63 and P:S:0.83 for 7.5 and 15 mg/d of glucagon

treatment groups, respectively) periods (Figure 6B). There was a significant time effect on

milk fat concentration during the treatment period (P:S:0.0031 ). Milk protein concentration

declined rapidly since the beginning of lactation until it reached a base line in the range

between 2.50 and 2.75% (Figure 6C). There were no significant differences in milk protein

concentration between glucagon-treated groups and the control group during the treatment

(P:S:0.13 and P:S:0.20 for 7.5 and 15 mg/d of glucagon treatment groups, respectively) and

posttreatment (P:S:0.64 and P:S:0.83 for 7.5 and 15 mg/d of glucagon treatment groups,

respectively) periods. There were significant time effects on milk protein concentration

during the treatment (P<0.0001) and posttreatment (P:S:0.028) periods. Milk urea

concentration was significantly higher in the control group as compared with that of the

glucagon-treated groups (P:S:0.0208 and P:S:0.0018 for 7.5 and 15 mg/d of glucagon treatment

groups) during the treatment period (Figure 6D). There were no significant differences in

milk urea concentration between the glucagon-treated groups and the control group during

the posttreatment period.

Feed intake and BCS. Feed intake of dairy cows was not affected by glucagon

administration during the treatment period (P:S:0.99 and P:S:0.95 for 7.5 and 15 mg/d of

glucagon treatment groups, respectively, Figure 7). However, cows given 7.5 mg/d glucagon

tended to have higher feed intake around d 9 postpartum. There were no differences in BCS

80

~70 .. Q) ~

~ 60 c: ·-"'C Q) Q) 50

LL

43

Treatment

a

0 2 4 6 8 10 12 14 16 18

Time postpartum, d

Figure 7. Feed intake of cows treated with saline, 7.5, and 15 mg/d of glucagon. Effects of 7.5 and 15 mg/d of glucagon administration during treatment period are P:::;0.99 and P:::;0.95, respectively. SEM=4.28 to 6.12.

44

between cows from all treatment groups (Figure 8). Precalving BCS were not as high as in

an earlier study from our research group (Hippen et al., I999a), which slightly decreased the

severity of fatty liver development in the present study.

Discussion

Subcutaneous glucagon injections administered for I 4 d beginning at d 2 postpartum

tended to prevent liver total lipid accumulation (Figure IA). However, the differences in liver

total lipids between the glucagon-treated groups and the control group were not statistically

significant because of the small numbers of cows in the treatment groups and the high

variability among cows within treatment groups. The peak of lipid accumulation by liver was

around d 9 postpartum, which agrees with data of others (Greenfield et al., 2000b).

Glucagon administration at I5 mg/d dosage prevented liver TAG accumulation as

compared with that of the control group (Figure IB). However, the decrease in liver TAG

accumulation provided by the 7 .5 mg/d dosage of glucagon was smaller and statistically

insignificant.

Liver phospholipid (Figure IC) and total cholesterol (Figure ID) concentrations

tended to be higher in control cows as compared to glucagon-treated cows. Our data disagree

with earlier published work (Bobe et al., 2003a). An increase in liver phospholipid and total

cholesterol concentrations in control cows was caused by liver TAG accumulation. The

percentages of liver TAG, phospholipid, and total cholesterol nearly added up to the

percentages of liver total lipids determined gravimetrically, showing indirectly that analysis

of liver lipid composition included all major lipids.

45

Figure 8. Body condition scores of cows from saline, 7.5, and 15 mg/d of glucagon treatment groups. Difference in body condition score between saline and 7.5 mg/d of glucagon treatment groups, saline and 15 mg/d of glucagon treatment groups, and 7.5 mg/d and 15 mg/d of glucagon treatment groups throughout the study are P<0.9971, P<0.9703, and P<0.9525, respectively. SEM=0.12 to 0.23.

46

Liver glycogen concentration declined from d 0 until d 6 postpartum in cows from all

treatment groups and then started to rise, reaching precalving values around d 32 postpartum

(Figure 2). There were no significant treatment differences in liver glycogen concentrations

during the treatment period. This suggests that plasma glucose concentration did not increase

in glucagon-treated groups at the expense of glycogen degradation. Moreover, glucagon had

an inhibitory effect on glycogen synthesis, because, despite a higher plasma glucose

concentration in cows from the glucagon-treated groups (Figure 3A), liver glycogen

concentration was not significantly different between treatments during the treatment period.

The inhibitory effect of glucagon on glycogen synthesis observed in our study agrees with

data from the literature (Jiang and Zhang, 2003). After glucagon administration, liver

glycogen concentration started to rise very steeply, reaching very high levels at d 20 and 27

postpartum, which were significantly different from the values in the control group. A huge

increase in liver glycogen concentration after the end of glucagon administration can be

explained by the fact that there were no significant differences in plasma glucose

concentration after the end of treatment period and most extra glucose produced via

gluconeogenesis in glucagon-treated groups after the end of treatment period was used for

glycogen synthesis. Evidently, enzymes of gluconeogenesis were still up-regulated in cows

from glucagon-treated groups after the end of glucagon administration, which caused extra

glucose production.

Plasma glucose concentration was increased during glucagon administration in a

dosage-dependent manner (Figure 3A). The fact that glucagon administered to dairy cows

causes an increase in blood glucose concentration agrees with the data of others (De Boer et

al., 1986; Hippen et al., 1999a). The increase in plasma glucose concentration probably

47

occurred because of the activation of gluconeogenesis and/or repartitioning of glucose from

the use in glycolysis. Glucagon administration did not increase plasma glucose concentration

at the expense of glycogen degradation; there were no significant differences in liver

glycogen concentration during glucagon administration (Figure 2).

Plasma NEFA concentration declined substantially during the time of glucagon

administration (Figure 3B), which is in agreement with our earlier published work (Hippen et

al., 1999a). Our data support the findings of recent studies that suggest that glucagon has no

direct effect on the rates of lipolysis in human subcutaneous adipose tissue in vivo (Bertin et

al., 2001; Jensen et al., 1991), although some earlier studies indicated glucagon lipolytic

effects in vivo and in vitro (Carlson et al., 1993; Perea et al., 1995). Moreover, changes in the

insulin to glucagon ratio have no effect on the rates of lipolysis as shown in rats (Ubukata et

al., 1996). Most likely, an increase in plasma insulin concentration after glucagon

administration suppresses lipolysis (Elks and Manganiello, 1985) and/or enhances

reesterification of NEFA in adipose tissue into TAG (Campbell et al., 1992). High blood

glucose concentration (Figure 3A) can be responsible for enhanced antilipolytic action of

insulin (Amer et al., 1983).

There were no significant differences in the concentrations of plasma BHBA (Figure

3C) and urinary acetone and acetoacetate (Figure 9) between either of the glucagon treated

groups and the control group. The concentration of ketone bodies in plasma and urine

however, tended to be higher in both the 15 mg/d of glucagon and control (saline) treatment

groups. Plasma BHBA concentration in the control group was elevated around d 9

postpartum probably because of high liver TAG (Figure lB) and low liver glycogen (Figure

2) concentrations in those cows around that time (Drackley et al., 1992). Cows from the 15

48

mg/d glucagon treatment group also had elevated plasma BHBA concentrations, which might

be explained by possible activation of liver carnitine palmitoyltransferase 1 (LCPT-1) by

glucagon (Louet et al., 2002). Cows from the 7 .5 mg/d of glucagon treatment group did not

have increased ketone body production, because glucagon dosage probably was not high

enough to induce ketogenesis.

Plasma urea tended to decrease in the glucagon-treated groups (Figure 3D), but the

difference between the control and either of the glucagon-treated groups were not significant,

which agrees with other studies (Hippen et al., 1999a, b ). The data provide indirect evidence

that glucagon does not increase utilization of amino acids as gluconeogenic precursors.

Around d 9 postpartum, plasma urea concentration was the lowest in the glucagon-treated

groups and the highest in the control group.

Glucagon injections caused a large increase in plasma insulin concentration, which

was sustained until the end of the treatment period (Figure 4A). The data agree with earlier

published work from our research group (She et al., 1999b). There was a steep rise in plasma

insulin concentration above 900 pg/ml after d 8 postpartum in the 15 mg/d glucagon­

treatment group, which led to a substantial decrease in plasma NEF A. On the bases of

Figures 3B and 4A, the reduction in plasma NEF A concentration below 400 µM can be

achieved if plasma insulin concentration is above 900 pg/ml. Insulin inhibitory effect on

lipolysis had been shown earlier.

Plasma glucagon concentrations were around 4000 pg/ml and were not significantly

different between the control group and either of the treatment groups throughout the study

(Figure 4B). The fact that we did not observe any increase in plasma glucagon concentration

after glucagon administration might be explained by a short half-life (5 min) of glucagon in

49

bovine blood (De Boer et al., 1986). Since we collected blood samples at 1 hr after glucagon

administration, this indicates that most of the injected glucagon was degraded by that time.

However, in comparison with the De Boer study, we used subcutaneous glucagon injections

instead of intravenous application, which can produce lower glucagon peaks in blood after

the injection and prolong slightly elevated glucagon concentrations in blood similar to what

was observed in a human study (Muhlhauser et al., 1985).

Milk production (Figure 5) was not affected by glucagon administration and was not

significantly different between either of the glucagon-treated groups and the control group

throughout the study. There was a rapid jump in milk production after parturition, which

reached 27 kg/d by d 4 postpartum in all treatment groups. Then, milk production increased

gradually in all treatment groups until the end of the study.

Lactose concentration (Figure 4A) was not significantly different between either of

the glucagon-treated groups and the control group throughout the study. However, lactose

concentration in milk from 7.5 mg/d of glucagon treatment was observed to be slightly

higher than in milk from the other treatment groups. Aside from variability among the cows,

it is difficult to explain the cause of increase in lactose concentration in 7.5 mg/d of glucagon

treatment group, but our data suggests that there is no direct relationship between blood

glucose concentration and lactose biosynthesis.

There were no significant differences in milk fat concentration between either of the

glucagon-treated groups and the control group throughout the study (Figure 6B). Milk fat

concentration was slightly higher in the control group around d 6 postpartum, and those

control cows also exhibited higher plasma NEFA concentrations. In general, in cows with the

50

same milk production, milk fat concentration might be correlated positively with plasma

NEF A concentration.

Milk protein concentrations (Figure 6C) in all treatment groups declined during the

first two weeks postpartum until it reached a plateau. There were no significant differences in

milk protein concentrations between either of the glucagon-treatment groups and the control

group, which disagrees with an earlier study from our group (Hippen et al., 1999a). Our data

suggest that glucagon administration has no effect on milk protein concentration, which

suggests the fact that glucagon does not facilitate the utilization of amino acids as

gluconeogenic precursors during periparturient period.

Milk urea concentration was significantly higher in the control group compared to

glucagon-treated groups during the treatment period (Figure 6D), which indicates that

treatment with glucagon may improve protein utilization.

Feed intake of dairy cows (Figure 7) rose slowly throughout the treatment period, but

feed intake was not significantly different between either of the glucagon-treated groups and

the control group. Cows from the 7.5 mg/d of glucagon treatment group tended to have

slightly higher feed intake around d 9 postpartum, which can be explained by lower ketone

body concentration in blood and in urine around that time. Subcutaneous glucagon injections

did not decrease feed intake as observed during intravenous glucagon administration (Hippen

et al., 1999a). Surprisingly, increases in plasma glucose concentration caused by glucagon

administration were not accompanied by increases in feed intake, which raises questions

about (1) whether cows do not have enough gluconeogenic precursors around parturition

(Gerloff, 2000) and (2) whether suppression of gluconeogenesic activity limits the use of

glucose precursors available in feed (Rukkwamsuk et al., 1999a). We think that it is possible

51

that glucagon increases plasma glucose concentration not only by activating hepatic

gluconeogenesis, but also by repartitioning glucose from use for energy production to milk

production. Otherwise, it is difficult to explain why such a large increase in plasma glucose

concentration after glucagon administration was not accompanied by increase in feed intake.

Conclusions

The results of our study show that subcutaneous injections of 15 mg/d of glucagon for

14 d beginning at d 2 postpartum help to prevent fatty liver development in transition dairy

cows. Glucagon administration at 7 .5 mg/d dosage shows a potential for fatty liver

prevention, but possibly at a lower level. Subcutaneous glucagon injections increased plasma

glucose and insulin concentrations, which decreased the rates of lipolysis from adipose tissue

and possibly increased the rates of blood NEFA reesterification, causing a reduction in

plasma NEFA concentration. As a result, liver TAG accumulation was prevented in the 15

mg/d of glucagon treatment group. Glucagon administration at 7.5 mg/d dosage showed a

potential for the prevention of liver TAG accumulation, but possibly at a reduced level.

Increase in plasma glucose concentration in response to glucagon injections was mainly

because of an activation of gluconeogenesis and possibly an increase in glucose

repartitioning from utilization as an energy source. Glucagon did not cause liver glycogen

depletion. There were no negative effects of glucagon administration on feed intake and milk

production and composition of dairy cows. Glucagon injections decreased milk urea

concentration and tended to decrease plasma urea concentration, which suggests a role of

glucagon in improvement of protein utilization. There were no significant effects of glucagon

52

administration on the rates of ketogenesis. Considered together, these changes indicate that

glucagon could be very useful tool for the control of fatty liver disease in transition dairy

cows.

Acknowledgments

The authors thank Swiss Valley Farms (Davenport, IA) for analysis of milk samples

and Eli Lilly (Indianapolis, IN) for provision of glucagon. The research was partly supported

by grant number 99-35005-8576 from the US Department of Agriculture and was part of the

regional research project NC-185. Appreciation is also extended to the management and staff

of the Iowa State University Dairy Farm for provision of cows and to many undergraduate,

graduate and veterinary medical students for assistance with collection and analysis of liver

and blood samples.

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expression by camp in combination with hnf4 alpha and camp-response element­

binding protein (creb). J Biol Chem 277: 37991-38000.

Muhlhauser, I., J. Koch, and M. Berger. 1985. Pharmacokinetics and bioavailability of

injected glucagon: Differences between intramuscular, subcutaneous, and intravenous

administration. Diabetes Care 8: 39-42.

Overton, T. R. et al. 1999. Substrate utilization for hepatic gluconeogenesis is altered by

increased glucose demand in ruminants. J Anim Sci 77: 1940-1951.

Perea, A., F. Clemente, J. Martinell, M. L. Villanueva-Penacarrillo, and I. Valverde. 1995.

Physiological effect of glucagon in human isolated adipocytes. Horm Metab Res 27:

372-375.

56

Richter, W. 0., H. Robl, and P. Schwandt. 1989. Human glucagon and vasoactive intestinal

polypeptide (vip) stimulate free fatty acid release from human adipose tissue in vitro.

Peptides 10: 333-335.

Rukkwamsuk, T., T. Wensing, and M. J. Geelen. 1999a. Effect of fatty liver on hepatic

gluconeogenesis in periparturient dairy cows. J Dairy Sci 82: 500-505.

Rukkwamsuk, T., T. Wensing, and T. A. Kruip. 1999b. Relationship between triacylglycerol

concentration in the liver and first ovulation in postpartum dairy cows.

Theriogenology 51: 1133-1142.

She, P., G. L. Lindberg, A. R. Hippen, D. C. Beitz, and J. W. Young. 1999. Regulation of

messenger ribonucleic acid expression for gluconeogenic enzymes during glucagon

infusions into lactating cows. J Dairy Sci 82: 1153-1163.

Ubukata, E., 0. Mokuda, Y. Sakamoto, and N. Shimizu. 1996. Effect of various

glucagon/insulin molar ratios on blood ketone body levels in rats by use of osmotic

minipumps. Diabetes Res Clin Pract 34: 1-6.

Van Winden, S. C., R. Jorritsma, K. E. Muller, and J.P. Noordhuizen. 2003. Feed intake,

milk yield, and metabolic parameters prior to left displaced abomasum in dairy cows.

J Dairy Sci 86: 1465-1471.

Wentink, G. H., T. S. van den Ingh, V. P. Rutten, K. E. Muller, and T. Wensing. 1999.

Reduced lymphoid response to skin allotransplants in cows with hepatic lipidosis. Vet

Q 21: 68-69.

57

Zerbe, H. et al. 2000. Altered functional and immunophenotypical properties of neutrophilic

granulocytes in postpartum cows associated with fatty liver. Theriogenology 54: 771-

786.

Zhu, L. H., L. E. Armentano, D. R. Bremmer, R. R. Grummer, and S. J. Bertics. 2000.

Plasma concentration of urea, ammonia, glutamine around calving, and the relation of

hepatic triglyceride, to plasma ammonia removal and blood acid-base balance. J

Dairy Sci 83: 734-740.

58

GENERAL CONCLUSIONS

General Discussion

The objective of the current study was to test whether multiple subcutaneous

glucagon injections of 7.5 and/or 15 mg/d dosages for 14 d beginning at d 2 postpartum

could prevent the development of fatty liver disease in transition dairy cows. The idea of

using glucagon for fatty liver prevention came from earlier work done in our research group

(Hippen et al., 1999a), which demonstrated the use of 14-d intravenous infusion of glucagon

to treat fatty liver and ketosis induced by feed restriction and diet supplementation with 1,3-

butanediol in lactating dairy cows. The results of the current study show that 14-d

subcutaneous injections of 15 mg/d of glucagon beginning at d 2 postpartum prevent fatty

liver development in transition dairy cows. Glucagon injections at 7 .5 mg/d dosage show a

potential for fatty liver prevention.

Subcutaneous glucagon injections increased plasma glucose and insulin and

decreased plasma NEPA concentrations in transition dairy cows predisposed to fatty liver

development postpartum because of overfeeding during the dry period. These beneficial

effects of subcutaneous glucagon administration are thought to be responsible for decrease in

liver total lipid and TAG concentrations and consequently fatty liver prevention in transition

dairy cows. There were no significant effects of glucagon administration on plasma BHBA

concentration. However, cows given the 15 mg/d of glucagon dosage tended to show

59

increase in plasma BHBA concentration around d 9 postpartum, which was similar to an

increase in BHBA concentration in the control group around the same time.

A mechanism by which glucagon is able to prevent fatty liver development is

unknown. It is possible that glucagon indirectly inhibits the rate of lipolysis and stimulated

uptake and reesterification of NEFA by adipose tissue because of increases in plasma insulin

and glucose concentrations. Glucagon also can have an effect on depletion of liver TAG pool

and/or increase of fatty acid utilization in liver by activating camitine palmitoyltransferase-1,

which leads to increased ~-oxidation and/or ketogenesis. It seems like glucagon has no effect

on the rates of VLDL synthesis and secretion from liver. This statement is supported

indirectly by our data showing that glucagon has no effect on plasma TAG concentration.

The effect of glucagon in this study was not confounded with the differences in body

condition score between cows from different treatment groups, because there were no

significant differences in body condition score between the cows from different treatment

groups throughout the study.

A use of glucagon as a preventive for fatty liver development did not cause any

negative effects on feed intake, milk production, and milk composition of dairy cows, which

makes glucagon a very useful tool for the disease prevention. Glucagon administration

decreased milk urea concentration, which suggests that it has a beneficial effect on improving

protein utilization in transition dairy cows.

60

Recommendations for Future Research

The current study demonstrated that subcutaneous glucagon injections could be used

for fatty liver prevention in transition dairy cows. However, there are still some questions

that have to be answered in order to optimize the use of glucagon for fatty liver prevention or

to come up with better ways to prevent and/or treat the disease. One of the questions is what

is the best model for fatty liver disease in transition dairy cows? Earlier studies conducted in

our research group used feed restriction and diet supplementation with 1,3-butanediol to

induce fatty liver and ketosis in lactating dairy cows. Such an approach produced a relatively

high incidence of fatty liver and ketosis in lactating dairy cows, but the etiology of naturally

occurring fatty liver disease could be completely different from what was induced by those

conditions. In the current study, we used diet supplementation with cracked corn to increase

energy density of a dry cow ration during the last 30 d of a dry period to make cows more

susceptible for fatty liver development postpartum because of over conditioning. It is

believed that cows with higher body condition scores have a higher incidence of fatty liver

disease. However, this approach was not very successful, because most of the cows in our

study did not develop severe fatty livers. A possible explanation for a failure to develop a

severe fatty liver disease in our study is that feed intake of dairy cows can start declining

during the last month of a dry period, and, in this case, extra corn feeding might not be able

to increase cows' body condition scores. Better models of fatty liver disease should be

created in the future. One of the approaches that can be used is overfeeding of cows during

the entire dry period or during the end of the lactation period plus during the dry period. Fatty

61

liver development is not only associated with high body condition scores of dairy cows but

also could be developed by feeding large amounts of grain during a dry period. So, other

alternatives than over conditioning should be considered for the disease development.

More research has to be done to study the physiology of transition and lactating dairy

cows. It would be very helpful to know what type of metabolic changes occur under normal

and pathological conditions, because in the literature you can find contradictory statements

about what is happening. For example, many authors claim that after calving transition dairy

cows experience hypoglycemic conditions while the others suggest the opposite. None of

them say what normal glucose concentration in blood of dairy cows should be around that

time. That is why greater knowledge about the physiology of transition and lactating dairy

cows would be very useful for data interpretation. Moreover, disagreements among results

from different publications sometimes happen only because of differences in approaches

used to analyze samples. Developing standardized assays would facilitate better data

comparison across different studies.

According to a current theory of fatty liver development in transition dairy cows, high

blood NEFA concentration is highly correlated with postpartum disease incidence. Future

studies of regulation of lipolysis in dairy cows can provide very useful information about the

etiology and other possible ways of fatty liver treatment and prevention.

Recent studies established a relationship between fatty liver and reproduction and

between fatty liver and immune function of dairy cows. More studies have to be done to

understand those relationships and to check whether glucagon administration has any effects

on them. Some authors are trying to relate infertility problems seen in dairy cows to low

blood insulin concentration around breeding time. More study of possible improvements of

62

reproductive performance of transition dairy cows related to the ability of glucagon

administration to increase blood insulin concentration should be done.

In addition, subcutaneous glucagon injections for 14 days are not practical for on

farm use. Other alternatives such as a slow-release formulation of glucagon should be

developed in the future and tested on transition dairy cows.

63

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72

ACKNOWLEDGMENTS

Completion of the M. S. degree accomplishes one of my goals on the way to getting a

Ph.D. degree and becoming a university professor. Many people have helped me to achieve

this goal, and I apologize to those whose names are not mentioned here, but I offer them my

gratitude and best wishes.

I express sincere appreciations to my parents and my sister without whose support

and love I would not have achieved anything in this life. I thank Dr. Donald Beitz for his

support and friendship as my major professor. Recognitions and thanks are given to Dr.

Ronald Horst, Dr. Kenneth Koehler, and Dr. James Reecy for serving as my Program of

Study Committee members.

Special thanks are given to Dr. Dennis Marple for his support during my application

process to graduate school in the Animal Science Department at Iowa State University. I also

thank Dr. Mykhailo Zubets, the President of Ukrainian Academy of Agrarian Sciences, for

serving as my major professor, when I was in graduate school at the National Agricultural

University of Ukraine, and for supporting me in graduate school and during an application

process to Iowa State University.