Ncb5or, Iron and Diabetes - Research Paper

7/30/2019 Ncb5or, Iron and Diabetes - Research Paper

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Can Iron Supplementation Delay Early-onset Diabetes?

Molecular Studies on a Novel Ncb5or-null Diabetes Mouse Model

A Science Paper

Presented to

Kansas BioGENEius Challenge

by

Vivek Menon


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Abstract

NADH cytochrome b5 oxidoreductase (Ncb5or) is a flavohemoprotein associated with the

endoplasmic reticulum and widely expressed in animal tissues. Ncb5or-null (KO) mice are

observed to develop early-onset lean diabetes around 7 weeks of age due to -cell loss. In

addition, Ncb5or is involved in fatty acid metabolism by influencing the functionality ofstearoyl-CoA desaturase (SCD), an iron-containing enzyme. Pancreatic -cells and hepatocytes

in prediabetic Ncb5or KO mice accumulate increased levels of free fatty acids and reactive

oxygen species (ROS) or oxidative stress. It is hypothesized that Ncb5or acts upstream of SCD

to prevent these cellular defects. This study focuses on the effects of iron supplementation on

Ncb5or KO mice, its potential in delaying diabetes, and the changes of molecular markers of

cellular stress. Our preliminary study show that Ncb5or KO mice receiving weekly Ferrlecit

injection since 5 weeks of age exhibit normal blood glucose levels at age 8 weeks as wild-type

(WT) counterparts, indicating delayed onset of diabetes iron supplementation. This study shows

that the markers of SCD defects, mitochondrial function and oxidative stress in the liver of these

treated KO mice are brought down to the same levels as WT control. This is in contrast to

increased marker expression in the liver of untreated KO mice, whose iron content is lower than

WT. We conclude that rescuing iron homeostasis in KO hepatocytes through iron

supplementation reduces SCD defects and oxidative stress. We speculate that iron

supplementation has a similar effect on -cells of prediabetic Ncb5or KO mice, thereby reducing

oxidative stress and increasing -cell viability.


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Table of Contents

Abstract......................................................................................................................................................... 1

Table of Contents.......................................................................................................................................... 2

1 Introduction........................................................................................................................................... 3

1.1 Ncb5or: ......................................................................................................................................... 3

1.2 Ncb5or and Diabetes:.................................................................................................................... 4

1.3 Ncb5or and SCD Function:........................................................................................................... 4

1.4 Ferrlecit Preliminary Study:.......................................................................................................... 5

1.5 Objectives: .................................................................................................................................... 7

1.6

Hypothesis:................................................................................................................................... 7

2 Materials and Methods.......................................................................................................................... 8

2.1 Mouse Lines:................................................................................................................................. 8

2.2 Experimental Mice Subjects: ........................................................................................................ 8

2.3 RNA Extraction/Preparation from Mice Liver: ............................................................................ 8

2.4 Reverse Transcription for cDNA Synthesis:................................................................................. 9

2.5 Quantitative Real-time PCR assay and Data Collection: ............................................................ 10

3 Results................................................................................................................................................. 12

3.1 Quantitation Data (Threshold Values): ....................................................................................... 12

3.2 Data Analysis: ............................................................................................................................. 14

4 Discussion........................................................................................................................................... 18

4.1 Conclusion .................................................................................................................................. 18

4.2 Future .......................................................................................................................................... 19

5 Acknowledgements ............................................................................................................................. 20

References ................................................................................................................................................... 21


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1 Introduction

1.1 Ncb5or:

Ncb5or (NADH cytochrome b(5) oxidoreductase) is a multi-domain redox enzyme found in all

animal tissue associated with the endoplasmic reticulum.[1][3]

According to previous research by

Dr. Hao Zhu and Dr. Bin Deng, Ncb5or contains: an N-terminal region, the b5 domain, the CS

domain, and the b5R domain. Ncb5or-b5 is a heme-binding domian homologous to microsomal

cytochrome b5 and belongs to the cytochrome b5 superfamily. Ncb5or-b5R, the FAD binding

domain, is homologous to cytochrome b5 reductase (Cyb5R3) and belongs to the ferredoxin

NADP+ reductase superfamily. Both superfamilies are of great biological significance whose

members have important functions. The b5 domain is homologous to microsomal cytochrome b5

and the b5R is homologous to cytochrome b5 reductase. NAD(P)H binding to the b5R domain

allows electron flow from NAD(P)H to FAD, and subsequently onto heme in the b5 domain,

reducing ferric iron ions to ferrous ions. The heme center of the protein was determined to have a

low redox potential, indicating that Ncb5or would serve as a potent electron donor. The CS

domain was found to be crucial in the interactions between the electron transfer between the b5

and b5R domains. The Ncb5or domains were sequenced in various combinations and

conformations and were used to genetically transform E. Coli bacteria via plasmids. The bacteria

were used to express Ncb5or domains in conjugation (e.g. b5+b5R and b5+CS+b5R). Protein

purification was done through ion affinity chromatography. It was found that the addition of the

CS domain in the protein substantially increased redox potential of the protein.[8]


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1.2 Ncb5or and Diabetes:

This section of the review aims to aims to establish the relationship between the diabetes,

Ncb5or. Type I diabetes is characterized by the loss of-cells, leading to a decreased level of

insulin production. Usually, in Type I patients, -cell apoptosis is an autoimmune response

within pancreatic islets. Islets from mice, from which Ncb5or was removed (Ncb5or-/-

), have

markedly impaired glucose- or arginine-stimulated insulin secretion.[2]

Subsequently, it has been

demonstrated that Ncb5or-/-

mice develop early-onset diabetes. Between 4 and 6 weeks of age

these mice develop a progressive loss of -cells in pancreatic islets, along with severe

hyperglycemia and decreased serum insulin levels, while insulin tolerance remains normal. By 7

weeks of age, these mice develop severe hyperglycemia with markedly decreased serum insulin

levels and nearly normal insulin tolerance.Ncb5or-/-

mice with diabetes are sensitive to insulin,

as shown by insulin tolerance tests, or ITT.[2]

In situ insulin staining reveals a progressive loss of

-cells, as well as in increase in oxidative- and ER-stress during the development of diabetes in

Ncb5or null mice, although the genetic mechanism behind this is still not known.

[6]

1.3 Ncb5or and SCD Function:

Ncb5or deficiency also results in lipodystrophy and increased hepatocyte sensitivity to

cytotoxic effects of saturated fatty acids.[5]

Despite increased fatty acid uptake and synthesis and

higher stearoyl-CoA desaturase (SCD1) expression, Ncb5or-/-

mice livers accumulate higher

levels of intracellular free fatty acids than wild-type (WT) mice. It has been demonstrated that

SCD1 requires Ncb5or in order to fulfill its function in converting saturated fatty acids (SFA)

into mono-unsaturated fatty acids (MUFA). As a result of free fatty acid accumulation, increased

fatty acid catabolism was observed in hepatocytes of Ncb5or-/-

. Consequently, levels of reactive


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oxygen species (ROS) were higher in Ncb5or-/-

hepatocytes, upregulating markers for oxidative

stress (MT1, MT2, HMOX1, GSTT3), as well as markers for mitochondrial biogenesis such as

PGC-1. These changes in gene expression mirrored the changes that occurred in SCD1-/-

mice,

further demonstrating that Ncb5or was crucial to SCD1 function and that impaired fatty acid

desaturation leads to oxidative stress.[5]

1.4 Ferrlecit Preliminary Study:

In an unpublished, preliminary study conducted at the University of Kansas Medical Center by

Dr. WengFang Wang and Dr. Hao Zhu, mice were given injections of an iron supplement known

as Ferrlecit), a common iron replacement product for the treatment of iron deficiency anemia.

Injections were performed weekly, along with body weight measurement, starting 5 weeks after

the date of birth. Mice were then euthanized and sacked after 8 weeks of treatment and all organs

were immediately placed into liquid nitrogen and stored at -80oC. The final body weight and

glucose levels were recorded at the 8 week mark as well.

As indicated by the data table below (Table 1), the iron injected Ncb5or-/-

mice do not

exhibit the glucose levels (mg/dL) typical of a hyperglycemic/diabetic mouse (Figure 1).

Although lipodystrophy was not prevented in Ncb5or-/-

mice, blood-glucose levels were normal,

indicating that the iron injections counteracted the typical formation of hyperglycemia found in

Ncb5or-/-

mice (Figure 2). Iron content was measured in the liver to verify the efficacy of iron

supplementation (Figure 3).


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Table 1: Preliminary Study Mouse Data

*Wild-type mice are indicated by WT and Ncb5or-/-

mice are indicated by KO (knock-out)

Figure 1: Figure 2:

Figure 3:


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However, the mechanism behind the lowering of blood-glucose levels and delay of early-

onset diabetes are unknown. Because Ferrlecit injections in Ncb5or-/-

have produced a similar

phenotype as wild-type (WT) mice, this study is aimed to determine the extent to which Ferrlecit

can reproduce the effects of Ncb5or presence. It should be noted that these procedures were not

conducted for the purpose of this study. Preserved mice livers were obtained from the storage

facility at the laboratory of Dr. Hao Zhu.

1.5 Objectives:

The purpose of this study is to use a genetic mouse model for studies of molecular mechanisms

of diabetes in humans and potential treatment. Specifically, this study investigates the cellular

pathways affected by iron injections and to evaluate relationship between iron supplementation

and fatty acid desaturation, mitochondrial function, and oxidative stress in hepatocytes, which

can help reflect possible changes that occur in -cells and globally in the mouse. This can help

establish a model for increased oxidative stress in -cells.

1.6 Hypothesis:

It is hypothesized that iron supplementation will increase SCD1 functionality, reducing FFA

accumulation, fatty acid catabolism, and oxidative stress. As a result, iron injections are expected

to normalize (down-regulate) gene expression levels in Ncb5or-/-

mice of fatty acid desaturation

(SCD1, SCD2), mitochondrial biogenesis (PGC-1), and oxidative stress (MT1, MT2, HMOX1,

GSTT3) to levels typical of wild type (WT) mice.


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2 Materials and Methods

2.1 Mouse Lines:

The two lines of mice utilized in the study are WT and Ncb5or KO mice (C57BL/6). The WT

mice (Ncb5or+/+

) have undergone no genetic alteration, whereas the KO mice (Ncb5or-/-

) lack a

functional Ncb5or gene through exon 4 disruption.

2.2 Experimental Mice Subjects:

Iron supplementation was performed in male mice as specified in a protocol approved by the

Institutional Animal Care and Use Committee at the KU Medical Center. Livers of 3 KO mice

(NC3554, NC3595, and NC3605) and 3 WT (NC3571, NC3591, and NC3607) were used in this

study.

2.3 RNA Extraction/Preparation from Mice Liver:

Kits used: Invitrogen TRIzol Reagent (15596-026)

In order to extract mRNA to quantify and compare gene expression, RNA was extracted using

the trizol method. A consistently small piece of each stored mouse liver was ground in a 15mL

conical tube containing 2mL of TRIzol Reagent. These TRIzol-sample mixtures were allowed to

sit at room temperature for 10 minutes. These mixture samples were then separated as necessary

into sterile 1.5mL Eppendorf tubes. 0.2mL of chloroform per 1 mL TRIzol was then added to

each mixture, inverting the tube 20 times to mix the solution. This allowed for separation from

the other organic material of RNA. The Trizol-chloroform mixture was then left at room

temperature for 15-30 minutes to allow for good phase separation, after which the samples were


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spun at 12000g in a centrifuge for 10 minutes at 4oC to completely separate the phases. Another

chloroform precipitation was then conducted in order to obtain a better quality of RNA. The

supernatant of the resulting tube, of clear color, was then carefully pipetted out and transferred

into a new sterile 1.5mL tubes. The tubes containing the other organic material were disposed of.

At this step, isopropanol was added to the clear supernatant in the new tubes (0.5 mL per

1 mL TRIzol), inverting the tubes to mix. The resulting solution was then kept at room

temperature for 10 minutes. Subsequent centrifuging of the samples at 12000g for 10 minutes at

4oC was conducted to form the RNA pellet. The supernatant was poured out (avoiding the RNA

pellet) and was washed with 70% EtOH. After a brief spin (9000 rpm for 5 minutes), all liquid

was removed using a micropippete, ensuring that nothing remained except the RNA pellet. The

pellet was air-dired for 5 minutes and then resuspended with 50 l DEPC-H2O.

The concentration of RNA in the samples was determined with a spectrophotometer by

diluting each sample 300-fold (add 1 ul sample to 299 ul H2O). Calculate by following: 1

OD260 unit = 0.04 ug/ul RNA. Note: All materials used, including micropipette tips, conicals,

and tubes were RNase free.

2.4 Reverse Transcription for cDNA Synthesis:

Kits used: Bio-Rad iScript cDNA Synthesis Kit (170-8891)

The RNA extracted from the liver samples had to subsequently be converted into cDNA in order

to quantify it using the quantitative PCR method. Thus, a Reverse Transcription kit was used to

convert the RNA to cDNA via the reverse-transcriptase enzyme. First, the RNA/primer/dNTP

mix was made by combining the following components in a sterile RNase-free microfuge tube:


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Total RNA 1variable volume (2 g) 5x iScript Reaction Mix8 L Nuclease-free watervariable volume (total reaction volume 40L)

Samples were then mixed well by pipetting up and down. The 40 l cDNA synthesis reaction

was then incubated at 25C for 5 minutes, at 42C for 30 minutes, and 85C for 5 minutes. The

yield of each cDNA sample was quantified using a spectrophotometer.

2.5 Quantitative Real-time PCR assay and Data Collection:

Kit used: Bio-Rad iQ SYBR Green Supermix.

The purpose of the quantitative real-time PCR assay was to compare the levels of mRNA of the

test genes that were present in the liver cells. In order to do that, the cDNA generated from the

Reverse Transcription reaction is amplified through heat cycles that denature the DNA, anneal

primers, and then replicates the gene that the primers were designed for. In addition, a

fluorescent marker, in this case SYBR Green, is also added to the PCR reaction. SYBR Green

attaches to the DNA in such a way that its fluorescence increases as the DNA is being amplified.

Thus, the qPCR machine measures and records the absorbance values of the samples in real time

as the DNA is being amplified. The machine and software also allows one to set an absorbance

threshold value and will tell you the number of cycles (C(t)) that it took for all of sample to reach

this threshold. The C(t) values generated from a this step were from a threshold of 0.2 RFU.

iQ SYBR Green supermix (containing fluorescent SYBR Green, polymerase and dNTPs),

template cDNA, the appropriate primers and RNase-free water were set to thaw on ice.

Afterwards, each individual solution was vortexed. The reaction mix for each well in the PCR

plate was prepared according to the following: (Reactions were performed in duplicate, i.e. each

cDNA sample was tested twice for gene expression levels.)


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iQ SYBR Green Supermix10L Forward Primer1 L (from 10M stock) Reverse Primer1 L (from 10M stock) Template cDNA1 L Nuclease Free Water7 L

The primers used in this experiment were the primers for 18s (internal control, SCD1, SCD2,

PGC-1, MT1, MT2, HMOX1, and GSTT3. In order to minimize pipetting mistakes and other

experimental errors, a master mix of the iQ SYBR Green Supermix, forward and reverse primer

was prepared for each individual gene. After mixing the reaction mix thoroughly, appropriate

volumes were dispensed into PCR tubes. The template cDNA was added last to the individual

PCR wells.

The quantitative PCR thermocycler (Bio-rad MiniOpticon) was programmed to following

temperatures optimized for the iQ SYBR Green Supermix:

1 Cycle: Initial Denaturing and enzyme activation95oC, 3:00 40 Cycles:

o Denaturing95oC, 0:10o Annealing - 60oC, 0:15o Extension72oC, 0:30

1 Cycle: Melt Curve55-95oC (in 0.5oC increments), 0:30 secsThe PCR tubes were then added to the thermocycler and the thermocycler was set to run. The

machine ran for about 2 hours and 15 minutes, determining the absorbance levels in each DNA-

containing well as it continually amplifies due to the thermocycler.


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3 Results

3.1 Quantitation Data (Threshold Values):

The threshold for absorbance levels was set to 0.2 RFU on the quantitative PCR machine

software in order to determine the amount of cycles of qPCR required for each sample to reach

the threshold (C(t) value). The raw data from the machine is depicted in Table 2.

Table 2: C(t) Values

Mouse

ID

Gene Ct Delta Ct Transcript Normalized

(*10^6)1KO 18s 8.07 0.00000000000000 1.00000000000000000 1000000

1KO SCD1 20.58 12.5123902315297 0.00017115721959087 171.1572196

1KO SCD2 29.30 21.232303041264 0.00000040591949311 0.405919493

1KO PGC-1a 27.80 19.7287734052323 0.00000115092606959 1.15092607

1KO MT1 19.79 11.7135832941509 0.00029775569158558 297.7556916

1KO HMOX1 27.10 19.0250454432295 0.00000187452247871 1.874522479

1KO GSTT3 24.65 16.5794352585224 0.00001021157369736 10.2115737

1KO 18s 7.98 0.00 1.00000000000000000 1000000

1KO SCD1 20.54 12.56 0.00016573706202763 165.737062

1KO SCD2 30.10 22.12 0.00000021980771232 0.219807712

1KO PGC-1a 27.57 19.59 0.00000126728544482 1.267285445

1KO MT1 20.15 12.17 0.00021698801033802 216.9880103

1KO HMOX1 26.47 18.49 0.00000271369968233 2.713699682

1KO GSTT3 24.66 16.68 0.00000950355547707 9.503555477

2KO 18s 7.59 0.00 1.0000000000000000 1000000

2KO SCD1 20.12 12.52 0.0001700553439192 170.0553439

2KO SCD2 30.36 22.77 0.0000001401007601 0.14010076

2KO PGC-1a 26.68 19.08 0.0000017997441340 1.799744134

2KO MT1 18.98 11.39 0.0003737721771698 373.7721772

2KO HMOX1 26.61 19.01 0.0000018885878425 1.888587842

2KO GSTT3 25.71 18.11 0.0000035307718120 3.5307718122KO 18s 8.44 0.00 1.0000000000000000 1000000

2KO SCD1 21.65 13.21 0.0001054498339734 105.449834

2KO SCD2 29.77 21.34 0.0000003779672067 0.377967207

2KO PGC-1a 26.26 17.82 0.0000043163607714 4.316360771

2KO MT1 18.74 10.30 0.0007940081982884 794.0081983

2KO HMOX1 25.82 17.39 0.0000058404952805 5.84049528

2KO GSTT3 25.40 16.96 0.0000078283519131 7.828351913


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3KO 18s 8.01 0.00 1.00000000000000 1000000

3KO SCD1 20.72 12.71 0.00014909954582 149.0995458

3KO SCD2 30.16 22.15 0.00000021438975 0.214389752

3KO PGC-1a 26.58 18.57 0.00000256302955 2.563029552

3KO MT1 18.83 10.82 0.00055354619651 553.5461965

3KO HMOX1 27.16 19.15 0.00000172391703 1.7239170323KO GSTT3 27.38 19.37 0.00000147834591 1.478345905

3KO 18s 8.28 0.00 1.00000000000000 1000000

3KO SCD1 20.33 12.05 0.00023632538220 236.3253822

3KO SCD2 29.96 21.68 0.00000029757612 0.297576117

3KO PGC-1a 26.80 18.52 0.00000265306494 2.653064945

3KO MT1 19.35 11.07 0.00046612075432 466.1207543

3KO HMOX1 26.86 18.58 0.00000255404614 2.554046139

3KO GSTT3 26.94 18.66 0.00000240863437 2.408634372

4WT 18s 8.961887 0 1 1000000

4WT SCD1 22.37535 13.4134584 9.1653E-05 91.65299838

4WT SCD2 29.14547 20.1835865 8.39721E-07 0.8397210554WT PGC-1a 27.44962 18.4877371 2.72042E-06 2.720423892

4WT MT1 22.14371 13.18182596 0.000107616 107.61554

4WT HMOX1 26.47226 17.51037022 5.35616E-06 5.356157372

4WT GSTT3 25.91582 16.95393179 7.87695E-06 7.876948173

4WT 18s 9.77885 0 1 1000000

4WT SCD1 22.60423 12.82538325 0.000137776 137.7764331

4WT SCD2 30.2828 20.50395028 6.72506E-07 0.672505648

4WT PGC-1a 27.66859 17.88973595 4.11768E-06 4.117682578

4WT MT1 22.92091 13.14205557 0.000110623 110.6234169

4WT HMOX1 25.75544 15.97659 1.55084E-05 15.50840668

4WT GSTT3 26.15861 16.37976376 1.17273E-05 11.72734725

5WT 18s 9.317409 0 1 1000000

5WT SCD1 21.7762 12.45878719 0.000177636 177.6361476

5WT SCD2 30.57091 21.25350041 3.99999E-07 0.399998962

5WT PGC-1a 27.3765 18.0590925 3.6616E-06 3.661604756

5WT MT1 21.13566 11.81825254 0.000276918 276.9181926

5WT HMOX1 27.48418 18.16676615 3.39827E-06 3.398274569

5WT GSTT3 27.6229 18.30549188 3.08672E-06 3.086724431

5WT 18s 9.164269 0 1 1000000

5WT SCD1 22.54259 13.37832119 9.39126E-05 93.91263557

5WT SCD2 30.39885 21.2345821 4.05279E-07 0.405278765WT PGC-1a 27.60174 18.43747399 2.81687E-06 2.81687311

5WT MT1 20.86218 11.69790677 0.000301009 301.0087883

5WT HMOX1 27.15615 17.99187853 3.83623E-06 3.836232171

5WT GSTT3 32.28673 23.12246109 1.09508E-07 0.109507947

6WT 18s 9.905972 0 1 1000000

6WT SCD1 22.97913 13.0731609 0.000116034 116.0343122

6WT SCD2 29.38013 19.47416077 1.37307E-06 1.373072492


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6WT PGC-1a 27.6537 17.74772845 4.54361E-06 4.543613502

6WT MT1 20.92169 11.01571787 0.00048299 482.9903999

6WT HMOX1 28.28154 18.37556743 2.94038E-06 2.940376992

6WT GSTT3 28.31737 18.41140101 2.86824E-06 2.868243611

6WT 18s 10.19675 0 1 1000000

6WT SCD1 22.39513 12.19837791 0.000212776 212.77585876WT SCD2 29.90961 19.71285761 1.16369E-06 1.163693371

6WT PGC-1a 27.55147 17.3547191 5.96635E-06 5.966353552

6WT MT1 21.60989 11.41313374 0.000366695 366.6945042

6WT HMOX1 29.15626 18.95950429 1.96165E-06 1.961645401

6WT GSTT3 28.21661 18.01986346 3.76254E-06 3.762535289

Figure 4: Amplification Curves

3.2 Data Analysis:

Calculating Relative Gene Expression:

Measured Ct data from the quantitative PCR machine was used to calculate relative gene

expression levels. The 18s expression level was used as an internal control for gene expression

because it is known to stay constant regardless of treatment. Because each cycle in qPCR

represents a doubling of initial DNA, 0.5 was raised to Ct (gene C(t)18s C(t)) value in order

to determine the initial amount of cDNA in the reaction, which is proportional to the amount of

mRNA of the particular gene in the sample and indicative of the level of expression of the gene.

These values were then arbitrarily multiplied by a constant value of 106

in order to be able to


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compare the results of this qPCR study to the results in the paperNcb5or deficiency increases

fatty acid catabolism and oxidative stress.5


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Figure 5: Fatty Acid Desaturation

Figure 6: Mitochondrial Biogenesis

Figure 7: Metal Chelation

Figure 8: Oxidative Stress Response


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Signficance Test:

Table 3: Expression Summary

NS = Not significant, i.e. p-value > 0.05

* = Significant, i.e. p-value


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4 Discussion

4.1 Conclusion

Oxidative stress is a known factor in -cell dysfunction in Ncb5or-/-

mice.[6]

Previous studies

have demonstrated that genes involved in fatty acid desaturation (SCD1 and SCD2),

mitochondrial biogenesis (Pgc-1), and oxidative stress response (MT1, MT2, HMOX1, GSTT3)

are all upregulated in Ncb5or deficient mice. In addition, these genetic alterations were

exacerbated by increased saturated fatty acid (SFA) levels and weakened by the presence of

mono-unsaturated fatty acids (MUFA). As previously stated, Ncb5or deficiency and SCD1

deficiency produce similar biochemical defects.[5]

This current study shows that iron-treatment

rescues SCD levels and increases its functionality in Ncb5or-/-

livers, with knock-out

expression levels much closer to wild-type expression levels. Thus, this study concludes that

iron-treatment does indeed improve SCD catalytic activity in Ncb5or-/-

mice, thereby reducing

SFA levels and increasing MUFA production in Ncb5or-defficient mice. The downregulation of

PGC-1 in treated mice compared to untreated Ncb5or-/-

mice imply that there is less fatty acid

catabolism in the hepatocytes, which would prevent a buildup of reactive oxygen species in the

cell. This is further proven in this study by the decrease in oxidative stress response markers

(MT1, MT2, HMOX1, GSTT3). It is speculated that a very similar process occurs in the -cells

of iron-treated Ncb5or-/-

mice, in which iron supplementation eventually lowers levels of

oxidative stress and delays -cell death in the pancreas.


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4.2 Future

Future studies should include a more thorough investigation of Ferrlecit injections interactions

with genes in pancreatic cells, as genetic analysis could shed more light as to the inner

mechanisms and functions of Ncb5or. In order to test the new hypothesized mechanism of delay

in -cell death, the same genes should be quantified in the -cells of iron-treated Ncb5or-/-

mice.

Additionally, because its functional similarity has now been identified, Ferrlecit could be used to

model biochemical reactions with Ncb5orin vivo. Finally, a bigger sample size would be useful

to verify the validity of this study.


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5 Acknowledgements

I would like to thank Dr. Hao Zhu and Dr. WenFang Wang at the KU Medical Center for

providing me with technical training, protocols and mouse tissues that are needed to conduct this

experiment, as well as guiding me through my independent research. I sincerely thank Mr. Joe

Whalen for support and enthusiasm and helping this project come to fruition. I thank the Blue

Valley CAPS program and the Zhu Diabetes Research Lab (funded by the National Institutes of

Health) for providing a professional and state-of-the-art research environment.


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References

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Ncb5or, Iron and Diabetes - Research Paper

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