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

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 of stearoyl-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.

Table of Contents
Abstract ......................................................................................................................................................... 1 Table of Contents .......................................................................................................................................... 2 1 Introduction ........................................................................................................................................... 3 1.1 1.2 1.3 1.4 1.5 1.6 2 Ncb5or: ......................................................................................................................................... 3 Ncb5or and Diabetes: .................................................................................................................... 4 Ncb5or and SCD Function: ........................................................................................................... 4 Ferrlecit Preliminary Study: .......................................................................................................... 5 Objectives: .................................................................................................................................... 7 Hypothesis: ................................................................................................................................... 7

Materials and Methods .......................................................................................................................... 8 2.1 2.2 2.3 2.4 2.5 Mouse Lines: ................................................................................................................................. 8 Experimental Mice Subjects: ........................................................................................................ 8 RNA Extraction/Preparation from Mice Liver: ............................................................................ 8 Reverse Transcription for cDNA Synthesis: ................................................................................. 9 Quantitative Real-time PCR assay and Data Collection: ............................................................ 10

Results ................................................................................................................................................. 12 3.1 3.2 Quantitation Data (Threshold Values): ....................................................................................... 12 Data Analysis: ............................................................................................................................. 14

Discussion ........................................................................................................................................... 18 4.1 4.2 Conclusion .................................................................................................................................. 18 Future .......................................................................................................................................... 19

Acknowledgements ............................................................................................................................. 20

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

1
1.1

Introduction
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]

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
4

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).

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:

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

2
2.1

Materials and Methods

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

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:

Total RNA 1 variable volume (2 g) 5x iScript Reaction Mix 8 L Nuclease-free water variable 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 Supermix 10L Forward Primer 1 L (from 10M stock) Reverse Primer 1 L (from 10M stock) Template cDNA 1 L Nuclease Free Water 7 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 activation 95oC, 3:00 40 Cycles: o Denaturing 95oC, 0:10 o Annealing - 60oC, 0:15 o Extension 72oC, 0:30 1 Cycle: Melt Curve 55-95oC (in 0.5oC increments), 0:30 secs

The 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 DNAcontaining well as it continually amplifies due to the thermocycler.

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3
3.1

Results
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 Delta Ct Transcript 0.00000000000000 12.5123902315297 21.232303041264 19.7287734052323 11.7135832941509 19.0250454432295 16.5794352585224 0.00 12.56 22.12 19.59 12.17 18.49 16.68 0.00 12.52 22.77 19.08 11.39 19.01 18.11 0.00 13.21 21.34 17.82 10.30 17.39 16.96
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Mouse ID 1KO 1KO 1KO 1KO 1KO 1KO 1KO 1KO 1KO 1KO 1KO 1KO 1KO 1KO 2KO 2KO 2KO 2KO 2KO 2KO 2KO 2KO 2KO 2KO 2KO 2KO 2KO 2KO

Gene 18s SCD1 SCD2 PGC-1a MT1 HMOX1 GSTT3 18s SCD1 SCD2 PGC-1a MT1 HMOX1 GSTT3 18s SCD1 SCD2 PGC-1a MT1 HMOX1 GSTT3 18s SCD1 SCD2 PGC-1a MT1 HMOX1 GSTT3

Ct 8.07 20.58 29.30 27.80 19.79 27.10 24.65 7.98 20.54 30.10 27.57 20.15 26.47 24.66 7.59 20.12 30.36 26.68 18.98 26.61 25.71 8.44 21.65 29.77 26.26 18.74 25.82 25.40

1.00000000000000000 0.00017115721959087 0.00000040591949311 0.00000115092606959 0.00029775569158558 0.00000187452247871 0.00001021157369736 1.00000000000000000 0.00016573706202763 0.00000021980771232 0.00000126728544482 0.00021698801033802 0.00000271369968233 0.00000950355547707 1.0000000000000000 0.0001700553439192 0.0000001401007601 0.0000017997441340 0.0003737721771698 0.0000018885878425 0.0000035307718120 1.0000000000000000 0.0001054498339734 0.0000003779672067 0.0000043163607714 0.0007940081982884 0.0000058404952805 0.0000078283519131

Normalized (*10^6) 1000000 171.1572196 0.405919493 1.15092607 297.7556916 1.874522479 10.2115737 1000000 165.737062 0.219807712 1.267285445 216.9880103 2.713699682 9.503555477 1000000 170.0553439 0.14010076 1.799744134 373.7721772 1.888587842 3.530771812 1000000 105.449834 0.377967207 4.316360771 794.0081983 5.84049528 7.828351913

3KO 3KO 3KO 3KO 3KO 3KO 3KO 3KO 3KO 3KO 3KO 3KO 3KO 3KO 4WT 4WT 4WT 4WT 4WT 4WT 4WT 4WT 4WT 4WT 4WT 4WT 4WT 4WT 5WT 5WT 5WT 5WT 5WT 5WT 5WT 5WT 5WT 5WT 5WT 5WT 5WT 5WT 6WT 6WT 6WT

18s SCD1 SCD2 PGC-1a MT1 HMOX1 GSTT3 18s SCD1 SCD2 PGC-1a MT1 HMOX1 GSTT3 18s SCD1 SCD2 PGC-1a MT1 HMOX1 GSTT3 18s SCD1 SCD2 PGC-1a MT1 HMOX1 GSTT3 18s SCD1 SCD2 PGC-1a MT1 HMOX1 GSTT3 18s SCD1 SCD2 PGC-1a MT1 HMOX1 GSTT3 18s SCD1 SCD2

8.01 20.72 30.16 26.58 18.83 27.16 27.38 8.28 20.33 29.96 26.80 19.35 26.86 26.94 8.961887 22.37535 29.14547 27.44962 22.14371 26.47226 25.91582 9.77885 22.60423 30.2828 27.66859 22.92091 25.75544 26.15861 9.317409 21.7762 30.57091 27.3765 21.13566 27.48418 27.6229 9.164269 22.54259 30.39885 27.60174 20.86218 27.15615 32.28673 9.905972 22.97913 29.38013

0.00 12.71 22.15 18.57 10.82 19.15 19.37 0.00 12.05 21.68 18.52 11.07 18.58 18.66 0 13.4134584 20.1835865 18.4877371 13.18182596 17.51037022 16.95393179 0 12.82538325 20.50395028 17.88973595 13.14205557 15.97659 16.37976376 0 12.45878719 21.25350041 18.0590925 11.81825254 18.16676615 18.30549188 0 13.37832119 21.2345821 18.43747399 11.69790677 17.99187853 23.12246109 0 13.0731609 19.47416077
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1.00000000000000 0.00014909954582 0.00000021438975 0.00000256302955 0.00055354619651 0.00000172391703 0.00000147834591 1.00000000000000 0.00023632538220 0.00000029757612 0.00000265306494 0.00046612075432 0.00000255404614 0.00000240863437 1 9.1653E-05 8.39721E-07 2.72042E-06 0.000107616 5.35616E-06 7.87695E-06 1 0.000137776 6.72506E-07 4.11768E-06 0.000110623 1.55084E-05 1.17273E-05 1 0.000177636 3.99999E-07 3.6616E-06 0.000276918 3.39827E-06 3.08672E-06 1 9.39126E-05 4.05279E-07 2.81687E-06 0.000301009 3.83623E-06 1.09508E-07 1 0.000116034 1.37307E-06

1000000 149.0995458 0.214389752 2.563029552 553.5461965 1.723917032 1.478345905 1000000 236.3253822 0.297576117 2.653064945 466.1207543 2.554046139 2.408634372 1000000 91.65299838 0.839721055 2.720423892 107.61554 5.356157372 7.876948173 1000000 137.7764331 0.672505648 4.117682578 110.6234169 15.50840668 11.72734725 1000000 177.6361476 0.399998962 3.661604756 276.9181926 3.398274569 3.086724431 1000000 93.91263557 0.40527876 2.81687311 301.0087883 3.836232171 0.109507947 1000000 116.0343122 1.373072492

6WT 6WT 6WT 6WT 6WT 6WT 6WT 6WT 6WT 6WT 6WT

PGC-1a MT1 HMOX1 GSTT3 18s SCD1 SCD2 PGC-1a MT1 HMOX1 GSTT3

27.6537 20.92169 28.28154 28.31737 10.19675 22.39513 29.90961 27.55147 21.60989 29.15626 28.21661

17.74772845 11.01571787 18.37556743 18.41140101 0 12.19837791 19.71285761 17.3547191 11.41313374 18.95950429 18.01986346

4.54361E-06 0.00048299 2.94038E-06 2.86824E-06 1 0.000212776 1.16369E-06 5.96635E-06 0.000366695 1.96165E-06 3.76254E-06

4.543613502 482.9903999 2.940376992 2.868243611 1000000 212.7758587 1.163693371 5.966353552 366.6945042 1.961645401 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

14

compare the results of this qPCR study to the results in the paper Ncb5or deficiency increases fatty acid catabolism and oxidative stress.5

15

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 <0.05 Error in Table 2 and Figures 5-8 is given in S.E.M. One-way analysis of variance (ANOVA) was used to compare the mean expression levels of SCD1, SCD2, PGC-1, MT1, MT2, HMOX1, and GSTT3 (determined from the previous calculations) between wild-type (WT) and Ncb5or-/- (KO) mice. Expression levels and error (standard error of mean) are summarized in Table 2. In addition, results of significance tests are indicated on the superscripts of the KO column. At an = 0.05 significance level, all of the tested genes expression levels are demonstrated to have no significant difference in treated WT and KO mice. Expression data under the Treated column is from the paper Ncb5or deficiency increases fatty acid catabolism and oxidative stress5 and is used to compare the ratio of gene expression between KO and WT mice.

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4
4.1

Discussion
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 Ncb5or in vivo. Finally, a bigger sample size would be useful to verify the validity of this study.

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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
[1] Zhu, H., Qiu, H., Yoon, H. W., Huang, S., and Bunn, H. F. (1999) Identification of a cytochrome b-type NAD(P)H oxidoreductase ubiquitously expressed in human cells, Proc Natl Acad Sci U S A 96, 14742-14747. [2] Xie, J., Zhu, H., Larade, K., Ladoux, A., Seguritan, A., Chu, M., Ito, S., Bronson, R. T., Leiter, E. H., Zhang, C. Y., Rosen, E. D., and Bunn, H. F. (2004) Absence of a reductase, NCB5OR, causes insulin-deficient diabetes, Proc Natl Acad Sci U S A 101, 10750-10755. [3] Zhu, H., Larade, K., Jackson, T. A., Xie, J., Ladoux, A., Acker, H., Berchner-Pfannschmidt, U., Fandrey, J., Cross, A. R., Lukat-Rodgers, G. S., Rodgers, K. R., and Bunn, H. F. (2004) NCB5OR is a novel soluble NAD(P)H reductase localized in the endoplasmic reticulum, J Biol Chem 279, 30316-30325. [4] Morahan, G., Mehta, M., James, I., Chen, W. M., Akolkar, B., Erlich, H. A., Hilner, J. E., Julier, C., Nerup, J., Nierras, C., Pociot, F., Todd, J. A., and Rich, S. S. (2011) Tests for genetic interactions in type 1 diabetes: linkage and stratification analyses of 4,422 affected sib-pairs, Diabetes 60, 1030-1040. [5] Xu, M., Wang, W. F., Frontera, J. R., Neely, M. C., Lu, J. J., Aires, D., Hsu, F.-F., Turk, J., Swerdlow, R. H., Carlson, S. E., and Zhu, H. (2011) Ncb5or deficiency increases fatty acid catabolism and oxidative stress, J Biol Chem 286, 11141-11154. [6] Wang, W. F., Guo, Y., Xu, M., Huang, H.-H., Novikova, L., Larade, K., Jiang, Z.-G., Thayer, T. C., Frontera, J. R., Aires, D., Ding, H., Turk, J., Mathews, C. E., Bunn, H. F., Stehno-Bittel, L., and Zhu, H. (2011) Development of diabetes in Ncb5or-null mice is associated with
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manifestations of endoplasmic reticulum and oxidative stress in beta cells, Biochimica et Biophysica Acta 1812, 1532-1541. [7] Guo, Y., Xu, M., Deng, B., Frontera, J. R. Kover, K.L.,Aires, D., Ding, H., Carlson, S.E., Turk, J., Wang, W.F., and Zhu, H. (2012) Beta-cell injury in Ncb5or-null mice is exacerbated by consumption of a high-fat diet. Eur. J. Lipid Sci. Technol. 114, 233243. [8] Deng, B. (2011). Structural basis of inter-domain electron transfer in ncb5or, a redox enzyme implicated in diabetes and lipid metabolism. (Doctoral dissertation, University of Kansas Medical Center).

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