ncb5or, iron and diabetes - research paper
TRANSCRIPT
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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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