Accepted Manuscript

Vitamin D supplementation inhibits oxidative stress and upregulate SIRT1/AMPK/

GLUT4 cascade in high glucose-treated 3T3L1 adipocytes and in adipose tissue of
high fat diet-fed diabetic mice

Prasenjit Manna, Arunkumar E. Achari, Sushil K. Jain

PII: S0003-9861(16)30417-9
DOI: 10.1016/j.abb.2017.01.002
Reference: YABBI 7422

To appear in: Archives of Biochemistry and Biophysics

Received Date: 17 October 2016

Revised Date: 14 December 2016
Accepted Date: 3 January 2017

Please cite this article as: P. Manna, A.E. Achari, S.K. Jain, Vitamin D supplementation inhibits oxidative
stress and upregulate SIRT1/AMPK/GLUT4 cascade in high glucose-treated 3T3L1 adipocytes and in
adipose tissue of high fat diet-fed diabetic mice, Archives of Biochemistry and Biophysics (2017), doi:
10.1016/j.abb.2017.01.002.

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 ACCEPTED MANUSCRIPT

VITAMIN D SUPPLEMENTATION INHIBITS OXIDATIVE STRESS AND

UPREGULATE SIRT1/AMPK/GLUT4 CASCADE IN HIGH GLUCOSE-TREATED
3T3L1 ADIPOCYTES AND IN ADIPOSE TISSUE OF HIGH FAT DIET-FED
DIABETIC MICE

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Prasenjit Manna, Arunkumar E. Achari, Sushil K. Jain

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Department of Pediatrics
Louisiana State University Health Sciences Center

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Shreveport, LA 71103

Running Head: Vitamin D, oxidative stress, and glucose metabolism

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Address for Correspondence: Dr. Sushil K. Jain, Department of Pediatrics, LSU Health
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Sciences Center, 1501 Kings Highway, Shreveport, LA 71103,TEL: 1-318-675-6086, FAX:

1-318-675-6059, E-MAIL: sjain@lsuhsc.edu
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ABSTRACT
This study examined the hypothesis that vitamin-D prevents oxidative stress and upregulates
glucose metabolism via activating insulin-independent signaling molecules in 3T3-L1 adipocytes
and in high fat diet (HFD)-fed mice. To investigate the mechanism 3T3L1 adipocytes were

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treated with high glucose (HG, 25 mM) and 1,25(OH)2D3 (1,25-dihydroxyvitamin D3) (0-50
nM). Results showed that 1,25(OH)2D3 supplementation decreased NOX4 expression, ROS

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production, NF-κB phosphorylation, and increased the expression of Nrf2 and Trx in HG-treated
cells. 1,25(OH)2D3 supplementation upregulated SIRT1 expression and AMPK phosphorylation

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and stimulated the IRS1/PI3K/PIP3/AKT/PKCζ signaling cascade, GLUT4 expression, and
glucose uptake in HG-treated adipocytes. The effect of 1,25(OH)2D3 on the phosphorylation of
both AMPK and IRS1, GLUT4 expression, and glucose uptake was significantly inhibited in

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SIRT1-knockdown adipocytes. This suggests the role of insulin-independent signaling molecules
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(SIRT1, AMPK) in mediating the effect of 1,25(OH)2D3 on the signaling cascade of glucose
uptake. In addition, cholecalciferol supplementation significantly upregulated pAMPK, SIRT-1
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and GLUT-4 levels in adipose tissue of mice fed with HFD. This study demonstrates a novel
molecular mechanism by which vitamin-D can prevent oxidative stress and upregulates glucose
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uptake via SIRT1/AMPK/IRS1/GLUT4 cascade in HG-treated adipocytes and in adipose tissue

of HFD diabetic mice.
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KEY WORDS: High glucose, oxidative stress, vitamin D, SIRT1/AMPK/IRS1/GLUT4,

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and glucose metabolism

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HIGHLIGHTS:

• 1,25(OH)2D3 improved glucose metabolism via SIRT-1/AMPK/GLUT-4 cascade

• 1,25(OH)2D3 ameliorates HG induced oxidative stress through NOX4/Nrf2/Trx pathway
• VDR signaling plays a key role in mediating the effect of 1,25(OH)2D3

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ABBREVIATIONS
1,25(OH)2D3, 1,25-dihydroxyvitamin D3; AKT, serine/threonine protein kinase; AMPK, AMP
activated protein kinase; GLUT1, glucose transporter 1; GLUT4, glucose transporter 4; IR,
insulin receptor; IRS1, insulin receptor substrate 1; NF-κB, nuclear factor kappa B; NOX4,
NADPH-oxidase 4; Nrf2: nuclear factor erythroid 2-related factor 2; PI3K, phosphoinositide 3-

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kinase; PIP3, phosphatidylinositol-3,4,5-trisphosphate; PKCζ/λ, protein kinase C ζ/λ; PTEN,
phosphatase and tensin homolog; PTP1B, protein tyrosine phosphatase 1B; SIRT1, sirtuin 1;

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Trx, thioredoxin; VDR, vitamin D receptor.

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INTRODUCTION
Epidemiological studies demonstrate that higher concentrations of circulating 25 hydroxy
vitamin D [25(OH)VD] are associated with better health outcomes, including a lower risk of
type 2 diabetes and cardiovascular disease [1]. Among patients with impaired glucose
metabolism plasma 25(OH)VD status is inversely associated with the circulatory markers of

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oxidative stress [2]. The molecular mechanism by which 1,25(OH)2D3 stimulates glucose
homeostasis is not clear. Activation of NADPH oxidases (NOX) has been widely accepted as a
major contributor of elevated ROS production in diabetes [3, 4]. Oxidative stress plays an
important role in impaired glucose metabolism via inhibition of the transcription factor nuclear

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factor (erythroid-derived 2) factor 2 (Nrf2) and downregulation of the transcription of
antioxidant proteins such as glutathione S-transferase (GST), thioredoxin (Trx), etc. [5, 6].
Whether 1,25(OH)2D3 can regulate NOX/Nrf2 signaling pathway and prevent oxidative stress

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and impaired glucose metabolism in diabetes is not known.
Glucose transporter 4 (GLUT4) plays a critical role in the regulation of glucose
metabolism and maintenance of body glucose homeostasis [7, 8]. Using a specific knockout
animal model as well as siRNA mediated gene silencing experiments, various studies suggest

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that both the insulin-dependent and the insulin-independent signaling molecules play an
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important role in the regulation of GLUT4 expression and glucose metabolism [7, 9]. The
insulin-dependent signaling cascade is initiated by the binding of insulin with its receptor (IR),
which undergoes receptor autophosphorylation and enhanced tyrosine kinase activity.
Subsequent phosphorylation of intracellular substrates (e.g., IRS1) on tyrosine residues
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stimulates the downstream signaling cascade of glucose metabolism [10]. Sirtuin 1 (SIRT1) and
AMP-activated protein kinase (AMPK) are emerging as important insulin-independent signaling
molecules which can also stimulate insulin sensitivity and glucose metabolism [11, 12]. Both
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SIRT1 and AMPK have been found to improve glucose metabolism via regulating the activation
of IRS1 in an insulin independent manner [13, 14]. There is no study in the literature examining
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whether the effect of 1,25(OH)2D3 on GLUT4 expression and glucose utilization can be
mediated via the insulin-dependent or insulin-independent signaling molecules. 1,25(OH)2D3 is
the most activated form of vitamin D in the body. It is crucial in maintaining calcium and
phosphorus homeostasis [15]. Using murine 3T3L1 adipocyte cell culture model and specific
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signal silencing approaches this study for the first time demonstrates that 1,25(OH)2D3
supplementation via vitamin D receptor (VDR)-mediated pathway can prevent oxidative stress
by regulating NOX4/Nrf2/Trx signaling pathway and improves GLUT4 expression and glucose
uptake via SIRT1/AMPK/IRS1 signaling cascade in high glucose (HG)-treated cells.
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Interestingly, 1,25(OH)2D3 supplementation coupled with insulin significantly boosted the

GLUT4 expression and glucose uptake compared to treatment with either insulin or 1,25(OH)2D3
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alone in HG-treated adipocytes. In addition, cholecalciferol supplementation significantly

upregulated pAMPK, SIRT-1 and GLUT-4 in adipose tissue of HFD-fed diabetic mice. This
suggests the contribution of insulin-independent signaling molecules to the higher glucose
metabolism observed in 1,25(OH)2D3 and insulin-treated cells.

MATERIALS AND METHODS

Materials – Anti-AMPK, anti-phosphorylated AMPK (Thr 172), anti-IR, anti-PI3K (p85α), anti-
AKT (AKT2), anti-phosphorylated AKT (Ser 473), anti-PKCζ, and anti-phosphorylated PKCζ/λ
(Thr 410/403) were purchased from Cell Signaling Technology (Beverly, MA). Anti-GLUT4,

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anti-GLUT1, anti-phosphorylated NF-κB (p65) (Ser 276), anti-VDR, anti-PTP1B, anti-NOX4,

anti-SIRT1, ant-Nrf2, and anti-Trx primary antibodies were purchased from Abcam, Inc.
(Cambridge, MA). Anti-IRS1 and anti-phospho IRS1 (Tyr 941) primary antibodies were
purchased from Upstate (Millipore) (Temecula, CA). Anti-NF-κB (p65) was purchased from
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti- Na+-K+-ATPase was purchased from
Proteintech Group, Inc. (Chicago, IL). All chemicals were purchased from Sigma Chemical Co.
(St. Louis, MO) unless otherwise mentioned.

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Animals: Male C57BL/6J mice (5 weeks old, 20-24 g) were purchased from The Jackson
Laboratory (Bar Harbor, ME, USA). The animals were fed either a standard chow diet (Harlan

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TD.08485, providing 5.2% calories as fat) (control), a high fat diet (HFD, Harlan TD.88137,
providing 42% calories as fat), or a VD (vitamin-D) deficient high fat diet (V-HFD-, Harlan
TD.140881, providing 42% calories as fat). The model of dietary-induced insulin resistance

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created both fasting hyperglycemia and hyperinsulinemia and is thus a reasonable model for the
human condition. All animals were kept in our animal care facility where ambient environmental
conditions (12:12-h light-dark cycle, 22–24°C) were maintained. All procedures were followed
in accordance with the ethical standards of the institution and after approval from the

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Institutional Animal Committee.
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Animal experimental design: Mice were be divided into various groups by computer-generated
randomization, so that each group contains 8 animals, and then housed and labeled in individual
cages. They were be fasted overnight and then weighed. The mice were be tested for
hyperglycemia by measuring their blood glucose concentration. Control animals were fed a
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normal diet (low fat diet). Diabetic control animals were fed a high fat diet for 16 wks. For
studies with VD deficient animals, mice were be fed a VD deficient high fat diet for 16 wks and
cholecalciferol were be gavaged to the animals at the doses 67 IU VD/kg body weight daily by
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oral gavage for last 8 wks. At the end of 16 weeks the animals were be fasted overnight and then
euthanized for analysis by exposure to isoflurane (Webster Veterinary Supply Inc., Devens,
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MA). The epididymal adipose tissue were collected immediately, weighed, quickly diced and
stored at -80˚C.

Justification of vitamin D deficient diet: Exogenous VD supplementation has been found to be

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beneficial in the regulation of glucose tolerance in a diabetic animal model. Knockout mice for
the gene encoding the receptor for 1,25(OH)2D3 [VDR)] are more susceptible to developing
insulin resistance [16, 17] however, this model do not mimic in vivo VD deficiency as seen in
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humans. Our study were examine the effect of cholecalciferol supplementation on glucose
metabolism in a HFD-induced VD-deficient diabetic animal model that mimics what is seen in
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AA diabetic patients.

Justification for selection of VD dose - According to the NIH, the safe dose of VD can reach up
to 4000 IU/day in a 60 kg adult, i.e. 67 IU/kg/day. The VD dose of 12.5 µg/kg (99-101) body
weight in STZ treated rats is equivalent to 30,000 IU for a 60 kg human. We decided to use 67
IU/kg/day, which is equivalent to 1.67 µg/kg/day (40 IU corresponds to 1 µg VD).
Cholecalciferol was dissolved in 0.1% olive oil and a stock solution of 1.67µg/mL was prepared.
An aliquot of 0.1 mL of the stock solution was given per 100 g BW. Treatment was carried out
using oral gavage alternate day for 8 wks. For alternate day gavaging supplementation dose was

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kept at double to maintain similar dose per day. Olive oil control group is included in treatment
groups. Special diets and details of treatment and sacrifice is given previously.

3T3L1 adipocyte cell culture and differentiation - The murine 3T3L1 fibroblast cell line was
obtained from American Type Culture Collection (ATCC, Manassas, VA). These cells were
cultured and differentiated into adipocytes following the method described earlier [18, 19]

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Treatment with control glucose (CG), high glucose (HG), and 1,25(OH)2D3 - Cells were
exposed to normal glucose (5.5 mM) or HG (25 mM) with or without 1,25(OH)2D3, the active
form of vitamin D. In high glucose studies, cells were exposed to a high glucose concentration of
25 mM. Many previous studies have reported that glucose concentrations as high as 50 mM have

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been found in the blood of patients with uncontrolled diabetes [20]. It is true that blood glucose
levels in patients are not likely to stay as high as 25 mM for 24 h. However, tissue damage in
diabetic patients occurs over many years of countless hyperglycemic episodes. Thus, the glucose

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concentration of 25 mM used in this cell culture study does not seem unreasonable. Cells were
pretreated with different concentrations (25 or 50 nM) of 1,25(OH)2D3 for 2 h, followed by HG
exposure for the next 20 h in DMEM medium. In the control group cells were treated with or
without 1,25(OH)2D3 (50 nM) for 22 h in normal glucose DMEM medium. The doses of

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1,25(OH)2D3 used in this study are in the physiological range [21]. Mannitol was used as an
osmolarity control. The cell viability was determined using the Alamar Blue reduction bioassay
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(Alamar Biosciences, Sacramento, CA).

siRNA transient transfection studies – SIRT1 siRNA, IR siRNA, VDR siRNA, and control
siRNA were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Cells were
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transiently transfected with 100 nM siRNA complex using LipofectamineTM2000 transfection

reagent (Invitrogen, Carlsbad, CA) following the method described earlier [22, 23]. Control
siRNA is a scrambled nonspecific RNA duplex that shares no sequence homology with any of
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the genes and was used as a negative control.

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Immunoblotting - For the immunoblotting, after treatment, cells were lysed in

radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris pH 8, 150 mM NaCl, 1% NP-40,
0.5% deoxycholic acid, 0.1% SDS) supplemented with protease and phosphatase inhibitors (1
mM PMSF, 5 µg/mL leupeptin, 2 µg/mL aprotinin, 1 mM EDTA, 10 mM NaF, and 1 mM
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NaVO4). The tissue homogenates were processed for the immunoblotting studies to investigate
the expression of different signaling molecules involved in glucose metabolism. To extract
protein from adipose tissue, ∼200 mg of tissue was homogenized in RIPA buffer on ice using a
rotor-stator. The homogenates were centrifuged at 10,000 g for 10 min and the subnatant (whole
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tissue extract) below the lipid cake was aspirated. Lysates were cleared by centrifugation and
total protein concentrations were determined using a BCA assay kit (Pierce/Thermo Scientific,
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Rockford, IL). All samples contained approximately the same amount of protein (~20-30 µg) and
were used for immunoblotting with either anti-NOX4 (1:1000 dilution), anti-Nrf2 (1:1000), anti-
Trx (1:1000), anti-VDR (1:1000), anti-IR (1:1000), anti-IRS1 (1:1000), anti-phospho IRS1 (Tyr
941), anti-SIRT1 (1:1000), anti-AMPK (1:2000), anti-phospho AMPK (Thr 172) (1:1000), anti-
PI3K (p85α) (1:1000), anti-PTP1B (1:5000 dilution), anti-AKT (AKT2) (1:2000 dilution), anti-
phosphorylated AKT (Ser 473) (1:500), anti-PKCζ (1:1000), anti-phosphorylated PKCζ/λ
(1:500) (Thr 410/403), anti-GLUT4 (1:1000), anti-GLUT1 (1:1000), anti-NF-κB (p65) (1:1000
dilution), or anti-phosphorylated NF-κB (p65) (Ser 276) (1:500), primary antibody, and the
appropriate HRP conjugated secondary antibody (1:5000 dilution). Different amount of protein
extracts of the experimental samples were run on the same gel and the results demonstrated that

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the ratio of the target protein and internal control was similar in each different amount of protein
(data not shown). The intensity of each immunoblotting band was measured using the histogram
tool of Adobe Photoshop CS5.

Glucose transporter plasma membrane expression assay - GLUT4 protein expression in

plasma membrane was determined using both flow cytometry [24] and immunoblotting studies.
For flow cytometric studies, after-treatment cells were washed in FACS buffer (PBS without

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Mg2+ and Ca2+, with the addition of 10% fetal bovine serum and 0.1% sodium azide),
centrifuged, suspended in FACS buffer and incubated for 2 h at 4°C with anti-GLUT4 (Santa
Cruz Biotechnology, sc-1606) primary antibody at a 1:50 dilution. The cells were then washed in
washing buffer for FACS (PBS containing 1% BSA and 0.1% sodium azide) and incubated with

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a FITC-conjugated appropriate secondary antibody (Abcam) at a 1:40 dilution on ice for 30 min
in the dark. After the incubation, 1 mL washing buffer for FACS was added to each sample. The
samples were then vortexed, centrifuged, the supernatant removed, and 0.5 mL FACS buffer

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added. In each experiment, a minimum of 15,000 cells was analyzed (per treatment condition) by
FACS Calibur flow cytometer (Becton–Dickinson, San Diego, CA) equipped with multicolor
analysis capability. Gates were set to exclude nonviable cells, cell debris, and cells of abnormal
size and shape. Results were expressed as mean fluorescence intensity (MFI) per 15,000 cells.

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For immunoblotting studies, after-treatment plasma membrane protein fractions were isolated
from the experimental samples using a commercially available plasma membrane protein
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extraction kit from Abcam and then performed immunoblotting using anti-GLUT4 primary
antibody following the method as described above. Plasma membrane protein fraction was also
used to examine the GLUT1 protein expression under experimental condition using anti-GLUT1
primary antibody. Plasma membrane (PM) enriched fractions was verified by noting Na+-K+-
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ATPase α-subunit expression by immunoblotting (1:1000).

Glucose uptake, PIP3 and ROS assay - The glucose uptake assay was performed using 6-[N-
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(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (6-NBDG; Invitrogen), a

fluorescent analogue of 2-deoxyglucose, following the method as described earlier [25, 26].
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Briefly, after treatment, cells were incubated with normal glucose DMEM medium containing 6-
NBDG (200 µM) for 1 h. After the incubation, cells were washed with PBS, then lysed with 0.1
M potassium phosphate buffer pH 10 containing 1% TritonX-100, and kept in the dark for 10
min. Following the addition of 30 µL DMSO to each sample, which was homogenized by
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pipetting up and down, the plate was read immediately using a microplate reader at
excitation/emission wave lengths of 466/540 nm. Results are expressed as % control. The
cellular PIP3 level was measured using the PIP3 Mass ELISA Kit (Echelon Biosciences, Inc.,
Salt Lake City, UT) [18, 27]. Appropriate controls and standards (specified by each
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manufacturer’s kit) were used each time. Intracellular ROS levels were measured using the
fluorescent dye H2DCFDA (2′, 7′ dichlorofluorescein diacetate [DCFH-DA]). Details of the
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ROS assay are as given before [28].

Data were analyzed statistically using one way ANOVA with Sigma Stat software (Jandel
Scientific, San Rafael, CA). When data passed a normality test, all groups were compared using
the Student–Newman–Keuls method. Values of p<0.05 were considered significant.

RESULTS
At the end of the study mice were sacrificed and blood glucose was measured in each
groups. HFD group (208.5 ± 18.2) showed a significant (p = 0.01) increase in blood glucose
level compared to control (135.8 ± 12.8). The VD group (185.6 ± 14.3) showed low blood

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glucose level compared to VD-deficient HFD-fed group (201.8 ± 4.8) and olive oil group (208.9
± 14.8). Though, the blood glucose level was decreased in VD group compared to VD-deficient
HFD-fed group and olive oil group, the decrease is not statistically significant (VD-deficient
HFD-fed group vs VD group p = 0.3; olive oil group vs VD group p=0.27). In this study, mice
were treated with VD for last 2 months. It seems VD treatment for more period is needed to
bring a significant reduction in blood glucose level or larger sample size may be required to get
the significant difference in blood glucose levels between diabetic control and VD treatment

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groups. Oxidative stress plays an important role in impaired glucose metabolism among diabetes
patients population [29]. To test whether cholecalciferol improves glucose metabolism in
diabetic mouse models, we investigated the levels of proteins such as AMPK, SIRT-1and
GLUT-4 in adipose tissue of animals fed with high fat diet and vitamin D-deficient high fat diet

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by Western blotting. The result found that phospho-AMPK (1A), SIRT-1 (1B), and GLUT-4
levels (1C) were significantly decreased in HFD and V-HFD. Treatment with cholecalciferol
showed increased the levels of phospho-AMPK, SIRT-1 and GLUT-4 in V-HFD.

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Figure 2 demonstrates the beneficial role of 1,25(OH)2D3 in preventing HG-induced oxidative
stress in 3T3L1 adipocytes. Results show that HG treatment increased the NOX4 expression
(2A), ROS production (2B), and NF-κB phosphorylation (2C), and decreased the expression of

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Nrf2 (2D) and Trx (2E) in adipocytes. 1,25(OH)2D3 supplementation decreased the NOX4
expression, ROS production, and NF-κB phosphorylation, and increased the expression of Nrf2
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and Trx suggesting a beneficial role of in preventing HG-induced oxidative stress.

Both insulin-dependent (IR) and insulin-independent (SIRT1, AMPK) signaling molecule

activate intracellular substrates (e.g. IRS1), which stimulates the downstream signaling cascade,
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PI3K/PIP3/AKT/PKCζ/λ, leading to GLUT4 translocation and glucose uptake. Figures 3-4 show
the effect of 1,25(OH)2D3 supplementation on the signaling cascade of GLUT4 translocation and
glucose uptake in HG-treated 3T3L1 adipocytes. HG exposure decreased the SIRT1 expression
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(3A) and the phosphorylation of AMPK (3B), and increased PTP1B expression (3C) followed by
a decrease in tyrosine phosphorylation of IRS1 (3D,) PI3K expression (3E), intracellular PIP3
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concentration (3F), and phosphorylation of both AKT (3G) and PKCζ/λ (3H). Though the level
of pAKT in 50nM (HG along with 1,25(OH)2D3 group) is decreased compared to 25nM (HG
along with 1,25(OH)2D3 group), but still the value in 50nM treatment in HG group showed
significant difference in pAKT status compared to HG alone. Although the level of total AKT in
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50nm (HG along with 1,25(OH)2D3 group) seems like slight increase compared to other groups,
the pAKT/AKT ratio in this group is still showed significant increase compared to HG alone.

GLUT4 translocation was examined by investigating its protein expression in both

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plasma membrane fraction and total cell lysate (4A). Plasma membrane enriched fractions was
also verified by noting Na+-K+-ATPase α-subunit expression. GLUT4 expression on plasma
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membrane was also verified by flow cytometry (4B). Results demonstrate that HG treatment
decreased the GLUT4 expression in both plasma membrane fraction and total cell lysate and
caused a reduction in glucose uptake (4D). Treatment with 1,25(OH)2D3 upregulated the SIRT1
expression and AMPK phosphorylation and stimulated the IRS1/PI3K/PIP3/AKT/PKCζ/λ
signaling cascade followed by an increase in GLUT4 expression and glucose uptake in HG-
treated adipocytes. In addition to GLUT4, GLUT1 is also playing an important role in basal
glucose transport in 3T3L1 adipocytes. However, no significant difference in GLUT1 total
protein expression or in its plasma membrane expression (4C) was observed among the different
experimental groups. Treatment with mannitol (an osmolarity control) did not affect GLUT4

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expression or glucose uptake in adipocytes (data not shown). Different treatments did not cause
any change in cell viability level (data not given).

Figure 5 shows the effect of 1,25(OH)2D3 on AMPK phosphorylation, IRS1

phosphorylation, GLUT4 expression, and glucose uptake in HG-treated adipocytes transfected
with either SIRT1 siRNA. Interestingly, in SIRT1 knockdown cells (5A), 1,25(OH)2D3
supplementation did not cause any increase in AMPK phosphorylation (5B), IRS1

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phosphorylation (5C), GLUT4 expression (5D), and glucose uptake (5E) in either control
glucose or HG-treated adipocytes. The level of AMPK phosphorylation was increased by
1,25(OH)2 D3 supplementation in SIRT-1 knockdown cells under HG condition. But, at the
same time, there was a slight increase in the level of SIRT-1 in SIRT-1 knockdown cells by

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1,25(OH)2 D3 supplementation under HG condition. This suggests the increase in AMPK
phosphorylation in HG condition by 1,25(OH)2 D3 was due to increase in SIRT-1 levels which
showed AMPK phosphorylation could be dependent on SIRT-1.

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However, Figure 6 shows that in IR knockdown cells (6A) 1, 25(OH)2D3 treatment
upregulated the IRS1 phosphorylation (6B), GLUT4 expression (6C) and glucose uptake (6D) in
HG-treated cells. Figures 5-6 demonstrate the role of insulin-independent signaling molecules

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(SIRT1, AMPK) in mediating the effect of 1,25(OH)2D3 on GLUT4 expression and glucose
uptake. In addition, we have also investigated the effect of insulin (25 nM) either alone or in
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combination with 1,25(OH)2D3 supplementation (50 nM) on GLUT4 expression and glucose
uptake HG-treated aipocytes. Results show that treatment with either insulin or 1,25(OH)2D3
alone increased the GLUT4 expression (7A) and glucose uptake (7B) in HG-treated cells.
However, 1,25(OH)2D3 supplementation along with insulin significantly boosted GLUT4
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expression (7A) and glucose uptake (7B) compared to supplementation with either insulin or
1,25(OH)2D3 alone in HG-treated adipocytes. The outcome of this study suggests that insulin-
independent signaling molecules (SIRT1, AMPK) can contribute to the higher glucose
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metabolism observed in 1,25(OH)2D3 and insulin-treated cells and this is in agreement with the
previous signal silencing studies.
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The beneficial effect of 1,25(OH)2D3 are now thought to be mediated via binding with its
specific receptor, VDR. Using VDR knockdown cells this study also examined the role of VDR
in mediating the effect of 1,25(OH)2D3 against HG-induced oxidative stress and impaired
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glucose metabolism in adipocyte cell culture model. Figure 8 shows that HG-induced increases
in NOX4 expression (8B) and ROS production (8C) and decreases in Nrf2 expression (8D) and
Trx expression (8E) could not be prevented by 1,25(OH)2D3 supplementation in VDR
knockdown cells. Additionally, 1,25(OH)2D3 supplementation was unable to increase SIRT1
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expression (8F), AMPK phosphorylation (8G), GLUT4 expression (8H), and glucose uptake (8I)
in HG-treated VDR knockdown cells suggesting a specific receptor mediated role for
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1,25(OH)2D3 in executing its beneficial effect against HG-induced oxidative stress and impaired
glucose metabolism.

DISCUSSION
Various cross-sectional studies in the literature support a link between low vitamin D
status and insulin resistance in healthy and diabetic subjects [1, 30, 31]. Vitamin D
supplementation has been found to decrease hyperglycemia in both type 1 and type 2 diabetic
patients [32, 33]. The beneficial effect of vitamin D on insulin resistance may be mediated by
stimulating the expression of insulin receptors, regulating the calcium pool, modulating cytokine
expression, and improving the action of insulin in various tissues [1, 30, 31, 34]. This study
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demonstrates that 1,25(OH)2D3 supplementation prevents oxidative stress by regulating

NOX4/Nrf2/Trx signaling pathway and improves the glucose uptake via stimulating the
SIRT1/AMPK/IRS1/GLUT4 cascade in HG-treated 3T3L1 adipocyte cells.
GLUT4 is known to play an important role in the regulation of glucose metabolism and
maintenance of body glucose homeostasis [7, 8]. Using GLUT4 heterozygous knockout mice,
various studies have shown that a decrease in GLUT4 expression caused an increase in the levels
of serum glucose and insulin, and a decrease in muscle glucose uptake, hypertension, and other

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complications similar to those seen in the diabetic population [7, 8, 35]. Activation of GLUT4
has been suggested as a therapeutic target for pharmacological intervention strategies to control
diabetic hyperglycemia [7]. GLUT4 translocation can be mediated by both the insulin-dependent

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and insulin-independent signaling molecules [9]. In the insulin-dependent signaling cascade of
glucose metabolism, GLUT4 translocation and glucose utilization is mediated via the tyrosine
phosphorylation of IR and its substrate (IRS1), followed by stimulation of the

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PI3K/PTEN/PIP3/AKT/PKCζ/λ signaling cascade [10].
Sirtuins, the mammalian homologue of SIR2, are a group of NAD(+) dependent enzymes
that post-translationally modify histones and other proteins [36, 37]. They have emerged as
broad regulators of many important processes, such as organ metabolism and function, calorie

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restriction, age-related diseases, DNA damage repair, etc. [36, 37]. SIRT1 is widely expressed in
various mammalian tissues such as brain, kidney, adipose tissue, muscle, and liver [38]. Among
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these, adipose tissue plays an important role in the maintenance of glucose homeostasis in the
human body. Yoshizaki et al. [14] demonstrate that SIRT1 knockdown in 3T3L1 adipocytes
inhibits the insulin-stimulated glucose uptake and GLUT4 translocation mediated by a decrease
in tyrosine phosphorylation of IRS1, and serine phosphorylation of AKT (with a subsequent
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increase in JNK phosphorylation as well as serine phosphorylation of IRS1). Increased

expression of SIRT1 has also been found to downregulate both protein and mRNA levels of
PTP1B, which acts as a negative regulator of insulin signaling mainly via the downregulation of
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tyrosine phosphorylation of IR or IRS1 [39]. In addition to sirtuins, AMP-activated protein

kinase has also been found to regulate insulin sensitivity and glucose transport [40]. Activation
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of AMPK promotes tyrosine phosphorylation of IR and IRS-1, serine/threonine phosphorylation

of Akt, and increases glucose uptake in primary rat cardiac myocytes [13]. Whilst AMPK is not a
tyrosine kinase, it is hypothesized that serine/threonine phosphorylation of IR and IRS-1 by
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AMPK promotes allosteric activation by tyrosine kinases [13]. Studies in the literature also
demonstrate the role of SIRT1 in the activation of AMPK [41]. Suchankova et al. demonstrated
that incubation with the SIRT1 inhibitor NAM downregulated the activity of both AMPK and
SIRT1, whereas incubation with the SIRT1 activator quercertin increased both of their activities
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[42]. All of these studies demonstrate a positive role for SIRT1 and AMPK in the regulation of
glucose metabolism via activating IRS1 in an insulin independent manner [13, 14].
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Our previous study reported that treatment with 1,25(OH)2D3 increased the GLUT4
expression and glucose utilization in HG-treated 3T3L1 adipocytes [19]. However, there is no
study in the literature describing whether the effect of vitamin D on GLUT4 expression and
glucose uptake is mediated via the insulin-dependent or insulin-independent signaling molecules.
This study demonstrates for the first time that 1,25(OH)2D3 supplementation upregulates SIRT1
expression and AMPK phosphorylation, inhibits PTP1B, and stimulates the
IRS1/PI3K/PIP3/AKT/PKCζ/λ signaling cascade, GLUT4 expression, and glucose uptake in
HG-treated adipocytes. Comparative signal silencing studies revealed that SIRT1 inhibition by
anti-sense mRNA significantly blocked the effect of 1,25(OH)2D3 on the phosphorylation of both

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AMPK and IRS1, GLUT4 expression, and glucose uptake in HG-treated adipocytes. However,
IR depletion could not prevent the effect of 1,25(OH)2D3 on IRS1 phosphorylation, GLUT4
expression, or glucose uptake in HG-treated adipocytes. This results suggest the role of insulin-
independent signaling molecules (SIRT1, AMPK) in mediating the effect of 1,25(OH)2D3 on the
signaling cascade of glucose uptake in 3T3L1 adipocytes. Additional studies demonstrate that
1,25(OH)2D3 supplementation coupled with insulin significantly upregulated GLUT4 expression
and glucose uptake compared to treatment with either insulin or 1,25(OH)2D3 alone in both HG-

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treated adipocytes. This results also suggest the contribution of insulin-independent signaling
molecules in the higher glucose metabolism observed in 1,25(OH)2D3 and insulin-treated cells.
Besides mitochondria, the family of NOX enzymes has been widely recognized as one of

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the major contributors of intracellular ROS production [43, 44]. NOX are membrane-bound
enzyme complexes that transfer electrons from NADPH to oxygen, producing superoxide and
H2O2 [44]. There are different isoforms of this enzyme and the expression of different subtypes

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varies amongst tissue types [44]. NOX4 is the major NOX isoform expressed in differentiated
3T3L1 adipocytes [45]. The involvement of NOX in hyperglycemia-induced ROS production is
well documented [3, 4]. Han et al. demonstrated that both high glucose and free fatty acid
supplementation generate ROS via NOX4 rather than by mitochondrial oxidation in 3T3L1

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adipocytes [45]. Consistent with the previous findings, this study also observed an increase in
NOX4 expression and ROS production in HG-supplemented differentiated 3T3L1 adipocytes. In
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order to regulate the intracellular ROS production, the transcription factor Nrf2 plays a vital role
in the activation of several antioxidant enzymes such as GST, TRx, etc. by stimulating their
transcription [6, 46]. Under normal physiological condition low concentration of prooxidants can
induce the expression of Nrf2 whereas under diseased condition high concentration of
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prooxidants causes a decreased expression of Nrf2 and Nrf2-regulated antioxidant proteins [47-
49]. This study demonstrates that treatment with excess glucose caused an increase in ROS
production and a decrease in the expression of Nrf2 and Trx in adipocytes. Supplementation with
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1,25(OH)2D3 decreased the NOX4 expression and ROS production and increased the expression
of Nrf2 and Trx in HG-treated cells suggesting a beneficial role of 1,25(OH)2D3 in preventing
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HG-induced oxidative stress via modulating NOX4/Nrf2/Trx signaling pathway in 3T3L1

adipocytes.
The biological effects of 1,25(OH)2D3 are now thought to me mediated via binding with
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VDR, a member of the nuclear receptor super-family of ligand activated transcription factors
[50]. Using VDR knockdown adipocytes this study examined the role of VDR in mediating the
beneficial effect of 1,25(OH)2D3 against HG-induced oxidative stress and impaired glucose
metabolism. Results demonstrate that in VDR knockdown cells 1,25(OH)2D3 supplementation
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could not downregulate NOX4 expression and ROS production and also unable to upregulate
Nrf2 expression and Trx expression in HG-treated cells. The beneficial effect of 1,25(OH)2D3 on
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the upregulation of SIRT1/AMPK/GLUT4 cascade and glucose uptake against HG exposure was
also inhibited in VDR knockdown adipocytes. This suggests that VDR plays an important role in
mediating the effect of 1,25(OH)2D3 on oxidative stress, GLUT4 upregulation, and glucose
uptake in HG-treated adipocytes.

In addition we have also demonstrated that cholecalciferol administration improved

glucose metabolism in high fat diet-fed mice. In general, rodents challenged with high fat diet
reported low AMPK activity in muscle and liver and develops insulin resistance/type2diabetes
[51]. SIRT1 is a energy sensor responds to overfeeding, starvation, changes in energy

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expenditure and exercise [52]. AMPK and SIRT-1 helps in glucose homeostasis by regulating
GLUT-4 proteins. SIRT1 can activate AMPK by deacetylating the upstream kinase LKB1, which
promotes LKB1 translocation from the nucleus to the cytosol, where it is activated and in turn
phosphorylates and activates AMPK [53-55]. Likewise, AMPK can activate SIRT1 by increasing
the NAD/NADH ratio or the expression/activity of NAMPT [56]. Collectively, these findings
suggest the existence of an AMPK-SIRT1 cycle that links the cell’s energy and redox states [52].
Here, we observed that treatment of vitamin D deficient high fat diet with cholecalciferol

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increased the level of SIRT-1, AMPK phosphorylation and GLUT-4 expression in adipose tissue.
These events in turn led to improve glucose metabolism in high fat diet-fed mice.

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Using cell culture and animal model, this study demonstrates that 1,25(OH)2D3
supplementation can prevent oxidative stress by regulating NOX4/Nrf2/Trx signaling pathway
and improve glucose metabolism via SIRT1/AMPK/IRS1/GLUT4 cascade in HG-treated cells

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(Figure 9). Signal silencing studies demonstrate that inhibition of the insulin-independent
signaling molecule, SIRT1, significantly blocked the effect of 1,25(OH)2D3 on AMPK
phosphorylation, IRS1 phosphorylation, GLUT4 expression, and glucose uptake in adipocytes.
1,25(OH)2D3 supplementation in combination with insulin also enhanced GLUT4 expression and

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glucose uptake compared to supplementation with either insulin or 1,25(OH)2D3 alone in HG-
treated adipocytes, which suggests that insulin-independent signaling molecules can contribute to
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the higher glucose metabolism observed in 1,25(OH)2D3 and insulin-treated cells. This study
demonstrates a novel molecular mechanism by which 1,25(OH)2D3 via receptor-mediated
pathway prevents oxidative stress by modulating NOX4/Nrf2/Trx signaling pathway and
upregulates glucose uptake through the SIRT1/AMPK/IRS1/GLUT4 cascade in HG-treated
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adipocytes.

ACKNOWLEDGEMENT
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The authors thank Ms Georgia Morgan for excellent editing of this manuscript.
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GRANTS
The authors are supported by grants from NCCAM (RO1 AT00742) and the Malcolm
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Feist Endowed Chair in Diabetes. This study is also funded by a fellowship from the Malcolm
Feist Cardiovascular Research Endowment, LSU Health Sciences Center, Shreveport.
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DISCLOSURE
The authors have declared that no conflict of interest exists.
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FIGURE LEGENDS

FIGURE 1. Effect of cholecalciferol on AMPK phosphorylation, SIRT-1 and GLUT-4 levels in

mice fed with vitamin D-deficient high fat diet. A: pAMPK/AMPK, B: SIRT1 and C: GLUT4.
Data are expressed as mean ± SE for three mice.

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FIGURE 2. Effect of 1,25(OH)2D3 supplementation on the signaling pathways of oxidative
stress in 3T3L1 adipocytes exposed to control glucose (CG) or high glucose (HG). A: NOX4, B:

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ROS, C: pNF-κB/NF-κB, D: Nrf2, and E: Trx. Adipocytes were pretreated with 1,25(OH)2D3 for
2 h followed by CG (7 mM) or HG (25 mM) exposure for the next 20 h. Values are mean±SE
(n=3).

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FIGURE 3. Effect of 1,25(OH)2D3 supplementation on the signaling cascade of glucose uptake
in 3T3L1 adipocytes exposed to control glucose (CG) or high glucose (HG). A: SIRT1, B:
pAMPK/AMPK, C: PTP1B, D: pIRS1/IRS1, E: PI3K, F: PIP3, G: pAKT/AKT, and H:

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pPKCζ/λ/PKCζ. Adipocytes were pretreated with 1,25(OH)2D3 for 2 h followed by CG (7 mM)
or HG (25 mM) exposure for the next 20 h. Values are mean±SE (n=3).
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FIGURE 4. Effect of 1,25(OH)2D3 supplementation on the GLUT4 expression and glucose
uptake in 3T3L1 adipocytes exposed to control glucose (CG) or high glucose (HG). A: GLUT4
protein expression (GLUT4-PM: GLUT4 protein expression in plasma membrane and GLUT4:
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GLUT4 protein expression in whole cell lysate), B: GLUT4 surface expression (flow cytometry),
C: GLUT1 protein expression (GLUT1-PM: GLUT1 protein expression in plasma membrane
and GLUT1: GLUT1 protein expression in whole cell lysate), and F: glucose uptake. Plasma
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membrane enriched fractions was verified by noting Na+-K+-ATPase α-subunit expression.

Adipocytes were pretreated with 1,25(OH)2D3 for 2 h followed by CG (7 mM) or HG (25 mM)
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exposure for the next 20 h. Values are mean±SE (n=3).

FIGURE 5. Effect of 1,25(OH)2D3 supplementation on AMPK phosphorylation, IRS1

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phosphorylation, GLUT4 expression, and glucose uptake in 3T3L1 adipocytes exposed to

control glucose (CG) or high glucose (HG) after transfection with SIRT1 siRNA (100 nM). A:
SIRT1, B: pAMPK/AMPK, C: pIRS1/IRS1, D: GLUT4, and E: glucose uptake. Adipocytes
transfected with SIRT1siRNA were treated with 1,25(OH)2D3 for 2 h followed by CG (7 mM) or
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HG (25 mM) exposure for the next 20 h. Control siRNA is a scrambled nonspecific RNA duplex
that shares no sequence homology with any of the genes. Values are mean±SE (n=3).
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FIGURE 6. Effect of 1,25(OH)2D3 supplementation on IRS1 phosphorylation, GLUT4

expression, and glucose uptake in 3T3L1 adipocytes exposed to control glucose (CG) or high
glucose (HG) after transfection with IR siRNA (100 nM). A: IR, B: pIRS1/IRS1, C: GLUT4, and
D: glucose uptake. Adipocytes transfected with IR siRNA were treated with 1,25(OH)2D3 for 2 h
followed by CG (7 mM) or HG (25 mM) exposure for the next 20 h. Control siRNA is a
scrambled nonspecific RNA duplex that shares no sequence homology with any of the genes.
Values are mean±SE (n=3).

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FIGURE 7. Effect of insulin and 1,25(OH)2D3 supplementation either alone or in combination

on the GLUT4 expression and glucose uptake in 3T3L1 adipocytes exposed to control glucose
(CG) or high glucose (HG). A: GLUT4 and B: glucose uptake. Adipocytes were pretreated with
1,25(OH)2D3 (50 nM) for 2 h followed by CG (7 mM) or HG (25 mM) exposure for the next 20
h. In the insulin group cells were treated with insulin (25 nM) for last 1 h of the experiment.
Values are mean±SE (n=3).

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FIGURE 8. Effect of 1,25(OH)2D3 supplementation on NOX4/Nrf/Trx signaling pathway and
SIRt1/AMPK/GLUT4 cascade of glucose uptake in 3T3L1 adipocytes exposed to control
glucose (CG) or high glucose (HG) after transfection with VDR siRNA (100 nM). A: VDR

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expression, B: NOX4, C: ROS, D: Nrf2, E: Trx, F: SIRT1, G: pAMPK/AMPK, G: GLUT4, and
I: glucose uptake. Adipocytes transfected with VDR siRNA were treated with 1,25(OH)2D3 for 2
h followed by CG (7 mM) or HG (25 mM) exposure for the next 20 h. Control siRNA is a

SC
scrambled nonspecific RNA duplex that shares no sequence homology with any of the genes.
Values are mean±SE (n=3).

FIGURE 9. Schematic diagram of the proposed mechanism by which 1,25(OH)2D3 prevents

U
oxidative stress and improve glucose uptake in HG-treated 3T3L1 adipocytes.
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