Vitamin D deficiency causes insulin resistance by provoking oxidative stress in hepatocytes

Vitamin D deficiency could cause insulin resistance. However, the underlying mechanisms are unclear. The 1α-Hydroxylase [“1α(OH)ase”] is a key enzyme for activate vitamin D3 synthesis. Here, we show that 1α(OH)ase stable knockdown by targeted shRNA led to vitamin D3 depletion in L02 hepatocytes. 1α(OH)ase silence also inhibited insulin-induced downstream signaling (IRS-1, ERK and AKT) transduction and glucose transporter 4 expression. Further, 1α(OH)ase shRNA in L02 hepatocytes led to significant reactive oxygen species production, p53-p21 activation and DNA damages. Such effects were almost completely reversed with co-treatment of n-acetylcysteine, which is an established anti-oxidant. Remarkably, insulin-induced downstream signaling transduction and glucose transporter 4 expression were recovered with n-acetylcysteine co-treatment in 1α(OH)ase-silenced L02 hepatocytes. Together, our results suggest that vitamin D deficiency-induced insulin resistance is possibly caused by oxidative stress in hepatocytes.


INTRODUCTION
Insulin resistance is a major reason of type-II diabetes [1,2]. It is also a characteristic manifestation of a wide range of other clinical diseases [3][4][5]. A number of genetic and/or environmental factors could cause insulin resistance [3][4][5]. Epidemic studies have suggested that that vitamin D deficiency is also associated with insulin resistance [6]. The supplementation of active vitamin D3 may help to improve the insulin resistance [6]. However, the underlying mechanism is largely unknown [6].
The function of reactive oxygen species (ROS) in insulin resistance has been well established [7][8][9]. Studies have demonstrated that ROS level is significantly increased in both clinical samples and experimental settings of insulin resistance [10][11][12]. Meanwhile, exogenous oxidative stress would lead to insulin resistance. These results suggest that oxidative stress might play a key function in insulin resistance [7,9]. Inhibition of ROS, on the other hand, could improve insulin sensitivity and glucose homeostasis in insulinresistant mice [10][11][12]. Clinical studies have also shown that supplement with anti-oxidant may improve insulin sensitivity [10][11][12].
25-Hydroxyvitamin D3 1α-Hydroxylase ["1α(OH) ase"] is a key enzyme for activate vitamin D3 synthesis [13,14]. In the current study, 1α(OH)ase was silenced in human L02 hepatocytes to mimic vitamin D deficiency. Our results suggest that vitamin D deficiency induces insulin resistance probably by provoking oxidative stress.

Knockdown of 1α(OH)ase leads to insulin resistance in L02 hepatocytes
The aim of this study is to test the potential effect of vitamin D deficiency on insulin resistance. The 1α(OH)ase-silenced L02 hepatocytes (See Figure 1) were thereby treated with insulin. Western blotting assay was performed to test insulin signalings [15,16]. Results in Figure 2A demonstrated that insulin (1 μg/mL, 10 min)induced activation of downstream signalings, including IRS-1 (insulin receptor substrate-1), ERK1/2 and AKT, was largely inhibited with 1α(OH)ase knockdown. Phosphorylated ("p-") IRS-1, p-AKT and p-ERK1/2 by insulin were all dramatically reduced in 1α(OH) ase-depleted L02 hepatocytes (Figure 2A). Expression of above total kinases was yet unchanged (Figure 2A). Quantified results summarizing three sets of repeated blot data further confirmed inhibition of insulin-induced IRS-1, ERK1/2 and AKT activations with 1α(OH)ase silence in L02 hepatocytes ( Figure 2B). Meanwhile, as shown in Figure 2C, expression of glucose transporter 4 (GLUT4), a key glucose transporter protein, was also downregulated in 1α(OH)ase-silenced L02 hepatocytes ( Figure 2C). Together, these results imply that knockdown of 1α(OH) ase might lead to insulin resistance in L02 hepatocytes.
In the current study, we proposed that oxidative stress could be the main reason of insulin resistance in vitamin D-deficient cells. Vitamin D depletion via stably silencing 1α (OH)ase in L02 hepatocytes led to significant ROS production, as well as subsequent p53-p21 activation and DNA damage. Such effects were almost completely nullified with co-treatment of NAC, the known antioxidant. Remarkably, insulin resistance was also abolished with co-treatment of NAC in 1α(OH)ase-depleted L02 hepatocytes. Together, our results suggest that vitamin D deficiency-induced insulin resistance is possibly caused by oxidative stress in L02 hepatocytes. against human 1α-Hydroxylase ["sh-1α(OH)ase-1"], were also exposed to n-acetylcysteine (NAC, 500 μM, renewed daily) or PBS; After 10 days, cells were treated with insulin (1 μg/mL) for 10 min, expressions of listed proteins were tested by Western blotting assay, and results of three sets of repeats were quantified (A); expressions of GLUT4 and tubulin were also tested. (B) Quantified results summarizing three sets of repeated blot data were also shown). Data were expressed as mean ± SD (n=3). * p <0.05 vs. "PBS" cells. www.impactjournals.com/oncotarget

Chemicals and reagents
Insulin, n-acetylcysteine (NAC) and puromycin were provided by Sigma Aldrich Chemicals (Nanjing, China). The antibodies of this study were provided by Cell Signaling Technology (Danvers, MA) and Abcam (Shanghai, China). The reagents for cell culture were obtained from Gibco Co. (Nantong, China).

Cell culture
Human L02 hepatocytes were provided by the Cell Bank of Shanghai Biological Institute (Shanghai, China). L02 cells were cultured in RPMI 1640 with 10% FBS and antibiotics, in a humidified atmosphere at 37 °C with 5% CO 2 . Cells were subjected to mycoplasma and microbial contamination examination every 2-3 months. Population doubling time, colony forming efficiency, and morphology were routinely checked to confirm the genotype.

1α(OH)ase shRNA
The two lentiviral 1α(OH)ase shRNAs with nonoverlapping sequences were designed and verified by Genepharm (Shanghai, China). The shRNA (10 μL/mL medium, per well) was added to L02 hepatocytes for 24 hours. Stable cells were selected by puromycin (0.5 μg/ mL, Sigma) for a total of 10 days. Puromycin-containing medium was renewed every two days. Silence of 1α(OH) ase in the stable L02 hepatocytes was confirmed by both qRT-PCR assay and Western blotting assay. Control cells were infected with lentiviral scramble control shRNA (Santa Cruz Biotech).

Vitamin D3 assay
The level of vitamin D3 in the conditional medium of L02 hepatocytes were tested via a commercial available enzyme linked fluorescent assay (ELFA) kit from Roche (Shanghai, China), according to the manufacturer's guidelines.

RNA isolation and qRT-PCR
As described in our previous studies [34,35], Trizol reagents (Invitrogen) was applied to extract the total RNA of L02 hepatocytes. RNA was then reversetranscribed (RT) with RT-PCR kit (Toyobo, Osaka, Japan). Quantitative Real-time PCR ("qRT-PCR") assay using the SYBR green kit was performed using the ABI-7600 PCR system (Applied Biosystems, Shanghai, China). mRNA primers of 1α(OH)ase and GAPDH were described previously [36,37]. We utilized the 2 ΔΔCt method to calculate relative 1α(OH)ase mRNA expression (vs. GAPDH). All the primers were synthesized by Genepharm (Shanghai, China).

Western blotting assay
First, the lysis buffer (Biyuntian, Wuxi, China) was applied to achieve protein lysates from L02 hepatocytes. For each condition, 30 μg total lysate proteins per lane were separated by the SDS-PAGE gels (10-12%) [16,38], which were then transferred to the polyvinylidene difluoride (PVDF, Millipore, Suzhou, China) membranes. Afterwards, the blots were blocked (in 10% of milk), and were incubated with designated primary and corresponding secondary antibodies. Enhanced chemiluminescence (ECL) reagents (Pierce, Nantong, China) were utilized to visual the interested bands [39][40][41]. Total gray of each band was quantified via the ImageJ software, and the value was normalized to that of loading control (ERK1/2 or β-Tubulin).

SOD activity assay
The superoxide dismutase (SOD) activity in L02 hepatocytes was assayed by the NWLSS kit, which is an extremely sensitive SOD kit, using WST-1 to generate a water-soluble formazan dye upon reduction with superoxide anion. The detailed procedure was described previously [42]. The final mixture was subjected to spectrophotometer detection at the absorbance at 560 nm.

Reactive oxygen species (ROS) assay
ROS content was tested by the DCFH-DA fluorescent dye assay (Invitrogen). The detailed protocol was described in our previous studies [34,35,41]. Briefly, after applied treatment, L02 hepatocytes were incubated with 10 μM of DCFH-DA for 30 min under the dark, which were thereafter tested for fluorescence under a Fluorescence Microplate Reader (Synergy 2, BioTek, Winooski, VT).

Lipid peroxidation assay
As described previously [43], cellular lipid peroxidation was evaluated by the thiobarbituric acid reactive substances (TBAR) assay [44]. Briefly, after the indicated treatment, L02 hepatocytes protein lysates (20 μg per condition) were mixed with 20% of acetic acid and thiobarbituric acid solution. After heating, the mixtures were centrifuged, and then in the supernatant the red pigment dye was tested via a microplate reader. TBAR activity was expressed as nM of malondialdehyde per mg protein. The values of treatment group were always normalized to control. γ-H2AX FACS assay γ-H2AX assay was performed to test DNA damage. Briefly, L02 hepatocytes were first trypsinized and fixed in ice-cold ethanol, which were then exposed to the antiγ-H2AX antibody (Cellular Signaling Tech) for 6 hours. Cells were then incubated with the FITC-conjugated secondary antibody. FACS assay was performed to determine γ-H2AX percentage, as the quantitative measurement of DNA damage [45].

Statistical analysis
The results were expressed as mean ± standard deviation (SD). The statistical analysis among different groups was done using one-way ANOVA with Scheffe's test [46,47].

Author contributions
All authors carried out the experiments, participated in the design of the study and performed the statistical analysis, participated in its design and coordination and helped to draft the manuscript.