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

Abstract

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.

Keywords: N-acetylcysteine (NAC); hepatocytes; insulin resistance; oxidative stress; vitamin D.

Figure 1. Knockdown of 1α(OH)ase leads to vitamin D3 depletion in L02 hepatocytes

Puromycin-selected stable L02 hepatocytes, expressing shRNA against human 1α-Hydroxylase [“sh-1α(OH)ase-1/-2”] or the scramble control shRNA (“sh-SCR”), were subjected to Western blotting assay (A) and qRT-PCR assay (B) to test 1α(OH)ase expression; Vitamin D3 content in the conditional medium was also analyzed (C). Relative 1α(OH)ase expression (vs. loading control ERK1/2) was quantified (A). Data were expressed as mean ± SD (n=5). * p <0.05 vs. “sh-SCR” cells. Experiments in this and all following figures were repeated three times, and similar results were obtained.

Figure 2. Knockdown of 1α(OH)ase leads to insulin resistance in L02 hepatocytes

Puromycin-selected stable L02 hepatocytes, expressing shRNA against human 1α-Hydroxylase [“sh-1α(OH)ase-1/-2”] or the scramble control shRNA (“sh-SCR”), were treated with insulin (1 μg/mL) for 10 min, expressions of listed proteins were tested by Western blotting assay (A); quantified results summarizing three sets of repeated blot data were also shown (B); expressions of GLUT4 and (β-) tubulin were also tested, results were also quantified (C). Data were expressed as mean ± SD (n=3). * p <0.05 vs. “sh-SCR” cells.

Figure 3. Knockdown of 1α(OH)ase leads to ROS production, p53-p21 activation and DNA damage in L02 hepatocytes

Puromycin-selected stable L02 hepatocytes, expressing shRNA against human 1α-Hydroxylase [“sh-1α(OH)ase-1/-2”] or the scramble control shRNA (“sh-SCR”), were subjected to listed assays to test SOD activity (A), ROS content (DCFH-DA assay) (B), lipid peroxidation level (TBAR assay) (C), p53-p21 signaling (Western blotting assay) (D), and DNA damage (γ-H2AX FACS assay) (E). Data were expressed as mean ± SD (n=5). * p <0.05 vs. “sh-SCR” cells.

Figure 4. N-acetylcysteine blocks ROS production, p53-p21 activation and DNA damage in 1α(OH)ase-silenced L02 hepatocytes

L02 hepatocytes, infected with shRNA 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, relative ROS content (DCFH-DA assay) (A), lipid peroxidation level (TBAR assay) (B) p53-p21 signaling (Western blotting assay) (C), and DNA damage (γ-H2AX FACS assay) (D) were tested. Data were expressed as mean ± SD (n=5). * p <0.05 vs. “PBS” cells.

Figure 5. NAC restores insulin sensitivity in 1α(OH)ase-silenced L02 hepatocytes

L02 hepatocytes, infected with shRNA 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.