Effects Of Vitamin D Deficiency On High-fat Diet-Induced Insulin Resistance And Hepatic Lipid Accumulation In Male Mice

Background It is increasingly recognized that vitamin D deficiency is associated with increased risks of metabolic disorders among overweight children. A recent study showed that vitamin D deficiency exacerbated inflammation in nonalcoholic fatty liver disease (NAFLD) through activating toll-like receptor (TLR)4 in a high-fat diet (HFD) rat model. Nevertheless, it is unknown whether vitamin D deficiency affects HFD-induced insulin resistance and hepatic lipid accumulation.Objective To explore the effects of vitamin D deficiency on HFD-induced insulin resistance and hepatic lipid accumulation.Methods Male ICR mice (35 days old) were randomly assigned into four groups as follows. In CD and VDD groups, mice were fed with AIN93M diets. In HFD and VDD+HFD groups, mice were fed with HFD. In VDD and VDD+HFD groups, vitamin D in feed was depleted. Mice were inspected daily for food intake and weighted weekly. Four weeks after diet intervention, fasting blood glucose was measured every 2 weeks. Glucose tolerance test (GTT) and insulin tolerance test (ITT) were assessed 12 weeks after diet intervention. Fourteen weeks after diet intervention, mice were killed. Serum was collected for detecting. Serum 25(OH)D was measured by radioimmunoassay. Serum insulin, leptin and adiponectin levels were measured by ELISA. Serum triglyceride, total cholesterol, very low density lipoprotein (VLDL) cholesterol and alanine transaminase (ALT) were determined by routine laboratory methods using an auto-analyzer. Liver tissue was collected and weighed. Western blotting analysis was used to detect proteins expression of pAkt and Akt 5 minutes after injection of insulin in liver. Real-time RT-PCR was used to detect expression of lipid metabolism related genes in liver. Hepatic triglyceride (TG) content was measured and hepatic lipid accumulation was evaluated by staining liver sections with hematoxylin& eosin (H&E) and oil red O. Histological scoring (NAFLD activity scores) was performed by a blinded hepatopathologist for steatosis, lobular inflammation and ballooning. Then perinephric and epididymis fats were collected and weighed. Real-time RT-PCR was used to detect gene expression of lipid metabolism in white adipose tissue.Results Fourteen weeks after diet intervention, serum 25(OH)D were reduced to less than 5 ng/mL in VDD and VDD+HFD groups (0.46±0.24 and 1.32±0.45 ng/mL, respectively), significantly lower than those in CD and HFD groups (16.65±2.65 and 13.77±0.94 ng/mL, respectively). (1) Effects of vitamin D deficiency on food intake and caloric intake:vitamin D deficiency induced slight elevation of food intakes and caloric intakes; vitamin D deficiency induced significant elevation of food intakes and caloric intakes in HFD fed mice. (2) Effects of vitamin D deficiency on body weight and hepatic lipid accumulation:feeding mice with vitamin D deficiency diet did not induce obesity and hepatic lipid accumulation; vitamin D deficiency markedly alleviated HFD-induced overweight and hepatic lipid accumulation. (3) Effects of vitamin D deficiency on serum lipids:no difference was observed in serum TG and VLDL cholesterol among different groups. (4) Effects of vitamin D deficiency on lipid metabolism-related genes in liver:vitamin D deficiency significantly attenuated HFD-induced up-regulation of hepatic peroxisome proliferator-activated receptor (Ppar)y, which promoted hepatic lipid uptake and lipid droplet formation, and its target gene Cd36. However, vitamin D deficiency had little effect on hepatic fatty acid synthesis and lipolysis-related genes among different groups. (5) Effects of vitamin D deficiency on blood glucose and insulin resistance:feeding mice with vitamin D deficiency diet did not induce hyperglycemia, hyperinsulinemia and insulin resistance. The glucose AUC was markedly reduced in VDD group as compared with CD group during the GTT. There was a downward trend on fasting blood glucose, glucose AUC during the GTT and ITT in VDD+HFD group as compared with HFD group. Meanwhile there was a upward trend on HFD-induced reduction of hepatic pAkt level in VDD+HFD group as compared with HFD group in investigating hepatic insulin signal transduction. (6) Effects of vitamin D deficiency on weight of white adipose tissue:no difference was observed in weight of perinephric fat or epididymis fat among different groups. (7) Effects of vitamin D deficiency on lipid metabolism-related genes in white adipose tissue:vitamin D deficiency significantly attenuated HFD-induced down-regulation of Fasn, Acc, and Scd-1 in white adipose tissue. No significant difference on the expression of genes for lipid transport in white adipose tissue was observed among different groups. In addition, vitamin D deficiency up-regulated carnitine palmitoyltrans (Cpt)2, the key enzyme for fatty acid β-oxidation, and uncoupling protein (Ucp)3, which separated oxidative phosphorylation from ATP production, in adipose tissue. (8) Effects of vitamin D deficiency on expression of uncoupling proteins in brown adipose tissue:the expression of Ucp3 mRNA in brown adipose tissue was significantly up-regulated in VDD and VDD+HFD groups.Conclusion (1) Vitamin D deficiency is not a direct risk factor for obesity, insulin resistance and hepatic lipid accumulation. (2)Vitamin D deficiency alleviates HFD-induced overweight, hyperinsulinemia and hepatic lipid accumulation through promoting fatty acid β-oxidation and elevating energy expenditure in adipose tissue.