Vitamin D3 affects liver expression of pro-/anti-inflammatory cytokines and nitric oxide synthases in type 2 diabetes

Abstract

Our objective was to study the effect of vitamin D₃ (VD) on hepatocellular oxidative-nitrosative stress and pro/anti-inflammatory cytokines in relation to nitric oxide (NO) formation and NO synthase (NOS) levels in type 2 diabetes mellitus (T2DM). After T2DM induction by high-fat diet and a single streptozotocin injection (25 mg/kg b. w.), male Wistar rats were treated with/without VD (1,000 IU/kg b. w., 30 days). Oxidative stress/inflammation and NOS/NO were assessed by flow cytometry, RT-qPCR, western blotting, and ELISA. A 3.3-fold decrease in serum 25(OH)D₃ was established in diabetic rats, suggesting their VD deficient status. T2DM was associated with excess reactive oxygen species (ROS; 2.4-fold) and NO (2.5-fold) production in hepatocytes paralleled by elevated levels of myeloperoxidase (1.7-fold), carbonylated (2.8-fold) and nitrotyrosylated (1.7-fold) proteins in liver tissue vs. control, indicative of oxidative-nitrosative stress. Low-grade inflammation in diabetic liver was confirmed by increased NF-κB transcriptional activity (1.24-fold) and mRNA expression of proinflammatory cytokines TNF-α (3.5-fold) and IL-1β (2.2-fold) with alleviating mRNAs of anti-inflammatory cytokines IL-4 (1.7-fold) and IL-10 (2.6-fold), while TGF-β1 expression raised 1.4-fold vs. control. Higher iNOS and eNOS mRNAs (2.7- and 3.3-fold, respectively) and protein (2.1- and 3.2-fold, respectively) levels, as well as NOS activity (1.6-fold) were found in diabetic liver. VD supplementation restored 25(OH)D₃, partially normalized NF-κB transcriptional activity and pro/anti-inflammatory cytokines, lowered hepatocellular ROS/NO, and oxidative protein modifications. However, VD had no effect on eNOS, IL-10 and TGF-β1 mRNAs. It also led to a further increase in myeloperoxidase, eNOS and iNOS proteins and NOS activity compared to diabetes. In conclusion, abnormal oxidative metabolism in T2DM is associated with enhanced NF-κB/NOS/NO response, which can be partially attenuated by VD treatment via normalization of pro-oxidative/pro-inflammatory processes. The paradoxical sustained increase in NOS expression in the presence of VD antioxidant activity likely improves hepatocellular NO bioavailability, ultimately reducing T2DM-associated liver injury.

Keywords: inflammatory cytokines; liver dysfunction; nitric oxide; oxidative-nitrosative stress; type 2 diabetes mellitus; vitamin D3.

FIGURE 1

The effect of vitamin D₃ treatment (1,000 IU/kg body weight, 30 days) on the insulin tolerance test in male Wistar rats after 3-month HFD and a single injection of streptozotocin (25 mg/kg). (A) The concentration of blood glucose in dynamics after intraperitoneal injection of insulin (0.7 units/kg) to rats; (B) Bar graphs represent the area under the “time-glucose concentration” curve. The data are presented as means ± SD, n = 7; *p < 0.05, the T2DM group vs. the control group, ^# p < 0.05, the T2DM + vitamin D₃ group vs. the T2DM group.

FIGURE 2

The serum level of 25-hydroxyvitamin D₃ (25(OH)D₃) of diabetic rats and after supplementation of vitamin D₃ (1,000 IU/kg body weight, 30 days). The data are presented as means ± SD, n = 7; *p < 0.05, the T2DM group vs. the control group, ^# p < 0.05, the T2DM + vitamin D₃ vs. the T2DM group.

FIGURE 3

Reactive oxygen species formation and oxidative protein modifications in type 2 diabetic liver. Quantification of DCFH-DA oxidation in isolated rat hepatocytes documented by flow cytometry analysis (A) and representative histograms of DCF fluorescence (B) are shown. Relative content of carbonylated proteins (C) and MPO (D) in the liver of diabetic rats and after supplementation of vitamin D₃ (1,000 IU/kg body weight, 30 days) was determined by western blot analysis. Representative immunoblots (E) are shown. The data are presented as means ± SD, n = 7; *p < 0.05, the T2DM group vs. the control group, ^# p < 0.05, the T2DM + vitamin D₃ vs. the T2DM group.

FIGURE 4

Nitric oxide formation in isolated rat hepatocytes in type 2 diabetic liver and effect of vitamin D₃ administration. Quantification of DAF oxidation documented by flow cytometry analysis (A) and representative histograms of DAF fluorescence (B) are shown. Relative protein level of 3-nitrotyrosine (C), iNOS and eNOS mRNA (D) and protein content (F), as well as their representative immunoblots (E) are shown in the liver of diabetic rats and after vitamin D₃ treatment (1,000 IU/kg body weight, 30 days). The data are presented as means ± SD, n = 7; *p < 0.05, the T2DM group vs. the control group, #p < 0.05, the T2DM + vitamin D₃ group vs. the T2DM group.

FIGURE 5

The enzymatic NOS activity in the liver of diabetic rats and after supplementation of vitamin D₃ (1,000 IU/kg body weight, 30 days). The data are presented as means ± SD, n = 7; *p < 0.05, the T2DM group vs. the control group, #p < 0.05, the T2DM + vitamin D₃ group vs. the T2DM group.

FIGURE 6

The relative gene expression of pro/anti-inflammatory cytokines in the liver tissue of rats with T2DM and after vitamin D₃ treatment (1,000 IU/kg body weight, 30 days). Relative mRNA content of Tnf-α (A), Il-1β (B), Il-4 (C), Il-10 (D), and Tgf-β (E) was determined by RT-qPCR. mRNA levels were adjusted to β-actin and Mrpl32 expression. The data are presented as means ± SD, n = 7; *p < 0.05, the T2DM group vs. the control group, ^# p < 0.05, the T2DM + vitamin D₃ group vs. the T2DM group.

FIGURE 7

The NF-κB p65 transcription activity in the nuclear fraction of liver extracts of rats with T2DM and after vitamin D₃ treatment (1,000 IU/kg body weight, 30 days). Data are shown as means ± SD, n = 7; *p < 0.05, the T2DM group vs. the control group, ^# p < 0.05, the T2DM + vitamin D₃ group vs. the T2DM group.