Effects of Vitamin D Supplementation on Adipose Tissue Inflammation and NF-κB/AMPK Activation in Obese Mice Fed a High-Fat Diet

Abstract

Adipose tissue expansion is strongly associated with increased adipose macrophage infiltration and adipocyte-derived pro-inflammatory cytokines, contributing to obesity-associated low-grade inflammation. Individuals with vitamin D deficiency have an increased prevalence of obesity and increased circulating inflammatory cytokines. However, the effect of vitamin D supplementation on obesity-induced inflammation remains controversial. Male C57BL/6J mice received a low-fat (10% fat) or high-fat (HF, 60% fat diet) containing 1000 IU vitamin D/kg diet, or HF supplemented with 10,000 IU vitamin D/kg diet for 16 weeks (n = 9/group). Vitamin D supplementation did not decrease HF-increased body weight but attenuated obesity-induced adipose hypertrophy and macrophage recruitment as demonstrated by the number of crown-like structures. Vitamin D supplementation significantly reduced the mRNA expression of CD11c, CD68, and iNOS, specific for inflammatory M1-like macrophages, and decreased serum levels of NO. In addition, significant reductions in pro-inflammatory gene expression of IL-6, MCP-1, and TNFα and mRNA levels of ASC-1, CASP1, and IL-1β involved in NLRP3 inflammasome were found in obese mice supplemented with vitamin D. Vitamin D supplementation significantly increased obesity-decreased AMPK activity and suppressed HF-increased NF-κB phosphorylation in adipose tissue from obese mice. These observed beneficial effects of vitamin D supplementation on adipose tissue expansion, macrophage recruitment, and inflammation might be related to AMPK/NF-κB signaling.

Keywords: NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3); adenosine monophosphate-activated protein kinase (AMPK); adipose tissue; inflammation; nuclear factor-kappa B (NF-κB); obesity; vitamin D.

Figure 1

Effect of vitamin D supplementation on body weight and food intake. (A) Changes of body weight (g) during 16-week diet supplementation, (B) body weight gain (g), (C) food intake (g consumed/day), (D) food efficiency (g gain/g consumed) = body weight gain (g)/total food consumption (g), (E) energy intake (kcal/day), and (F) energy efficiency (g gain/kcal consumed) = body weight gain (g)/total energy intake (kcal). Values are expressed as the mean ± SEM (n = 9/group). NOR, 10% fat diet with 1000 IU vitamin D; HF, 60% fat diet with 1000 IU vitamin D; HF + HVD, 60% fat diet with 10,000 IU vitamin D. ** p < 0.01 compared to the HF group.

Figure 2

Effect of vitamin D supplementation on serum metabolic parameters. (A) Serum levels of TG, TC, HDL-C, and LDL-C. LDL-C = TC − HDL-C − (TG/5). (B) Serum ALT and AST activities. (C) Serum 25OHD amounts. Values are expressed as the mean ± SEM (n = 9/group). NOR, 10% fat diet with 1000 IU vitamin D; HF, 60% fat diet with 1000 IU vitamin D; HF + HVD, 60% fat diet with 10,000 IU vitamin D. ALT, alanine transaminase; AST, aspartate aminotransferase; HDL-C, high-density lipoprotein-cholesterol; LDL-C, low-density lipoprotein-cholesterol; TC, total cholesterol; TG, triglyceride. * p < 0.05; ** p < 0.01 compared to the HF group.

Figure 3

Effect of vitamin D supplementation on adipose tissue weight and adipocyte size. (A) White adipose tissue (WAT) weights. (B) Representative hematoxylin and eosin (H&E)-stained epidydimal sections (scale bars, 200 μm; magnification, 400×). (C) Average adipocyte diameter of epididymal WAT (eWAT). (D) Gene expression of adipogenesis in eWAT. Gene expression of β-actin, as a reference gene was used for normalization, and data were presented as fold change compared to the HF group. Values are expressed as the mean ± SEM (n = 9/group). NOR, 10% fat diet with 1000 IU vitamin D; HF, 60% fat diet with 1000 IU vitamin D; HF + HVD, 60% fat diet with 10,000 IU vitamin D. * p < 0.05; ** p < 0.01 compared to the HF group.

Figure 4

Influence of vitamin D supplementation on adipose tissue macrophage accumulation and polarization in epididymal white adipose tissue (eWAT) of obese mice. (A) Representative images of F4/80-immunostained eWAT (scale bars, 200 μm; magnification, 400×). The black arrows represent a crown-like structure (CLS). (B) The mean CLS number was observed under a microscope, quantified from multiple histologic sections, and expressed as CLS number per 100 adipocytes. (C) mRNA levels related to M1/M2 macrophage polarization were determined by RT-qPCR, normalized to that of β-actin, and expressed as the fold change compared to the HF group. (D) Serum nitric oxide (NO) amount was analyzed using a Griess reagent kit and expressed as the fold difference relative to the HF group. Values are expressed as the mean ± SEM (n = 9/group). NOR, 10% fat diet with 1000 IU vitamin D; HF, 60% fat diet with 1000 IU vitamin D; HF + HVD, 60% fat diet with 10,000 IU vitamin D. * p < 0.05; ** p < 0.01 compared to the HF group.

Figure 5

Effect of vitamin D supplementation on pro-inflammatory cytokines and NLRP3 inflammasome. mRNA levels related to (A) pro-inflammatory cytokines and (C) NLRP3 inflammasome components in epididymal white adipose tissue (eWAT) were measured by RT-qPCR, normalized to β-actin, and expressed as the fold change compared to the HF group. Serum levels of (B) TNFα and (D) IL-1β were quantified using commercial colorimetric ELISA kits, normalized to their respective protein concentrations, and presented as the fold change compared to the HF group. Values are expressed as the mean ± SEM (n = 9/group). NOR, 10% fat diet with 1000 IU vitamin D; HF, 60% fat diet with 1000 IU vitamin D; HF + HVD, 60% fat diet with 10,000 IU vitamin D. * p < 0.05; ** p < 0.01 compared to the HF group.

Figure 6

Influence of vitamin D supplementation on AMPK and NF-κB activities. Commercial ELISA kits were used to measure nuclear (A) NF-κB activity and (B) AMPK activation in epididymal WAT (eWAT) from obese mice. Results were normalized to their relative protein levels and presented as the fold difference compared to the HF group. Values are expressed as the mean ± SEM (n = 9/group). NOR, 10% fat diet with 1000 IU vitamin D; HF, 60% fat diet with 1000 IU vitamin D; HF + HVD, 60% fat diet with 10,000 IU vitamin D. * p < 0.05; ** p < 0.01 compared to the HF group.