Uncoupling of inflammation and insulin resistance by NF-kappaB in transgenic mice through elevated energy expenditure

Abstract

To study the metabolic activity of NF-kappaB, we investigated phenotypes of two different mouse models with elevated NF-kappaB activities. The transcriptional activity of NF-kappaB is enhanced either by overexpression of NF-kappaB p65 (RelA) in aP2-p65 mice or inactivation of NF-kappaB p50 (NF-kappaB1) through gene knock-out. In these models, energy expenditure was elevated in day and night time without a change in locomotion. The mice were resistant to adulthood obesity and diet-induced obesity without reduction in food intake. The adipose tissue growth and adipogenesis were inhibited by the elevated NF-kappaB activity. Peroxisome proliferator-activator receptor gamma expression was reduced by NF-kappaB at the transcriptional level. The two models exhibited elevated inflammatory cytokines (tumor necrosis factor-alpha and interleukin-6) in adipose tissue and serum. However, insulin sensitivity was not reduced by the inflammation in the mice on a chow diet. On a high fat diet, the mice were protected from insulin resistance. The glucose infusion rate was increased more than 30% in the hyperinsulinemic-euglycemic clamp test. Our data suggest that the transcription factor NF-kappaB promotes energy expenditure and inhibits adipose tissue growth. The two effects lead to prevention of adulthood obesity and dietary obesity. The energy expenditure may lead to disassociation of inflammation with insulin resistance. The study indicates that inflammation may prevent insulin resistance by eliminating lipid accumulation.

FIGURE 1.

p65 expression in aP2-p65 mice. A, p65 mRNA in epididymal fat and liver. B, p65 protein in fat and liver. The p65 protein was determined in the whole cell lysate in a Western blot. C, mRNA of NF-κB target genes in epididymal fat of 8-week-old mice. mRNA was determined by quantitative RT-PCR. D, mRNA of p65 and NF-κB target genes in differentiated MEFs of aP2-p65 mice. MEFs were made from embryos of aP2-p65 mice and differentiated into adipocytes in vitro. E, DNA binding activity of NF-κB in differentiated MEFs. The electrophoretic mobility shift assay (EMSA) result is shown. F, mRNA of p65 and NF-κB target genes in differentiated preadipocytes of epididymal fat pad. In the bar graph, values are the mean ± S.E. (n = 3). *, p < 0.05; **, p < 0.001 by Student's t test.

FIGURE 2.

Lean phenotype in aP2-p65 mice. Body weight (BW), fat content, food intake, and energy expenditure were monitored in the aP2-p65 mice. A, body weight. B, body fat content (percentage). This was determined by NMR. C, body lean mass. D, food intake. Food intake was monitored daily for 3 days at 10 weeks of age. The average daily food intake (g) was converted into kcal and normalized with body lean mass (kg). E, energy expenditure. The test was conducted at 12 weeks of age, and the unit is kcal/kg of body lean mass/h. F, oxygen consumption. The unit is volume (ml) of oxygen/kg of body lean mass/h. G, physical activity. Interruption of a horizontal laser beam (X) was used to indicate the locomotor activity. H, body temperature and cold response. The test was conducted at 24 weeks in age. I, energy expenditure in female aP2-p65 mice (n = 5). The test was conducted at 16 weeks in age. A–E, n = 8; G and H, n = 10 in WT or Tg group. Values are the mean ± S.E. *, p < 0.05; **, p < 0.001 by Student's t test. RER, respiratory exchange ratio.

FIGURE 3.

Adipose tissue inflammation. Fat tissue was collected at the age of 20 weeks and used in morphology and gene expression studies. A, epididymal fat pad. B, F4/80 immunostaining in WAT. The macrophages are shown in brown. C, adipocyte-specific genes. mRNA expression was determined by real time RT-PCR and expressed as -fold change. D, fatty acid genes, including genes related to fat synthesis and lipolysis. E, inflammatory genes. F, cytokines in serum. TNF-α and IL-6 were determined in serum using multiplex kits. G, macrophage markers. The mRNA expression was determined by real time RT-PCR. In this figure, values are means ± S.E. (n = 6). *, p < 0.05; **, p < 0.001 by Student's t test. Acdc, adiponectin; FAS, fatty acid synthase; SREBP, sterol response element-binding protein; HSL, hormone-sensitive lipase; LPL, lipoprotein lipase.

FIGURE 4.

Differentiation of preadipocytes. The intact epididymal fat pad was collected at the age of 5 weeks and used to prepare preadipocytes. The differentiation was induced in the classic adipogenic mixture supplemented with thiazolidinedione (2 μm). A, pref-1 expression in preadipocytes. B, mRNA at day 6 of differentiation. Adiponectin mRNA was examined together with leptin, PPARγ, and aP2. C, triglyceride (TG) content indicated by BODIPY-stained lipid droplets (green fluorescence) at day 8 of differentiation. Blue fluorescence dye of DAPI indicates nucleus. Triglyceride quantification by ratio of BODIBY and DAPI signals is shown. D, the PPARγ promoter activity. The transcription of PPARγ was analyzed in 3T3-L1 adipocytes using the PPARγ2-luciferase reporter system in the transient transfection. p65 was transiently cotransfected with the PPARγ-Luc vector. The cells were serum-starved and treated either with or without TNF-α (20 ng/ml) overnight. In the bar graphs, each point represents mean ± S.E. (n = 3). **, p < 0.001 by Student's t test.

FIGURE 5.

Adiposity and energy metabolism on HFD. Body weight, body composition, food intake, and energy expenditure were examined using the NMR and metabolic chamber. The energy metabolism and food intake were examined at 10–12 weeks of age (5–6 weeks on HFD). A, body weight. B, body fat content (percentage). This was determined by NMR. C, epididymal fat. The epididymal fat (EF) was determined in weight and percentage of body weight (BW). D, food intake. Food intake was monitored daily for 3 days. Average daily food intake (g) was converted into kcal and normalized with the body lean mass (kg) and 24 h. E, energy expenditure. The unit is kcal/kg of body lean mass/h. F, spontaneous physical activity. The frequency of horizontal movement was shown for day and night time. Values are the mean ± S.E. (n = 8). *, p < 0.05; **, p < 0.001 by Student's t test.

FIGURE 6.

Lean phenotype of p50-KO mice. The phenotype study was conducted in p50-KO mice at 8–12 weeks of age on chow diet. A, body weight and composition were examined using NMR. The weight (g) of body fat and lean mass was used in the calculation. The results are expressed relative to the control used as a standard. B, energy expenditure was monitored using the metabolic chamber and normalized with body lean mass. C, food intake was determined in mice at 10 weeks of age. The daily average food intake was expressed in kcal normalized by body lean mass and time. D, inflammation gene expression in epididymal fat pads in quantitative RT-PCR. E, PPARγ protein determined in fat tissue in a Western blot. F, mRNA of adipocyte-specific genes was determined in fat tissues by quantitative RT-PCR. G, adipogenesis of preadipocytes of p50-KO mice in vitro. H, the body weight of p50-KO mice before and after HFD feeding at 20 weeks. I, spontaneous physical activity of p50-KO mice monitored in the metabolic chamber. In this figure, values are the mean ± S.E. (n = 9). *, p < 0.05; **, p < 0.001 by Student's t test.

FIGURE 7.

Insulin sensitivity in aP2-p65 mice. Hyperinsulinemic-euglycemic clamps were conducted in conscious mice following 16 weeks of HFD (at 24 weeks of age). A, steady-state glucose infusion rates (GIR) during clamps. B, insulin-stimulated whole body glucose turnover. C, hepatic glucose production (HGP) in WT mice. D, hepatic glucose production in Tg mice. E, hepatic insulin action expressed as percentage suppression of basal HGP during clamps. F, whole body glycogen and lipid synthesis. G, insulin-stimulated glucose uptake in WAT. H, insulin-stimulated glucose uptake in skeletal muscle (quadriceps). I, insulin-stimulated glucose uptake in heart. Values are means ± S.E. (n = 6–9).