Chronic inflammation in fat plays a crucial role in the development of obesity-related insulin resistance

Abstract

Insulin resistance arises from the inability of insulin to act normally in regulating nutrient metabolism in peripheral tissues. Increasing evidence from human population studies and animal research has established correlative as well as causative links between chronic inflammation and insulin resistance. However, the underlying molecular pathways are largely unknown. In this report, we show that many inflammation and macrophage-specific genes are dramatically upregulated in white adipose tissue (WAT) in mouse models of genetic and high-fat diet-induced obesity (DIO). The upregulation is progressively increased in WAT of mice with DIO and precedes a dramatic increase in circulating-insulin level. Upon treatment with rosiglitazone, an insulin-sensitizing drug, these macrophage-originated genes are downregulated. Histologically, there is evidence of significant infiltration of macrophages, but not neutrophils and lymphocytes, into WAT of obese mice, with signs of adipocyte lipolysis and formation of multinucleate giant cells. These data suggest that macrophages in WAT play an active role in morbid obesity and that macrophage-related inflammatory activities may contribute to the pathogenesis of obesity-induced insulin resistance. We propose that obesity-related insulin resistance is, at least in part, a chronic inflammatory disease initiated in adipose tissue.

Figures

Figure 1

(a) The transcriptional regulation of inflammation genes in the WAT of mice with genetic or diet-induced obesity/diabetes by quantitative RT-PCR (TaqMan). For comparison, the expression level of these genes in lean mice was arbitrarily set at 1; error bars represent ± SE. LF, low fat (10% fat); HF, high fat (60% fat). ob/ob and db/db mice and appropriate controls (n = 5 per group) were obtained from The Jackson Laboratory, fed a standard chow diet (Farmer’s Exchange), and sacrificed at 15 weeks of age. DIO mice (C57BL/6J; The Jackson Laboratory) were obtained at 4 weeks of age and placed on the designated diet of 60% kcal from fat (Research Diets Inc.) for 16 weeks (n = 10 per group). y axes show arbitrary units representing relative expression levels of mRNAs. (b) Confirmation of inflammation-gene regulation by Northern blot analysis. An independent set of animals from the same experiment was used.

Figure 2

The transcriptional regulation of inflammation genes in progressively obese and insulin-resistant mice. Time is shown as weeks on diet. (a) Changes in body weight, fasting blood glucose, and fasting plasma insulin over 26 weeks on diets. *Statistically significant difference (P < 0.05, Student’s t test). (b) mRNA expression of ADAM8, MIP-1 For comparison, the expression level of these genes in lean mice was arbitrarily set at 1; error bars represent ± SE. C57BL/6J mice were obtained from The Jackson Laboratory and started on diets of 10% fat (low fat; LF) or 60% fat (high fat; HF) (Research Diets Inc.) at 4 weeks of age (0-week time point). Animals were sacrificed after the specified number of weeks on the diet (0 weeks, n = 10 per group; 3 weeks, n = 3 per group; 6 weeks, n = 10 per group; 8 weeks, n = 3 per group; 11 weeks, n = 3 per group; 16 weeks, n = 10 per group; 26 weeks, n = 10 per group). y axes show arbitrary units representing relative expression levels of mRNAs.

Figure 3

The transcriptional regulation of ADAM8, MIP-1α, MCP-1, MAC-1, F4/80, and CD68 in macrophage-rich tissues (WAT, lung, and spleen) of ob/ob and db/db mice. For comparison, the gene expression level in lean mice was arbitrarily set at 1; error bars represent ± SE. y axes show arbitrary units representing relative expression levels of mRNAs.

Figure 4

The mRNA expression of ADAM8, MIP-1α, MCP-1, MAC-1, F4/80, and CD68 in stromal-vascular and adipocyte fractions. For quality control, leptin was examined in these samples. To compare with the expression of known adipose inflammation genes, TNF-α and IKKβ expression was also examined. For genes predominantly expressed in the stromal-vascular fraction, the expression in adipocytes was set at 1; error bars represent ± SE. For leptin, the expression in stromal-vascular cells was set at 1. y axes show arbitrary units representing relative expression levels of mRNAs.

Figure 5

Histological comparison between wild-type and ob/ob WAT and stromal-vascular cells. For each panel, the wild type at ×100 is seen at the left, ob/ob at ×100 in the middle, and ob/ob at ×400 at the right. (a) WAT morphological differences at 3 months (toluidine blue O on paraffin sections). Note the presence of nucleated stromal cells in the high magnification of the ob/ob type at the right. (b) WAT morphological differences at 5 months (toluidine blue O on paraffin sections). The stromal multinucleated cells have increased in the ob/ob type seen at the right, with early features of lipolysis in the ob/ob adipocytes manifested by multifocal cell shrinkage. (c) WAT at 3 months probed with F4/80 antisense RNA (in situ hybridization on fresh frozen sections). (d) WAT at 3 months immunostained with anti–F4/80 antibody (immunohistochemistry on paraffin sections, brown staining). (e) Primary stromal-vascular cells from 5-month-old mice, immunostained with anti–F4/80 antibody (red staining). (f) Primary stromal-vascular cells from 5-month-old mice stained with oil red O.

Figure 6

The expression of ADAM8, MIP-1α, MCP-1, MAC-1, F4/80, and CD68 in ob/ob WAT treated with rosiglitazone. Twelve-week-old ob/ob mice were treated with either 15 mg/kg rosiglitazone (Rosi) or vehicle (sterile water; Veh) for 28 consecutive days (n = 10 in each group). For comparison, the expression level in rosiglitazone-treated ob/ob mice was arbitrarily set at 1; error bars represent ± SE; *P < 0.05. y axes show arbitrary units representing relative expression levels of mRNAs.

Figure 7

Hypothetical model of chronic inflammation and adipocyte insulin resistance. When adiposity reaches a certain threshold, factors derived from adipocytes induce macrophage activation and infiltration. Activated macrophages secrete cytokines that can impair adipocyte insulin sensitivity and stimulate further activation and infiltration of peripheral monocytes and macrophages into fat. Preadipocytes can also secrete chemokines under the stimulation of TNF-α, which can contribute to macrophage infiltration. These amplifying signals increasingly impair adipocyte insulin signaling and eventually cause systemic insulin resistance.