In this article, we show that transgenic mice with muscle-specific overexpression of IL-10 were protected from diet-induced insulin resistance in skeletal muscle, and this was associated with increased IRS-1 and Akt activities and reduced levels of macrophages and cytokines in skeletal muscle. These results are consistent with our observations that IL-10 treatment in normal mice prevents lipid-mediated insulin resistance in skeletal muscle. Taken together, our findings indicate that IL-10 is a positive regulator of muscle insulin sensitivity and that IL-10 prevents muscle insulin resistance by attenuating an obesity-associated macrophage and cytokine response in muscle.
Increasing evidence indicates the role of chronic low-grade inflammation and macrophage activation in insulin resistance (9
). A cohort of recent studies have demonstrated increases in macrophage infiltration and cytokine expression in adipose tissue and their association with insulin resistance in obese humans and animal models (40
). Obesity-induced macrophage infiltration is associated with increased adipocyte expression of MCP-1 and macrophage inflammatory protein-1α (37
). Additionally, mice with adipocyte-specific overexpression of MCP-1 (ap2-MCP-1) develop insulin resistance associated with increased macrophage infiltration in adipose tissue (44
). CCR2 binds to MCP-1 and regulates macrophage recruitment, and CCR2 knockout mice show increased insulin sensitivity with reduced macrophage level in adipose tissue (38
). Although these findings support the role of adipose infiltration of macrophages in insulin resistance, it remains unclear whether macrophage infiltration is a cause or consequence of insulin resistance. In this regard, macrophage infiltration in obese mice (e.g., leptin-deficient ob/ob
mice) may be triggered by adipocyte hypertrophy and apoptosis, which result from insulin resistance (46
). It is also unclear whether macrophages infiltrate into skeletal muscle in obesity and whether this directly contributes to muscle insulin resistance, an important primary event in the development of type 2 diabetes.
Here, we report for the first time that a short-term (3 weeks) feeding of HFD increased macrophage infiltration in skeletal muscle that was associated with increased muscle expression of CCR2. We also found that macrophage levels in adipose tissue were elevated after 3 weeks of HFD in wild-type mice. Since 3 weeks of HFD is the earliest inducible time point of diet-induced insulin resistance (29
), these results support the notion that macrophage infiltration is causally associated with obesity-associated insulin resistance in skeletal muscle. Diet-induced macrophage infiltration in muscle was associated with increased muscle expression of TNF-α and IL-6. TNF-α is a proinflammatory cytokine that is actively secreted by macrophages and adipocytes and shown to cause insulin resistance by downregulating AMP-activated protein kinase (12
). Furthermore, IL-6 is a proinflammatory cytokine that is elevated in obese, diabetic subjects and shown to cause insulin resistance by activating STAT3-SOCS3 expression and inhibiting the insulin signaling pathway in liver (11
). Thus, our findings indicate that diet-induced insulin resistance is in part due to increases in macrophage infiltration and local levels of TNF-α and IL-6 in skeletal muscle and subsequent deleterious effects of these cytokines on the muscle insulin signaling. Although recent studies have argued the role of IL-6 in insulin resistance, more studies are clearly needed to determine the metabolic role of IL-6 in diabetic skeletal muscle (50
). We should also point out that these results do not argue against the role of adipose tissue macrophages and inflammation in obesity-induced insulin resistance. Instead, our report implicates that local inflammation in skeletal muscle may be an additional mechanism by which modest obesity affects muscle insulin action.
The protective effects of IL-10 against diet-induced defects in muscle insulin signaling and glucose metabolism were associated with reductions in macrophage levels and local cytokine expressions in skeletal muscle of HFD-fed MCK-IL10 mice. Muscle IL-10 expression also blunted diet-induced increases in CCR2 protein expression in muscle. These results demonstrate that the anti-inflammatory action of IL-10 prevents the obesity-induced CCR2 expression and macrophage infiltration in skeletal muscle, which attenuates the deleterious effects of local cytokines on muscle insulin signaling and glucose metabolism. Taken together, our findings indicate a causative role of macrophage infiltration and local cytokines on obesity-associated insulin resistance, and they support a role of IL-10 in the regulation of glucose homeostasis. However, our findings do not rule out the role of local lipids or lipid metabolites in muscle insulin resistance following chronic high-fat feeding.
The ability of IL-10 secreted in skeletal muscle to attenuate several of the pathogenic insulin-signaling phenotypes associated with type 2 diabetes is interesting with respect to the design of future therapeutic treatments for this disease. While potential problems associated with chronically elevated levels of IL-10 would require further investigation, the relatively low levels of IL-10 (30% in plasma and 100% in skeletal muscle) tested in the present study did not appear deleterious to the mice. Numerous alternative versions of MCK-based regulatory gene cassettes have been designed and tested in mice in conjunction with both systemic and intramuscular delivery with adeno-associated virus vectors (52
). Since these cassettes are striated muscle-specific and have a range of transcriptional activities, it should, in principle, be feasible to design optimal combinations of MCK-IL10 gene expression constructs, adeno-associated virus titers, and vector delivery routes that would lead to the secretion of sufficient intramuscular or systemic levels of IL-10 to possibly improve insulin sensitivity in type 2 diabetes.