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. 2009 Nov;58(11):2525-35.
doi: 10.2337/db08-1261. Epub 2009 Aug 18.

Interleukin-10 prevents diet-induced insulin resistance by attenuating macrophage and cytokine response in skeletal muscle

Eun-Gyoung Hong  1 , Hwi Jin Ko, You-Ree Cho, Hyo-Jeong Kim, Zhexi Ma, Tim Y Yu, Randall H Friedline, Evelyn Kurt-Jones, Robert Finberg, Matthew A Fischer, Erica L Granger, Christopher C Norbury, Stephen D Hauschka, William M Philbrick, Chun-Geun Lee, Jack A Elias, Jason K Kim
Affiliations

Interleukin-10 prevents diet-induced insulin resistance by attenuating macrophage and cytokine response in skeletal muscle

Eun-Gyoung Hong et al. Diabetes. 2009 Nov.

Abstract

Objective: Insulin resistance is a major characteristic of type 2 diabetes and is causally associated with obesity. Inflammation plays an important role in obesity-associated insulin resistance, but the underlying mechanism remains unclear. Interleukin (IL)-10 is an anti-inflammatory cytokine with lower circulating levels in obese subjects, and acute treatment with IL-10 prevents lipid-induced insulin resistance. We examined the role of IL-10 in glucose homeostasis using transgenic mice with muscle-specific overexpression of IL-10 (MCK-IL10).

Research design and methods: MCK-IL10 and wild-type mice were fed a high-fat diet (HFD) for 3 weeks, and insulin sensitivity was determined using hyperinsulinemic-euglycemic clamps in conscious mice. Biochemical and molecular analyses were performed in muscle to assess glucose metabolism, insulin signaling, and inflammatory responses.

Results: MCK-IL10 mice developed with no obvious anomaly and showed increased whole-body insulin sensitivity. After 3 weeks of HFD, MCK-IL10 mice developed comparable obesity to wild-type littermates but remained insulin sensitive in skeletal muscle. This was mostly due to significant increases in glucose metabolism, insulin receptor substrate-1, and Akt activity in muscle. HFD increased macrophage-specific CD68 and F4/80 levels in wild-type muscle that was associated with marked increases in tumor necrosis factor-alpha, IL-6, and C-C motif chemokine receptor-2 levels. In contrast, MCK-IL10 mice were protected from diet-induced inflammatory response in muscle.

Conclusions: These results demonstrate that IL-10 increases insulin sensitivity and protects skeletal muscle from obesity-associated macrophage infiltration, increases in inflammatory cytokines, and their deleterious effects on insulin signaling and glucose metabolism. Our findings provide novel insights into the role of anti-inflammatory cytokine in the treatment of type 2 diabetes.

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Figures

FIG. 1.
FIG. 1.
IL-10 treatment protects against diet-induced insulin resistance in C57BL/6 mice. Mice were fed standard diet or HFD for 3 weeks, and IL-10 or saline (control) was treated for the last 3 days of diet period. A: Muscle IL-10 protein levels in standard diet– and HFD-fed C57BL/6 mice and in obese RCS10 mice. B: Plasma IL-10 levels following 3 days of chronic IL-10 delivery using osmotic pumps. C: IL-10 protein expression in gastrocnemius (GAS) and quadriceps (QD) muscle in IL-10–treated and control mice. □, Control; ■, IL-10 treated. D: Local IL-10 protein levels in heart, liver, and white adipose tissue (WAT) in IL-10–treated and control mice. □, Control; ■, IL-10 treated. E: Whole-body fat mass, measured using 1H-MRS, in standard diet– and HFD-fed mice following IL-10 or saline treatment. □, Control; ■, IL-10 treated. F: Glucose infusion rates during hyperinsulinemic-euglycemic clamps. G: Insulin-stimulated glucose uptake in skeletal muscle. H: Insulin-stimulated threonine phosphorylation of Akt and Akt protein levels in muscle. Muscle samples were obtained 15 min after insulin injection. I: Hepatic insulin action expressed as insulin-mediated percent suppression of basal hepatic glucose production (HGP). Values are means ± SE for five to eight mice in each experiment. *P < 0.05 vs. standard diet–fed mice or control mice without IL-10 treatment (A–D); *P < 0.05 vs. respective standard diet–fed mice (E–I). A Student's t test was used in A–D, and a two-way ANOVA was used in E–I for statistical analysis.
FIG. 2.
FIG. 2.
Transgenic mice with muscle-specific overexpression of IL-10 (MCK-IL10). A: IL-10R1 protein expression in C2C12 myoblast and myotube cells. B: Map of MCK-IL10 construct. C: Photographs of MCK-IL10 mice and WT littermates. D: Body weight. E: Whole body lean and fat mass. F: Hematoxylin-eosin staining of gastrocnemius (GAS) and quadriceps (QD) muscle. G: Muscle IL-10 protein levels. H: Plasma and local IL-10 protein levels in heart, liver, and white adipose tissue (WAT). I: STAT3 activity (Tyr705phosphorylation of STAT3) in skeletal muscle. Values are means ± SE for six to eight mice in each group. *P < 0.05 vs. WT mice. A Student's t test was used for statistical analysis. (A high-quality color digital representation of this figure is available in the online issue.)
FIG. 3.
FIG. 3.
MCK-IL10 mice are more insulin sensitive and are protected against diet-induced insulin resistance in skeletal muscle. MCK-IL10 and WT mice were fed HFD for 3 weeks, and hyperinsulinemic-euglycemic clamps were performed in conscious mice to measure insulin action. Energy balance was measured for 3 days using metabolic cages. A: Daily food intake in standard diet– and HFD-fed mice. □, WT; ■, MCK-IL10. B: Vo2 consumption. C: Physical activity. D: Whole-body fat mass. E: Basal (overnight-fasted) plasma glucose levels. F: Basal plasma insulin levels. G: Glucose infusion rates during hyperinsulinemic-euglycemic clamps. H: Insulin-stimulated whole-body glucose turnover. I: Insulin-stimulated glucose uptake in skeletal muscle. Values are means ± SE for six to eight mice in each experimental group. *P < 0.05 vs. WT mice fed respective diet. A two-way ANOVA was used for statistical analysis.
FIG. 4.
FIG. 4.
Muscle insulin signaling and hepatic insulin action in MCK-IL10 and WT mice fed standard or HFD for 3 weeks. A: Insulin-stimulated tyrosine phosphorylation of IRS-1 and IRS-1 protein levels in skeletal muscle (gastrocnemius). B: Hepatic insulin action. Values are means ± SE for four to six mice in each experimental group. *P < 0.05 vs. WT mice fed standard or HFD. A two-way ANOVA was used for statistical analysis.
FIG. 5.
FIG. 5.
HFD increases macrophage levels in WT skeletal muscle, and diet-induced inflammatory response in muscle is attenuated in MCK-IL10 mice. A: Macrophage-specific CD68 levels in skeletal muscle. B: Immunohistochemistry analysis using anti-CD68 and anti-F4/80 (arrows) in skeletal muscle. C: Skeletal muscle F4/80 levels in immunofluorescence staining. *P < 0.05 vs. WT mice fed a standard diet (A–C). D: Average number of macrophages isolated from muscle samples of standard diet– or HFD-fed WT and MCK-IL10 mice. *P < 0.05 vs. HFD-fed WT mice. E: Representative cytometric data showing the frequency of macrophages (CD11b+F4/80+) in each group. Values are means ± SE for five to seven mice in each experimental group. A two-way ANOVA was used for statistical analysis. (A high-quality color digital representation of this figure is available in the online issue.)
FIG. 6.
FIG. 6.
Immunofluorescence analysis using anti-F4/80 (green labeling) and nuclei labeling with DAPI (blue labeling) in skeletal muscle in WT and MCK-IL10 mice following standard diet or HFD for 3 weeks. (A high-quality color digital representation of this figure is available in the online issue.)
FIG. 7.
FIG. 7.
Inflammatory response in WT and MCK-IL10 mice following HFD. A: CCR2 protein expression in skeletal muscle. B: Phospho-JNK1 normalized to JNK1 protein levels in skeletal muscle. *P < 0.05 vs. WT mice fed respective diet. Values are means ± SE for three to five samples in each experimental group. A two-way ANOVA was used for statistical analysis.
FIG. 8.
FIG. 8.
Serum and tissue cytokine levels in WT and MCK-IL10 mice following standard diet or HFD for 3 weeks. A: Muscle IL-6 levels. B: Plasma IL-6 levels. C: Muscle TNF-α levels. D: Plasma TNF-α levels. E: Muscle IL-10 levels. F: Liver IL-10 levels. G: IL-10 levels in white adipose tissue (WAT). H: Intramuscular triglyceride levels in C57BL/6 mice following IL-10 or saline (control) treatment. I: Intramuscular triglyceride levels in MCK-IL10 and WT mice following standard diet or HFD. *P < 0.05 vs. standard diet. Values are means ± SE for four to seven mice in each experimental group. A two-way ANOVA was used for statistical analysis in all figures except H (Student's t test).
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