HSP72 protects against obesity-induced insulin resistance

Abstract

Patients with type 2 diabetes have reduced gene expression of heat shock protein (HSP) 72, which correlates with reduced insulin sensitivity. Heat therapy, which activates HSP72, improves clinical parameters in these patients. Activation of several inflammatory signaling proteins such as c-jun amino terminal kinase (JNK), inhibitor of kappaB kinase, and tumor necrosis factor-alpha, can induce insulin resistance, but HSP 72 can block the induction of these molecules in vitro. Accordingly, we examined whether activation of HSP72 can protect against the development of insulin resistance. First, we show that obese, insulin resistant humans have reduced HSP72 protein expression and increased JNK phosphorylation in skeletal muscle. We next used heat shock therapy, transgenic overexpression, and pharmacologic means to overexpress HSP72 either specifically in skeletal muscle or globally in mice. Herein, we show that regardless of the means used to achieve an elevation in HSP72 protein, protection against diet- or obesity-induced hyperglycemia, hyperinsulinemia, glucose intolerance, and insulin resistance was observed. This protection was tightly associated with the prevention of JNK phosphorylation. These findings identify an essential role for HSP72 in blocking inflammation and preventing insulin resistance in the context of genetic obesity or high-fat feeding.

Fig. 1.

Human obesity and insulin resistance are associated with decreased expression of HSP72 and increased phosphorylation of JNK in skeletal muscle. Representative immunoblots and quantification of HSP72 protein expression (A) and phosphorylation (Thr¹⁸³/Tyr¹⁸⁵)/total JNK in healthy humans and obese, insulin resistant humans (B) (healthy, n = 19; obese IR, n = 23; *, P < 0.05 compared with healthy).

Fig. 2.

Weekly heat therapy prevents JNK phosphorylation in the skeletal muscle of mice, which improves HFD-induced hyperinsulinemia and hyperglycemia. (A) Representative immunoblots of phosphorylation (Thr¹⁸³/Tyr¹⁸⁵)/total JNK in mixed gastrocnemius muscle from wild-type mice subjected to ST or HT. Fasting glucose (B), fasting insulin (C), HOMA-IR (D), and i.p. glucose tolerance (E) from wild type mice placed on a standard chow diet (black bars) or an HFD (gray bars) for 16 weeks while undergoing weekly ST or HT. Experiments were completed at least 72 h after ST or HT. (n = 7–12 mice per group; *, P < 0.05 ST HFD vs. all other conditions.)

Fig. 3.

HSP72 overexpression in skeletal muscle prevents high-fat-feeding-induced glucose intolerance and insulin resistance, JNK phosphorylation, and impaired insulin signaling. (A) Immunoblot showing enhanced HSP72 expression in mixed gastrocnemius muscle but not epididymal white adipose tissue or liver when comparing HSP72 transgenic (TG) with WT mice. I.p. insulin (B) and glucose tolerance (C) and representative immunoblots and quantification of phosphorylation (Thr¹⁸³/Tyr¹⁸⁵)/total JNK in mixed gastrocnemius muscles (D) from WT and HSP72 overexpression (HSP72+/+) mice placed on a standard chow (black bars) or high-fat diet (gray bars) for 16 weeks. (E) Representative immunoblots and quantification of phosphorylation of Akt (Thr³⁰⁸ and Ser⁴⁷³) in mixed gastrocnemius muscle excised 2–5 min after i.p. injection of saline (B) or 1.5 units/kg insulin (I) from WT (black bars) and HSP72+/+ (gray bars) mice after 16 weeks on the HFD. (n = 5–9 mice per group; *, P < 0.05 WT HFD vs. all other conditions for B–D; *, P < 0.05 HSP72+/+ insulin treated vs. all other groups for E.)

Fig. 4.

HSP72 overexpression in skeletal muscle prevents high-fat-feeding-induced increases in body weight and activation of IKK in liver and results in reduced fat pad weight, increased mitochondrial oxidative enzyme activity, and increased circulating adiponectin. Body weight (A), epididymal fat pad weight (B), food intake (C), CS and β-hydroxyacyl-CoA-dehydrogenase (β-HAD) in extensor digitorum longus muscle (D), representative immunoblots and quantification of phosphorylation (Ser¹⁸⁰/Ser¹⁸¹) of inhibitor of κB kinase (IKK)/β-actin in liver (E), and plasma adiponectin (F) from WT and HSP72 overexpression (HSP72+/+) mice placed on a standard chow diet (black bars) or an HFD (gray bars) for 16 weeks. (n = 4–7 mice per group; *, P < 0.05 HFD CON vs. other groups for A and E, *, P < 0.05 HSP72+/+ HFD vs. other groups for D; †, main effect for genotype for B, D, and F; ‡, main effect for diet for C.)

Fig. 5.

BGP-15 activates HSP72 in the skeletal muscles of ob/ob mice, preventing JNK phosphorylation and insulin resistance. Representative immunoblots and quantification of HSP72 (A) and phosphorylation (Thr-183/Tyr-185)/total JNK (B) in quadriceps muscles. Fasting glucose (C), insulin (D), and insulin-stimulated glucose disposal rate (IS-GDR) (E) during a hyperinsulinemic euglycemic clamp. (F) Hepatic glucose production (HGP) without insulin (basal) and after a hyperinsulinemic euglycemic clamp (clamp). All experiments were performed in leptin-deficient (ob/ob) mice treated with saline (black bars; control, 200 μl of saline) or BGP-15 (gray bars; 15 mg/kg per day in 200 μl of saline) for 15 days by oral gavage. (n = 5–7 mice per group; *, P < 0.05 compared with control; *†, P < 0.05 compared with basal for BGP-15-treated group.)