Role of resistin in diet-induced hepatic insulin resistance

Abstract

Resistin is an adipose-derived hormone postulated to link adiposity to insulin resistance. To determine whether resistin plays a causative role in the development of diet-induced insulin resistance, we lowered circulating resistin levels in mice by use of a specific antisense oligodeoxynucleotide (ASO) directed against resistin mRNA and assessed in vivo insulin action by the insulin-clamp technique. After 3 weeks on a high-fat (HF) diet, mice displayed severe insulin resistance associated with an approximately 80% increase in plasma resistin levels. In particular, the rate of endogenous glucose production (GP) increased more than twofold compared with that in mice fed a standard chow. Treatment with the resistin ASO for 1 week normalized the plasma resistin levels and completely reversed the hepatic insulin resistance. Importantly, in this group of mice, the acute infusion of purified recombinant mouse resistin, designed to acutely elevate the levels of circulating resistin up to those observed in the HF-fed mice, was sufficient to reconstitute hepatic insulin resistance. These results provide strong support for a physiological role of resistin in the development of hepatic insulin resistance in this model.

Figure 1

Treatment with resistin ASO normalized plasma resistin levels in HF-fed mice. (A) Experimental design for treatment with resistin ASO and clamp studies. Mice on HF diet for 3 weeks received an i.p. injection of resistin ASO (RsASO) or control ASO (ConASO) 7 and 3 days before the insulin-clamp studies. Intravenous catheters were inserted into the jugular vein 3 days before the clamp procedure. (B) Plasma insulin levels at the end of insulin clamp. Insulin levels were similar in all experimental groups. (C) Plasma resistin levels. Circulating resistin levels are significantly increased in mice on HF diet (black bar) as compared with mice on SC (white bar). Treatment with resistin ASO significantly decreased circulating resistin levels to those of SC-fed mice. Finally, infusion of recombinant mouse resistin acutely restored circulating resistin levels (HF + RsASO + i.v. Rs) to those observed in the HF-fed mice treated with control ASO. (D) Increased liver TGs with HF diet. Hepatic TG content was increased twofold by HF diet, whereas treatment with resistin ASO or acute infusion of recombinant resistin did not significantly altered hepatic TG levels. *P < 0.01 vs. SC group; #P < 0.01 vs. HF + ConASO; P < 0.01 vs. HF + RsASO. ww, wet weight.

Figure 2

Circulating resistin is required for diet-induced hepatic insulin resistance. (A) Schematic representation of the insulin-clamp procedure. The infusion studies lasted a total of 90 minutes. Mice received a primed-constant infusion of HPLC-purified [3H-3]-glucose (0.1 μCi/min) and insulin (3.6 mU/kg/min) at t = 0 minutes for the duration of the study. A variable infusion of a 10% glucose solution was started and periodically adjusted (glucose as needed) to maintain the plasma glucose concentration at approximately 8 mM for the rest of the study. (B) Rates of glucose uptake (Rd) during the insulin-clamp studies. (C) Rates of endogenous glucose production (GP) during the insulin-clamp studies. (D) Effect of resistin ASO and resistin infusion on total glucose output (in vivo flux-through G6Pase). P < 0.05 vs. SC group; *P < 0.01 vs. SC group; #P < 0.01 vs. HF + ConASO; ##P < 0.01 vs. HF + RsASO.

Figure 3

Effect of resistin antisense and resistin infusion on hepatic glucose fluxes during insulin-clamp studies. (A) Effect of resistin antisense and resistin infusion on the rates of PEP-gluconeogenesis (GNG) and glucose cycling (GC). (B) Effect of resistin antisense and resistin infusion on the rate of glycogenolysis. (C) Effect of resistin antisense and resistin infusion on hepatic G6Pase and PEPCK mRNA. (D) Effect of resistin antisense and resistin infusion on hepatic AMPK phosphorylation (p-AMPK). (E) Effect of recombinant resistin on AMPK phosphorylation in isolated primary hepatocytes. *P < 0.01 vs. SC group; #P < 0.01 vs. HF + ConASO; P < 0.01 vs. HF + RsASO.

Figure 4

Effect of resistin antisense on phosphorylation of hepatic Akt and GSK3. (A) Experimental design for treatment with resistin ASO and acute stimulation with insulin (100 mU). (B) Effect of resistin ASO on phosphorylation of Akt on serine 473 (p-Akt473) and GSK3 (p-GSK3) in liver extracts from HF-fed mice treated with ConASO and RsASO. Unstimulated samples, saline alone, are included as negative controls. *P < 0.05 vs. HF + ConASO group.