Hemodynamic and metabolic actions of Adiponectin in muscle

Insulin resistance is characterised by an impaired ability of insulin to stimulate glucose uptake, especially into skeletal muscle. Insulin has hemodynamic actions, stimulating total blood flow as well as increasing microvascular perfusion. This stimulation of microvascular perfusion may account for up to half of insulin-mediated skeletal muscle glucose uptake and is impaired in insulin resistant states. Adiponectin exhibits a number of actions similar to insulin, including stimulation of glucose uptake in incubated muscle, and reduction of glucose release from hepatocytes. Recent studies have shown that adiponectin is also able to increase nitric oxide (NO) production by the endothelium and relax pre-constricted isolated aortic rings, suggesting that adiponectin may act as a vasodilator. It was hypothesised that adiponectin may have hemodynamic effects similar to insulin, which may be an important aspect of its insulin-sensitising ability by regulating access of insulin and glucose to myocytes. Therefore, there were three major aims of this thesis: i) to examine the acute vascular actions of adiponectin in a simplified but intact vascular system, the pump-perfused rat hindlimb; ii) to investigate whether acute adiponectin infusion alters hemodynamics or glucose homeostasis in the presence and absence of insulin in anaesthetised rats; and iii) to determine whether the vascular actions of adiponectin are altered in insulin resistance. The initial aim of the present study was to produce high purity, physiologically active recombinant adiponectin in sufficient quantity for use in perfused hindlimb and in vivo experiments. Full-length murine adiponectin was expressed in E. coli and purified by affinity chromatography. The adiponectin product was found to be present primarily as a trimer, similar to that reported in previous studies. The constant-flow pump-perfused rat hindlimb was used to evaluate the direct effects of adiponectin on the skeletal muscle vasculature. A physiological concentration of adiponectin (6.5˜í¬¿g.mL-1) alone had no observable vascular activity in this largely dilated system. Adiponectin pre-treatment and co-infusion inhibited the increase in perfusion pressure and associated metabolic stimulation caused by low-dose (1nM) endothelin-1 (ET-1), but not vasoconstriction caused by either high-dose (20nM) ET-1 or 50nM norepinephrine. This action of adiponectin was apparently independent of NO, suggesting a potential novel mechanism of adiponectin action. A high-fat fed rat model was used to determine whether the vascular actions of adiponectin are retained in insulin resistance. The ability of adiponectin to inhibit ET-1-mediated vasoconstriction was not apparent in animals fed a high-fat diet. However, the vasoconstrictor response to ET-1 (1 or 3nM) itself was found to be reduced following highfat feeding. The vasoconstrictor response was restored in the presence of the NO synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester, indicating increased NO bioavailability during hindlimb perfusion in the insulin resistant rats. Use of a specific inhibitor of the inducible NOS (iNOS) isoform demonstrated that this increased NO was derived from induction of iNOS. Systemic vascular and metabolic actions of adiponectin were then examined in anaesthetised rats, alone and during a hyperinsulinemic euglycemic clamp. No differences were observed between vehicle and adiponectin (24 or 96˜í¬¿g.min-1.kg-1) treated rats in macrovascular parameters, microvascular perfusion or glucose metabolism. This finding was consistent whether the rats were fed normal chow or a high-fat diet to induce insulin resistance. This study has identified a novel vascular action of adiponectin to specifically oppose ET-1- mediated vasoconstriction. This effect was not apparent in insulin resistance, possibly implicating loss of adiponectin activity in disease development. Additionally, the response to exogenous ET-1 was reduced in high-fat fed rats, due to upregulation of iNOS. The altered state of balance between ET-1 and NO in insulin resistance may have important implications for endothelial dysfunction. Meanwhile, no effect of acute adiponectin infusion, either hemodynamic or metabolic, was apparent in vivo. Whilst chronic hemodynamic actions of adiponectin may play a role in insulin sensitivity, they do not appear to be a major aspect of acute adiponectin action in vivo.