Impaired insulin-mediated muscle glucose uptake in vivo can be the direct result of reduced microvascular blood flow responses to insulin, and can result from small (two-fold) increases in dietary fat. Thus, microvascular insulin-resistance can occur independently to the development of myocyte insulin-resistance.
Insulin resistance plays a key role in the development of type 2 diabetes. Skeletal muscle is the major storage site for glucose following a meal and as such has a key role in maintenance of blood glucose concentrations. Insulin resistance is characterised by impaired insulin-mediated glucose disposal in skeletal muscle. Multiple mechanisms can contribute to development of muscle insulin resistance and our research has demonstrated an important role for loss of microvascular function within skeletal muscle. We have shown that insulin can enhance blood flow to the microvasculature in muscle thus improving the access of glucose and insulin to the myocytes to augment glucose disposal. Obesity, insulin resistance and ageing are all associated with impaired microvascular responses to insulin in skeletal muscle. Impairments in insulin-mediated microvascular perfusion in muscle can directly cause insulin resistance, and this event can occur early in the aetiology of this condition. Understanding the mechanisms involved in the loss of microvascular function in muscle has the potential to identify novel treatment strategies to prevent or delay progression of insulin resistance and type 2 diabetes.
Objective-To investigate the effects of activation of the AMP-activated protein kinase (AMPK) on muscle perfusion and to elucidate the mechanisms involved. Methods and Results-In a combined approach, we studied the vasoactive actions of AMPK activator by 5-aminoimidazole-4-carboxamide-1--D-ribofuranoside (AICAR) on rat cremaster muscle resistance arteries (Ϸ100 m) ex vivo and on microvascular perfusion in the rat hindlimb in vivo. In isolated resistance arteries, AICAR increased Thr172 phosphorylation of AMPK in arteriolar endothelium, which was predominantly located in microvascular endothelium. AICAR induced vasodilation (19Ϯ4% at 2 mmol/L, PϽ0.01), which was abolished by endothelium removal, inhibition of NO synthase (with N-nitro-L-arginine), or AMPK (with compound C). Smooth muscle sensitivity to NO, determined by studying the effects of the NO donor S-nitroso-N-acetylpenicillamine (SNAP), was not affected by AICAR except at the highest dose. AICAR increased endothelial nitric oxide synthase activity, as indicated by Ser1177 phosphorylation. In vivo, infusion of AICAR markedly increased muscle microvascular blood volume (Ϸ60%, PϽ0.05), as was evidenced by contrast-enhanced ultrasound, without effects on blood pressure, femoral blood flow, or hind leg glucose uptake. Conclusion-Activation
Bradley EA, Richards SM, Keske MA, Rattigan S. Local NOS inhibition impairs vascular and metabolic actions of insulin in rat hindleg muscle in vivo. Am J Physiol Endocrinol Metab 305: E745-E750, 2013. First published July 30, 2013 doi:10.1152/ajpendo.00289.2013.-Insulin stimulates microvascular recruitment in skeletal muscle, and this vascular action augments muscle glucose disposal by ϳ40%. The aim of the current study was to determine the contribution of local nitric oxide synthase (NOS) to the vascular actions of insulin in muscle. Hooded Wistar rats were infused with the NOS inhibitor N -nitro-L-arginine methylester (L-NAME, 10 M) retrogradely via the epigastric artery in one leg during a systemic hyperinsulinemiceuglycemic clamp (3 mU·min Ϫ1 ·kg Ϫ1 ϫ 60 min) or saline infusion. Femoral artery blood flow, microvascular blood flow (assessed from 1-methylxanthine metabolism), and muscle glucose uptake (2-deoxyglucose uptake) were measured in both legs. Local L-NAME infusion did not have any systemic actions on blood pressure or heart rate. Local L-NAME blocked insulin-stimulated changes in femoral artery blood flow (84%, P Ͻ 0.05) and microvascular recruitment (98%, P Ͻ 0.05), and partially blocked insulin-mediated glucose uptake in muscle (reduced by 34%, P Ͻ 0.05). L-NAME alone did not have any metabolic effects in the hindleg. We conclude that insulinmediated microvascular recruitment is dependent on local activation of NOS in muscle and that this action is important for insulin's metabolic actions. muscle blood flow; microvascular blood flow; hyperinsulinemiceuglycemic clamp; nitric oxide; nitric oxide synthase INSULIN STIMULATES TOTAL LIMB blood flow and recruits flow to the microvasculature in skeletal muscle (21,31,32,34). These vascular actions occur in both humans (6, 33) and experimental animals (21,31,32,34). We (4), and others (1, 2), have proposed that insulin-mediated vascular responses facilitate insulin and glucose delivery to the myocyte and thereby enhance the rate of glucose disposal. Insulin-mediated increases in skeletal muscle blood flow and microvascular responses are blunted in insulinresistant rats (26, 36) and humans (6, 13), suggesting that reduced muscle perfusion may contribute to the insulin resistance. Recently, we demonstrated that insulin resistance induced by increased dietary fat can originate from impaired microvascular insulin responses and occur before the development of myocyte insulin resistance (18). Therefore, it is important to understand the mechanism by which insulin stimulates microvascular recruitment under healthy circumstances to identify the basis of vascularderived insulin resistance.Studies by Steinberg et al. (27) and Scherrer et al. (23) have demonstrated that local infusion of a nitric oxide synthase (NOS) inhibitor completely abolishes insulin-in-
There is evidence that reactive oxygen species (ROS) contribute to the regulation of skeletal muscle glucose uptake during highly fatiguing ex vivo contraction conditions via AMP-activated protein kinase (AMPK). In this study we investigated the role of ROS in the regulation of glucose uptake and AMPK signaling during low-moderate intensity in situ hindlimb muscle contractions in rats, which is a more physiological protocol and preparation. Male hooded Wistar rats were anesthetized, and then N-acetylcysteine (NAC) was infused into the epigastric artery (125 mg.kg(-1).h(-1)) of one hindlimb (contracted leg) for 15 min before this leg was electrically stimulated (0.1-ms impulse at 2 Hz and 35 V) to contract at a low-moderate intensity for 15 min. The contralateral leg did not receive stimulation or local NAC infusion (rest leg). NAC infusion increased (P<0.05) plasma cysteine and cystine (by approximately 360- and 1.4-fold, respectively) and muscle cysteine (by 1.5-fold, P=0.001). Although contraction did not significantly alter muscle tyrosine nitration, reduced (GSH) or oxidized glutathione (GSSG) content, S-glutathionylation of protein bands at approximately 250 and 150 kDa was increased (P<0.05) approximately 1.7-fold by contraction, and this increase was prevented by NAC. Contraction increased (P<0.05) skeletal muscle glucose uptake 20-fold, AMPK phosphorylation 6-fold, ACCbeta phosphorylation 10-fold, and p38 MAPK phosphorylation 60-fold, and the muscle fatigued by approximately 30% during contraction and NAC infusion had no significant effect on any of these responses. This was despite NAC preventing increases in S-glutathionylation with contraction. In conclusion, unlike during highly fatiguing ex vivo contractions, local NAC infusion during in situ low-moderate intensity hindlimb contractions in rats, a more physiological preparation, does not attenuate increases in skeletal muscle glucose uptake or AMPK signaling.
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