Stephen Rattigan

Hemodynamic Actions of Insulin in Rat Skeletal Muscle: Evidence for Capillary Recruitment

Insulin-induced increases in blood flow are hypothesized to enhance overall glucose uptake by skeletal muscle. Whether the insulin-mediated changes in blood flow are associated with altered blood flow distribution and increased capillary recruitment in skeletal muscle is not known. In the present study, the effects of insulin on hemodynamic parameters in rat skeletal muscle in vivo were investigated. Mean arterial blood pressure, heart rate, femoral blood flow, hind leg vascular resistance, and glucose uptake were measured in control and euglycemic insulin-clamped (10 mU · min−1 · kg−1) anesthetized rats. Blood flow distribution within the hind leg muscles was assessed by measuring the metabolism of 1-methylxanthine (1-MX), an exogenously added substrate for capillary xanthine oxidase. Insulin treatment had no effect on heart rate but significantly increased arterial blood pressure (12 mmHg) and femoral blood flow (80%) and decreased hind leg vascular resistance (31%). Changes were similar in magnitude and in time of onset to those reported in humans. Insulin treatment increased hind leg glucose uptake approximately fourfold and also increased hind leg 1-MX metabolism by 50%, suggesting increased exposure to endothelial xanthine oxidase. To ascertain whether the increased 1-MX metabolism was simply due to increased bulk femoral blood flow, epinephrine was infused at a dose (0.125 pg · min−1 · kg−1) chosen to match the insulin-induced increase in femoral blood flow. This dose of epinephrine had no significant effects on arterial blood pressure or heart rate but increased femoral blood flow and lowered hind leg vascular resistance to a similar extent as insulin. Epinephrine did not significantly alter 1-MX metabolism as compared with control animals. These results demonstrate that insulin increases total hind leg blood flow and metabolism of 1-MX, suggesting a recruitment of capillary blood flow in rat hind leg not mimicked by epinephrine.

Contraction‐associated translocation of protein kinase C in rat skeletal muscle

Electrical stimulation of the sciatic nerve of the anaesthetized rat in vivo led to a time‐dependent translocation of protein kinase C from the muscle cytosol to the particulate fraction. Maximum activity of protein kinase C in the particulate fraction occurred after 2 min of intermittent short tetanic contractions of the gastrocnemius‐plantaris‐soleus muscle group and coincided with the loss of activity from the cytosol. Translocation of protein kinase C may imply a role for this kinase in contraction‐initiated changes in muscle metabolism.

The vasodilatory actions of insulin on resistance and terminal arterioles and their impact on muscle glucose uptake

Whether a discrete vascular action of insulin in skeletal muscle integrally participates in insulin‐mediated glucose disposal has been extensively examined but remains a contentious issue. Here, we review some of the data both supporting and questioning the role of insulin‐mediated increases in limb blood flow in glucose metabolism. We advance the hypothesis that controversy has arisen, at least in part, from a failure to recognize that insulin exerts at least three separate actions on the peripheral vasculature, each with its own characteristic dose and time responsiveness. We summarize how, viewed in this manner, certain points of contention can be resolved. We also advance the hypothesis that an action on the precapillary arteriole may play the dominant role in mediating perfusion‐dependent effects of insulin on glucose metabolism in muscle. Copyright

Vasopressin and angiotensin II stimulate oxygen uptake in the perfused rat hindlimb

Vasopressin and angiotensin II markedly stimulated oxygen uptake in the perfused rat hindlimb. The increase due to each agent approached 70% of the basal rate, and was greater than that produced by a maximal concentration of norepinephrine. Half-maximal stimulation occurred at 60 pM vasopressin, 0.5 nM angiotensin II and 10 nM norepinephrine. Angiotensins I and III were less potent than angiotensin II. For each agent, the dose-dependent increase in oxygen uptake coincided with a dose-dependent increase in perfusion pressure. The effects of both vasopressin and angiotensin to increase oxygen uptake and pressure were not inhibited by either phentolamine, propranolol or a combination of the two, but were completely inhibited by the vasodilator, nitroprusside. Nitroprusside also inhibited flow-induced increases in hindlimb oxygen uptake and perfusion pressure. The findings indicate a key role for the vascular system in the control of hindlimb oxygen uptake.

Local Nitric Oxide Synthase Inhibition Reduces Skeletal Muscle Glucose Uptake but Not Capillary Blood Flow During In Situ Muscle Contraction in Rats

OBJECTIVE—We have previously shown in humans that local infusion of a nitric oxide synthase (NOS) inhibitor into the femoral artery attenuates the increase in leg glucose uptake during exercise without influencing total leg blood flow. However, rodent studies examining the effect of NOS inhibition on contraction-stimulated skeletal muscle glucose uptake have yielded contradictory results. This study examined the effect of local infusion of an NOS inhibitor on skeletal muscle glucose uptake (2-deoxyglucose) and capillary blood flow (contrast-enhanced ultrasound) during in situ contractions in rats. RESEARCH DESIGN AND METHODS—Male hooded Wistar rats were anesthetized and one hindleg electrically stimulated to contract (2 Hz, 0.1 ms) for 30 min while the other leg rested. After 10 min, the NOS inhibitor NG-nitro-l-arginine methyl ester (l-NAME) (arterial concentration of 5 μmol/l) or saline was infused into the epigastric artery of the contracting leg. RESULTS—Local NOS inhibition had no effect on blood pressure, heart rate, or muscle contraction force. Contractions increased (P < 0.05) skeletal muscle NOS activity, and this was prevented by l-NAME infusion. NOS inhibition caused a modest significant (P < 0.05) attenuation of the increase in femoral blood flow during contractions, but importantly there was no effect on capillary recruitment. NOS inhibition attenuated (P < 0.05) the increase in contraction-stimulated skeletal muscle glucose uptake by ∼35%, without affecting AMP-activated protein kinase (AMPK) activation. CONCLUSIONS—NOS inhibition attenuated increases in skeletal muscle glucose uptake during contraction without influencing capillary recruitment, suggesting that NO is critical for part of the normal increase in skeletal muscle fiber glucose uptake during contraction.

Active role for the vasculature in the delivery of insulin to skeletal muscle

1. In the 80+ years since insulins discovery, an enormous amount of literature has accumulated relating to its actions on body fat, glucose and protein metabolism. In particular, skeletal muscle has been extensively studied because of its major role as a site of insulin‐mediated glucose disposal. Liver and adipose tissue are two other extensively studied sites of insulin action. Much less investigation has been directed towards delineating insulins actions on cells other than myocytes, adipocytes and hepatocytes.

Insulin-like action of catecholamines and Ca2+ to stimulate glucose transport and GLUT4 translocation in perfused rat heart

The uptake of 2-deoxyglucose by perfused rat hearts was compared to the distribution of the insulin-regulatable glucose transporter (GLUT4) in membrane preparations from the same hearts. The hearts were treated with the alpha-adrenergic combination of epinephrine + propranolol, the beta-adrenergic agonist isoproterenol, high (8 mM) Ca2+ concentrations, insulin and the alpha adrenergic combination or insulin alone. Epinephrine (1 microM) + propranolol (10 microM), isoproterenol (10 microM), high Ca2+, insulin (1 microM) + epinephrine (1 microM) + propranolol (10 microM) and insulin (1 microM) each led to an increase in 2-deoxyglucose uptake and a shift in the recovery of the GLUT4 from a high-speed pellet membrane fraction (putatively intracellular) to a low-speed pellet membrane fraction (putatively sarcolemmal). There were significant correlations (r = -0.673, P less than 0.001) between the stimulation of 2-deoxyglucose uptake and the loss of GLUT4 from the intracellular membrane fraction, or the increase in the sarcolemmal fraction. The data provide evidence that the GLUT4 is translocated by agents that stimulate glucose transport in heart, and therefore this mechanism is not restricted to insulin.

Insulin‐Induced Microvascular Recruitment in Skin and Muscle are Related and Both are Associated with Whole‐Body Glucose Uptake

Please cite this paper as: Meijer RI, de Boer MP, Groen MR, Eringa EC, Rattigan S, Barrett EJ, Smulders YM, Serne EH. Insulin‐induced microvascular recruitment in skin and muscle are related and both are associated with whole‐body glucose uptake. Microcirculation 19: 494–500, 2012.

Activation of AMP-Activated Protein Kinase by 5-Aminoimidazole-4-Carboxamide-1-β-d-Ribofuranoside in the Muscle Microcirculation Increases Nitric Oxide Synthesis and Microvascular Perfusion

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-&bgr;-d-ribofuranoside (AICAR) on rat cremaster muscle resistance arteries (≈100 &mgr;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 (19±4% at 2 mmol/L, P<0.01), 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 (≈60%, P<0.05), as was evidenced by contrast-enhanced ultrasound, without effects on blood pressure, femoral blood flow, or hind leg glucose uptake. Conclusion—Activation of AMPK by AICAR activates endothelial nitric oxide synthase in arteriolar endothelium by increasing its Ser1177 phosphorylation, which leads to vasodilation of resistance arteries and recruitment of microvascular perfusion in muscle.

A new method to study changes in microvascular blood volume in muscle and adipose tissue: real-time imaging in humans and rat

We employed and evaluated a new application of contrast-enhanced ultrasound for real-time imaging of changes in microvascular blood volume (MBV) in tissues in females, males, and rat. Continuous real-time imaging was performed using contrast-enhanced ultrasound to quantify infused gas-filled microbubbles in the microcirculation. It was necessary to infuse microbubbles for a minimum of 5-7 min to obtain steady-state bubble concentration, a prerequisite for making comparisons between different physiological states. Insulin clamped at a submaximal concentration (∼75 μU/ml) increased MBV by 27 and 39% in females and males, respectively, and by 30% in female subcutaneous adipose tissue. There was no difference in the ability of insulin to increase muscle MBV in females and males, and microvascular perfusion rate was not increased significantly by insulin. However, perfusion rate of the microvascular space was higher in females compared with males. In rats, insulin clamped at a maximal concentration increased muscle MBV by 60%. Large increases in microvascular volume and perfusion rate were detected during electrical stimulation of muscle in rats and immediately after exercise in humans. We have demonstrated that real-time imaging of changes in MBV is possible in human and rat muscle and in subcutaneous adipose tissue and that the method is sensitive enough to pick up relatively small changes in MBV when performed with due consideration of steady-state microbubble concentration. Because of real-time imaging, the method has wide applications for determining MBV in different organs during various physiological or pathophysiological conditions.