Decreased microvascular vasomotion and myogenic response in rat skeletal muscle in association with acute insulin resistance.

In addition to increased glucose uptake, insulin action is associated with increased total and microvascular blood flow, and vasomotion in skeletal muscle. The aim of this study was to determine the effect of acute insulin resistance caused by the peripheral vasoconstrictor alpha-methylserotonin (alphaMT) on microvascular vasomotion in muscle. Heart rate (HR), mean arterial pressure (MAP), femoral blood flow (FBF), whole body glucose infusion (GIR) and hindleg glucose uptake (HGU) were determined during control and hyperinsulinaemic euglycaemic clamp conditions in anaesthetized rats receiving alphaMT infusion. Changes in muscle microvascular perfusion were measured by laser Doppler flowmetry (LDF) and vasomotion was assessed by applying wavelet analysis to the LDF signal. Insulin increased GIR and HGU. Five frequency bands corresponding to cardiac, respiratory, myogenic, neurogenic and endothelial activities were detected in the LDF signal. Insulin infusion alone increased FBF (1.18 +/- 0.10 to 1.78 +/- 0.12 ml min(-1), P < 0.05), LDF signal strength (by 16% compared to baseline) and the relative amplitude of the myogenic component of vasomotion (0.89 +/- 0.09 to 1.18 +/- 0.06, P < 0.05). When infused alone alphaMT decreased LDF signal strength and the myogenic component of vasomotion by 23% and 27% respectively compared to baseline, but did not affect HGU or FBF. Infusion of alphaMT during the insulin clamp decreased the stimulatory effects of insulin on GIR, HGU, FBF and LDF signal and blocked the myogenic component of vasomotion. These data suggest that insulin action to recruit microvascular flow may in part involve action on the vascular smooth muscle to increase vasomotion in skeletal muscle to thereby enhance perfusion and glucose uptake. These processes are impaired with this model of alphaMT-induced acute insulin resistance.

Contrast-enhanced ultrasound measurement of microvascular perfusion relevant to nutrient and hormone delivery in skeletal muscle: a model study in vitro.

Contrast-enhanced ultrasound (CEU) has been used to measure muscle microvascular perfusion in vivo in response to exercise and insulin. In the present study we address whether CEU measurement of capillary volume is influenced by bulk flow and if measured capillary filling rate allows discrimination of different flow pattern changes within muscle. Three in vitro models were used: (i) bulk flow rate was varied within a single length of capillary tubing; (ii) at constant bulk flow, capillary volume was increased 3-fold by joining lengths of capillary in series, and compared to a single length; and (iii) at constant bulk flow, capillary volume was increased by sharing flow between a number of lengths of identical capillaries in parallel. The contrast medium for CEU was gas-filled albumin microbubbles. Pulsing interval (time) versus acoustic-intensity curves were constructed and from these, capillary volume and capillary filling rate were calculated. CEU estimates of capillary volume were not affected by changes in bulk flow. Furthermore, as CEU estimates of capillary volume increased, measures of capillary filling rate decreased, regardless of whether capillaries were connected in series or parallel. Therefore, CEU can detect a change in filling rate of the microvascular volume under measurement, but it can not be used to discriminate between different flow patterns within muscle that might account for capillary recruitment in vivo.

Insulin and contraction increase nutritive blood flow in rat muscle in vivo determined by microdialysis of L-[14C]glucose.

In the present study, a mathematical model using the microdialysis outflow: inflow (O/I) ratio of the novel analogue L-[14C]glucose has been developed which allows the calculation of the nutritive (and non-nutritive) flow in muscle as a proportion of total blood flow. Anaesthetized rats had microdialysis probes carrying L-[14C]glucose inserted through a calf muscle group (tibialis/plantaris/gastrocnemius). The nutritive fraction of total blood flow was determined under basal conditions and in response to contraction (electrical field stimulation), insulin (hyperinsulinaemic euglycaemic clamp with 10 mU min(-1) kg(-1) insulin) or saline control from limb blood flow and the microdialysis O/I ratio of L-[14C]glucose. Both contraction and insulin infusion decreased the O/I ratio of L-[14C]glucose and increased total limb blood flow. Calculations based on mathematical models using L-[14C]glucose O/I and limb blood flow revealed that during basal conditions, the nutritive fraction of total flow was 0.38 +/- 0.06, indicating that basal flow was predominantly non-nutritive. Contraction and insulin increased the nutritive fraction to 0.82 +/- 0.24 (P < 0.05) and 0.52 +/- 0.12 (P < 0.05). Thus the increase in limb blood flow from insulin was fully accommodated by nutritive flow, while contraction increased nutritive flow at the expense of non-nutritive flow. This novel method using microdialysis and the O/I ratio of L-[14C]glucose allows the determination of the nutritive fraction of total flow in muscle as well as the proportion of total flow that may be redistributed in response to contraction and insulin.

Graded occlusion of perfused rat muscle vasculature decreases insulin action.

Insulin increases capillary recruitment in vivo and impairment of this may contribute to muscle insulin resistance by limiting either insulin or glucose delivery. In the present study, the effect of progressively decreased rat muscle perfusion on insulin action using graded occlusion with MS (microspheres; 15 mum in diameter) was examined. EC (energy charge), PCr/Cr (phosphocreatine/creatine ratio), AMPK (AMP-activated protein kinase) phosphorylation on Thr(172) (P-AMPKalpha/total AMPK), oxygen uptake, nutritive capacity, 2-deoxyglucose uptake, Akt phosphorylation on Ser(473) (P-Akt/total Akt) and muscle 2-deoxyglucose uptake were determined. Arterial injection of MS (0, 9, 15 and 30 x 10(6) MS/15 g of hindlimb muscle, as a bolus) into the pump-perfused (0.5 ml x min(-1) x g(-1) of wet weight) rat hindlimb led to increased pressure (-0.5+/-0.8, 15.9+/-2.1, 28.7+/-4.6 and 60.3+/-9.4 mmHg respectively) with minimal changes in oxygen uptake. Nutritive capacity was decreased from 10.6+/-1.0 to 3.8+/-0.9 micromol x g(-1) of muscle x h(-1) (P<0.05) with 30 x 10(6) MS. EC was unchanged, but PCr/Cr was decreased dose-dependently to 61% of basal with 30 x 10(6) MS. Insulin-mediated increases in P-Akt/total Akt decreased from 2.15+/-0.35 to 1.41+/-0.23 (P<0.05) and muscle 2-deoxyglucose uptake decreased from 130+/-19 to 80+/-12 microg x min(-1) x g(-1) of dry weight (P<0.05) with 15 x 10(6) MS; basal P-AMPKalpha in the absence of insulin was increased, but basal P-Akt/total Akt and muscle 2-deoxyglucose uptake were unaffected. In conclusion, partial occlusion of the hindlimb muscle has no effect on basal glucose uptake and marginally impacts on oxygen uptake, but markedly impairs insulin delivery to muscle and, thus, insulin-mediated Akt phosphorylation and glucose uptake.

Microvascular flow routes in muscle controlled by vasoconstrictors.

Vasoconstrictors can either increase or decrease metabolism of the constant flow pump-perfused rat hindlimb. In addition, there is indirect evidence from vascular casts, surface fluorometry, dye entrapment studies, and fluorescent microsphere mapping of flow that this may be due to redistribution of flow between putatively nutritive and non-nutritive routes within muscle. In the present study, we used two methods in an attempt to identify perfused nutritive and non-nutritive vessels in muscle sections: (i) a combination of perfusion fixation with glutaraldehyde and post-perfusion Griffonia simplicifolia lectin and (ii) perfusion with rhodamine-dextran70 (lysine fixable) and post-fixation with formaldehyde. Perfusions involved vehicle only (control, a mix of nutritive and non-nutritive flow), 15 nM angiotensin II (AII) to increase, or 1 microM serotonin (5-HT) to decrease nutritive flow. Microscopic examination of muscle sections following AII showed an increase in perfused capillaries with fewer areas of under-perfusion, relative to control. In contrast, 5-HT caused a marked decrease in perfused capillaries relative to control and evidence that flow was carried by connective tissue vessels that on average were of greater diameter and were more sparsely distributed than capillaries. It is concluded that vasoconstrictors that alter hindlimb metabolism do so by intra-muscle redistribution between capillaries (nutritive) and non-nutritive (connective tissue) vessels within each muscle.

Axially symmetric semi-infinite domain models of microdialysis and their application to the determination of nutritive flow in rat muscle.

Theoretical models for the description of microdialysis outflow:inflow (O/I) ratio for 3H2O and [14C]ethanol were developed, taking into account the nutritive fraction of total blood flow in muscle. The models yielded an approximately exponential decay expression for the O/I ratio, dependent on the physical dimensions of a linear probe (length and radius), the flow rate through the probe, muscle blood flow (including the nutritive fraction) and the diffusion coefficients for the tracer in the probe and muscle. The models compared favourably with experimental data from the constant-flow perfused rat hindlimb. Estimates of the nutritive fraction of total blood flow from experimental data were determined by minimizing the error between model and experimental data. The nutritive fraction was found to be 0.22 +/- 0.04 under basal perfusion conditions. When 70 nM noradrenaline (norepinephrine) was included in the perfusion medium, the nutritive fraction was 0.91 +/- 0.06 (P < 0.05). The inclusion of 300 nM serotonin, decreased the nutritive fraction to 0.05 +/- 0.01 (P < 0.05). This model can be applied to the determination of nutritive fraction of skeletal muscle blood flow in physiologically relevant microvascular conditions such as during exercise and in disease states.

Nutritive blood flow improves interstitial glucose and lactate exchange in perfused rat hindlimb.

Microdialysis was used to assess the interstitial concentrations of glucose and lactate in the constant-flow-perfused rat hindlimb under varying levels of nutritive flow controlled by vasoconstrictors. Increased nutritive flow was achieved by norepinephrine (NE) or angiotensin II (ANG II) and decreased nutritive flow by serotonin (5-HT). NE and ANG II increased oxygen and glucose uptake as well as hindlimb lactate release by 50%. 5-HT decreased oxygen uptake by 15% but had no significant effect on glucose uptake or hindlimb lactate release. Microdialysis recovery of glucose and lactate was significantly elevated by NE and ANG II and decreased by 5-HT. The calculated interstitial concentration of glucose was increased by NE and ANG II but decreased by 5-HT. The interstitial concentration of lactate was decreased by NE and ANG II but increased by 5-HT. In all cases, nitroprusside reversed the effects of the vasoconstrictors. These data indicate that increased nutritive blood flow enhances the exchange of glucose and lactate by improving the supply of glucose to and the removal of lactate from the interstitium.