John M. B. Newman

Contribution of mitochondrial proton leak to respiration rate in working skeletal muscle and liver and to SMR

Proton pumping across the mitochondrial inner membrane and proton leak back through the natural proton conductance pathway make up a futile cycle that dissipates redox energy. We measured respiration and average mitochondrial membrane potential in perfused rat hindquarter with maximal tetanic contraction of the left gastrocnemius-soleus-plantaris muscle group, and we estimate that the mitochondrial proton cycle accounted for 34% of the respiration rate of the preparation. Similar measurements in rat hepatocytes given substrates to cause a high rate of gluconeogenesis and ureagenesis showed that the proton cycle accounted for 22% of the respiration rate of these cells. Combining these in vitro values with literature values for the contribution of skeletal muscle and liver to standard metabolic rate (SMR), we calculate that the proton cycle in working muscle and liver may account for 15% of SMR in vivo. Although this value is less than the 20% of SMR we calculated previously using data from resting skeletal muscle and hepatocytes, it is still large, and we conclude that the futile proton cycle is a major contributor to SMR.Proton pumping across the mitochondrial inner membrane and proton leak back through the natural proton conductance pathway make up a futile cycle that dissipates redox energy. We measured respiration and average mitochondrial membrane potential in perfused rat hindquarter with maximal tetanic contraction of the left gastrocnemius-soleus-plantaris muscle group, and we estimate that the mitochondrial proton cycle accounted for 34% of the respiration rate of the preparation. Similar measurements in rat hepatocytes given substrates to cause a high rate of gluconeogenesis and ureagenesis showed that the proton cycle accounted for 22% of the respiration rate of these cells. Combining these in vitro values with literature values for the contribution of skeletal muscle and liver to standard metabolic rate (SMR), we calculate that the proton cycle in working muscle and liver may account for 15% of SMR in vivo. Although this value is less than the 20% of SMR we calculated previously using data from resting skeletal muscle and hepatocytes, it is still large, and we conclude that the futile proton cycle is a major contributor to SMR.

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 α‐methylserotonin (αMT) 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 αMT 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 αMT 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 αMT 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 αMT‐induced acute insulin resistance.

Insulin-Induced Changes in Microvascular Vasomotion and Capillary Recruitment are Associated in Humans

Insulin‐induced capillary recruitment is considered a significant regulator of overall insulin‐stimulated glucose uptake. Insulins action to recruit capillaries has been hypothesized to involve insulin‐induced changes in vasomotion. Data directly linking vasomotion to capillary perfusion, however, are presently lacking. We, therefore, investigated whether insulins actions on capillary recruitment and vasomotion were interrelated in a group of healthy individuals. We further assessed the role of capillary recruitment in the association between vasomotion and insulin‐mediated glucose uptake.

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.

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.

Relationship of MTT reduction to stimulants of muscle metabolism.

MTT, a positively charged tetrazolium salt, is widely used as an indicator of cell viability and metabolism and has potential for histochemical identification of tissue regions of hypermetabolism. In the present study, MTT was infused in the constant-flow perfused rat hindlimb to assess the effect of various agents and particularly vasoconstrictors that increase muscle metabolism. Reduction of MTT to the insoluble formazan in muscles assessed at the end of experiments was linear over a 30 min period and production rates were greater in red fibre types than white fibre types. The vasoconstrictors, norepinephrine (100 nM) and angiotensin (10 nM) decreased MTT formazan production in all muscles but increased hindlimb oxygen uptake and lactate efflux. Veratridine, a Na(+) channel opener that increases hindlimb oxygen uptake and lactate efflux without increases in perfusion pressure, also decreased MTT formazan production. Membrane stabilizing doses (100 microM) of (+/-)-propranolol reversed the inhibitory effects of angiotensin and veratridine on MTT formazan production. Muscle contractions elicited by stimulation of the sciatic nerve, reversed the norepinephrine-mediated inhibitory effects on MTT formazan production, even though oxygen consumption and lactate efflux were further stimulated. Stimulation of hindlimb muscle oxygen uptake by pentachlorophenol, a mitochondrial uncoupler, was not associated with alterations in MTT formazan production. It is concluded that apart from muscle contractions MTT formazan production does not increase with increased muscle metabolism. Since the vasoconstrictors angiotensin and norepinephrine as well as veratridine activate Na(+) channels and the Na(+)/K(+) pump, energy required for Na(+) pumping may be required for MTT reduction. It is unlikely that vasoconstrictors that stimulate oxygen uptake do so by uncoupling respiration.

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.

Reply from J. Newman, R. Dwyer, P. St-Pierre, S. Richards, M. Clark and S. Rattigan

Thank you for allowing us a reply to the letter by Wiernsperger and Bouskela regarding our recently published paper (Newman et al. 2009). We also thank Wiernsperger and Bouskela for their interest in our paper and apologise for the apparent misunderstanding of the statements referred to in their letter. Our statements regarding determining vasomotion in skeletal muscle were made within the context of the laser Doppler flowmetry (LDF) technique and in particular the spectral analysis of LDF signals for investigating flowmotion (and vasomotion).