Succinate recovers mitochondrial oxygen consumption in septic rat skeletal muscle

OBJECTIVE: Mitochondrial dysfunction, particularly affecting complex I of the respiratory chain, could play a fundamental role in the development of multiple organ failure during sepsis. Increasing electron flow through complex II by addition of succinate may improve mitochondrial oxygen utilization and thus adenosine triphosphate production. DESIGN: Ex vivo animal study. SETTING: University research laboratory. SUBJECTS: Male adult Wistar rats. INTERVENTIONS: Fecal peritonitis was induced in conscious, fluid-resuscitated, hemodynamically-monitored rats. Sham-operation and na?ve animals acted as controls. At 48 hrs, clinical severity was graded. Soleus muscle was taken for measurement of mitochondrial complex activities and oxygen consumption. The effect of glutamate plus malate (complex I substrates) and succinate (complex II substrate) on mitochondrial respiration was assessed. MEASUREMENTS AND MAIN RESULTS: In the presence of glutamate plus malate, mitochondrial oxygen consumption was abnormally low in skeletal muscle tissue from moderately-to-severely septic animals as compared with na?ve and sham-operation controls (both p < .01). On addition of succinate, mitochondrial respiration was augmented in all groups, particularly in moderately-to-severely septic animals (39% +/- 6% increase) as compared with na?ve (11% +/- 5%; p < .01) and sham-operation controls (10% +/- 5%; p < .01). In the presence of succinate, mitochondrial oxygen consumption was similar between the groups. CONCLUSIONS: Succinate increases mitochondrial oxygen consumption in ex vivo skeletal muscle taken from septic animals, bypassing the predominant inhibition occurring at complex I. This warrants further exploration in vivo as a putative therapeutic modality.     

Contrast‐enhanced ultrasound using bolus injections of contrast agent for assessment of postprandial microvascular blood volume in human skeletal muscle

Methods capable of measuring blood flow in a tissue‐specific manner are needed. The purpose of this study was to investigate whether contrast‐enhanced ultrasound (CEUS) using bolus injections of SonoVue^® is an useful method for assessing postprandial changes in microvascular perfusion in the vastus lateralis muscle. Ten healthy, young subjects were recruited for this study. Six subjects participated in washout and reproducibility protocols to assess washout time of SonoVue^® and the reproducibility of the method when measuring microvascular blood volume (MBV). Six subjects (two of which also participated in the washout and reproducibility protocols) participated in exercise and nutrition protocols, to assess the ability of the method to detect changes in MBV in response to these interventions. Intraday variation (coefficients of variation) for MBV indices, as assessed by peak signal intensity (PI) or mean plateau signal intensity (mPI), was high (PI: 19 ± 4·2%; mPI: 23 ± 3·3%). The exercise protocol induced significant increases in MBV indices (PI:+113%, P?0·0001; mPI:+218%, P?0·0001) acutely after exercise cessation. There were no changes in MBV indices in response to feeding during the nutrition protocol (PI: P = 0·51; mPI: P = 0·51). We conclude that CEUS using bolus injections of SonoVue^® is not capable of detecting changes in MBV of vastus lateralis in response to feeding. This is probably due to the low reproducibility of the method. However, the method is capable of measuring changes in MBV in response to exercise. This method could therefore be used when investigating exercise‐induced changes in microvascular perfusion.     

Short-term effects of terlipressin bolus infusion on sublingual microcirculatory blood flow during septic shock

Purpose  

Terlipressin bolus infusion may contribute to overshooting increases in systemic vascular resistance with concomitant reductions in systemic blood flow and oxygen delivery. Whether these effects negatively impact on microcirculatory perfusion is still not known. The objective of the present study was, therefore, to elucidate the effects of a single terlipressin bolus dose of 0.5 mg on microcirculatory perfusion in patients with catecholamine-dependent septic shock.     

Overexpression of uncoupling protein 3 in skeletal muscle protects against fat-induced insulin resistance

Insulin resistance is a major factor in the pathogenesis of type 2 diabetes and is strongly associated with obesity. Increased concentrations of intracellular fatty acid metabolites have been postulated to interfere with insulin signaling by activation of a serine kinase cascade involving PKCtheta in skeletal muscle. Uncoupling protein 3 (UCP3) has been postulated to dissipate the mitochondrial proton gradient and cause metabolic inefficiency. We therefore hypothesized that overexpression of UCP3 in skeletal muscle might protect against fat-induced insulin resistance in muscle by conversion of intramyocellular fat into thermal energy. Wild-type mice fed a high-fat diet were markedly insulin resistant, a result of defects in insulin-stimulated glucose uptake in skeletal muscle and hepatic insulin resistance. Insulin resistance in these tissues was associated with reduced insulin-stimulated insulin receptor substrate 1- (IRS-1-) and IRS-2-associated PI3K activity in muscle and liver, respectively. In contrast, UCP3-overexpressing mice were completely protected against fat-induced defects in insulin signaling and action in these tissues. Furthermore, these changes were associated with a lower membrane-to-cytosolic ratio of diacylglycerol and reduced PKCtheta activity in whole-body fat-matched UCP3 transgenic mice. These results suggest that increasing mitochondrial uncoupling in skeletal muscle may be an excellent therapeutic target for type 2 diabetes mellitus.     

Effect of motor neuromuscular electrical stimulation on microvascular perfusion of stimulated rat skeletal muscle

The purpose of this study was to determine the effect of neuromuscular electrical stimulation (NMES) (2,500-pps sine wave interrupted at 50 bps) on the degree of microvascular perfusion in stimulated skeletal muscle. The tibialis anterior (TA) and extensor digitorum longus (EDL) muscles of 36 male rats were treated with NMES for 30 minutes at current amplitudes sufficient to produce a sustained muscle contraction (motor NMES). Muscle tissue was removed at 0, 5, 10, 15, and 30 minutes after NMES. The perfused vessel/muscle fiber ratio (PV/F) of the stimulated animals at time 0 minutes was greater than that of the unstimulated control animals. A gradual decrease in the magnitude of the PV/F increase was noted over time. Depending on the muscle's fiber-type composition, the PV/F values returned to control levels by 10 to 30 minutes after motor NMES. The results indicate (1) that motor NMES significantly increases the degree of microvascular perfusion in stimulated rat skeletal muscle and (2) that the increased degree of perfusion persists for various lengths of time, depending on the fiber-type composition of the muscle. Thus, if responses in an animal model can be used as indicators of similar human responses, then the results of this study suggest that NMES can be used to increase the degree of microvascular perfusion in human skeletal muscle.     

Impaired nutritive skeletal muscle blood flow in patients with chronic renal failure

1. The possibility that abnormalities of skeletal muscle may limit the exercise tolerance of patients with chronic renal failure was investigated in patients undergoing regular haemodialysis. 2. Blood flow to the calf, a vascular bed consisting predominantly of skeletal muscle, was measured in six patients before and after exercise and compared with values obtained from 12 control subjects. 3. The patients were limited on exertion and had an abnormal response of calf blood flow to bicycle exercise. Resting calf blood flow was similar in patients and control subjects, but the mean increase in calf blood flow in response to submaximal exercise was 0.55 (SEM 0.12) ml min-1 100 ml-1 in the patients and 1.43 (SEM 0.17) ml min-1 100 ml-1 in the control subjects. The increase after symptom-limited maximal exercise was 1.50 (SEM 0.80) ml min-1 100 ml-1 in the patients and 4.20 (SEM 0.40) ml min-1 100 ml-1 in the control subjects. 4. Skeletal muscle biopsies from eight haemodialysis patients were studied by histochemistry and electron microscopy. 5. Oxidative enzyme activity was increased and there were large subsarcolemmal aggregates of structurally normal mitochondria. Necrotic capillaries were observed as empty basement membrane tubes containing fragments of degenerating endothelium. 6. The changes were compatible with a response to a chronic reduction in skeletal muscle blood flow.     

Control of skeletal muscle blood flow during postural changes in acute myocardial infarction

Central and local regulation of skeletal muscle blood flow in the leg was studied in the acute phase of myocardial infarction. Blood flow was measured by the local 133Xe washout technique in the anterior tibial muscle. The vasoconstrictor response to increase in venous transmural pressure was not present on day 1 after coronary occlusion but gradually returned during days 2, 3 and 7. A normal response to decrease in arterial perfusion pressure was observed, suggesting that intrinsic vascular reactions responsible for autoregulation of blood flow were not affected in the acute phase of myocardial infarction. Passive head-up tilt (30 degrees) induced vasoconstriction in skeletal muscle tissue. There were no differences in this response at any time during the course of acute myocardial infarction. The abolition of the vasoconstrictor response to increase in venous transmural pressure on day 1 is most likely due to centrally elicited increase in sympathetic activity as a normal vasoconstrictor response was obtained the following days. Decrease in baroreceptor activity induced by head-up tilt enabled a marked neurogenically mediated vasoconstriction in skeletal muscle tissue which was almost unaffected during the course of myocardial infarction.     

Effects of severe smoke inhalation injury and septic shock on global hemodynamics and microvascular blood flow in sheep

This prospective, randomized, controlled experimental study looks at the effects on global and regional microvascular blood flow (RMBF) in an ovine model of septic shock after severe smoke inhalation injury. Sixteen sheep were randomized into two groups, a control group (no injury, n = 8) and a smoke/sepsis (SS) group (n = 8), which received an insufflation of 4 sets of 12 breaths of cotton smoke (<40 degrees C) followed by instillation of live Pseudomonas aeruginosa into both lung lobes, according to an established protocol. All sheep were mechanically ventilated with 100% oxygen, and fluid resuscitated with lactated Ringer's solution for the entire duration of the 24-h experimental period to maintain hematocrit at baseline (BL) levels. Healthy control animals were not subjected to the injury and received only 4 x 12 breaths of room air and instillation of the vehicle (normal saline). Blood flow was analyzed using colored microspheres. Control animals remained hemodynamically stable and had no statistical changes from BL in visceral or cerebral blood flow during the entire experimental period. All SS animals developed a hypotensive, hyperdynamic circulation, characterized by a significant increase in heart rate and cardiac output with a simultaneous significant fall in mean arterial pressure, which, in combination, led to a fall in systemic vascular resistance index versus BL (P < 0.001, each). In visceral organs, the trachea showed a significant increase in RMBF (P < 0.001). In addition, skeletal muscle significantly increased versus BL and versus controls over time (P < 0.01). Whereas the pancreas displayed a significant drop in RMBF versus BL and controls (P < 0.05), no statistical differences occurred in the renal cortex, spleen, and ileum. All investigated cerebral structures, such as the cortex cerebri, basal ganglia, thalamus, hippocampus, pons, medulla oblongata, and cerebellum showed a significant increase in RMBF versus BL and versus control animals (P < 0.05, each). These data differ in areas of normal, increased, and decreased RMBF during septic shock after smoke inhalation injury and show differences to former studies of our group investigating RMBF in ovine models of either smoke inhalation or P. aeruginosa infusion. The results of this study reflect the complex pathophysiological variances of the combined injury and may provide a basis for future investigations for the treatment of this kind of injury.     

NO-mediated alterations in skeletal muscle nutritive blood flow and lactate metabolism in fibromyalgia

The purpose of these investigations was to determine if differences exist in skeletal muscle nutritive blood flow and lactate metabolism in women with fibromyalgia (FM) compared to healthy women (HC); furthermore, to determine if differences in nitric oxide-mediated systems account for any detected alterations in blood flow and lactate metabolism and contribute to exertional fatigue in FM. FM (n = 8) and HC (n = 8) underwent a cycle ergometry test of aerobic capacity, a muscle biopsy for determination of nitric oxide synthase (eNOS, nNOS, iNOS) content, and microdialysis for investigation of muscle nutritive blood flow and lactate metabolism. During prolonged (3h) resting conditions, the ethanol outflow/inflow ratio (inversely related to blood flow) increased in FM over time compared to HC (P < 0.05). FM also exhibited a reduced nutritive blood flow response to aerobic exercise (P < 0.05). There was an increase in dialysate lactate in response to acetylcholine in FM, and to sodium nitroprusside in both groups, with a greater rise in dialysate lactate in FM (P < 0.05). The iNOS protein content was higher in FM and was negatively correlated with total exercise time (r(2) = 0.462, P < 0.05). In conclusion: (1) There is reduced nutritive flow response to aerobic exercise and reduced maximal exercise time in FM that might relate to higher iNOS protein content and contribute to exertional fatigue in FM; (2) The increased dialysate lactate in FM in response to stimulation of NOS or a nitric oxide donor suggest that FM may be more sensitive than HC to the suppressive effect of nitric oxide on oxidative phosphorylation.     

Upregulation of myocellular DGAT1 augments triglyceride synthesis in skeletal muscle and protects against fat-induced insulin resistance

Increased fat deposition in skeletal muscle is associated with insulin resistance. However, exercise increases both intramyocellular fat stores and insulin sensitivity, a phenomenon referred to as "the athlete's paradox". In this study, we provide evidence that augmenting triglyceride synthesis in skeletal muscle is intrinsically connected with increased insulin sensitivity. Exercise increased diacylglycerol (DAG) acyltransferase (DGAT) activity in skeletal muscle. Channeling fatty acid substrates into TG resulted in decreased DAG and ceramide levels. Transgenic overexpression of DGAT1 in mouse skeletal muscle replicated these findings and protected mice against high-fat diet-induced insulin resistance. Moreover, in isolated muscle, DGAT1 deficiency exacerbated insulin resistance caused by fatty acids, whereas DGAT1 overexpression mitigated the detrimental effect of fatty acids. The heightened insulin sensitivity in the transgenic mice was associated with attenuated fat-induced activation of DAG-responsive PKCs and the stress mediator JNK1. Consistent with these changes, serine phosphorylation of insulin receptor substrate 1 was reduced, and Akt activation and glucose 4 membrane translocation were increased. In conclusion, upregulation of DGAT1 in skeletal muscle is sufficient to recreate the athlete's paradox and illustrates a mechanism of exercise-induced enhancement of muscle insulin sensitivity. Thus, increasing muscle DGAT activity may offer a new approach to prevent and treat insulin resistance and type 2 diabetes mellitus.     

缺氧对大鼠骨骼肌毛细血管密度和血流量的影响

背景:组织毛细血管增多,可缩短氧从毛细血管向组织细胞弥散的距离,改善组织供氧。但组织毛细血管密度与组织的代谢水平密切相关,单纯缺氧能否引起骨骼肌毛细血管密度增加尚有争议。目的:观察不同缺氧时间大鼠骨骼肌毛细血管密度和血流供应的变化规律。设计:非随机对照实验。对动物缺氧前体质量加以选择,使各组动物的体质量在缺氧末期基本接近。地点和对象:实验地点:第三军医大学病理生理学与高原生理学教研室。清洁级纯种健康Wistar大鼠64只,雌雄各半,分为4组:对照组、缺氧5d组、缺氧15d组和缺氧30d组。干预:缺氧5d组、15d组和30d组大鼠在模拟海拔5000m的减压舱中分别连续减压缺氧5,15和30d后(23h/d),对照组大鼠在舱外饲养。用肌球蛋白ATP酶(mATPase)组织化学方法显示骨骼肌Ⅰ,Ⅱ型纤维和毛细血管并进行图像分析,测定骨骼肌纤维横断面积和毛细血管密度以及单位面积内毛细血管数与肌纤维数的比值;用放射性微球法测定骨骼肌血流量和血管阻力。主要观察指标:①各组大鼠血球压积和骨骼肌血流量、血管阻力的变化。②大鼠腓肠肌Ⅰ,Ⅱ型纤维横断面积。③各组大鼠骨骼肌毛细血管密度和单位面积内毛细血管数与肌纤维数的比值。结果:缺氧5d组大鼠与对照组相比,骨骼肌纤维横断面积即出现显著缩小犤I型分别为(2254±     

Urea clearance: a new method to register local changes in blood flow in rat skeletal muscle based on microdialysis

Increasing evidence suggests that local blood flow should be monitored during microdialysis (MD) as the recovery of analytes is affected by local blood flow. At present ethanol clearance is the standard technique for this purpose, but it is not functional at very low perfusion velocities. Here, we introduce a technique for MD whereby local tissue blood flow is recorded by the use of urea clearance (changes inflow/outflow concentration), in conjunction with measurements of tissue metabolism (glucose, lactate and puruvate). MD probes were inserted into the gracilis muscle of 15 rats and perfused with a medium containing urea (20 mmol l^?1). Changes in muscle blood flow were made by addition of noradrenaline (5 μg ml^?1) to the perfusion medium at two perfusion velocities (0·6 and 0·4 μl min^?1). The clearance of urea from the perfusion medium was then calculated and examined in relation to the dose of noradrenaline and to the coexisting changes in extracellular metabolites. The results showed reproducible and dose‐dependent changes in blood flow that were induced by noradrenaline. These were characterized by dose‐dependent changes in the urea clearance as well as blood‐flow‐specific changes in the MD metabolic markers (reduction in glucose and increase in lactate). The sensitivity for blood flow changes as assessed by urea clearance (MD) was increased at 0·4 compared with the 0·6 μl min^?1 perfusion speed. The results indicate that inclusion of urea to the perfusion medium may be used to monitor changes in skeletal muscle blood flow at low perfusion velocities and in parallel assess metabolic variables with a high recovery (>90%).     

Basal and exercise-induced skeletal muscle blood flow is augmented in type I diabetes mellitus

Hyperaemia occurs early in the renal and retinal microcirculation of patients with type I (insulin-dependent) diabetes mellitus, and may be critical in the development of nephropathy and retinopathy. We therefore sought to determine whether resting and exercise-induced hyperaemia was also apparent in the skeletal muscle circulation of young subjects with type I diabetes. Blood flow was assessed by venous occlusion plethysmography in 18 diabetic (DM) subjects and 20 matched controls. Exercise entailed 2 min of isotonic exercise against no load. Endothelium-dependent and -independent vasodilator function was assessed following intra-arterial infusion of acetylcholine and sodium nitroprusside respectively. Forearm blood flow (FBF) was higher in DM subjects than in controls (3.3+/-0.3 and 2.2+/-0.2 ml x min(-1) x 100 ml(-1) forearm respectively; P<0.005). This was not due to differences in forearm or body size, blood pressure, heart rate, lipid status or glycaemic control. Peripheral insulin levels were higher in DM subjects than in controls (48.5+/-8 and 15.5+/-1.5 micro-units/ml respectively; P<0.005). Resting FBF was closely correlated with insulin levels (r(2)=0.4; P<0.005). Parameters of exercise-induced hyperaemia [including peak flow (16.4+/-1.4 and 12.0+/-0.7 ml x min(-1) x 100 ml(-1) forearm in DM and control subjects respectively; P<0.01) and the volume repaid to the forearm at 5 min post-exercise (32.1+/-3.1 and 23.1+/-1.4 ml x 100 ml(-1) forearm respectively; P<0.05)] were also significantly greater in DM subjects, even when differences in resting FBF were taken into account. Peak hyperaemic blood flow and the volume repaid at 5 min were also related to insulin levels (r(2)=0.16; P<0.05 and r(2)=0.27; P<0.005 respectively). The vasodilator response to acetylcholine was reduced in DM subjects (P<0.05; analysis of variance), and the slope of this dose-flow relationship was inversely related to insulin levels (r(2)=0.2; P<0.05). These data show that both resting and exercise-induced skeletal muscle blood flow are augmented in young patients with type I diabetes, possibly due to the vasodilatory effect of increased insulin levels. Diminished vasodilator responses to acetylcholine may also, in part, be a consequence of insulin-augmented resting muscle blood flow.