Red Gastrocnemius Research Articles

Novel role for thioredoxin reductase‐2 in mitochondrial redox adaptations to obesogenic diet and exercise in heart and skeletal muscle

Increased fatty acid availability and oxidative stress are physiological consequences of exercise (Ex) and a high-fat, high-sugar (HFHS) diet. Despite these similarities, the global effects of Ex are beneficial, whereas HFHS diets are largely deleterious to the cardiovascular system. The reasons for this disparity are multifactorial and incompletely understood. We hypothesized that differences in redox adaptations following HFHS diet in comparison to exercise may underlie this disparity, particularly in mitochondria. Our objective in this study was to determine mechanisms by which heart and skeletal muscle (red gastrocnemius, RG) mitochondria experience differential redox adaptations to 12 weeks of HFHS diet and/or exercise training (Ex) in rats. Surprisingly, both HFHS feeding and Ex led to contrasting effects in heart and RG, in that mitochondrial H2O2 decreased in heart but increased in RG following both HFHS diet and Ex, in comparison to sedentary animals fed a control diet. These differences were determined to be due largely to increased antioxidant/anti-inflammatory enzymes in the heart following the HFHS diet, which did not occur in RG. Specifically, upregulation of mitochondrial thioredoxin reductase-2 occurred with both HFHS and Ex in the heart, but only with Ex in RG, and systematic evaluation of this enzyme revealed that it is critical for suppressing mitochondrial H2O2 during fatty acid oxidation. These findings are novel and important in that they illustrate the unique ability of the heart to adapt to oxidative stress imposed by HFHS diet, in part through upregulation of thioredoxin reductase-2. Furthermore, upregulation of thioredoxin reductase-2 plays a critical role in preserving the mitochondrial redox status in the heart and skeletal muscle with exercise.

Short-term caloric restriction, resveratrol, or combined treatment regimens initiated in late-life alter mitochondrial protein expression profiles in a fiber-type specific manner in aged animals

Aging is associated with a loss in muscle known as sarcopenia that is partially attributed to apoptosis. In aging rodents, caloric restriction (CR) increases health and longevity by improving mitochondrial function and the polyphenol resveratrol (RSV) has been reported to have similar benefits. In the present study, we investigated the potential efficacy of using short-term (6weeks) CR (20%), RSV (50mg/kg/day), or combined CR+RSV (20% CR and 50mg/kg/day RSV), initiated at late-life (27months) to protect muscle against sarcopenia by altering mitochondrial function, biogenesis, content, and apoptotic signaling in both glycolytic white and oxidative red gastrocnemius muscle (WG and RG, respectively) of male Fischer 344×Brown Norway rats. CR but not RSV attenuated the age-associated loss of muscle mass in both mixed gastrocnemius and soleus muscle, while combined treatment (CR+RSV) paradigms showed a protective effect in the soleus and plantaris muscle (P<0.05). Sirt1 protein content was increased by 2.6-fold (P<0.05) in WG but not RG muscle with RSV treatment, while CR or CR+RSV had no effect. PGC-1α levels were higher (2-fold) in the WG from CR-treated animals (P<0.05) when compared to ad-libitum (AL) animals but no differences were observed in the RG with any treatment. Levels of the anti-apoptotic protein Bcl-2 were significantly higher (1.6-fold) in the WG muscle of RSV and CR+RSV groups compared to AL (P<0.05) but tended to occur coincident with elevations in the pro-apoptotic protein Bax so that the apoptotic susceptibility as indicated by the Bax to Bcl-2 ratio was unchanged. There were no alterations in DNA fragmentation with any treatment in muscle from older animals. Additionally, mitochondrial respiration measured in permeabilized muscle fibers was unchanged in any treatment group and this paralleled the lack of change in cytochrome c oxidase (COX) activity. These data suggest that short-term moderate CR, RSV, or CR+RSV tended to modestly alter key mitochondrial regulatory and apoptotic signaling pathways in glycolytic muscle and this might contribute to the moderate protective effects against aging-induced muscle loss observed in this study.

Abstract 427: Apoliporotein A-I Improves Glucose Tolerance and Enhances Insulin-Dependent and Insulin-Independent Glucose Uptake in the db/db Mouse Model of Type 2 Diabetes

Introduction Type 2 diabetes is characterised by decreased high-density lipoprotein (HDL) levels, as well as the level of apolipoprotein A-I (apoA-I), the main apolipoprotein of HDL. Pharmacological elevation of HDL levels is associated with improved glycemic control in patients with type 2 diabetes and HDL also improves glucose uptake into skeletal muscle in vitro. With the current interest in HDL raising therapeutics, understanding how apoA-I/HDL improve glucose tolerance in type 2 diabetes is important. Objective To determine the impact of increasing plasma apoA-I levels on glycemic control and glucose uptake in the db/db mouse model of type 2 diabetes. Methods Fasted 6-7 week old male db/db mice received a single intraperitoneal injection of human apoA-I (40 mg/kg) or PBS. Two hours after the infusion mice were randomly assigned into 3 groups (n=7-8/group) and subjected to a glucose tolerance test (1 g/kg D-glucose), an insulin tolerance test (3 U/kg human insulin) or used to determine glucose uptake into skeletal muscle. For the glucose and insulin tolerance tests blood glucose levels were measured with a glucometer. For the glucose uptake experiments, gastrocnemius muscles were incubated with 1 mM [3H]2-deoxy-glucose and 7 mM D-[14C]mannitol in the presence or absence of insulin, then homogenized. 3H and 14C content of the homogenates was determined by liquid scintillation counting. Results Plasma apoA-I levels reached 65.2±16.6 μg/mL by 2 h post-injection, and remained elevated for &gt;10 h. Mice that received apoA-I had significantly improved glucose tolerance (AUC apoA-I=2574±70; PBS=2927±137, p&lt;0.05) and insulin sensitivity (AUC apoA-I=388.8±23.8; PBS=194.1±19.6, p&lt;0.001). In the absence of insulin, apoA-I increased glucose uptake by 1.8 and 1.7 fold in red and white gastrocnemius muscle, respectively. In the presence of insulin, apoA-I increased glucose uptake 2.5 and 1.8 fold, in red and white gastrocnemius muscle, respectively. Conclusion A single infusion of apoA-I significantly increases glucose tolerance, insulin sensitivity and skeletal muscle glucose uptake in db/db mice. Insulin and apoA-I work synergistically to improve skeletal muscle glucose uptake.

An amino acid mixture improves glucose tolerance and lowers insulin resistance in the obese Zucker rat

The purpose of this investigation was to test an amino acid mixture on glucose tolerance in obese Zucker rats [experiment (Exp)-1] and determine whether differences in blood glucose were associated with alterations in muscle glucose uptake [experiment (Exp)-2]. Exp-1 rats were gavaged with either carbohydrate (OB-CHO), carbohydrate plus amino acid mixture (OB-AA-1), carbohydrate plus amino acid mixture with increased leucine concentration (OB-AA-2) or water (OB-PLA). The glucose response in OB-AA-1 and OB-AA-2 were similar, and both were lower compared to OB-CHO. This effect of the amino acid mixtures did not appear to be solely attributable to an increase in plasma insulin. Rats in Exp-2 were gavaged with carbohydrate (OB-CHO), carbohydrate plus amino acid mixture (OB-AA-1) or water (OB-PLA). Lean Zuckers were gavaged with carbohydrate (LN-CHO). Fifteen minutes after gavage, a radiolabeled glucose analog was infused through a catheter previously implanted in the right jugular vein. Blood glucose was significantly lower in OB-AA-1 compared to OB-CHO while the insulin responses were similar. Glucose uptake was greater in OB-AA-1 compared with OB-CHO, and similar to that in LN-CHO in red gastrocnemius muscle (5.15 ± 0.29, 3.8 ± 0.27, 5.18 ± 0.34 µmol/100 g/min, respectively). Western blot analysis showed that Akt substrate of 160 kDa (AS160) phosphorylation was enhanced for OB-AA-1 and LN-CHO compared to OB-CHO. These findings suggest that an amino acid mixture improves glucose tolerance in an insulin resistant model and that these improvements are associated with an increase in skeletal muscle glucose uptake possibly due to improved intracellular signaling.

Mammalian target of rapamycin pathway is up-regulated by both acute endurance exercise and chronic muscle contraction in rat skeletal muscle

This study examined changes in the expression of translation initiation regulatory proteins and mRNA following both an acute bout of endurance exercise and chronic muscle contractile activity. Female Sprague Dawley rats ran for 2 h at 15 m·min(-1) followed by an increase in speed of 5 m·min(-1) every 5 min until volitional fatigue. The red gastrocnemius muscle was harvested from nonexercised animals (control; n = 6) and from animals that exercised either immediately after exercise (n = 6) or following 3 h of recovery from exercise (n = 6). Compared with control, ribosomal protein S6 (rpS6) mRNA was elevated (p < 0.05) at both 0 h (+32%) and 3 h (+47%). Both a catalytic subunit of eukaryotic initiation factor 2B (eIF2Bε) (+127%) and mammalian target of rapamycin (mTOR) mRNA (+44%) were increased at 3 h, compared with control. Phosphorylation of mTOR (+40%) and S6 kinase 1 (S6K1) (+266%) were increased immediately after exercise (p < 0.05). Female Sprague Dawley rats also underwent chronic stimulation of the peroneal nerve continuously for 7 days. The red gastrocnemius muscle was removed 24 h after cessation of the stimulation. Chronic muscle stimulation increased (p < 0.05) mTOR protein (+74%), rpS6 (+31%), and eukaryotic initiation factor 2α (+44%, p = 0.069), and this was accompanied by an increase in cytochrome c (+31%). Increased resting phosphorylation was observed for rpS6 (+51%) (p < 0.05) but not for mTOR or eukaryotic initiation factor 4E binding protein 1. These experiments demonstrate that both acute and chronic contractile activity up-regulate the mTOR pathway and mitochondrial content in murine skeletal muscle. This up-regulation of the mTOR pathway may increase translation efficiency and may also represent an important control point in exercise-mediated mitochondrial biogenesis.

Exhaustive Training Increases Uncoupling Protein 2 Expression and Decreases Bcl-2/Bax Ratio in Rat Skeletal Muscle

This work investigates the effects of oxidative stress due to exhaustive training on uncoupling protein 2 (UCP2) and Bcl-2/Bax in rat skeletal muscles. A total of 18 Sprague-Dawley female rats were randomly divided into three groups: the control group (CON), the trained control group (TC), and the exhaustive trained group (ET). Malondialdehyde (MDA), superoxide dismutase (SOD), xanthine oxidase (XOD), ATPase, UCP2, and Bcl-2/Bax ratio in red gastrocnemius muscles were measured. Exhaustive training induced ROS increase in red gastrocnemius muscles, which led to a decrease in the cell antiapoptotic ability (Bcl-2/Bax ratio). An increase in UCP2 expression can reduce ROS production and affect mitochondrial energy production. Thus, oxidative stress plays a significant role in overtraining.

The Novel Angiotensin II Receptor Blocker Azilsartan Medoxomil Ameliorates Insulin Resistance Induced by Chronic Angiotensin II Treatment in Rat Skeletal Muscle

Angiotensin receptor (type 1) blockers (ARBs) can reduce both hypertension and insulin resistance induced by local and systemic activation of the renin-angiotensin-aldosterone system. The effectiveness of azilsartan medoxomil (AZIL-M), a novel imidazole-based ARB, to facilitate metabolic improvements in conditions of angiotensin II (Ang II)-associated insulin resistance is currently unknown. The aim of this study was to determine the impact of chronic AZIL-M treatment on glucose transport activity and key insulin signaling elements in red skeletal muscle of Ang II-treated rats. Male Sprague-Dawley rats were treated for 8 weeks with or without Ang II (200 ng/kg/min) combined with either vehicle or AZIL-M (1 mg/kg/day). Ang II induced significant (p < 0.05) increases in blood pressure, which were completely prevented by AZIL-M. Furthermore, Ang II reduced insulin-mediated glucose transport activity in incubated soleus muscle, and AZIL-M co-treatment increased this parameter. Moreover, AZIL-M treatment of Ang II-infused animals increased the absolute phosphorylation of insulin signaling molecules, including Akt [both Ser⁴⁷³ (81%) and Thr³⁰⁸ (23%)] and AS160 Thr⁶⁴² (42%), in red gastrocnemius muscle frozen in situ. Absolute AMPKα (Thr¹⁷²) phosphorylation increased (98%) by AZIL-M treatment, and relative Thr³⁸⁹ phosphorylation of p70 S6K1, a negative regulator of insulin signaling, decreased (51%) with AZIL-M treatment. These results indicate that ARB AZIL-M improves the in vitro insulin action on glucose transport in red soleus muscle and the functionality of the Akt/AS160 axis in red gastrocnemius muscle in situ in Ang II-induced insulin-resistant rats, with the latter modification possibly associated with enhanced AMPKα and suppressed p70 S6K1 activation.

Not only accumulation, but also saturation status of intramuscular lipids is significantly affected by PPARγ activation

Intramuscular lipid accumulation has been associated with insulin resistance, and after thiazolidinediones (TZD) treatment, it was shown to be reduced in some, but not all, studies. This work was undertaken to investigate the relationships between intramuscular lipids [free fatty acids (FFA), diacylglycerols (DAG), triacylglycerol (TAG) and phospholipids] and plasmalemmal expression of fatty acid (FA) transporter [FAT/CD36 and FABPpm] in the muscles of varying oxidative capacity, after peroxisome proliferator-activated receptors gamma (PPARγ) activation (rosiglitazone) in an animal model of high-fat-diet-induced insulin resistance. Endurance training was also included to further explore the differences in these relationships. We have used gas liquid chromatography to estimate FA content and composition in each lipid fraction. For sarcolemmal expression of FA transporters, subfractionation of skeletal muscles with subsequent western blot technique was applied. High-fat diet induced intramuscular accumulation of FFA, DAG and TAG, irrespective of muscle's fibre composition. PPARγ activation (rosiglitazone) and, to a lesser extent, endurance training further increased TAG accumulation, while it reduced DAG in oxidative muscles (soleus and red gastrocnemius). Aforementioned interventions increased also sarcolemmal FAT/CD36 and FABPpm expressions in particular muscles. Irrespective of diet, rosiglitazone and exercise decreased significantly FA saturation status favouring proportionate enhancement in monounsaturated FA (rosiglitazone) or polyunsaturated FAs (endurance training). These findings support the conclusion that not only the change in total lipid content (DAG and TAG), but also FA composition is affected by rosiglitazone in an animal model of high-fat-diet-induced insulin resistance.

Differential Adaptations to High‐calorie, Western‐pattern Diet and Exercise Training in Redox Status and Mitochondria of Heart and Skeletal Muscle

Two interventions known to cause profound changes in cellular metabolism and redox enzyme expression/activity in heart and skeletal muscle are exercise (EX), and high‐fat, high sugar “Western pattern” diet (HFHS). To determine whether heart and skeletal muscle are differentially impacted by HFHS diet and EX, Sprague‐Dawley rats were fed either standard chow (CON) or a HFHS diet for 12 weeks (HFHS). A sub‐group of HFHS rats exercise‐trained 5 days/week for 2 hours/day (HFHS+EX). Following these interventions, mitochondrial H2O2 emission (mH2O2) and Ca2+ tolerance (mCa2+) supported by pyruvate + malate + succinate, and O2 consumption (mO2) supported by glutamate+malate (GM) and palmitoyl‐carnitine (PC) was determined in permeabilized red gastrocnemius (RG) and cardiac fibers. HFHS diet increased mH2O2 ~2‐fold, and HFHS+EX decreased in RG (P&lt;0.05) compared to CON. Surprisingly, the HFHS diet decreased mH2O2 ~2‐fold in heart compared to CON (P&lt;0.05), and was even further decreased in HFHS+EX. Maximal PC‐supported mO2 increased in RG in both HFHS and HFHS+Ex compared to CON (P&lt;0.05), but in heart this was only increased in HFHS+EX. Maximal GM‐supported mO2 decreased in both RG and heart with HFHS, and this decrease was prevented in HFHS+EX. Total mCa2+ increased only with HFHS+EX in both RG and heart compared to CON. These findings suggest that heart and skeletal muscle mitochondria are differentially affected by diet and exercise, which point to adaptations in cellular redox state. (Supported by NIH grant HL098780 to EJA)

Doxorubicin impairs skeletal muscle mitochondrial respiratory capacity in skeletal muscle

Debilitating muscle weakness and fatigue are common side effects of chemotherapy in cancer patients, limiting recovery and increasing morbidity. Our previous work shows that doxorubicin, a common chemotherapy drug, elevates reactive oxygen species (ROS) and decreases contractile force in skeletal muscle. As mitochondria represent a primary source of oxidant generation in muscle, we hypothesized that doxorubicin may compromise mitochondrial respiratory control and increase ROS production. 72 hrs following a single doxorubicin injection (20 mg/kg), maximal ADP‐stimulated O2 consumption was decreased during respiration supported by palmitoylcarnitine (PC) (−46 ± 26 %, p&lt;0.05) and pyruvate/malate (−43 ± 21 %, p&lt;0.05, n=6/group) in permeabilized fiber bundles (PmFB) from red gastrocnemius muscle. PmFB from doxorubicin treated animals also display increased rates of mitochondrial H2O2 emission with PC (23 ± 2 %) and 3‐phosphoglycerate (49 ± 2 %, p=0.1). Our data indicate that doxorubicin impairs both maximal State 3, and fatty‐acid‐supported respiration. In addition, doxorubicin promotes mitochondrial ROS generation, a potential underlying cause of doxorubicin‐induced muscle dysfunction.Supported by DK073488

Low Intensity Exercise Attenuates Acute Lipid Loading‐Induced Alterations in Mitochondrial Function in Rat Skeletal Muscle

A single high‐fat meal acutely increases skeletal muscle mitochondrial H2O2 emitting potential (mEH2O2), shifts the intracellular redox environment to a more oxidized state, and increases circulating markers of oxidative stress. Bioenergetically, this implies an acute lipid load may elevate the reducing pressure/membrane potential (Δ Ψm) within mitochondria and, conversely, that even a mild increase in energy expenditure may be sufficient to prevent these effects. To test this hypothesis, male Sprague‐Dawley rats received an oral lipid gavage (20% intralipid, 45 Kcal/kg lean body mass) or water followed either by 2h of rest or 1h of rest plus 1h of low intensity treadmill exercise (15 m/min, 0% grade). Permeabilized fiber bundles were prepared from red gastrocnemius muscle for testing mitochondrial function. In rats receiving lipid, Δ Ψm and mEH2O2 were higher (P&lt;0.05) and calcium retention capacity (mCa2+ RC, an index of resistance to mitochondrial permeability transition) was lower under state IV and/or “clamped” ADP‐stimulated state III conditions. All three effects were prevented when lipid gavage was followed by low‐intensity exercise. Respiratory capacity was unaffected by any of the interventions. These findings provide evidence that mitochondrial Δ Ψm, mEH2O2, and mCa2+ RC are acutely affected by nutritional overload in skeletal muscle, but can be prevented by low intensity exercise. NIH DK073488

Attenuation of Age-Dependent Lipid Profile by Treadmill Running in Different Skeletal Muscle Fibers of Old Rats

The goal of this study was to distinguish the impact of regular treadmill running on age-induced altered lipid profiles in different skeletal muscle fiber types of rats. Wistar strain male albino rats of two age groups, young (3 months, n = 12) and aged/old (18 months, n = 12) were divided into two groups such as, control (Con) and exercise trained (ExT; 23 m/min, 30 min/day, 5 days/week for 12-week). After completion of the last training session, lipid metabolic profiles, including total cholesterol (TC), triglycerides (TG), phospholipids and malondialdehyde (MDA, lipid peroxidation marker) levels were monitored in soleus (SOL), red gastrocnemius (RG) and white gastrocnemius (WG) muscle fibers of rat. The TC, TG and MDA contents were significantly (P ＜ 0.05) increased with advancement of age, while, phospholipid content was decreased with age in all muscle fiber types. However, here we found significantly (P ＜ 0.05) decreased TC and TG levels, and increased phospholipid contents after treadmill exercise in all muscle fiber types of aged rats. The decreased TC content was more in young muscles (WG 31%), and the increased phospholipid content was more in old muscles fibers after treadmill running compared to their respective counter age group rats. However, increased MDA levels in old muscle fibers (SOL-132.13 ±2, RG-129 ±2 and WG-147 ±2.5 μmoles/g) were not attenuated by exercise training (SOL- 151 ±2.4, RG-148 ±2.7 and WG-157 ±3 μmoles/g) in this study. These results demonstrated that decreased TC and TG, and increased phospholipid contents by regular treadmill running might be beneficial to counteract the age-associated malfunctions in different muscle fiber types and also avoid the musculoskeletal disorders in aged rats.

Interaction between overtraining and the interindividual variability may (not) trigger muscle oxidative stress and cardiomyocyte apoptosis in rats.

Severe endurance training (overtraining) may cause underperformance related to muscle oxidative stress and cardiomyocyte alterations. Currently, such relationship has not been empirically established. In this study, Wistar rats (n = 19) underwent eight weeks of daily exercise sessions followed by three overtraining weeks in which the daily frequency of exercise sessions increased. After the 11th training week, eight rats exhibited a reduction of 38% in performance (nonfunctional overreaching group (NFOR)), whereas eleven rats exhibited an increase of 18% in performance (functional overreaching group (FOR)). The red gastrocnemius of NFOR presented significantly lower citrate synthase activity compared to FOR, but similar to that of the control. The activity of mitochondrial complex IV in NFOR was lower than that of the control and FOR. This impaired mitochondrial adaptation in NFOR was associated with increased antioxidant enzyme activities and increased lipid peroxidation (in muscle and plasma) relative to FOR and control. Cardiomyocyte apoptosis was higher in NFOR. Plasma creatine kinase levels were unchanged. We observed that some rats that presented evidence of muscle oxidative stress are also subject to cardiomyocyte apoptosis under endurance overtraining. Blood lipid peroxides may be a suitable biomarker for muscle oxidative stress that is unrelated to severe muscle damage.

The Effects of Altitude Training on the AMPK-Related Glucose Transport Pathway in the Red Skeletal Muscle of Both Lean and Obese Zucker Rats

The skeletal muscle AMP-activated protein kinase (AMPK)-related glucose transport pathway is involved in glucose homeostasis. In this study, we examined whether obese control Zucker rats had abnormal expression of proteins in the LKB1-AMPK-AS160-GLUT4 pathway in red gastrocnemius muscle compared to that in lean (normal) control Zucker rats. We also compared the chronic training effects of exercise, hypoxia, and altitude training on this pathway in lean and obese rats. At sea level, lean and obese rats were divided into 4 groups for 6 weeks training as follows: 1) control; 2) exercise (progressive daily swimming-exercise training with comparable exercise signals between the two groups); 3) hypoxia (8 hours of daily 14% O2 exposure); and 4) exercise plus hypoxia (also called altitude training). Seven animals were used for each group. The obese rats in the control group had higher body weights, elevated fasting insulin and glucose levels, and higher baseline levels of muscle AMPK and AS160 phosphorylation compared with those of lean control rats. For obese Zucker rats in the exercise or hypoxia groups, the muscle AMPK phosphorylation level was significantly decreased compared with that of the control group. For obese Zucker rats in the altitude training group, the levels of AMPK, AS160 phosphorylation, fasting insulin, and fasting glucose were decreased concomitant with an approximate 50% increase in the muscle GLUT4 protein level compared with those of the control group. In lean rats, the altitude training efficiently lowered fasting glucose and insulin levels and increased muscle AMPK and AS160 phosphorylation as well as GLUT4 protein levels. Our results provide evidence that long-term altitude training may be a potentially effective nonpharmacological strategy for treating and preventing insulin resistance based on its effects on the skeletal muscle AMPK-AS160-GLUT4 pathway.

Fasting increases palmitic acid incorporation into rat hind-limb intramuscular acylglycerols while short-term cold exposure has no effect

The aim of the study was to investigate the palmitic acid incorporation into intramuscular acylglycerols in perfused hind-limb skeletal muscles of different fibre types in rats either fasted for 48 h or exposed to cold (6 °C) for 12 h. Hind-limb preparations of fasted and cold exposed rats were perfused with buffers containing tritium labelled and cold palmitic acid. Palmitic acid incorporation into intracellular lipid pools in the soleus, plantaris, red and white gastrocnemius and red and white quadriceps was measured. It was found that fasting increased approximately 2-fold palmitic acid incorporation in all muscles examined regardless of the fibre type composition of the muscle. On the other hand, exposure to cold had no effect on the palmitic acid incorporation into intramuscular acylglycerols regardless the muscle fibre type. The increased incorporation of palmitic acid into acylglycerols in fasted animals is in line with data showing that 48 h fasting stimulates the expression of plasma membrane proteins putatively facilitating fatty acid uptake. It appears that although 12 h cold exposure increases the use of fatty acids as energy substrates it does not alter the incorporation of palmitic acid into intramuscular acylglycerols in the perfused rat hind-limb.

Neuropeptide Y overflow and metabolism in skeletal muscle arterioles

The purpose of this study was to characterize neuropeptide Y (NPY) overflow and metabolism from isolated skeletal muscle arterioles of female rats. Gastrocnemius first-order arterioles were removed from young (2 months), young adult (6 months) and middle-aged (12 months) F344 female rats. Arterioles were isolated, cannulated and pressurized in a microvessel bath with field stimulation electrodes. NPY overflow from isolated arterioles was assessed at 0 s and 30 s post-field stimulation. Dipeptidyl peptidase IV (DPPIV) activity was quantified via fluorometric assay of whole vessel homogenate. In young adult and middle-aged rats, NPY overflow increased 0 s and 30 s following field stimulation. In young adult rats, DPPIV inhibition resulted in an increase in NPY overflow at 30 s, while middle-aged rats had no increase in NPY overflow with DPPIV inhibition (P <0.05). DPPIV activity was influenced by factors such as age, vessel type, and endothelium (P <0.05). The present data suggest that DPPIV plays a significant role in modulating the actions of NPY in arterioles of young adult females; however, this role appears to diminish with age.

Enhanced Ca2+ transport and muscle relaxation in skeletal muscle from sarcolipin-null mice

Sarcolipin (SLN) inhibits sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) pumps. To evaluate the physiological significance of SLN in skeletal muscle, we compared muscle contractility and SERCA activity between Sln-null and wild-type mice. SLN protein expression in wild-type mice was abundant in soleus and red gastrocnemius (RG), low in extensor digitorum longus (EDL), and absent from white gastrocnemius (WG). SERCA activity rates were increased in soleus and RG, but not in EDL or WG, from Sln-null muscles, compared with wild type. No differences were seen between wild-type and Sln-null EDL muscles in force-frequency curves or maximum rates of force development (+dF/dt). Maximum relaxation rates (-dF/dt) of EDL were higher in Sln-null than wild type across a range of submaximal stimulation frequencies, but not during a twitch or peak tetanic contraction. For soleus, no differences were seen between wild type and Sln-null in peak tetanic force or +dF/dt; however, force-frequency curves showed that peak force during a twitch and 10-Hz contraction was lower in Sln-null. Changes in the soleus force-frequency curve corresponded with faster rates of force relaxation at nearly all stimulation frequencies in Sln-null compared with wild type. Repeated tetanic stimulation of soleus caused increased (-dF/dt) in wild type, but not in Sln-null. No compensatory responses were detected in analysis of other Ca(2+) regulatory proteins using Western blotting and immunohistochemistry or myosin heavy chain expression using immunofluorescence. These results show that 1) SLN regulates Ca(2+)-ATPase activity thereby regulating contractile kinetics in at least some skeletal muscles, 2) the functional significance of SLN is graded to the endogenous SLN expression level, and 3) SLN inhibitory effects on SERCA function are relieved in response to repeated contractions thus enhancing relaxation rates.

Effects of Different High Fat Diets on FABP Content in Rat Skeletal Muscles

Long-term feeding of high fat diet increases both oxidative enzymes in skeletal muscle and endurance capacity in rats, whereas it is well-known to cause obesity. PURPOSE: The purpose of this study was to examine the effects of high fat diet containing long-chain diacylglycerol (DAG), medium-chain triacylglycerol (MTG) or long-chain triacylglycerol (TAG) on oxidative enzyme activities and fatty acid binding protein (FABP) contents in rat skeletal muscles. METHODS: Male Wistar rats, 5 weeks old, were assigned to 4 groups; low fat diet (LF; n=6, 12% calories as fat), TAG high fat diet (n=6, 60% calories as fat), DAG high fat diet (n=6, 60% calories as fat) and MTG high fat diet (n=7, 60% calories as fat) groups. Animals were fed their diets and water ad libitum for 5 weeks. Citrate synthase (CS) activity was measured by a spectrophotometric assay, and FABP content was measured with an ELISA kit. RESULTS: Epidydimal fat pad was much smaller in MTG group than in HTG and DAG groups (P<0.05). TAG and MTG groups were increased CS activity (TAG, 40±2.5 mol·min-1·g-1; MTG, 44±2.8 mol·min-1·g-1) in soleus muscle (Sol) compared with LF (35±4.1 mol·min-1·g-1) group (p < 0.01). FABP contents of Sol and red gastrocnemius muscle (Gas) were significantly higher in TAG (Sol, 62 ±7.3; red Gas, 59±21.8 ng/ml) and DAG (Sol, 58±9.1; red Gas, 51±5.8 ng/ml) groups than in MTG (Sol, 28±2.8; red Gas, 28±6.2 ng/ml) group (p < 0.05). On the other hand, FABP contents of white Gas were, albeit small, significantly higher in TAG (1.8±0.3 ng/ml), DAG (2.4±0.3 ng/ml) and MTG (1.5±0.2 ng/ml) groups than in LF (0.3±0.1 ng/ml) group (p < 0.05). CONCLUSION: Thus, the 5-week-feeding of DAG high fat diet increased FABP content in Sol and red/white Gas, and also the MTG high fat diet increased both FABP content in white Gas and oxidative enzymes without fat accumulation, suggesting that "MTG plus DAG" high fat diet may enhance fat metabolism and exercise performance without obesity.

Cordyceps sinensis promotes exercise endurance capacity of rats by activating skeletal muscle metabolic regulators

Cordyceps sinensis is a traditional Chinese medicine used for promotion of health, longevity and athletic power. However, the molecular mechanism for anti-fatigue activity and physical fitness has not yet been reported. The present study was conducted to evaluate the exercise endurance promoting activities of fungal traditional Chinese medicine (FTCM) Cordyceps sinensis cultured whole mycelium (CS) and the underlying mechanisms. CS was orally supplemented (200mg/kg body weight/day) to rats for 15days with or without swimming exercise along with exercise and placebo groups. Both CS supplementation and supplementation concurrent with exercise improved exercise endurance by 1.79- (P<0.05) and 2.9-fold (P<0.01) respectively as compared to placebo rats. CS supplementation concurrent with exercise also increased the swimming endurance by 1.32-fold (P<0.05) over the exercise group. To study the molecular mechanism of the observed effect, we measured the expression levels of endurance responsive skeletal muscle metabolic regulators AMPK, PGC-1α and PPAR-δ as well as endurance promoting and antioxidant genes like MCT1, MCT4, GLUT4, VEGF, NRF-2, SOD1 and TRX in red gastrocnemius muscle. Our results indicate that CS supplementation significantly upregulates the skeletal muscle metabolic regulators, angiogenesis, better glucose and lactate uptake both in exercised and non-exercised rats. We have also observed increased expression of oxidative stress responsive transcription factor NRF-2 and its downstream targets SOD1 and TRX by CS supplementation. CS supplementation with or without exercise improves exercise endurance capacity by activating the skeletal muscle metabolic regulators and a coordinated antioxidant response. Consequently, CS can be used as a potent natural exercise mimetic.

Pre‐junctional potentiation of ATP overflow is age‐dependent

The purpose was to evaluate the role of α2‐pre‐junctional receptors in ATP overflow in the resistance arterioles from adult and middle aged rats. Using F344 rats (6 months old: n=5 female; n=6 males) and (12 months old: n=5 females; n=5 males), the 1A arterioles from the red gastrocnemius muscle were dissected and cannulated with glass pipettes in a tissue bath. The ATP and null probes were positioned such that the tips of the probes were touching the arteriole in similar anatomical locations and a normal ATP curve was generated (0.78μM to 25 μM ATP). Field stimulation resulted in an increase in ATP overflow in arterioles from 6‐month old rats (1.88±6.14 μM ATP) and 12‐month old rats (34.95±91.30 μM ATP). Adding the α2‐agonist, UK14,304, resulted in no change in 6‐month olds (3.34±3.10 μM ATP), but increased ATP overflow in some 12‐month olds (119.09±256.71 μM). Blocking α2‐receptors using idozoxan, had little effect on 6‐month olds (1.81±1.59 μM ATP), but blocked the action of UK14,304 in 12‐month olds (11.74±23.04 μM ATP). There were no sex‐related differences in ATP overflow. These data indicate that there are significant age‐related changes in mechanisms modulating ATP overflow in skeletal muscle resistance vessels and that α2‐pre‐junctional activation may not act in the way previously described in the heart, brain and tail. This research was supported by NIA (R03AG033245) and the Arkansas Biosciences Institute.