Randall W. Bryner

Apoptotic adaptations from exercise training in skeletal and cardiac muscles

The effect of exercise on apoptosis in postmitotic tissues is not known. In this study, we investigated the effect of regular moderate physical activity (i.e., exercise training) on the extent of apoptosis in rat skeletal and cardiac muscles. Adult Sprague Dawley rats were trained (TR) 5 days weekly for 8 wk on treadmill. Sedentary rats served as controls (CON). An ELISA was used to detect mono‐ and oligonucleosome fragmentation as an indicator of apoptosis. Bcl‐2, Bax, Apaf‐1, AIF, cleaved PARP, cleaved caspase‐3, cleaved/active caspase‐9, heat shock protein (HSP)70, Cu/Zn‐SOD, and Mn‐SOD protein levels were determined by Western analyses. Bcl‐2 and Bax transcript contents were estimated by RT‐PCR. A spectrofluorometric assay was used to determine caspase‐3 activity. DNA fragmentation in ventricles of the TR group decreased by 15% whereas that in soleus of the TR group tended to decrease (P=0.058) when compared with CON group. Protein contents of Bcl‐2, HSP70, and Mn‐SOD increased in both soleus and ventricle muscles of TR animals when compared with CON animals. Apaf‐1 protein content in the soleus of TR animals was lower than that of CON animals. Bcl‐2 mRNA levels increased in both ventricle and soleus muscles of TR animals, and Bax mRNA levels decreased in the soleus of TR animals when compared with CON animals. Furthermore, HSP70 protein content was negatively correlated to Bax mRNA content and was positively correlated to Bcl‐2 protein and mRNA contents. Mn‐SOD protein content was negatively correlated to the apoptotic index, and caspase‐3 activity and was positively correlated to Bcl‐2 transcript content and HSP70 protein content. These data suggest that exercise training attenuates the extent of apoptosis in cardiac and skeletal muscles.

Satellite cell proliferation is reduced in muscles of obese Zucker rats but restored with loading

The obese Zucker rat (OZR) is a model of metabolic syndrome, which has lower skeletal muscle size than the lean Zucker rat (LZR). Because satellite cells are essential for postnatal muscle growth, this study was designed to determine whether reduced satellite cell proliferation contributes to reduced skeletal mass in OZR vs. LZR. Satellite cell proliferation was determined by a constant-release 5-bromo-2-deoxyuridine (BrdU) pellet that was placed subcutaneously in each animal. Satellite cell proliferation, as determined by BrdU incorporation, was significantly attenuated in control soleus and plantaris muscles of the OZR compared with that shown in the LZR. To determine whether this attenuation of satellite cell activity could be rescued in OZR muscles, soleus and gastrocnemius muscles were denervated, placing a compensatory load on the plantaris muscle. In the LZR and the OZR after 21 days of loading, increases of approximately 25% and approximately 30%, respectively, were shown in plantaris muscle wet weight compared with that shown in the contralateral control muscle. The number of BrdU-positive nuclei increased similarly in loaded plantaris muscles from LZR and OZR. Myogenin, MyoD, and Akt protein expressions were lower in control muscles of OZR than in those of the LZR, but they were all elevated to similar levels in the loaded plantaris muscles of OZR and LZR. These data indicate that metabolic syndrome may reduce satellite cell proliferation, and this may be a factor that contributes to the reduced mass in control muscles of OZR; however, satellite cell proliferation can be restored with compensatory loading in OZR.

Bax signaling regulates palmitate-mediated apoptosis in C2C12 myotubes

Insulin resistance is a primary characteristic of type 2 diabetes. Several lines of evidence suggest that accumulation of free fatty acids in skeletal muscle may at least in part contribute to insulin resistance and may be linked to mitochondrial dysfunction, leading to apoptosis. Palmitate treatment of several cell lines in vitro results in apoptosis and inhibits protein kinase B (Akt) activity in response to insulin. However, the role of Bax and Bcl-2 in regulating palmitate-induced apoptosis has not been well studied. Therefore, the purpose of this study was to determine whether palmitate-induced apoptosis in C(2)C(12) myotubes is dependent on Bax to Bcl-2 binding. An additional purpose of this study was to determine whether the changes in Bax to Bcl-2 binding corresponded to decreases in Akt signaling in palmitate-treated myoblasts. Apoptotic signaling proteins were examined in C(2)C(12) myotubes treated overnight with palmitate. Bax to Bcl-2 binding was determined through a coimmunoprecipitation assay that was performed in myotubes after 2 h of serum starvation, followed by 10 min of serum reintroduction. This experiment evaluated whether temporal Akt activity coincided with Bax to Bcl-2 binding. Last, the contribution of Bax to palmitate-induced apoptosis was determined by treatment with Bax siRNA. Palmitate treatment increased apoptosis in C(2)C(12) myotubes as shown by a twofold increase in DNA fragmentation, an approximately fivefold increase in caspase-3 activity, and a 2.5-fold increase in caspase-9 activity. Palmitate treatment significantly reduced Akt protein expression and Akt activity. In addition, there was a fourfold reduction in Bax to Bcl-2 binding with palmitate treatment, which mirrored the reduction in Akt(Ser473) phosphorylation. Furthermore, treatment of the C(2)C(12) myotubes with Bax siRNA attenuated the apoptotic effects of palmitate treatment. These data show that palmitate induces Bax-mediated apoptosis in C(2)C(12) myotubes and that this effect corresponds to reductions in Akt(Ser473) phosphorylation.

Mitochondrial apoptotic signaling is elevated in cardiac but not skeletal muscle in the obese Zucker rat and is reduced with aerobic exercise

Mitochondrial apoptosis and apoptotic signaling modulations by aerobic training were studied in cardiac and skeletal muscles of obese Zucker rats (OZR), a rodent model of metabolic syndrome. Comparisons were made between left ventricle, soleus, and gastrocnemius muscles from OZR (n = 16) and aged-matched lean Zucker rats (LZR; n = 16) that were untrained (n = 8) or aerobically trained on a treadmill for 9 wk (n = 8). Cardiac Bcl-2 protein expression levels were approximately 50% lower in the OZR compared with the LZR, with no difference in either of the skeletal muscles. Bax protein expression levels were similar in skeletal muscles of the OZR compared with the LZR. Furthermore, mitochondrial apoptotic signaling was not different in skeletal muscles of OZR and LZR groups. However, there was an approximate sevenfold increase in the Bax protein accumulation in the myocardial mitochondrial-rich protein fraction of the OZR compared with the LZR. Additionally, there was an increase in cytosolic cytochrome c released from the mitochondria, caspase-9 and caspase-3 activity, with a corresponding elevation in DNA fragmentation in the cardiac muscles of the OZR compared with the LZR. Exercise training reduced cardiac Bax protein levels, the mitochondrial localization of Bax, cytosolic cytochrome c, caspase activity, and DNA fragmentation in cardiac muscles of the OZR after exercise, with no change in the skeletal muscles. These data show that mitochondrial apoptosis is elevated in the cardiac but not skeletal muscles of the OZR, but aerobic exercise training was effective in reducing cardiac mitochondrial apoptotic signaling.

Docosahexaenoic Acid protects muscle cells from palmitate-induced atrophy.

Background. Accumulation of free fatty acids leads to lipid-toxicity-associated skeletal muscle atrophy. Palmitate treatment reduces myoblast and myotube growth and causes apoptosis in vitro. It is not known if omega-3 fatty acids will protect muscle cells against palmitate toxicity. Therefore, we examined the effects of docosahexaenoic acid (DHA) on skeletal muscle growth. Methods. Mouse myoblasts (C2C12) were differentiated to myotubes, and then treated with 0 or 0.5 mM palmitic acid or 0 or 0.1 mM DHA. Results. Intramyocellular lipid was increased in palmitate-treated cells but was prevented by DHA-palmitate cotreatment. Total AMPK increased in DHA+ palmitate-treated compared to palmitate only cells. RpS6 phosphorylation decreased after palmitate (−55%) and this was blunted by DHA+ palmitate (−35%) treatment. Palmitate treatment decreased PGC1α protein expression by 69%, but was increased 165% with DHA+ palmitate (P = 0.017) versus palmitate alone. While palmitate induced 25% and 90% atrophy in myotubes (after 48 hours and 96 hours, resp.), DHA+ palmitate treatment caused myotube hypertrophy of ~50% and 100% after 48 and 96 hours, respectively. Conclusion. These data show that DHA is protective against palmitate-induced atrophy. Although DHA did not activate the AMPK pathway, DHA treatment restored growth-signaling (i.e., rpS6) and rescued palmitate-induced muscle atrophy.

Effects of exercise and obesity on UCP3 content in rat hindlimb muscles.

UNLABELLED Uncoupling protein 3 (UCP3) is a mitochondrial inner membrane protein, which is hypothesized to shuttle nonmetabolized fatty acids, particularly when excessive fatty acids are present. PURPOSE Obese Zucker rats (OZR) have systematically elevated levels of fatty acids, with decreased fatty acid metabolism. We hypothesized that basal UCP3 protein expression levels would be elevated in the skeletal muscles of the OZR compared with the lean Zucker rats (LZR). In addition, because aerobic exercise training has been shown to elevate the ability of skeletal muscle to metabolize lipids, we also hypothesized that aerobic exercise training would decrease skeletal muscle UCP3 protein expression and that this would be more pronounced in the skeletal muscles of the OZR. METHODS OZR and LZR were aerobically trained on a motorized treadmill for 55 min x d(-1), 5 d x wk(-1), for 9 wk. UCP3 and oxidative enzymes were measured in plantaris, gastrocnemius, and soleus muscles. RESULTS Basal UCP3 protein expression was elevated approximately eightfold in the plantaris muscles and threefold in the gastrocnemius muscles of the OZR compared with the LZR (P < 0.05). However, there was no difference in UCP3 protein expression in the soleus muscles of the OZR compared with the LZR (P = 0.34). Furthermore, aerobic exercise training did not significantly alter UCP3 protein expression in the soleus, plantaris, or gastrocnemius muscles of the LZR; however, UCP3 protein expression levels decreased in trained OZR soleus and gastrocnemius muscles compared with controls. CONCLUSIONS The decrease in UCP3 with aerobic exercise training was most notable in the soleus of the OZR. These data demonstrate that the exercise-induced adaptations of UCP3 protein levels are muscle specific in obese animals compared with lean animals.

Loss of Adipocyte Vegf Impairs Endurance Exercise Capacity in Mice

PURPOSE Reducing vascular endothelial growth factor (VEGF) in adipose tissue alters adipose vascularity and metabolic homeostasis. We hypothesized that this would also affect metabolic responses during exercise-induced stress and that adipocyte-specific VEGF-deficient (adipoVEGF-/-) mice would have impaired endurance capacity. METHODS Endurance exercise capacity in adipoVEGF-/- (n = 10) and littermate control (n = 11) mice was evaluated every 4 wk between 6 and 24 wk of age using a submaximal endurance run to exhaustion at 20 m·min(-1) at 10° incline. Maximal running speed, using incremental increases in speed at 30-s intervals, was tested at 25 and 37 wk of age. RESULTS White and brown adipose tissue capillarity were reduced by 40% in adipoVEGF-/-, and no difference in skeletal muscle capillarity was observed. Endurance run time to exhaustion was 30% lower in adipoVEGF-/- compared with that in controls at all time points (P < 0.001), but no difference in maximal running speed was observed between the groups. After exercise (1 h at 50% maximum running speed), adipoVEGF-/- mice displayed lower circulating insulin (P < 0.001), lower glycerol (P < 0.05), and tendency for lower blood glucose (P = 0.06) compared with controls. There was no evidence of altered oxidative damage or changes in carnitine palmitoyltransferase-1β expression in skeletal muscle of adipoVEGF-/- mice. CONCLUSIONS These data suggest that VEGF-mediated deficits in adipose tissue blunt the availability of lipid substrates during endurance exercise, which likely reduced endurance performance. Surprisingly, we also found an unchanged basal blood glucose despite lower circulating insulin in adipoVEGF-/- mice, suggesting that loss of adipocyte VEGF can blunt insulin release and/or increase basal insulin sensitivity.

Respiratory muscle training positively affects vasomotor response in young healthy women

Vasomotor response is related to the capacity of the vessel to maintain vascular tone within a narrow range. Two main control mechanisms are involved: the autonomic control of the sympathetic neural drive (global control) and the endothelial smooth cells capacity to respond to mechanical stress by releasing vasoactive factors (peripheral control). The aim of this study was to evaluate the effects of respiratory muscle training (RMT) on vasomotor response, assessed by flow-mediated dilation (FMD) and heart rate variability, in young healthy females. The hypothesis was that RMT could enhance the balance between sympathetic and parasympathetic neural drive and reduce vessel shear stress. Thus, twenty-four women were randomly assigned to either RMT or SHAM group. Maximal inspiratory mouth pressure and maximum voluntary ventilation were utilized to assess the effectiveness of the RMT program, which consisted of three sessions of isocapnic hyperventilation/ week for eight weeks, (twenty-four training sessions). Heart rate variability assessed autonomic balance, a global factor regulating the vasomotor response. Endothelial function was determined by measuring brachial artery vasodilation normalized by shear rate (%FMD/SR). After RMT, but not SHAM, maximal inspiratory mouth pressure and maximum voluntary ventilation increased significantly (+31% and +16%, respectively). Changes in heart rate variability were negligible in both groups. Only RMT exhibited a significant increase in %FMD/SR (+45%; p<0.05). These data suggest a positive effect of RMT on vasomotor response that may be due to a reduction in arterial shear stress, and not through modulation of sympatho-vagal balance.

Psychological stress‐induced cerebrovascular dysfunction: the role of metabolic syndrome and exercise

What is the central question of this study? How does chronic stress impact cerebrovascular function and does metabolic syndrome accelerate the cerebrovascular adaptations to stress? What role does exercise training have in preventing cerebrovascular changes to stress and metabolic syndrome? What is the main finding and its importance? Stressful conditions lead to pathological adaptations of the cerebrovasculature via an oxidative nitric oxide pathway, and the presence of metabolic syndrome produces a greater susceptibility to stress‐induced cerebrovascular dysfunction. The results also provide insight into the mechanisms that may contribute to the influence of stress and the role of exercise in preventing the negative actions of stress on cerebrovascular function and structure.