Epinephrine and AICAR-induced PGC-1α mRNA expression is intact in skeletal muscle from rats fed a high-fat diet.

Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) is a master regulator of mitochondrial biogenesis and is controlled, at least in part, through AMP-activated protein kinase and p38-dependent pathways. There is evidence demonstrating that activation of these kinases and induction of PGC-1α in skeletal muscle are regulated by catecholamines. The purpose of the present study was to determine if consumption of a high-fat diet (HFD) impairs epinephrine and 5-aminoimidazole-4-carboxamide-1ß-d-ribofuranoside (AICAR) signaling and induction of PGC-1α in rat skeletal muscle. Male Wistar rats were fed chow or a HFD for 6 wk and then given a weight-adjusted bolus injection of epinephrine (20, 10, or 5 µg/100 g body wt sc) or saline, and triceps muscles were harvested 30 min (signaling) or 2 and 4 h (gene expression) postinjection. Despite blunted increases in p38 phosphorylation, the ability of epinephrine to induce PGC-1α was intact in skeletal muscle from HFD-fed rats and was associated with normal increases in activation of PKA and phosphorylation of cAMP response element-binding protein, reputed mediators of PGC-1α expression. The attenuated epinephrine-mediated increase in p38 phosphorylation was independent of increases in MAPK phosphatase 1. At 2 h following AICAR treatment (0.5 g/kg body wt sc), AMP-activated protein kinase and acetyl-CoA carboxylase phosphorylation were similar in skeletal muscle from chow- and HFD-fed rats. Surprisingly, AICAR-induced increases in PGC-1α mRNA levels were greater in skeletal muscle from HFD-fed rats. Our results demonstrate that the ability of epinephrine and AICAR to induce PGC-1α remains intact in skeletal muscle from HFD-fed rats. These results question the existence of reduced ß-adrenergic responsiveness in diet-induced obesity and demonstrate that increases in p38 phosphorylation are not required for induction of PGC-1α in muscle from obese rats.

Epinephrine induces PDK4 mRNA expression in adipose tissue from obese, insulin resistant rats.

Thiazolidinediones (TZDs) are a commonly prescribed class of insulin sensitizing drugs that increase fatty acid re-esterification, in part through the induction of pyruvate dehydrogenase kinase 4 (PDK4). Owing to the deleterious side effects of TZDs the identification of alternative approaches with which to increase PDK4 is essential. We recently demonstrated that epinephrine increases PDK4 expression through p38 and peroxisome proliferator-activated receptor γ (PPARγ) dependent pathways in cultured adipose tissue from lean rats. The purpose of this study was to determine whether acute epinephrine treatment, in vivo, can induce PDK4 mRNA expression in adipose tissue from obese, insulin resistant rats and if the reputed signaling pathways mediating this effect are intact. To this end we fed male Wistar rats a chow or high-fat diet (HFD, 60% kcals from fat) for 6 weeks. Rats were then injected with a weight-adjusted bolus of epinephrine and tissue harvested. Despite a blunted activation of p38 epinephrine increased PDK4 mRNA expression to a similar extent in adipose tissue from chow and HFD rats. 5'AMP-activated protein kinase (AMPK) signaling was not altered by the HFD. Similar to epinephrine, 2 h of swim exercise, an intervention that increases plasma catecholamines, also increased PDK4 mRNA levels to a similar extent in adipose tissue from both lean and HFD rats. Collectively these findings demonstrate, for the first time, that acute elevations in catecholamines induce PDK4 in adipose tissue from HFD rats, that this effect is likely independent of p38, a reputed mediator of PDK4 expression and that exercise, similar to TZDs can induce PDK4 in adipose tissue from obese, insulin resistant rats.

Dietary supplementation with vitamin E and C attenuates dexamethasone-induced glucose intolerance in rats.

Glucocorticoid excess induces marked insulin resistance and glucose intolerance. A recent study has shown that antioxidants prevent dexamethasone (DEX)-induced insulin resistance in cultured adipocytes. The purpose of this investigation was to examine the effects of dietary vitamin E and C (Vit E/C) supplementation on DEX-induced glucose intolerance in rats. We hypothesized that feeding rats a diet supplemented with Vit E/C would improve glucose tolerance and restore insulin signaling in skeletal muscle, adipose, and liver and prevent alterations in AMPK signaling in these tissues. Male Wistar rats received either a control or Vit E/C-supplemented diet (0.5 g/kg diet each of L-ascorbate and DL-all rac-alpha-tocopherol) for 9 days prior to, and during, 5 days of daily DEX treatment (subcutaneous injections 0.8 mg/g body wt). DEX treatment resulted in increases in the glucose and insulin area under the curve (AUC) during an intraperitoneal glucose tolerance test. The glucose, but not insulin, AUC was lowered with Vit E/C supplementation. Improvements in glucose tolerance occurred independent of a restoration of PKB phosphorylation in tissues of rats stimulated with an intraperitoneal injection of insulin but were associated with increases in AMPK signaling in muscle and reductions in AMPK signaling and the expression of fatty acid oxidation enzymes in liver. There were no differences in mitochondrial enzymes in triceps muscles between groups. This study is the first to report that dietary Vit E/C supplementation can partially prevent DEX-induced glucose intolerance in rats.

Reductions in RIP140 are not required for exercise- and AICAR-mediated increases in skeletal muscle mitochondrial content.

Receptor interacting protein 1 (RIP140) has recently been demonstrated to be a key player in the regulation of skeletal muscle mitochondrial content. We have shown that ß-guanadinopropionic acid (ß-GPA) feeding reduces RIP140 protein content and mRNA levels concomitant with increases in mitochondrial content (Williams DB, Sutherland LN, Bomhof MR, Basaraba SA, Thrush AB, Dyck DJ, Field CJ, Wright DC. Am J Physiol Endocrinol Metab 296: E1400-E1408, 2009). Since ß-GPA feeding reduces high-energy phosphate levels and activates AMPK, alterations reminiscent of exercise, we hypothesized that exercise training would reduce RIP140 protein content. We further postulated that an acute bout of exercise, or interventions known to induce the expression of mitochondrial enzymes or genes involved in mitochondrial biogenesis, would result in decreases in nuclear RIP140 content. Two weeks of daily swim training increased markers of mitochondrial content in rat skeletal muscle independent of reductions in RIP140 protein. Similarly, high-intensity exercise training in humans failed to reduce RIP140 content despite increasing skeletal muscle mitochondrial enzymes. We found that 6 wk of daily 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR) injections had no effect on RIP140 protein content in rat skeletal muscle while RIP140 content from LKB1 knockout mice was unaltered despite reductions in mitochondria. An acute bout of exercise, AICAR treatment, and epinephrine injections increased the mRNA levels of PGC-1α, COXIV, and lipin1 independent of decreases in nuclear RIP140 protein. Surprisingly these interventions increased RIP140 mRNA expression. In conclusion our results demonstrate that decreases in RIP140 protein content are not required for exercise and AMPK-dependent increases in skeletal muscle mitochondrial content, nor do acute perturbations alter the cellular localization of RIP140 in parallel with the induction of genes involved in mitochondrial biogenesis.

Interactions between the consumption of a high-fat diet and fasting in the regulation of fatty acid oxidation enzyme gene expression: an evaluation of potential mechanisms.

The consumption of high-fat diets (HFDs) and fasting are known to increase the expression of enzymes involved in fatty acid oxidation (FAO). However, it has been reported that the ability of physiological stressors to induce enzymes of FAO in skeletal muscle is blunted with obesity. In this regard, we sought to explore the effects and potential mechanisms of an HFD on the expression of FAO enzymes in the fed and fasted state. The consumption of an HFD increased the mRNA expression or protein content of medium-chain acyl-CoA dehydrogenase (MCAD), uncoupling protein-3 (UCP3), and pyruvate dehydrogenase kinase 4 (PDK4) in the fed state. Fasting increased the mRNA expression of PDK4, MCAD, and UCP-3, and the protein content of UCP-3 in chow but not HFD rats. HFDs did not increase carnitine palmitoyl transfer-1 (CPT-1) mRNA levels in the fed state and the effects of fasting were markedly reduced compared with chow-fed rats. The expression of peroxisome-proliferator-activated receptor-γ coactivator-1ß (PGC-1ß) was increased in muscle from HFD rats in the fed state, while PGC-1-related coactivator (PRC) was increased with fasting in chow-fed but not HFD rats. Plasma fatty acid levels were elevated in the fed state from HFD rats but not increased further with fasting, whereas fasting increased plasma fatty acids in chow-fed animals. Fasting-mediated increases in plasma epinephrine, and the activation of PKA and AMPK in skeletal muscle were similar between chow and HFD rats. p38 MAPK phosphorylation was increased with fasting in chow-fed but not HFD rats. Our findings suggest that a blunted effect of fasting on the induction of PDK4, MCAD, and UCP3 in skeletal muscle from HFD rats is likely a result of already elevated levels of these enzymes, the induction of which is associated with increases in plasma fatty acid and PGC-1ß. On the other hand, a blunted induction of PRC and CPT-1 mRNA may be explained by decreases in p38 MAPK signaling.

Epinephrine-mediated regulation of PDK4 mRNA in rat adipose tissue.

Fatty acid reesterification in adipose tissue is dependent on the generation of glycerol 3-phosphate, and, at least in rodent adipose tissue, this appears to occur primarily through glyceroneogenesis. A key enzyme in this process is pyruvate dehydrogenase kinase 4 (PDK4). PDK4 is induced in white adipose tissue by thiazolidinediones (TZDs) and the inhibition or knockdown of PDK4 inhibits TZD-induced increases in glyceroneogenesis. Since TZDs have many unwanted side effects, we were interested in identifying alternative mechanisms that could regulate PDK4 mRNA expression in white adipose tissue. In this regard we hypothesized that exercise, fasting, and epinephrine would increase PDK4 mRNA levels in rat epididymal adipose tissue. We further postulated that the p38 mitogen-activated protein kinase (MAPK) and 5'-AMP-activated protein kinase (AMPK) signaling pathways would control PDK4 mRNA expression in cultured adipose tissue. Exercise, fasting, and in or ex vivo epinephrine treatment increased PDK4 mRNA levels. These perturbations did not increase the expression of PDK1, -2, or -3. Pyruvate dehydrogenase phosphorylation was increased after an overnight fast and 4 h after the cessation of exercise. In cultured adipose tissue, epinephrine increased p38 and AMPK signaling; however, the direct activation of AMPK by AICAR or metformin led to reductions in PDK4 mRNA levels. The p38 inhibitor SB202190 reduced epinephrine-mediated increases in p38 MAPK activation without altering hormone-sensitive lipase or AMPK phosphorylation or attenuating epinephrine-induced increases in lipolysis. Reductions in p38 MAPK signaling were associated with decreases in PDK4 mRNA expression. The inhibition of peroxisome proliferator-activated receptor-γ (PPARγ) also attenuated the induction of PDK4. Our results are the very first to demonstrate an epinephrine-mediated regulation of PDK4 mRNA levels in white adipose tissue and suggest that p38 MAPK and PPARγ could be involved in this pathway.

Rapid in vivo whole body composition of rats using cone beam µCT.

Precise, noninvasive analysis and quantification of in vivo body composition is essential for research involving longitudinal, small-animal disease models. We investigated the feasibility and precision of a rapid, flat-panel µCT scanner to report whole body adipose tissue volume (ATV), lean tissue volume (LTV), skeletal tissue volume (STV), and bone mineral content (BMC) in 25 postmortem female and 52 live male Sprague-Dawley rats. µCT images, acquired in three 90-mm segments and reconstructed with 308 µm of isotropic voxel spacing, formed contiguous image volumes of each entire rat specimen. Three signal-intensity thresholds (determined to be -186, 5, and 155 HU) were used to classify each voxel as adipose, lean, or skeletal tissue, respectively. Tissue masses from the volume fractions of ATV, LTV, and STV were calculated from assumed tissue densities of 0.95, 1.05, and 1.92 g/cm(-3), respectively. A CT-derived total mass was calculated for each rat and compared with the gravimetrically measured mass, which differed on average for the postmortem female and the live male group by 2.5 and 1.1%, respectively. To evaluate the accuracy of the CT-derived body composition technique, following the live male study excised muscle tissue in the lower right leg of all rats in group B were compared with the image-derived LT measurement of the same regional compartment and found to differ on average by 2.2%. Through repeated CT measurements of postmortem specimens, the whole body ATV, LTV, STV, and BMC measurement analysis gave a precision value of ±0.6, 1.9, 1.7, and 0.5% of the average value, respectively.

The effects of apelin treatment on skeletal muscle mitochondrial content.

Adipose tissue is recognized as a key player in the regulation of whole body metabolism. Apelin, is a recently identified adipokine that when given to mice results in increases in skeletal muscle uncoupling protein 3 (UCP3) content. Similarly, acute apelin treatment has been shown to increase the activity of 5'-AMP-activated protein kinase (AMPK), a reputed mediator of skeletal muscle mitochondrial biogenesis. Given these findings, we sought to determine the effects of apelin on skeletal muscle mitochondrial content. Male Wistar rats were given daily intraperitoneal injections of apelin-13 (100 nmol/kg) for 2 wk. We made the novel observation that the activities of citrate synthase, cytochrome c oxidase, and beta-hydroxyacyl coA dehydrogenase (betaHAD) were increased in triceps but not heart and soleus muscles from apelin-treated rats. When confirming these results we found that both nuclear and mitochondrial-encoded subunits of the respiratory chain were increased in triceps from apelin-treated rats. Similarly, apelin treatment increased the protein content of components of the mitochondrial import and assembly pathway. The increases in mitochondrial marker proteins were associated with increases in proliferator-activated receptor-gamma coactivator-1 (PGC-1beta) but not PGC-1alpha or Pgc-1-related co-activator (PRC) mRNA expression. Chronic and acute apelin treatment did not increase the protein content and/or phosphorylation status of AMPK and its downstream substrate acetyl-CoA carboxylase. These findings are the first to demonstrate that apelin treatment can induce skeletal muscle mitochondrial content. Given the lack of an effect of apelin on AMPK signaling and PGC-1alpha mRNA expression, these results suggest that apelin increases skeletal muscle mitochondrial content through a mechanism that is distinct from that of more robust physiological stressors.

Diabetes-induced atrophy is associated with a muscle-specific alteration in NF-kappaB activation and expression.

NF-kappaB is a transcription factor implicated in pathological responses that develop during diabetes mellitus, including skeletal muscle atrophy. Given that NF-kappaB activation, protein composition, and content within diabetic skeletal muscle remain generally uncharacterized, a streptozotocin (STZ) model was used to assess NF-kappaB activation, composition, and content. Sprague-Dawley rats were injected with STZ (55 mg/kg) and after 30 days the soleus (SOL), plantaris (PL), red gastrocnemius (RG), and white gastrocnemius (WG) muscles were assessed by electrophoresis mobility shift assay and western blotting. NF-kappaB activation was detected in all muscles examined, but was reduced in RG muscles from diabetic animals. Supershifts indicated NF-kappaB was composed primarily of p50 in diabetic and control animals. The content of both p65 and p52 was elevated in SOL and PL muscles, while p52 was decreased in RG. The coactivating protein, Bcl-3, was increased in WG and RG, but decreased in PL. Both p50 and RelB remained unchanged in all tissues examined. All muscles from diabetic animals demonstrated reduced mass when compared to controls, but only the gastrocnemius demonstrated atrophy as reflected by a reduced muscle-to-body mass ratio. In conclusion, diabetic alterations to the contents and activation of the NF-kappaB protein were tissue-specific, but did not appear to alter dimer composition of constitutively bound NF-kappaB. These results indicate that diabetes may alter NF-kappaB activity and expression in a muscle-specific manner.

Respiratory muscle training and the performance of a simulated anti-G straining maneuver.

INTRODUCTION: Prolonged +G,-exposure eventually decreases a pilot's ability to maintain an effective anti-G straining maneuver (AGSM). Previous studies have implicated the respiratory muscles (RMs) as main contributors to this AGSM-induced fatigue. Thus, this study aimed to investigate if respiratory muscle training (RMT) may be of benefit to improve RM strength, endurance, and performance of the AGSM. METHODS: Subjects (N=14; 27 +/- 5.3 yrs) trained with a commercially available RM trainer for 6 wk, 4 times/wk 20 min per session. Data collection consisted of pulmonary function tests (PFTs) and a RM testing protocol simulating the AGSM. Testing occurred every 2 wk for the duration of RMT, and similarly during the 6-wk control (CON) phase where subjects did not train. The simulated AGSM performance was evaluated through measures of peak respiratory pressures, peak systolic arterial pressure (SAP), mean arterial pressure (MAP), and tidal volumes. RESULTS: Training significantly improved (P < 0.05) RM strength after 6 wk of RMT measured in maximal expiratory pressures (RMT = 207.8 +/- 15.8 cmH2O; CON = 181.3 +/- 13.7 cmH2O) and maximal inspiratory pressures (RMT = -154.7 +/- 8.9 cmH2O; CON = -141.9 +/- 8.5 cmH2O). All other PFTs were unchanged. During performance of the AGSM, only peak expiratory pressure demonstrated an increased performance benefit (RMT = 91.5 +/- 5.9 cmH2O; CON = 82.8 +/- 4.3 cmH2O). Peak inspiratory pressure, SAP, MAP, and tidal volumes remained unchanged. CONCLUSION: Without evident translation of the increased RM strength to performance of the AGSM at +1 Gz, the benefits of RMT for ameliorating AGSM-induced fatigue within the high +G, environment are limited.

Heat stress inhibits skeletal muscle hypertrophy.

Heat shock proteins (Hsps) are molecular chaperones that aid in protein synthesis and trafficking and have been shown to protect cells/tissues from various protein damaging stressors. To determine the extent to which a single heat stress and the concurrent accumulation of Hsps influences the early events of skeletal muscle hypertrophy, Sprague-Dawley rats were heat stressed (42 degrees C, 15 minutes) 24 hours prior to overloading 1 plantaris muscle by surgical removal of the gastrocnemius muscle. The contralateral plantaris muscles served as controls. Heat-stressed and/or overloaded plantaris muscles were assessed for muscle mass, total muscle protein, muscle protein concentration, Type I myosin heavy chain (Type I MHC) content, as well as Hsp72 and Hsp25 content over the course of 7 days following removal of the gastrocnemius muscle. As expected, in non-heat-stressed animals, muscle mass, total muscle protein and MHC I content were significantly increased (P < 0.05) following overload. In addition, Hsp25 and Hsp72 increased significantly after 2 and 3 days of overload, respectively. A prior heat stress-elevated Hsp25 content to levels similar to those measured following overload alone, but heat stress-induced Hsp72 content was increased significantly greater than was elicited by overload alone. Moreover, overloaded muscles from animals that experienced a prior heat stress showed a lower muscle mass increase at 5 and 7 days; a reduced total muscle protein elevation at 3, 5, and 7 days; reduced protein concentration; and a diminished Type I MHC content accumulation at 3, 5, and 7 days relative to nonheat-stressed animals. These data suggest that a prior heat stress and/or the consequent accumulation of Hsps may inhibit increases in muscle mass, total muscle protein content, and Type I MHC in muscles undergoing hypertrophy.