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-1β-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 β-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.     

Effect of exercise intensity and AICAR on isoform-specific expressions of murine skeletal muscle PGC-1α mRNA: a role of β₂-adrenergic receptor activation

There are three isoforms of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) mRNA, which promotes mitochondrial biogenesis in skeletal muscles. Compared with PGC-1α-a mRNA, PGC-1α-b or PGC-1α-c mRNA is transcribed by a different exon 1 of the PGC-1α gene. In this study, effects of exercise intensity and 5-aminoimidazole-4-carboxamide-1β-d-ribofuranoside (AICAR) on isoform-specific expressions of PGC-1α were investigated. All isoforms were increased in proportion to exercise intensity of treadmill running (10-30 m/min for 30 min). Preinjection of β?-adrenergic receptor (AR) antagonist (ICI 118551) inhibited the increase in PGC-1α-b and PGC-1α-c mRNAs, but not the increase in PGC-1α-a mRNA, in response to high-intensity exercise. Although high-intensity exercise activated α2-AMP-activated protein kinase (α2-AMPK) in skeletal muscles, inactivation of α2-AMPK activity did not affect high-intensity exercise-induced mRNA expression of all PGC-1α isoforms, suggesting that activation of α2-AMPK is not mandatory for an increase in PGC-1α mRNA by high-intensity exercise. A single injection in mice of AICAR, an AMPK activator, increased mRNAs of all PGC-1α isoforms. AICAR increased blood catecholamine concentrations, and preinjection of β?-AR antagonist inhibited the increase in PGC-1α-b and PGC-1α-c mRNAs but not the increase in PGC-1α-a mRNA. Direct exposure of epitrochlearis muscle to AICAR increased PGC-1α-a but not the -b isoform. These data indicate that exercise-induced PGC-1α expression was dependent on the intensity of exercise. Exercise or AICAR injection increased PGC-1α-b and PGC-1α-c mRNAs via β?-AR activation, whereas high-intensity exercise increased PGC-1α-a expression by a multiple mechanism in which α2-AMPK is one of the signaling pathways.     

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-1β (PGC-1β) 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-1β. On the other hand, a blunted induction of PRC and CPT-1 mRNA may be explained by decreases in p38 MAPK signaling.     

Sirtuin 1 (SIRT1) deacetylase activity is not required for mitochondrial biogenesis or peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) deacetylation following endurance exercise

The protein deacetylase, sirtuin 1 (SIRT1), is a proposed master regulator of exercise-induced mitochondrial biogenesis in skeletal muscle, primarily via its ability to deacetylate and activate peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). To investigate regulation of mitochondrial biogenesis by SIRT1 in vivo, we generated mice lacking SIRT1 deacetylase activity in skeletal muscle (mKO). We hypothesized that deacetylation of PGC-1α and mitochondrial biogenesis in sedentary mice and after endurance exercise would be impaired in mKO mice. Skeletal muscle contractile characteristics were determined in extensor digitorum longus muscle ex vivo. Mitochondrial biogenesis was assessed after 20 days of voluntary wheel running by measuring electron transport chain protein content, enzyme activity, and mitochondrial DNA expression. PGC-1α expression, nuclear localization, acetylation, and interacting protein association were determined following an acute bout of treadmill exercise (AEX) using co-immunoprecipitation and immunoblotting. Contrary to our hypothesis, skeletal muscle endurance, electron transport chain activity, and voluntary wheel running-induced mitochondrial biogenesis were not impaired in mKO versus wild-type (WT) mice. Moreover, PGC-1α expression, nuclear translocation, activity, and deacetylation after AEX were similar in mKO versus WT mice. Alternatively, we made the novel observation that deacetylation of PGC-1α after AEX occurs in parallel with reduced nuclear abundance of the acetyltransferase, general control of amino-acid synthesis 5 (GCN5), as well as reduced association between GCN5 and nuclear PGC-1α. These findings demonstrate that SIRT1 deacetylase activity is not required for exercise-induced deacetylation of PGC-1α or mitochondrial biogenesis in skeletal muscle and suggest that changes in GCN5 acetyltransferase activity may be an important regulator of PGC-1α activity after exercise.     

Rescue of dystrophic skeletal muscle by PGC-1α involves a fast to slow fiber type shift in the mdx mouse

Increased utrophin expression is known to reduce pathology in dystrophin-deficient skeletal muscles. Transgenic over-expression of PGC-1α has been shown to increase levels of utrophin mRNA and improve the histology of mdx muscles. Other reports have shown that PGC-1α signaling can lead to increased oxidative capacity and a fast to slow fiber type shift. Given that it has been shown that slow fibers produce and maintain more utrophin than fast skeletal muscle fibers, we hypothesized that over-expression of PGC-1α in post-natal mdx mice would increase utrophin levels via a fiber type shift, resulting in more slow, oxidative fibers that are also more resistant to contraction-induced damage. To test this hypothesis, neonatal mdx mice were injected with recombinant adeno-associated virus (AAV) driving expression of PGC-1α. PGC-1α over-expression resulted in increased utrophin and type I myosin heavy chain expression as well as elevated mitochondrial protein expression. Muscles were shown to be more resistant to contraction-induced damage and more fatigue resistant. Sirt-1 was increased while p38 activation and NRF-1 were reduced in PGC-1α over-expressing muscle when compared to control. We also evaluated if the use a pharmacological PGC-1α pathway activator, resveratrol, could drive the same physiological changes. Resveratrol administration (100 mg/kg/day) resulted in improved fatigue resistance, but did not achieve significant increases in utrophin expression. These data suggest that the PGC-1α pathway is a potential target for therapeutic intervention in dystrophic skeletal muscle.     

An acute bout of high-intensity interval training increases the nuclear abundance of PGC-1α and activates mitochondrial biogenesis in human skeletal muscle

Low-volume, high-intensity interval training (HIT) increases skeletal muscle mitochondrial capacity, yet little is known regarding potential mechanisms promoting this adaptive response. Our purpose was to examine molecular processes involved in mitochondrial biogenesis in human skeletal muscle in response to an acute bout of HIT. Eight healthy men performed 4 × 30-s bursts of all-out maximal intensity cycling interspersed with 4 min of rest. Muscle biopsy samples (vastus lateralis) were obtained immediately before and after exercise, and after 3 and 24 h of recovery. At rest, the majority of peroxisome proliferator-activated receptor γ coactivator (PGC)-1α, a master regulator of mitochondrial biogenesis, was detected in cytosolic fractions. Exercise activated p38 MAPK and AMPK in the cytosol. Nuclear PGC-1α protein increased 3 h into recovery from exercise, a time point that coincided with increased mRNA expression of mitochondrial genes. This was followed by an increase in mitochondrial protein content and enzyme activity after 24 h of recovery. These findings support the hypothesis that an acute bout of low-volume HIT activates mitochondrial biogenesis through a mechanism involving increased nuclear abundance of PGC-1α.     

Chronic AMPK stimulation attenuates adaptive signaling in dystrophic skeletal muscle

In the present study, we evaluated how a pharmacologically induced phenotype shift in dystrophic skeletal muscle would affect subsequent intracellular signaling in response to a complementary, adaptive physiological stimulus. mdx mice were treated with the AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR; 500 mg·kg(-1)·day(-1)) for 30 days, and then one-half of the animals were subjected to a bout of treadmill running to induce acute AMPK and p38 MAPK signaling. The mRNA levels of phenotypic modifiers, including peroxisome proliferator-activated receptor-δ (PPARδ), PPARγ coactivator-1α (PGC-1α), receptor interacting protein 140 (RIP 140), and silent information regulator two ortholog 1 (SIRT1) were assessed in skeletal muscle, as well as the expression of the protein arginine methyltransferase genes PRMT1 and CARM1. We found unique AMPK and p38 phosphorylation and expression signatures between dystrophic and healthy muscle. In dystrophic skeletal muscle, treadmill running induced PPARδ, PGC-1α, and SIRT1 mRNAs, three molecules that promote the slow, oxidative myogenic program. In the mdx animals that received the chronic AICAR treatment, running-elicited AMPK and p38 phosphorylation was attenuated compared with vehicle-treated mice. Similarly, acute stress-evoked expression of PPARδ, PGC-1α, and SIRT1 was also blunted by chronic pharmacological AMPK stimulation. Skeletal muscle PRMT1 and CARM1 protein contents were higher in mdx mice compared with wild-type littermates. The acute running-evoked induction of PRMT1 and CARM1 mRNAs was also attenuated by the AICAR treatment. Our data demonstrate that prior pharmacological conditioning is a salient determinant in how dystrophic muscle adapts to subsequent complementary, acute physiological stress stimuli. These results provide insight into possible therapeutic applications of synthetic agonists in neuromuscular diseases, such as during chronic administration to Duchenne muscular dystrophy patients.     

Targeting EP4 by curcumin through cross talks of AMP-dependent kinase alpha and p38 mitogen-activated protein kinase signaling: The role of PGC-1α and Sp1

Head and neck cancer is one of the most morbid human malignancies with an overall poor prognosis and severely compromised quality of life. As a result, there is significant interest in developing adjuvant therapies to augment currently available treatment protocols. Curcumin has been found to possess anti-cancer activities via its effect on a variety of biological pathways. In this study, we showed that curcumin inhibits head and neck cancer cell growth through reduction of PGE₂ receptor EP4 gene expression. Blockade of AMP-dependent kinase (AMPK), and p38 MAPK by either chemical inhibitors or siRNAs antagonized the inhibitory effect of curcumin on EP4 expression, which was reversed by metformin, an activator of AMPK. Curcumin induced PGC-1α protein that was blocked by compound C and SB239063. Silencing of PGC-1α reversed the effect of curcumin on EP4 protein. Overexpression of EP4 overcame the effect of curcumin on head and neck cancer cell growth. In addition, curcumin reduced Sp1 protein. Overexpression of Sp1 resisted the inhibitory effect of curcumin on EP4 promoter activity and protein expression. Interestingly, overexpression of PGC-1α further enhanced the inhibitory effect of curcumin on Sp1 protein expression that was blocked by SB239063. In conclusion, this study shows that curcumin inhibits EP4 gene expression dependent of AMPKα and p38 MAPK activation, this leads to reduction of Sp1 protein and binding to specific area in the EP4 gene promoter. The cross talks of AMPKα and p38 MAPK signaling, the kinase-mediated PGC-1α expression and reciprocity of PGC-1α and Sp1 enhance this process. This ultimately results in inhibition of head and neck cancer cell proliferation.     

The inhibitory effect of dexamethasone on platelet-derived growth factor-induced vascular smooth muscle cell migration through up-regulating PGC-1α expression

Dexamethasone has been shown to inhibit vascular smooth muscle cell (VSMC) migration, which is required for preventing restenosis. However, the mechanism underlying effect of dexamethasone remains unknown. We have previously demonstrated that peroxisome proliferator-activated receptor gamma (PPARγ) coactivator-1 alpha (PGC-1α) can inhibit VSMC migration and proliferation. Here, we investigated the role of PGC-1α in dexamethasone-reduced VSMC migration and explored the possible mechanism. We first examined PGC-1α expression in cultured rat aortic VSMCs. The results revealed that incubation of VSMCs with dexamethasone could significantly elevate PGC-1α mRNA expression. In contrast, platelet-derived growth factor (PDGF) decreased PGC-1α expression while stimulating VSMC migration. Mechanistic study showed that suppression of PGC-1α by small interfering RNA strongly abrogated the inhibitory effect of dexamethasone on VSMC migration, whereas overexpression of PGC-1α had the opposite effect. Furthermore, an analysis of MAPK signal pathways showed that dexamethasone inhibited ERK and p38 MAPK phosphorylation in VSMCs. Overexpression of PGC-1α decreased both basal and PDGF-induced p38 MAPK phosphorylation, but it had no effect on ERK phosphorylation. Finally, inhibition of PPARγ activation by a PPARγ antagonist GW9662 abolished the suppressive effects of PGC-1α on p38 MAPK phosphorylation and VSMC migration. These effects of PGC-1α were enhanced by a PPARγ agonist troglitazone. Collectively, our data indicated for the first time that one of the anti-migrated mechanisms of dexamethasone is due to the induction of PGC-1α expression. PGC-1α suppresses PDGF-induced VSMC migration through PPARγ coactivation and, consequently, p38 MAPK inhibition.     

The protective role of peroxisome proliferator-activated receptor γ coactivator-1α in hyperthyroid cardiac hypertrophy

Heart failure is a major cause of death throughout the world. Hyperthyroidism has been shown to induce cardiac hypertrophy, which is a contributing factor to heart failure. However, the mechanism underling effect of thyroid hormone is not completely clear. The present study investigates the role of peroxisome proliferator-activated receptor (PPAR) γ coactivator-1α (PGC-1α) in cardiac hypertrophy induced by Triiodothyronine (T3). We investigated PGC-1α mRNA expression in rat hearts exposed to T3 in vivo and ex vivo. Surprisingly, we found that the extended periods of T3 treatment led to an increase in PGC-1α expression compared to shorter treatment times, which resulted in a reduction of PGC-1α expression. Mechanistic studies showed that suppression of PGC-1α by small interfering RNA in cardiomyocytes amplified the cellular hypertrophic response to T3 stimulation, whereas overexpression of PGC-1α was protective. Furthermore, we presented evidence to show that T3 decreased PGC-1α expression via p38 mitogen-activated protein kinases (MAPK) pathway. Our studies also revealed that overexpression of PGC-1α in cardiomyocytes inhibited basal and T3-induced p38 MAPK phosphorylation. These data indicate for the first time that PGC-1α plays protective role in T3-induced cardiac hypertrophy and that hypertrophic growth induced by T3 involves a regulatory pathway between PGC-1α and p38 MAPK.     

Skeletal muscle PGC-1α is required for maintaining an acute LPS-induced TNFα response

Many lifestyle-related diseases are associated with low-grade inflammation and peroxisome proliferator activated receptor γ coactivator (PGC)-1α has been suggested to be protective against low-grade inflammation. However, whether these anti-inflammatory properties affect acute inflammation is not known. The aim of the present study was therefore to investigate the role of muscle PGC-1α in acute inflammation. Quadriceps muscles were removed from 10-week old whole body PGC-1α knockout (KO), muscle specific PGC-1α KO (MKO) and muscle-specific PGC-1α overexpression mice (TG), 2 hours after an intraperitoneal injection of either 0.8 μg LPS/g body weight or saline. Basal TNFα mRNA content was lower in skeletal muscle of whole body PGC-1α KO mice and in accordance TG mice showed increased TNFα mRNA and protein level relative to WT, indicating a possible PGC-1α mediated regulation of TNFα. Basal p65 phosphorylation was increased in TG mice possibly explaining the elevated TNFα expression in these mice. Systemically, TG mice had reduced basal plasma TNFα levels compared with WT suggesting a protective effect against systemic low-grade inflammation in these animals. While TG mice reached similar TNFα levels as WT and showed more marked induction in plasma TNFα than WT after LPS injection, MKO PGC-1α mice had a reduced plasma TNFα and skeletal muscle TNFα mRNA response to LPS. In conclusion, the present findings suggest that PGC-1α enhances basal TNFα expression in skeletal muscle and indicate that PGC-1α does not exert anti-inflammatory effects during acute inflammation. Lack of skeletal muscle PGC-1α seems however to impair the acute TNFα response, which may reflect a phenotype more susceptible to infections as also observed in type 2 diabetes patients.