Sean L. McGee

Similar metabolic adaptations during exercise after low volume sprint interval and traditional endurance training in humans

Low‐volume ‘sprint’ interval training (SIT) stimulates rapid improvements in muscle oxidative capacity that are comparable to levels reached following traditional endurance training (ET) but no study has examined metabolic adaptations during exercise after these different training strategies. We hypothesized that SIT and ET would induce similar adaptations in markers of skeletal muscle carbohydrate (CHO) and lipid metabolism and metabolic control during exercise despite large differences in training volume and time commitment. Active but untrained subjects (23 ± 1 years) performed a constant‐load cycling challenge (1 h at 65% of peak oxygen uptake before and after 6 weeks of either SIT or ET (n= 5 men and 5 women per group). SIT consisted of four to six repeats of a 30 s ‘all out’ Wingate Test (mean power output ∼500 W) with 4.5 min recovery between repeats, 3 days per week. ET consisted of 40–60 min of continuous cycling at a workload that elicited ∼65% (mean power output ∼150 W) per day, 5 days per week. Weekly time commitment (∼1.5 versus∼4.5 h) and total training volume (∼225 versus∼2250 kJ week−1) were substantially lower in SIT versus ET. Despite these differences, both protocols induced similar increases (P < 0.05) in mitochondrial markers for skeletal muscle CHO (pyruvate dehydrogenase E1α protein content) and lipid oxidation (3‐hydroxyacyl CoA dehydrogenase maximal activity) and protein content of peroxisome proliferator‐activated receptor‐γ coactivator‐1α. Glycogen and phosphocreatine utilization during exercise were reduced after training, and calculated rates of whole‐body CHO and lipid oxidation were decreased and increased, respectively, with no differences between groups (all main effects, P < 0.05). Given the markedly lower training volume in the SIT group, these data suggest that high‐intensity interval training is a time‐efficient strategy to increase skeletal muscle oxidative capacity and induce specific metabolic adaptations during exercise that are comparable to traditional ET.

Metabolic Adaptations to Short-term High-intensity Interval Training: A Little Pain for a Lot of Gain?

High-intensity interval training (HIT) is a potent time-efficient strategy to induce numerous metabolic adaptations usually associated with traditional endurance training. As little as six sessions of HIT over 2 wk or a total of only approximately 15 min of very intense exercise (~600 kJ), can increase skeletal muscle oxidative capacity and endurance performance and alter metabolic control during aerobic-based exercise.

AMP-Activated Protein Kinase Regulates GLUT4 Transcription by Phosphorylating Histone Deacetylase 5

OBJECTIVE—Insulin resistance associated with obesity and diabetes is ameliorated by specific overexpression of GLUT4 in skeletal muscle. The molecular mechanisms regulating skeletal muscle GLUT4 expression remain to be elucidated. The purpose of this study was to examine these mechanisms. RESEARCH DESIGN AND METHODS AND RESULTS—Here, we report that AMP-activated protein kinase (AMPK) regulates GLUT4 transcription through the histone deacetylase (HDAC)5 transcriptional repressor. Overexpression of HDAC5 represses GLUT4 reporter gene expression, and HDAC inhibition in human primary myotubes increases endogenous GLUT4 gene expression. In vitro kinase assays, site-directed mutagenesis, and site-specific phospho-antibodies establish AMPK as an HDAC5 kinase that targets S259 and S498. Constitutively active but not dominant-negative AMPK and 5-aminoimidazole-4-carboxamide-1-β-d-ribonucleoside (AICAR) treatment in human primary myotubes results in HDAC5 phosphorylation at S259 and S498, association with 14-3-3 isoforms, and H3 acetylation. This reduces HDAC5 association with the GLUT4 promoter, as assessed through chromatin immunoprecipitation assays and HDAC5 nuclear export, concomitant with increases in GLUT4 gene expression. Gene reporter assays also confirm that the HDAC5 S259 and S498 sites are required for AICAR induction of GLUT4 transcription. CONCLUSIONS—These data reveal a signal transduction pathway linking cellular energy charge to gene transcription directed at restoring cellular and whole-body energy balance and provide new therapeutic targets for the treatment and management of insulin resistance and type 2 diabetes.

Exercise-induced histone modifications in human skeletal muscle

Skeletal muscle adaptations to exercise confer many of the health benefits of physical activity and occur partly through alterations in skeletal muscle gene expression. The exact mechanisms mediating altered skeletal muscle gene expression in response to exercise are unknown. However, in recent years, chromatin remodelling through epigenetic histone modifications has emerged as a key regulatory mechanism controlling gene expression in general. The purpose of this study was to examine the effect of exercise on global histone modifications that mediate chromatin remodelling and transcriptional activation in human skeletal muscle in response to exercise. In addition, we sought to examine the signalling mechanisms regulating these processes. Following 60 min of cycling, global histone 3 acetylation at lysine 9 and 14, a modification associated with transcriptional initiation, was unchanged from basal levels, but was increased at lysine 36, a site associated with transcriptional elongation. We examined the regulation of the class IIa histone deacetylases (HDACs), which are enzymes that suppress histone acetylation and have been implicated in the adaptations to exercise. While we found no evidence of proteasomal degradation of the class IIa HDACs, we found that HDAC4 and 5 were exported from the nucleus during exercise, thereby removing their transcriptional repressive function. We also observed activation of the AMP‐activated protein kinase (AMPK) and the calcium–calmodulin‐dependent protein kinase II (CaMKII) in response to exercise, which are two kinases that induce phosphorylation‐dependent class IIa HDAC nuclear export. These data delineate a signalling pathway that might mediate skeletal muscle adaptations in response to exercise.

Altering dietary nutrient intake that reduces glycogen content leads to phosphorylation of nuclear p38 MAP kinase in human skeletal muscle: association with IL-6 gene transcription during contraction

To determine the effect of glycogen availability and contraction on intracellular signaling and IL‐6 gene transcription, eight males performed 60 min of exercise on two occasions: either with prior ingestion of a normal (Con) or low carbohydrate (LCHO) diet that reduced pre‐exercise muscle glycogen content. Muscle biopsies were obtained and analyzed for IL‐6 mRNA. In addition, nuclear proteins were isolated from the samples and analyzed for the mitogen‐ activated protein kinases (MAPK) c‐jun amino‐terminal kinase (JNK) 1 and 2 and p38 MAPK. Nuclear fractions were also analyzed for the phosphorylated forms of JNK (p‐JNK) and p38 MAPK (p‐ p38 MAPK) and the abundance of the nuclear transcription factors nuclear factor of activated T cells (NFAT) and nuclear factor kappa‐β (NF‐κβ). No differences were observed in the protein abundance of total JNK 1/2, p38 MAPK, NFAT, or NF‐κβ before exercise, but the nuclear abundance of p‐p38 MAPK was higher (P<0.05) in LCHO. Contraction resulted in an increase (P<0.05) in nuclear p‐JNK 1/2, but there were no differences when comparing CON with LCHO. The fold increase in IL‐6 mRNA with contraction was potentiated (P<0.05) in LCHO. A correlation between pre‐exercise nuclear phosphorylated p38 MAPK and contraction‐induced fold increase in IL‐6 mRNA was performed, revealing a highly significant correlation (r=0.96; P<0.01). We next incubated L6 myotubes in ionomycin (a compound known to induce IL‐6 mRNA) with or without the pyridinylimidazole p38 MAPK inhibitor SB203580. Treatments did not affect total nuclear p38 MAPK, but ionomycin increased (P<0.05) both nuclear p‐p38 MAPK and IL‐6 mRNA. The addition of SB203580 to ionomycin decreased (P<0.05) nuclear p‐ p38 MAPK and totally abolished (P<0.05) the ionomycin‐ induced increase in IL‐6 mRNA. These data suggest that reduced carbohydrate intake that results in low intramuscular glycogen leads to phosphorylation of p38 MAPK at the nucleus. Furthermore, phosphorylation of p38 MAPK in the nucleus appears to be an upstream target for IL‐6, providing new insights into the regulation of IL‐6 gene transcription.

Normal hypertrophy accompanied by phosphoryation and activation of AMP‐activated protein kinase α1 following overload in LKB1 knockout mice

The activation of the AMP‐activated protein kinase (AMPK) and inhibition of the mammalian target of rapamycin complex 1 (mTORC1) is hypothesized to underlie the fact that muscle growth following resistance exercise is decreased by concurrent endurance exercise. To directly test this hypothesis, the capacity for muscle growth was determined in mice lacking the primary upstream kinase for AMPK in skeletal muscle, LKB1. Following either 1 or 4 weeks of overload, there was no difference in muscle growth between the wild type (wt) and LKB1−/− mice (1 week: wt, 38.8 ± 7.75%; LKB1−/−, 27.8 ± 12.98%; 4 week: wt, 75.8 ± 15.2%; LKB1−/−, 85.0 ± 22.6%). In spite of the fact that the LKB1 had been knocked out in skeletal muscle, the phosphorylation and activity of the α1 isoform of AMPK were markedly increased in both the wt and the LKB1−/− mice. To identify the upstream kinase(s) responsible, we studied potential upstream kinases other than LKB1. The activity of both Ca2+–calmodulin‐dependent protein kinase kinase α(CaMKKα) (5.05 ± 0.86‐fold) and CaMKKβ (10.1 ± 2.59‐fold) increased in the overloaded muscles, and this correlated with their increased expression. Phosphorylation of TAK‐1 also increased 10‐fold following overload in both the wt and LKB1 mice. Even though the α1 isoform of AMPK was activated by overload, there were no increases in expression of mitochondrial proteins or GLUT4, indicating that the α1 isoform is not involved in these metabolic adaptations. The phosphorylation of TSC2, an upstream regulator of the TORC1 pathway, at the AMPK site (Ser1345) was increased in response to overload, and this was not affected by LKB1 deficiency. Taken together, these data suggest that the α1 isoform of AMPK is preferentially activated in skeletal muscle following overload in the absence of metabolic adaptations, suggesting that this isoform might be important in the regulation of growth but not metabolism.

Response of BRAF mutant melanoma to BRAF inhibition is mediated by a network of transcriptional regulators of glycolysis

UNLABELLED Deregulated glucose metabolism fulfills the energetic and biosynthetic requirements for tumor growth driven by oncogenes. Because inhibition of oncogenic BRAF causes profound reductions in glucose uptake and a strong clinical benefit in BRAF-mutant melanoma, we examined the role of energy metabolism in responses to BRAF inhibition. We observed pronounced and consistent decreases in glycolytic activity in BRAF-mutant melanoma cells. Moreover, we identified a network of BRAF-regulated transcription factors that control glycolysis in melanoma cells. Remarkably, this network of transcription factors, including hypoxia-inducible factor-1α, MYC, and MONDOA (MLXIP), drives glycolysis downstream of BRAF(V600), is critical for responses to BRAF inhibition, and is modulated by BRAF inhibition in clinical melanoma specimens. Furthermore, we show that concurrent inhibition of BRAF and glycolysis induces cell death in BRAF inhibitor (BRAFi)-resistant melanoma cells. Thus, we provide a proof-of-principle for treatment of melanoma with combinations of BRAFis and glycolysis inhibitors. SIGNIFICANCE BRAF is suppress glycolysis and provide strong clinical benefi t in BRAF V600 melanoma. We show that BRAF inhibition suppresses glycolysis via a network of transcription factors that are critical for complete BRAFi responses. Furthermore, we provide evidence for the clinical potential of therapies that combine BRAFis with glycolysis inhibitors.

EXERCISE AND SKELETAL MUSCLE GLUCOSE TRANSPORTER 4 EXPRESSION: MOLECULAR MECHANISMS

1 Skeletal muscle is a highly plastic tissue that has a remarkable ability to adapt to external demands, such as exercise. Many of these adaptations can be explained by changes in skeletal muscle gene expression. A single bout of exercise is sufficient to induce the expression of some metabolic genes. We have focused our attention on the regulation of glucose transporter isoform 4 (GLUT‐4) expression in human skeletal muscle. 2 Glucose transporter isoform 4 gene expression is increased immediately following a single bout of exercise, and the GLUT‐4 enhancer factor (GEF) and myocyte enhancer factor 2 (MEF2) transcription factors are required for this response. Glucose transporter isoform enhancer factor and MEF2 DNA binding activities are increased following exercise, and the molecular mechanisms regulating MEF2 in exercising human skeletal muscle have also been examined. 3 These studies find possible roles for histone deacetylase 5 (HDAC5), adenosine monophosphate–activated protein kinase (AMPK), peroxisome proliferator‐activated receptor gamma coactivator 1α (PGC‐1α) and p38 mitogen‐activated protein kinase (MAPK) in regulating MEF2 through a series of complex interactions potentially involving MEF2 repression, coactivation and phosphorylation. 4 Given that MEF2 is a transcription factor required for many exercise responsive genes, it is possible that these mechanisms are responsible for regulating the expression of a variety of metabolic genes during exercise. These mechanisms could also provide targets for the treatment and management of metabolic disease states, such as obesity and type 2 diabetes, which are characterized by mitochondrial dysfunction and insulin resistance in skeletal muscle.

AMPK and transcriptional regulation.

The AMP-activated protein kinase (AMPK) is an energy sensing enzyme that once activated, promotes energy production and limits energy utilisation to ensure cellular survival. In addition to targeting numerous metabolic enzymes for this purpose, it is becoming apparent that AMPK can also regulate a number of transcriptional processes. These processes ensure cell survival through the inhibition of cell cycle and growth mechanisms, and also prepare the cell for future perturbations in energy balance by increasing the capacity of the cell to produce ATP. While these adaptations might be inextricably linked through regulation of the proliferation-differentiation process, recent studies have identified a number of transcriptional regulators as AMPK substrates that give insights into the regulation of transcription by AMPK in a number of metabolically active tissues.

Activating HSP72 in rodent skeletal muscle increases mitochondrial number and oxidative capacity and decreases insulin resistance

Induction of heat shock protein (HSP)72 protects against obesity-induced insulin resistance, but the underlying mechanisms are unknown. Here, we show that HSP72 plays a pivotal role in increasing skeletal muscle mitochondrial number and oxidative metabolism. Mice overexpressing HSP72 in skeletal muscle (HSP72Tg) and control wild-type (WT) mice were fed either a chow or high-fat diet (HFD). Despite a similar energy intake when HSP72Tg mice were compared with WT mice, the HFD increased body weight, intramuscular lipid accumulation (triacylglycerol and diacylglycerol but not ceramide), and severe glucose intolerance in WT mice alone. Whole-body VO2, fatty acid oxidation, and endurance running capacity were markedly increased in HSP72Tg mice. Moreover, HSP72Tg mice exhibited an increase in mitochondrial number. In addition, the HSP72 coinducer BGP-15, currently in human clinical trials for type 2 diabetes, also increased mitochondrial number and insulin sensitivity in a rat model of type 2 diabetes. Together, these data identify a novel role for activation of HSP72 in skeletal muscle. Thus, the increased oxidative metabolism associated with activation of HSP72 has potential clinical implications not only for type 2 diabetes but also for other disorders where mitochondrial function is compromised.