Combined overexpression of SIRT1 and knockout of GCN5 in adult skeletal muscle does not affect glucose homeostasis or exercise performance in mice.

Sirtuin 1 (SIRT1) and general control of amino acid synthesis 5 (GCN5) regulate mitochondrial biogenesis via opposing modulation of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) acetylation status and activity. However, the combined contribution of SIRT1 and GCN5 to skeletal muscle metabolism and endurance performance in vivo is unknown. In this study, we investigated the impact of combined skeletal muscle-specific overexpression of SIRT1 and deletion of GCN5 on glucose homeostasis, skeletal muscle mitochondrial biogenesis and function, and metabolic adaptation to endurance exercise training in mice. We generated mice with combined and tamoxifen-inducible skeletal muscle-specific overexpression of SIRT1 and knockout of GCN5 (dTG) and floxed [wild type (WT)] littermates using a Cre-LoxP approach. All mice were treated with tamoxifen at 5-6 wk of age, and 4-7 wk later glucose homeostasis, skeletal muscle contractile function, mitochondrial function, and the effects of 14 days of voluntary wheel running on expression of metabolic proteins and exercise capacity were assessed. There was no difference in oral glucose tolerance, skeletal muscle contractile function, mitochondrial abundance, or maximal respiratory capacity between dTG and WT mice. Additionally, there were no genotype differences in exercise performance and markers of mitochondrial biogenesis after 14 days of voluntary wheel running. These results demonstrate that combined overexpression of SIRT1 and loss of GCN5 in vivo does not promote metabolic remodeling in skeletal muscle of sedentary or exercise-trained mice.

Acute inhibition of protein deacetylases does not impact skeletal muscle insulin action.

Whether the histone deacetylase (HDAC) and sirtuin families of protein deacetylases regulate insulin-stimulated glucose uptake, independent of their transcriptional effects, has not been studied. Our objective was to determine the nontranscriptional role of HDACs and sirtuins in regulation of skeletal muscle insulin action. Basal and insulin-stimulated glucose uptake and signaling and acetylation were assessed in L6 myotubes and skeletal muscle from C57BL/6J mice that were treated acutely (1 h) with HDAC (trichostatin A, panobinostat, TMP195) and sirtuin inhibitors (nicotinamide). Treatment of L6 myotubes with HDAC inhibitors or skeletal muscle with a combination of HDAC and sirtuin inhibitors increased tubulin and pan-protein acetylation, demonstrating effective impairment of HDAC and sirtuin deacetylase activities. Despite this, neither basal nor insulin-stimulated glucose uptake or insulin signaling was impacted. Acute reduction of the deacetylase activity of HDACs and/or sirtuins does not impact insulin action in skeletal muscle.

Germline or inducible knockout of p300 or CBP in skeletal muscle does not alter insulin sensitivity.

Akt is a critical mediator of insulin-stimulated glucose uptake in skeletal muscle. The acetyltransferases, E1A binding protein p300 (p300) and cAMP response element-binding protein binding protein (CBP) are phosphorylated and activated by Akt, and p300/CBP can acetylate and inactivate Akt, thus giving rise to a possible Akt-p300/CBP axis. Our objective was to determine the importance of p300 and CBP to skeletal muscle insulin sensitivity. We used Cre-LoxP methodology to generate mice with germline [muscle creatine kinase promoter (P-MCK and C-MCK)] or inducible [tamoxifen-activated, human skeletal actin promoter (P-iHSA and C-iHSA)] knockout of p300 or CBP. A subset of P-MCK and C-MCK mice were switched to a calorie-restriction diet (60% of ad libitum intake) or high-fat diet at 10 wk of age. For P-iHSA and C-iHSA mice, knockout was induced at 10 wk of age. At 13-15 wk of age, we measured whole-body energy expenditure, oral glucose tolerance, and/or ex vivo skeletal muscle insulin sensitivity. Although p300 and CBP protein abundance and mRNA expression were reduced 55%-90% in p300 and CBP knockout mice, there were no genotype differences in energy expenditure or fasting glucose and insulin concentrations. Moreover, neither loss of p300 or CBP impacted oral glucose tolerance or skeletal muscle insulin sensitivity, nor did their loss impact alterations in these parameters in response to a calorie restriction or high-fat diet. Muscle-specific loss of either p300 or CBP, be it germline or in adulthood, does not impact energy expenditure, glucose tolerance, or skeletal muscle insulin action.

SIRT1 regulates nuclear number and domain size in skeletal muscle fibers.

Skeletal muscle fibers are giant multinucleated cells wherein individual nuclei govern the protein synthesis in a finite volume of cytoplasm; this is termed the myonuclear domain (MND). The factors that control MND size remain to be defined. In the present study, we studied the contribution of the NAD+ -dependent deacetylase, sirtuin 1 (SIRT1), to the regulation of nuclear number and MND size. For this, we isolated myofibers from mice with tissue-specific inactivation (mKO) or inducible overexpression (imOX) of SIRT1 and analyzed the 3D organisation of myonuclei. In imOX mice, the number of nuclei was increased whilst the average MND size was decreased as compared to littermate controls. Our findings were the opposite in mKO mice. Muscle stem cell (satellite cell) numbers were reduced in mKO muscles, a possible explanation for the lower density of myonuclei in these mice; however, no change was observed in imOX mice, suggesting that other factors might also be involved, such as the functional regulation of stem cells/muscle precursors. Interestingly, however, the changes in the MND volume did not impact the force-generating capacity of muscle fibers. Taken together, our results demonstrate that SIRT1 is a key regulator of MND sizes, although the underlying molecular mechanisms and the cause-effect relationship between MND and muscle function remain to be fully defined.

Defining the contribution of skeletal muscle pyruvate dehydrogenase α1 to exercise performance and insulin action.

The pyruvate dehydrogenase complex (PDC) converts pyruvate to acetyl-CoA and is an important control point for carbohydrate (CHO) oxidation. However, the importance of the PDC and CHO oxidation to muscle metabolism and exercise performance, particularly during prolonged or high-intensity exercise, has not been fully defined especially in mature skeletal muscle. To this end, we determined whether skeletal muscle-specific loss of pyruvate dehydrogenase alpha 1 ( Pdha1), which is a critical subunit of the PDC, impacts resting energy metabolism, exercise performance, or metabolic adaptation to high-fat diet (HFD) feeding. For this, we generated a tamoxifen (TMX)-inducible Pdha1 knockout (PDHmKO) mouse, in which PDC activity is temporally and specifically ablated in adult skeletal muscle. We assessed energy expenditure, ex vivo muscle contractile performance, and endurance exercise capacity in PDHmKO mice and wild-type (WT) littermates. Additionally, we studied glucose homeostasis and insulin sensitivity in muscle after 12 wk of HFD feeding. TMX administration largely ablated PDHα in skeletal muscle of adult PDHmKO mice but did not impact energy expenditure, muscle contractile function, or low-intensity exercise performance. Additionally, there were no differences in muscle insulin sensitivity or body composition in PDHmKO mice fed a control or HFD, as compared with WT mice. However, exercise capacity during high-intensity exercise was severely impaired in PDHmKO mice, in parallel with a large increase in plasma lactate concentration. In conclusion, although skeletal muscle PDC is not a major contributor to resting energy expenditure or long-duration, low-intensity exercise performance, it is necessary for optimal performance during high-intensity exercise.

Muscle PGC-1α is required for long-term systemic and local adaptations to a ketogenic diet in mice.

Low-carbohydrate/high-fat (LCHF) diets are increasingly popular dietary interventions for body weight control and as treatment for different pathological conditions. However, the mechanisms of action are still poorly understood, in particular, in long-term administration. Besides liver, brain, and heart, skeletal muscle is one of the major organs involved in the regulation of physiological and pathophysiological ketosis. We assessed the role of the peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) in skeletal muscle of male wild-type control and PGC-1α muscle-specific knockout mice upon 12 wk of LCHF diet feeding. Interestingly, LCHF diet administration increased oxygen consumption in a muscle PGC-1α-dependent manner, concomitant with a blunted transcriptional induction of genes involved in fatty acid oxidation and impairment in exercise performance. These data reveal a new role for muscle PGC-1α in regulating the physiological adaptation to long-term LCHF diet administration.

Skeletal muscle PGC-1α modulates systemic ketone body homeostasis and ameliorates diabetic hyperketonemia in mice.

Ketone bodies (KBs) are crucial energy substrates during states of low carbohydrate availability. However, an aberrant regulation of KB homeostasis can lead to complications such as diabetic ketoacidosis. Exercise and diabetes affect systemic KB homeostasis, but the regulation of KB metabolism is still enigmatic. In our study in mice with either knockout or overexpression of the peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α in skeletal muscle, PGC-1α regulated ketolytic gene transcription in muscle. Furthermore, KB homeostasis of these mice was investigated during withholding of food, exercise, and ketogenic diet feeding, and after streptozotocin injection. In response to these ketogenic stimuli, modulation of PGC-1α levels in muscle affected systemic KB homeostasis. Moreover, the data demonstrate that skeletal muscle PGC-1α is necessary for the enhanced ketolytic capacity in response to exercise training and overexpression of PGC-1α in muscle enhances systemic ketolytic capacity and is sufficient to ameliorate diabetic hyperketonemia in mice. In cultured myotubes, the transcription factor estrogen-related receptor-α was a partner of PGC-1α in the regulation of ketolytic gene transcription. These results demonstrate a central role of skeletal muscle PGC-1α in the transcriptional regulation of systemic ketolytic capacity.-Svensson, K., Albert, V., Cardel, B., Salatino, S., Handschin, C. Skeletal muscle PGC-1α modulates systemic ketone body homeostasis and ameliorates diabetic hyperketonemia in mice.

Resveratrol and SRT1720 Elicit Differential Effects in Metabolic Organs and Modulate Systemic Parameters Independently of Skeletal Muscle Peroxisome Proliferator-activated Receptor γ Co-activator 1α (PGC-1α).

Resveratrol (RSV) and SRT1720 (SRT) elicit beneficial metabolic effects and are postulated to ameliorate obesity and related metabolic complications. The co-activator, peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α), has emerged as a major downstream effector responsible for metabolic remodeling of muscle and other metabolic tissues in response to RSV or SRT treatment. However, the requirement of PGC-1α in skeletal muscle for the systemic metabolic effects of these compounds has so far not been demonstrated. Using muscle-specific PGC-1α knock-out mice, we show that PGC-1α is necessary for transcriptional induction of mitochondrial genes in muscle with both RSV and SRT treatment. Surprisingly, the beneficial effects of SRT on glucose homeostasis and of both compounds on energy expenditure occur even in the absence of muscle PGC-1α. Moreover, RSV and SRT treatment elicit differential transcriptional effects on genes involved in lipid metabolism and mitochondrial biogenesis in liver and adipose tissue. These findings indicate that RSV and SRT do not induce analogous metabolic effects in vivo. Our results provide important insights into the mechanism, effects, and organ specificity of the caloric restriction mimetics RSV and SRT. These findings are important for the design of future therapeutic interventions aimed at ameliorating obesity and obesity-related metabolic dysfunction.

The coactivator PGC-1α regulates skeletal muscle oxidative metabolism independently of the nuclear receptor PPARß/δ in sedentary mice fed a regular chow diet.

AIMS/HYPOTHESIS: Physical activity improves oxidative capacity and exerts therapeutic beneficial effects, particularly in the context of metabolic diseases. The peroxisome proliferator-activated receptor (PPAR) γ coactivator-1α (PGC-1α) and the nuclear receptor PPARß/δ have both been independently discovered to play a pivotal role in the regulation of oxidative metabolism in skeletal muscle, though their interdependence remains unclear. Hence, our aim was to determine the functional interaction between these two factors in mouse skeletal muscle in vivo. METHODS: Adult male control mice, PGC-1α muscle-specific transgenic (mTg) mice, PPARß/δ muscle-specific knockout (mKO) mice and the combination PPARß/δ mKO + PGC-1α mTg mice were studied under basal conditions and following PPARß/δ agonist administration and acute exercise. Whole-body metabolism was assessed by indirect calorimetry and blood analysis, while magnetic resonance was used to measure body composition. Quantitative PCR and western blot were used to determine gene expression and intracellular signalling. The proportion of oxidative muscle fibre was determined by NADH staining. RESULTS: Agonist-induced PPARß/δ activation was only disrupted by PPARß/δ knockout. We also found that the disruption of the PGC-1α-PPARß/δ axis did not affect whole-body metabolism under basal conditions. As expected, PGC-1α mTg mice exhibited higher exercise performance, peak oxygen consumption and lower blood lactate levels following exercise, though PPARß/δ mKO + PGC-1α mTg mice showed a similar phenotype. Similarly, we found that PPARß/δ was dispensable for PGC-1α-mediated enhancement of an oxidative phenotype in skeletal muscle. CONCLUSIONS/INTERPRETATION: Collectively, these results indicate that PPARß/δ is not an essential partner of PGC-1α in the control of skeletal muscle energy metabolism.

Insights into post-translational regulation of skeletal muscle contractile function by the acetyltransferases, p300 and CBP.

Mice with skeletal muscle-specific inducible double knockout of the lysine acetyltransferases, p300 (E1A binding protein p300) and CBP (cAMP-response element-binding protein binding protein), referred to as i-mPCKO, demonstrate a dramatic loss of contractile function in skeletal muscle and ultimately die within 7 days. Given that many proteins involved in ATP generation and cross-bridge cycling are acetylated, we investigated whether these processes are dysregulated in skeletal muscle from i-mPCKO mice and thus could underlie the rapid loss of muscle contractile function. Just 4-5 days after inducing knockout of p300 and CBP in skeletal muscle from adult i-mPCKO mice, there was ∼90% reduction in ex vivo contractile function in the extensor digitorum longus (EDL) and a ∼65% reduction in in vivo ankle dorsiflexion torque, as compared to wildtype (WT; i.e. Cre negative) littermates. Despite the profound loss of contractile force in i-mPCKO mice, there were no genotype-driven differences in fatigability during repeated contractions, nor were there genotype differences in mitochondrial specific pathway enrichment of the proteome, intermyofibrillar mitochondrial volume or mitochondrial respiratory function. As it relates to cross-bridge cycling, remarkably, the overt loss of contractile function in i-mPCKO muscle was reversed in permeabilized fibers supplied with exogenous Ca 2+ and ATP, with active tension being similar between i-mPCKO and WT mice, regardless of Ca 2+ concentration. Actin-myosin motility was also similar in skeletal muscle from i-mPCKO and WT mice. In conclusion, neither mitochondrial abundance/function, nor actomyosin cross-bridge cycling, are the underlying driver of contractile dysfunction in i-mPCKO mice. New & Noteworthy: The mechanism underlying dramatic loss of muscle contractile function with inducible deletion of both p300 and CBP in skeletal muscle remains unknown. Here we find that impairments in mitochondrial function or cross-bridge cycling are not the underlying mechanism of action. Future work will investigate other aspects of excitation-contraction coupling, such as Ca 2+ handling and membrane excitability, as contractile function could be rescued by permeabilizing skeletal muscle, which provides exogenous Ca 2+ and bypasses membrane depolarization.

Insights into posttranslational regulation of skeletal muscle contractile function by the acetyltransferases, p300 and CBP.

Mice with skeletal muscle-specific and inducible double knockout of the lysine acetyltransferases, p300 (E1A binding protein p300) and CBP (cAMP-response element-binding protein binding protein), referred to as i-mPCKO, demonstrate a dramatic loss of contractile function in skeletal muscle and ultimately die within 7 days. Given that many proteins involved in ATP generation and cross-bridge cycling are acetylated, we investigated whether these processes are dysregulated in skeletal muscle from i-mPCKO mice and, thus, whether they could underlie the rapid loss of muscle contractile function. Just 4-5 days after inducing knockout of p300 and CBP in skeletal muscle from adult i-mPCKO mice, there was ∼90% reduction in ex vivo contractile function in the extensor digitorum longus (EDL) and a ∼65% reduction in in vivo ankle dorsiflexion torque, as compared with wild type (WT; i.e., Cre negative) littermates. Despite this profound loss of contractile force in i-mPCKO mice, there were no genotype-driven differences in fatigability during repeated contractions, nor were there genotype differences in mitochondrial-specific pathway enrichment of the proteome, intermyofibrillar mitochondrial volume, or mitochondrial respiratory function. As it relates to cross-bridge cycling, remarkably, the overt loss of contractile function in i-mPCKO muscle was reversed in permeabilized fibers supplied with exogenous Ca2+ and ATP, with active tension being similar between i-mPCKO and WT mice, regardless of Ca2+ concentration. Actin-myosin motility was also similar in skeletal muscle from i-mPCKO and WT mice. In conclusion, neither mitochondrial abundance/function, nor actomyosin cross-bridge cycling, are the underlying driver of contractile dysfunction in i-mPCKO mice.NEW & NOTEWORTHY The mechanism underlying dramatic loss of muscle contractile function with inducible deletion of both E1A binding protein p300 (p300) and cAMP-response element-binding protein binding protein (CBP) in skeletal muscle remains unknown. Here, we find that impairments in mitochondrial function or cross-bridge cycling are not the underlying mechanism of action. Future work will investigate other aspects of excitation-contraction coupling, such as Ca2+ handling and membrane excitability, as contractile function could be rescued by permeabilizing skeletal muscle, which provides exogenous Ca2+ and bypasses membrane depolarization.

p300 or CBP is required for insulin-stimulated glucose uptake in skeletal muscle and adipocytes.

While current thinking posits that insulin signaling to glucose transporter 4 (GLUT4) exocytic translocation and glucose uptake in skeletal muscle and adipocytes is controlled by phosphorylation-based signaling, many proteins in this pathway are acetylated on lysine residues. However, the importance of acetylation and lysine acetyltransferases to insulin-stimulated glucose uptake is incompletely defined. Here, we demonstrate that combined loss of the acetyltransferases E1A binding protein p300 (p300) and cAMP response element binding protein binding protein (CBP) in mouse skeletal muscle caused a complete loss of insulin-stimulated glucose uptake. Similarly, brief (i.e., 1 hour) pharmacological inhibition of p300/CBP acetyltransferase activity recapitulated this phenotype in human and rodent myotubes, 3T3-L1 adipocytes, and mouse muscle. Mechanistically, these effects were due to p300/CBP-mediated regulation of GLUT4 exocytic translocation and occurred downstream of Akt signaling. Taken together, we highlight a fundamental role for acetylation and p300/CBP in the direct regulation of insulin-stimulated glucose transport in skeletal muscle and adipocytes.

Attenuated mTOR signaling and enhanced autophagy in adipocytes from obese patients with type 2 diabetes.

Type 2 diabetes (T2D) is strongly linked to obesity and an adipose tissue unresponsive to insulin. The insulin resistance is due to defective insulin signaling, but details remain largely unknown. We examined insulin signaling in adipocytes from T2D patients, and contrary to findings in animal studies, we observed attenuation of insulin activation of mammalian target of rapamycin (mTOR) in complex with raptor (mTORC1). As a consequence, mTORC1 downstream effects were also affected in T2D: feedback signaling by insulin to signal-mediator insulin receptor substrate-1 (IRS1) was attenuated, mitochondria were impaired and autophagy was strongly upregulated. There was concomitant autophagic destruction of mitochondria and lipofuscin particles, and a dependence on autophagy for ATP production. Conversely, mitochondrial dysfunction attenuated insulin activation of mTORC1, enhanced autophagy and attenuated feedback to IRS1. The overactive autophagy was associated with large numbers of cytosolic lipid droplets, a subset with colocalization of perlipin and the autophagy protein LC3/atg8, which can contribute to excessive fatty acid release. Patients with diagnoses of T2D and overweight were consecutively recruited from elective surgery, whereas controls did not have T2D. Results were validated in a cohort of patients without diabetes who exhibited a wide range of insulin sensitivities. Because mitochondrial dysfunction, inflammation, endoplasmic-reticulum stress and hypoxia all inactivate mTORC1, our results may suggest a unifying mechanism for the pathogenesis of insulin resistance in T2D, although the underlying causes might differ.

p300 and cAMP response element-binding protein-binding protein in skeletal muscle homeostasis, contractile function, and survival.

BACKGROUND: Reversible ε-amino acetylation of lysine residues regulates transcription as well as metabolic flux; however, roles for specific lysine acetyltransferases in skeletal muscle physiology and function are unknown. In this study, we investigated the role of the related acetyltransferases p300 and cAMP response element-binding protein-binding protein (CBP) in skeletal muscle transcriptional homeostasis and physiology in adult mice. METHODS: Mice with skeletal muscle-specific and inducible knockout of p300 and CBP (PCKO) were generated by crossing mice with a tamoxifen-inducible Cre recombinase expressed under the human α-skeletal actin promoter with mice having LoxP sites flanking exon 9 of the Ep300 and Crebbp genes. Knockout of PCKO was induced at 13-15 weeks of age via oral gavage of tamoxifen for 5 days to both PCKO and littermate control [wildtype (WT)] mice. Body composition, food intake, and muscle function were assessed on day 0 (D0) through 5 (D5). Microarray and tandem mass tag mass spectrometry analyses were performed to assess global RNA and protein levels in skeletal muscle of PCKO and WT mice. RESULTS: At D5 after initiating tamoxifen treatment, there was a reduction in body weight (-15%), food intake (-78%), stride length (-46%), and grip strength (-45%) in PCKO compared with WT mice. Additionally, ex vivo contractile function [tetanic tension (kPa)] was severely impaired in PCKO vs. WT mice at D3 (~70-80% lower) and D5 (~80-95% lower) and resulted in lethality within 1 week-a phenotype that is reversed by the presence of a single allele of either p300 or CBP. The impaired muscle function in PCKO mice was paralleled by substantial transcriptional alterations (3310 genes; false discovery rate < 0.1), especially in gene networks central to muscle contraction and structural integrity. This transcriptional uncoupling was accompanied by changes in protein expression patterns indicative of impaired muscle function, albeit to a smaller magnitude (446 proteins; fold-change > 1.25; false discovery rate < 0.1). CONCLUSIONS: These data reveal that p300 and CBP are required for the control and maintenance of contractile function and transcriptional homeostasis in skeletal muscle and, ultimately, organism survival. By extension, modulating p300/CBP function may hold promise for the treatment of disorders characterized by impaired contractile function in humans.

Skeletal muscle mitochondrial function and exercise capacity are not impaired in mice with knockout of STAT3.

Signal transducer and activator of transcription 3 (STAT3) was recently found to be localized to mitochondria in a number of tissues and cell types, where it modulates oxidative phosphorylation via interactions with the electron transport proteins, complex I and complex II. Skeletal muscle is densely populated with mitochondria although whether STAT3 contributes to skeletal muscle oxidative capacity is unknown. In the present study, we sought to elucidate the contribution of STAT3 to mitochondrial and skeletal muscle function by studying mice with muscle-specific knockout of STAT3 (mKO). First, we developed a novel flow cytometry-based approach to confirm that STAT3 is present in skeletal muscle mitochondria. However, contrary to findings in other tissue types, complex I and complex II activity and maximal mitochondrial respiratory capacity in skeletal muscle were comparable between mKO mice and floxed/wild-type littermates. Moreover, there were no genotype differences in endurance exercise performance, skeletal muscle force-generating capacity, or the adaptive response of skeletal muscle to voluntary wheel running. Collectively, although we confirm the presence of STAT3 in skeletal muscle mitochondria, our data establish that STAT3 is dispensable for mitochondrial and physiological function in skeletal muscle.NEW & NOTEWORTHY Whether signal transducer and activator of transcription 3 (STAT3) can regulate the activity of complex I and II of the electron transport chain and mitochondrial oxidative capacity in skeletal muscle, as it can in other tissues, is unknown. By using a mouse model lacking STAT3 in muscle, we demonstrate that skeletal muscle mitochondrial and physiological function, both in vivo and ex vivo, is not impacted by the loss of STAT3.

Calorie Restriction-Induced Increase in Skeletal Muscle Insulin Sensitivity Is Not Prevented by Overexpression of the p55α Subunit of Phosphoinositide 3-Kinase.

Introduction: The Phosphoinositide 3-kinase (PI3K) signaling pathway plays an important role in skeletal muscle insulin-stimulated glucose uptake. While whole-body and tissue specific knockout (KO) of individual or combinations of the regulatory subunits of PI3K (p85α, p55α, and p50α or p85ß); increase insulin sensitivity, no study has examined whether increasing the expression of the individual regulatory subunits would inhibit insulin action in vivo. Therefore, the objective of this study was to determine whether skeletal muscle-specific overexpression of the p55α regulatory subunit of PI3K impairs skeletal muscle insulin sensitivity, or prevents its enhancement by caloric restriction. Methods: We developed a novel "floxed" mouse that, through the Cre-LoxP approach, allows for tamoxifen (TMX)-inducible and skeletal muscle-specific overexpression of the p55α subunit of PI3K (referred to as, 'p55α-mOX'). Beginning at 10 weeks of age, p55α-mOX mice and their floxed littermates (referred to as wildtype [WT]) either continued with free access to food (ad libitum; AL), or were switched to a calorie restricted diet (CR; 60% of AL intake) for 20 days. We measured body composition, whole-body energy expenditure, oral glucose tolerance and ex vivo skeletal muscle insulin sensitivity in isolated soleus and extensor digitorum longus muscles using the 2-deoxy-glucose (2DOG) uptake method. Results: p55α mRNA and protein expression was increased ∼2 fold in muscle from p55α-mOX versus WT mice. There were no differences in energy expenditure, total activity, or food intake of AL-fed mice between genotypes. Body weight, fat and lean mass, tissue weights, and fasting glucose and insulin were comparable between p55α-mOX and WT mice on AL, and were decreased equally by CR. Interestingly, overexpression of p55α did not impair oral glucose tolerance or skeletal muscle insulin signaling or sensitivity, nor did it impact the ability of CR to enhance these parameters. Conclusion: Skeletal muscle-specific overexpression of p55α does not impact skeletal muscle insulin action, suggesting that p85α and/or p50α may be more important regulators of skeletal muscle insulin signaling and sensitivity.

Muscle-specific knockout of general control of amino acid synthesis 5 (GCN5) does not enhance basal or endurance exercise-induced mitochondrial adaptation.

OBJECTIVE: Lysine acetylation is an important post-translational modification that regulates metabolic function in skeletal muscle. The acetyltransferase, general control of amino acid synthesis 5 (GCN5), has been proposed as a regulator of mitochondrial biogenesis via its inhibitory action on peroxisome proliferator activated receptor-γ coactivator-1α (PGC-1α). However, the specific contribution of GCN5 to skeletal muscle metabolism and mitochondrial adaptations to endurance exercise in vivo remain to be defined. We aimed to determine whether loss of GCN5 in skeletal muscle enhances mitochondrial density and function, and the adaptive response to endurance exercise training. METHODS: We used Cre-LoxP methodology to generate mice with muscle-specific knockout of GCN5 (mKO) and floxed, wildtype (WT) littermates. We measured whole-body energy expenditure, as well as markers of mitochondrial density, biogenesis, and function in skeletal muscle from sedentary mice, and mice that performed 20 days of voluntary endurance exercise training. RESULTS: Despite successful knockdown of GCN5 activity in skeletal muscle of mKO mice, whole-body energy expenditure as well as skeletal muscle mitochondrial abundance and maximal respiratory capacity were comparable between mKO and WT mice. Further, there were no genotype differences in endurance exercise-mediated mitochondrial biogenesis or increases in PGC-1α protein content. CONCLUSION: These results demonstrate that loss of GCN5 in vivo does not promote metabolic remodeling in mouse skeletal muscle.

Loss of Renal Tubular PGC-1α Exacerbates Diet-Induced Renal Steatosis and Age-Related Urinary Sodium Excretion in Mice.

The kidney has a high energy demand and is dependent on oxidative metabolism for ATP production. Accordingly, the kidney is rich in mitochondria, and mitochondrial dysfunction is a common denominator for several renal diseases. While the mitochondrial master regulator peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) is highly expressed in kidney, its role in renal physiology is so far unclear. Here we show that PGC-1α is a transcriptional regulator of mitochondrial metabolic pathways in the kidney. Moreover, we demonstrate that mice with an inducible nephron-specific inactivation of PGC-1α in the kidney display elevated urinary sodium excretion, exacerbated renal steatosis during metabolic stress but normal blood pressure regulation. Overall, PGC-1α seems largely dispensable for basal renal physiology. However, the role of PGC-1α in renal mitochondrial biogenesis indicates that activation of PGC-1α in the context of renal disorders could be a valid therapeutic strategy to ameliorate renal mitochondrial dysfunction.

mTORC2 sustains thermogenesis via Akt-induced glucose uptake and glycolysis in brown adipose tissue.

Activation of non-shivering thermogenesis (NST) in brown adipose tissue (BAT) has been proposed as an anti-obesity treatment. Moreover, cold-induced glucose uptake could normalize blood glucose levels in insulin-resistant patients. It is therefore important to identify novel regulators of NST and cold-induced glucose uptake. Mammalian target of rapamycin complex 2 (mTORC2) mediates insulin-stimulated glucose uptake in metabolic tissues, but its role in NST is unknown. We show that mTORC2 is activated in brown adipocytes upon ß-adrenergic stimulation. Furthermore, mice lacking mTORC2 specifically in adipose tissue (AdRiKO mice) are hypothermic, display increased sensitivity to cold, and show impaired cold-induced glucose uptake and glycolysis. Restoration of glucose uptake in BAT by overexpression of hexokinase II or activated Akt2 was sufficient to increase body temperature and improve cold tolerance in AdRiKO mice. Thus, mTORC2 in BAT mediates temperature homeostasis via regulation of cold-induced glucose uptake. Our findings demonstrate the importance of glucose metabolism in temperature regulation.

Apoptotic neutrophils augment the inflammatory response to Mycobacterium tuberculosis infection in human macrophages.

Macrophages in the lung are the primary cells being infected by Mycobacterium tuberculosis (Mtb) during the initial manifestation of tuberculosis. Since the adaptive immune response to Mtb is delayed, innate immune cells such as macrophages and neutrophils mount the early immune protection against this intracellular pathogen. Neutrophils are short-lived cells and removal of apoptotic cells by resident macrophages is a key event in the resolution of inflammation and tissue repair. Since anti-inflammatory activity is not compatible with effective immunity to intracellular pathogens, we therefore investigated how uptake of apoptotic neutrophils modulates the function of Mtb-activated human macrophages. We show that Mtb infection exerts a potent proinflammatory activation of human macrophages with enhanced gene activation and release of proinflammatory cytokines and that this response was augmented by apoptotic neutrophils. The enhanced macrophage response is linked to apoptotic neutrophil-driven activation of the NLRP3 inflammasome and subsequent IL-1ß signalling. We also demonstrate that apoptotic neutrophils not only modulate the inflammatory response, but also enhance the capacity of infected macrophages to control intracellular growth of virulent Mtb. Taken together, these results suggest a novel role for apoptotic neutrophils in the modulation of the macrophage-dependent inflammatory response contributing to the early control of Mtb infection.