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The biophysical environment of membrane phospholipids affects structure, function, and stability of membrane-bound proteins.1,2 Obesity can disrupt membrane lipids, and in particular, alter the activity of sarco/endoplasmic reticulum (ER/SR) Ca2+-ATPase (SERCA) to affect cellular metabolism.3-5 Recent evidence suggests that transport efficiency (Ca2+ uptake / ATP hydrolysis) of skeletal muscle SERCA can be uncoupled to increase energy expenditure and protect mice from diet-induced obesity.6,7 In isolated SR vesicles, membrane phospholipid composition is known to modulate SERCA efficiency.8-11 Here we show that skeletal muscle SR phospholipids can be altered to decrease SERCA efficiency and increase whole-body metabolic rate. The absence of skeletal muscle phosphatidylethanolamine (PE) methyltransferase (PEMT) promotes an increase in skeletal muscle and whole-body metabolic rate to protect mice from diet-induced obesity. The elevation in metabolic rate is caused by a decrease in SERCA Ca2+-transport efficiency, whereas mitochondrial uncoupling is unaffected. Our findings support the hypothesis that skeletal muscle energy efficiency can be reduced to promote protection from obesity.

Barth syndrome (BTHS) is a rare, infantile-onset, X-linked mitochondriopathy exhibiting a variable presentation of failure to thrive, growth insufficiency, skeletal myopathy, neutropenia, and heart anomalies due to mitochondrial dysfunction secondary to inherited TAFAZZIN transacetylase mutations. Although not reported in BTHS patients, male infertility is observed in several Tafazzin (Taz) mouse alleles and in a Drosophila mutant. Herein, we examined the male infertility phenotype in a BTHS-patient-derived D75H point-mutant knockin mouse (TazPM) allele that expresses a mutant protein lacking transacetylase activity. Neonatal and adult TazPM testes were hypoplastic, and their epididymis lacked sperm. Histology and biomarker analysis revealed TazPM spermatogenesis is arrested prior to sexual maturation due to an inability to undergo meiosis and the generation of haploid spermatids. Moreover, TazPM testicular mitochondria were found to be structurally abnormal, and there was an elevation of p53-dependent apoptosis within TazPM seminiferous tubules. Immunoblot analysis revealed that TazPM gamete genome integrity was compromised, and both histone γ-H2Ax and Nucleoside diphosphate kinase-5 protein expression were absent in juvenile TazPM testes when compared to controls. We demonstrate that Taz-mediated transacetylase activity is required within mitochondria for normal spermatogenesis, and its absence results in meiotic arrest. We hypothesize that elevated TazPM spermatogonial apoptosis causes azoospermia and complete infertility. [ABSTRACT FROM AUTHOR]

Cancers invoke various pathways to mitigate external and internal stresses to continue their growth and progression. We previously reported that the eIF2 kinase GCN2 and the integrated stress response are constitutively active in prostate cancer (PCa) and are required to maintain amino acid homeostasis needed to fuel tumor growth. However, although loss of GCN2 function reduces intracellular amino acid availability and PCa growth, there is no appreciable cell death. Here, we discovered that the loss of GCN2 in PCa induces prosenescent p53 signaling. This p53 activation occurred through GCN2 inhibition–dependent reductions in purine nucleotides that impaired ribosome biogenesis and, consequently, induced the impaired ribosome biogenesis checkpoint. p53 signaling induced cell cycle arrest and senescence that promoted the survival of GCN2-deficient PCa cells. Depletion of GCN2 combined with loss of p53 or pharmacological inhibition of de novo purine biosynthesis reduced proliferation and enhanced cell death in PCa cell lines, organoids, and xenograft models. Our findings highlight the coordinated interplay between GCN2 and p53 regulation during nutrient stress and provide insight into how they could be targeted in developing new therapeutic strategies for PCa. Editor's summary: The integrated stress response (ISR) enables tumor cells to adapt quickly to their tissue environment, thus supporting cell survival and tumor progression. Cordova et al. found that inhibiting the ISR kinase GCN2 kills prostate cancer cells deficient in the transcription factor p53. GCN2 inhibition decreased the availability of amino acids that are used in purine nucleotide synthesis. In turn, this nucleotide deficiency was survivable only with p53 signaling, which induced cell cycle arrest and senescence. In mice, although inhibiting GCN2 slowed the growth of p53-functional tumors, it did so to a greater extent and induced cell death in p53-deficient tumors. The loss of p53 typically makes killing tumor cells challenging, and these findings indicate a way to take advantage of those mutations. —Leslie K. Ferrarelli [ABSTRACT FROM AUTHOR]

Cardiomyopathy is the predominant defect in Barth syndrome (BTHS) and is caused by a mutation of the X-linked Tafazzin (TAZ) gene, which encodes an enzyme responsible for remodeling mitochondrial cardiolipin. Despite the known importance of mitochondrial dysfunction in BTHS, how specific TAZ mutations cause diverse BTHS heart phenotypes remains poorly understood. We generated a patient-tailored CRISPR/Cas9 knock-in mouse allele (TazPM) that phenocopies BTHS clinical traits. As TazPM males express a stable mutant protein, we assessed cardiac metabolic dysfunction and mitochondrial changes and identified temporally altered cardioprotective signaling effectors. Specifically, juvenile TazPM males exhibit mild left ventricular dilation in systole but have unaltered fatty acid/amino acid metabolism and normal adenosine triphosphate (ATP). This occurs in concert with a hyperactive p53 pathway, elevation of cardioprotective antioxidant pathways, and induced autophagy-mediated early senescence in juvenile TazPM hearts. However, adult TazPM males exhibit chronic heart failure with reduced growth and ejection fraction, cardiac fibrosis, reduced ATP, and suppressed fatty acid/amino acid metabolism. This biphasic changeover from a mild-to-severe heart phenotype coincides with p53 suppression, downregulation of cardioprotective antioxidant pathways, and the onset of terminal senescence in adult TazPM hearts. Herein, we report a BTHS genotype/phenotype correlation and reveal that absent Taz acyltransferase function is sufficient to drive progressive cardiomyopathy. [ABSTRACT FROM AUTHOR]

New Findings: What is the central question of this study?Skeletal muscle extracellular vesicles likely act as pro‐angiogenic signalling factors: does overexpression of peroxisome proliferator‐activated receptor γ coactivator 1‐α (PGC‐1α) alter skeletal muscle myotube extracellular vesicle release, contents and angiogenic potential?What is the main finding and its importance?Overexpression of PGC‐1α results in secretion of extracellular vesicles that elevate measures of angiogenesis and protect against acute oxidative stress in vitro. Skeletal muscle with high levels of PGC‐1α expression, commonly associated with exercise induced angiogenesis and high basal capillarization, may secrete extracellular vesicles that support capillary growth and maintenance. Skeletal muscle capillarization is proportional to muscle fibre mitochondrial content and oxidative capacity. Skeletal muscle cells secrete many factors that regulate neighbouring capillary endothelial cells (ECs), including extracellular vesicles (SkM‐EVs). Peroxisome proliferator‐activated receptor γ coactivator 1‐α (PGC‐1α) regulates mitochondrial biogenesis and the oxidative phenotype in skeletal muscle. Skeletal muscle PGC‐1α also regulates secretion of multiple angiogenic factors, but it is unknown whether PGC‐1α regulates SkM‐EV release, contents and angiogenic signalling potential. PGC‐1α was overexpressed via adenovirus in primary human myotubes. EVs were collected from PGC‐1α‐overexpressing myotubes (PGC‐EVs) as well as from green fluorescent protein‐overexpressing myotubes (GFP‐EVs), and from untreated myotubes. EV release and select mRNA contents were measured from EVs. Additionally, ECs were treated with EVs to measure angiogenic potential of EVs in normal conditions and following an oxidative stress challenge. PGC‐1α overexpression did not impact EV release but did elevate EV content of mRNAs for several antioxidant proteins (nuclear factor erythroid 2‐related factor 2, superoxide dismutase 2, glutathione peroxidase). PGC‐EV treatment of cultured human umbilical vein endothelial cells (HUVECs) increased their proliferation (+36.6%), tube formation (length: +28.1%; number: +25.7%) and cellular viability (+52.9%), and reduced reactive oxygen species levels (−41%) compared to GFP‐EVs. Additionally, PGC‐EV treatment protected against tube formation impairments and induction of cellular senescence following acute oxidative stress. Overexpression of PGC‐1α in human myotubes increases the angiogenic potential of SkM‐EVs. These angiogenic benefits coincided with increased anti‐oxidative capacity of recipient HUVECs. High PGC‐1α expression in skeletal muscle may prompt the release of SkM‐EVs that support vascular redox homeostasis and angiogenesis. [ABSTRACT FROM AUTHOR]

Metabolic control within skeletal muscle is designed to limit ADP accumulation even during conditions where ATP demand is out of balance with ATP synthesis. This is accomplished by the reactions of adenylate kinase (AK: ADP+ ADP [left and right arrow] AMP+ATP) and AMP deaminase (AMP+[H.sub.2]0 [right arrow] N[H.sub.3]+IMP), which limit ADP accumulation under these conditions. The purpose of this study was to determine whether AK deficiency (AK-/-) would result in sufficient ADP accumulation to be visible using [sup.31]P-NMRS during the high energy demands of frequent in situ tetanic contractions. To do this we examined the high-energy phosphates of the gastrocnemius muscle in the knockout mouse with AK1-/- and wild-type (WT) control muscle over the course of 64 rapid (2/s) isometric tetanic contractions. Near-complete depletion of phosphocreatine was apparent after 16 contractions in both groups. By ~40 contractions, ADP was clearly visible in AK1-/- muscle. This transient concentration of the NMR visible tree ADP was estimated to be ~1.7 mM, and represents the first time free ADP has been directly measured in contracting skeletal muscle. Such an increase in free ADP is severalfold greater than previously thought to occur. This large accumulation of tree ADP also represents a significant reduction in energy available from ATP, and has implications on cellular processes that depend on a high yield of energy from ATP such as calcium sequestration. Remarkably, the AK1-/- and WT muscles exhibited similar fatigue profiles. Our findings suggest that skeletal muscle is surprisingly tolerant to a large increase in ADP and by extension, a decline in energy from ATP. muscle energetics; muscle relaxation; magnetic resonance spectroscopy

Skeletal muscle fiber types differ in their contents of total phosphate, which includes inorganic phosphate ([P.sub.i]) and high-energy organic pools of ATP and phosphocreatine (PCr). At steady state, uptake of [P.sub.i] into the cell must equal the rate of efflux, which is expected to be a function of intracellular [P.sub.i] concentration. We measured [sup.32]P-labeled [P.sub.i] uptake rates in different muscle fiber types to determine whether they are proportional to cellular [P.sub.i] content. [P.sub.i] uptake rates in isolated, perfused rat hindlimb muscles were linear over time and highest in soleus (2.42 [+ or -] 0.17 [micro]mol * [g.sup.-1] * [h.sup.-1]), lower in red gastrocnemius (1.31 [+ or -] 0.11 [micro] mol * [g.sup.-1] * [h.sup.-1]), and lowest in white gastrocnemius (0.49 [+ or -] 0.06 [micro]mol * [g.sup.-1] * [h.sup.-1]). Reasonably similar rates were obtained in vivo. [P.sub.i] uptake rates at plasma [P.sub.i] concentrations of 0.3-1.7 mM confirm that the [P.sub.i] uptake process is nearly saturated at normal plasma [P.sub.i] levels. [P.sub.i]] uptake rate correlated with cellular [P.sub.i] content (r = 0.99) but varied inversely with total phosphate content. Sodium-phosphate cotransporter (PiT-1) protein expression in soleus and red gastrocnemius were similar to each other and seven- to eightfold greater than PiT-1 expression in white gastrocnemius. That the PiT-1 expression pattern did not match the pattern of P, uptake across fiber types implies that other factors are involved in regulating [P.sub.i] uptake in skeletal muscle. Furthermore, fractional turnover of the cellular [P.sub.i] pool (0.67, 0.57, and 0.33 [h.sup.-1] in soleus, red gastrocnemius, and white gastrocnemius, respectively) varies among fiber types, indicating differential management of intracellular [P.sub.i], likely due to differences in resistance to [P.sub.i] efflux from the fiber. inorganic phosphate; sodium-inorganic phosphate transporters; PiT-2; inorganic phosphate efflux

Total creatine ([Cr.sub.total] = phosphocreatine + creatine) concentrations differ substantially among mammalian skeletal muscle. Because the primary means to add [Cr.sub.total] to muscle is uptake of creatine through the sodium-dependent creatine transporter (CrT), differences in creatine uptake and CrT expression could account for the variations in [[Cr.sub.total]] among muscle fiber types. To test this hypothesis, hindlimbs of adult rats were perfused with 0.05-1 mM [[sup.14]C]creatine for up to 90 min. Creatine uptake rates at 1 mM creatine were greatest in the soleus (140 [+ or -] 8.8 nmol*[h.sub.-1]*[g.sub.-1]), less in the red gastrocnemius (117 [+ or -] 8.3), and least in the white gastrocnemius (97 [+ or -] 10.7). These rates were unaltered by time, insulin concentration, or increased perfusate sodium concentration. Conversely, creatine uptake rates were correspondingly decreased among fiber types by lower creatine and sodium concentrations. The CrT protein content by Western blot analysis was similarly greatest in the soleus, less in the red gastrocnemius, and least in the white gastrocnemius, whereas CrT mRNA was not different. Creatine uptake rates differ among skeletal muscle fiber sections in a manner reasonably assigned to the 58-kDa band of the CrT. Furthermore, creatine uptake rates scale inversely with creatine content, with the lowest uptake rate in the fiber type with the highest [Cr.sub.total] and vice versa. This suggests that the creatine pool fractional turnover rate is not common across muscle phenotypes and, therefore, is differentially regulated. phosphocreatine; insulin; creatine turnover

Short-term creatine supplementation is reported to result in a decreased ratio of phosphocreatine (PCr) to total creatine (TCr) in human skeletal muscle at rest. Assuming equilibrium of the creatine kinase reaction, this decrease in PCr:TCr implies increased cytoplasmic ADP and decreased Gibbs free energy of ATP hydrolysis in muscle, which seems contrary to the reported ergogenic benefits of creatine supplementation. This study measured changes in PCr and TCr in vastus lateralis muscle of adult men (N = 6, 21-35 y old) during and 1 day after 5 d of creatine monohydrate supplementation (0.43 g⋅kg body weight-1⋅d-1) using noninvasive 31P and ¹H magnetic-resonance spectroscopy (MRS). Plasma and red-blood-cell creatine increased by 10-fold and 2-fold, respectively, by the third day of supplementation. MRS-measured skeletal muscle PCr and TCr increased linearly and in parallel throughout the 5 d, and there was no significant difference in the percentage increase in muscle PCr (11.7% ± 2.3% after 5 d) vs. TCr (14.9% ± 4.1%) at any time point. The results indicate that creatine supplementation does not alter the PCr:TCr ratio, and hence the cytoplasmic Gibbs free energy of ATP hydrolysis, in human skeletal muscle at rest. [ABSTRACT FROM AUTHOR]

Key points: Exercise/exercise training can enhance insulin sensitivity through adaptations in skeletal muscle, the primary site of insulin‐mediated glucose disposal; however, in humans the range of improvement can vary substantially.The purpose of this study was to determine if obesity influences the magnitude of the exercise response in relation to improving insulin sensitivity in human skeletal muscle.Electrical pulse stimulation (EPS; 24 h) of primary human skeletal muscle myotubes improved insulin action in tissue from both lean and severely obese individuals, but responses to EPS were blunted with obesity.EPS improved insulin signal transduction in myotubes from lean but not severely obese subjects and increased AMP accumulation and AMPK Thr172 phosphorylation, but to a lesser degree in myotubes from the severely obese.These data reveal that myotubes of severely obese individuals enhance insulin action and stimulate exercise‐responsive molecules with contraction, but in a manner and magnitude that differs from lean subjects. Exercise/muscle contraction can enhance whole‐body insulin sensitivity; however, in humans the range of improvements can vary substantially. In order, to determine if obesity influences the magnitude of the exercise response, this study compared the effects of electrical pulse stimulation (EPS)‐induced contractile activity upon primary myotubes derived from lean and severely obese (BMI ≥ 40 kg/m2) women. Prior to muscle contraction, insulin action was compromised in myotubes from the severely obese as was evident from reduced insulin‐stimulated glycogen synthesis, glucose oxidation, glucose uptake, insulin signal transduction (IRS1, Akt, TBC1D4), and insulin‐stimulated GLUT4 translocation. EPS (24 h) increased AMP, IMP, AMPK Thr172 phosphorylation, PGC1α content, and insulin action in myotubes of both the lean and severely obese subjects. However, despite normalizing indices of insulin action to levels seen in the lean control (non‐EPS) condition, responses to EPS were blunted with obesity. EPS improved insulin signal transduction in myotubes from lean but not severely obese subjects and EPS increased AMP accumulation and AMPK Thr172 phosphorylation, but to a lesser degree in myotubes from the severely obese. These data reveal that myotubes of severely obese individuals enhance insulin action and stimulate exercise‐responsive molecules with contraction, but in a manner and magnitude that differs from lean subjects. Key points: Exercise/exercise training can enhance insulin sensitivity through adaptations in skeletal muscle, the primary site of insulin‐mediated glucose disposal; however, in humans the range of improvement can vary substantially.The purpose of this study was to determine if obesity influences the magnitude of the exercise response in relation to improving insulin sensitivity in human skeletal muscle.Electrical pulse stimulation (EPS; 24 h) of primary human skeletal muscle myotubes improved insulin action in tissue from both lean and severely obese individuals, but responses to EPS were blunted with obesity.EPS improved insulin signal transduction in myotubes from lean but not severely obese subjects and increased AMP accumulation and AMPK Thr172 phosphorylation, but to a lesser degree in myotubes from the severely obese.These data reveal that myotubes of severely obese individuals enhance insulin action and stimulate exercise‐responsive molecules with contraction, but in a manner and magnitude that differs from lean subjects. [ABSTRACT FROM AUTHOR]

Peroxisomes are indispensable organelles for lipid metabolism in humans, and their biogenesis has been assumed to be under regulation by peroxisome proliferator-activated receptors (PPARs). However, recent studies in hepatocytes suggest that the mitochondrial proliferator PGC-1α (peroxisome proliferator-activated receptor gamma coactivator-1α) also acts as an upstream transcriptional regulator for enhancing peroxisomal abundance and associated activity. It is unknown whether the regulatory mechanism(s) for enhancing peroxisomal function is through the same node as mitochondrial biogenesis in human skeletal muscle (HSkM) and whether fatty acid oxidation (FAO) is affected. Primary myotubes from vastus lateralis biopsies from lean donors (BMI = 24.0 ± 0.6 kg/m2; n = 6) were exposed to adenovirus encoding human PGC-1α or GFP control. Peroxisomal biogenesis proteins (peroxins) and genes (PEXs) responsible for proliferation and functions were assessed by Western blotting and real-time qRT-PCR, respectively. [1-14C] palmitic acid and [1-14C]lignoceric acid (exclusive peroxisomal-specific substrate) were used to assess mitochondrial oxidation of peroxisomal-derived metabolites. After overexpression of PGC-1α, 1) peroxisomal membrane protein 70 kDa (PMP70), PEX19, and mitochondrial citrate synthetase protein content were significantly elevated (P < 0.05), 2) PGC-1α, PMP70, key PEXs, and peroxisomal β-oxidation mRNA expression levels were significantly upregulated (P < 0.05), and 3) a concomitant increase in lignoceric acid oxidation by both peroxisomal and mitochondrial activity was observed (P < 0.05). These novel findings demonstrate that, in addition to the proliferative effect on mitochondria, PGC-1α can induce peroxisomal activity and accompanying elevations in long-chain and very-longchain fatty acid oxidation by a peroxisomal-mitochondrial functional cooperation, as observed in HSkM cells. [ABSTRACT FROM AUTHOR]