Cell selectivity in succinate receptor SUCNR1/GPR91 signaling in skeletal muscle.

Succinate is released by skeletal muscle during exercise and activates SUCNR1/GPR91. Signaling of SUCNR1 is involved in metabolite-sensing paracrine communication in skeletal muscle during exercise. However, the specific cell types responding to succinate and the directionality of communication are unclear. We aim to characterize the expression of SUCNR1 in human skeletal muscle. De novo analysis of transcriptomic datasets demonstrated that SUCNR1 mRNA is expressed in immune, adipose, and liver tissues, but scarce in skeletal muscle. In human tissues, SUCNR1 mRNA was associated with macrophage markers. Single-cell RNA sequencing and fluorescent RNAscope demonstrated that in human skeletal muscle, SUCNR1 mRNA is not expressed in muscle fibers but coincided with macrophage populations. Human M2-polarized macrophages exhibit high levels of SUCNR1 mRNA and stimulation with selective agonists of SUCNR1 triggered Gq- and Gi-coupled signaling. Primary human skeletal muscle cells were unresponsive to SUCNR1 agonists. In conclusion, SUCNR1 is not expressed in muscle cells and its role in the adaptive response of skeletal muscle to exercise is most likely mediated via paracrine mechanisms involving M2-like macrophages within the muscle.NEW & NOTEWORTHY Macrophages but not skeletal muscle cells respond to extracellular succinate via SUCNR1/GPR91.

Group IIA secreted phospholipase A2 (PLA2G2A) augments adipose tissue thermogenesis.

Group IIA secreted phospholipase A2 (PLA2G2A) hydrolyzes glycerophospholipids at the sn-2 position resulting in the release of fatty acids and lysophospholipids. C57BL/6 mice do not express Pla2g2a due to a frameshift mutation (wild-type [WT] mice). We previously reported that transgenic expression of human PLA2G2A in C57BL/6 mice (IIA+ mice) protects against weight gain and insulin resistance, in part by increasing total energy expenditure. Additionally, we found that brown and white adipocytes from IIA+ mice have increased expression of mitochondrial uncoupling markers, such as uncoupling protein 1 (UCP1), peroxisome proliferator-activated receptor-gamma coactivator, and PR domain containing 16, suggesting that the energy expenditure phenotype might be due to an increased thermogenic capacity in adipose tissue. Here, we further characterize the impact of PLA2G2A on thermogenic mechanisms in adipose tissue. Metabolic analysis of WT and IIA+ mice revealed that even when housed within their thermoneutral zone, IIA+ mice have elevated energy expenditure compared to WT littermates. Increased energy expenditure in IIA+ mice is associated with increased citrate synthase activity in brown adipose tissue (BAT) and increased mitochondrial respiration in both brown and white adipocytes. We also observed that direct addition of recombinant PLA2G2A enzyme to in vitro cultured adipocytes results in the marked induction of UCP1 protein expression. Finally, we report that PLA2G2A induces the expression of numerous transcripts related to energy substrate transport and metabolism in BAT, suggestive of an increase in substrate flux to fuel BAT activity. These data demonstrate that PLA2G2A enhances adipose tissue thermogenesis, in part, through elevated substrate delivery and increased mitochondrial content in BAT.

Three weeks of interrupting sitting lowers fasting glucose and glycemic variability, but not glucose tolerance, in free-living women and men with obesity.

We aimed to determine whether interrupting prolonged sitting improves glycemic control and the metabolic profile of free-living adults with obesity. Sixteen sedentary individuals {10 women/6 men; median [interquartile range (IQR)] age 50 (44-53) yr, body mass index (BMI) 32 (32-35.8) kg/m2} were fitted with continuous glucose and activity monitors for 4 wk. After a 1-wk baseline period, participants were randomized into habitual lifestyle (Control) or frequent activity breaks from sitting (FABS) intervention groups. Each day, between 0800 and 1800 h, FABS received smartwatch notifications to break sitting with 3 min of low-to-moderate-intensity physical activity every 30 min. Glycemic control was assessed by oral glucose tolerance test (OGTT) and continuous glucose monitoring. Blood samples and vastus lateralis biopsies were taken for assessment of clinical chemistry and the skeletal muscle lipidome, respectively. Compared with baseline, FABS increased median steps by 744 [IQR (483-951)] and walking time by 10.4 [IQR (2.2-24.6)] min/day. Other indices of activity/sedentary behavior were unchanged. Glucose tolerance and average 24-h glucose curves were also unaffected. However, mean (±SD) fasting glucose levels [-0.34 (±0.37) mmol/L] and daily glucose variation [%CV; -2% (±2.2%)] reduced in FABS, suggesting a modest benefit for glycemic control that was most robust at higher volumes of daily activity. Clinical chemistry and the skeletal muscle lipidome were largely unperturbed, although two long-chain triglycerides increased 1.25-fold in FABS, postintervention. All parameters remained stable in control. Under free-living conditions, FABS lowered fasting glucose and glucose variability. Larger volumes of activity breaks from sitting may be required to promote greater health benefits.NEW & NOTEWORTHY Under free-living conditions, breaking sitting modestly increased activity behavior. Breaking sitting was insufficient to modulate glucose tolerance or the skeletal muscle lipidome. Activity breaks reduced fasting blood glucose levels and daily glucose variation compared with baseline, with a tendency to also decrease fasting LDLc. This intervention may represent the minimal dose for breaking sedentary behavior, with larger volumes of activity possibly required to promote greater health benefits.

Comparative profiling of skeletal muscle models reveals heterogeneity of transcriptome and metabolism.

Rat L6, mouse C2C12, and primary human skeletal muscle cells (HSMCs) are commonly used to study biological processes in skeletal muscle, and experimental data on these models are abundant. However, consistently matched experimental data are scarce, and comparisons between the different cell types and adult tissue are problematic. We hypothesized that metabolic differences between these cellular models may be reflected at the mRNA level. Publicly available data sets were used to profile mRNA levels in myotubes and skeletal muscle tissues. L6, C2C12, and HSMC myotubes were assessed for proliferation, glucose uptake, glycogen synthesis, mitochondrial activity, and substrate oxidation, as well as the response to in vitro contraction. Transcriptomic profiling revealed that mRNA of genes coding for actin and myosin was enriched in C2C12, whereas L6 myotubes had the highest levels of genes encoding glucose transporters and the five complexes of the mitochondrial electron transport chain. Consistently, insulin-stimulated glucose uptake and oxidative capacity were greatest in L6 myotubes. Insulin-induced glycogen synthesis was highest in HSMCs, but C2C12 myotubes had higher baseline glucose oxidation. All models responded to electrical pulse stimulation-induced glucose uptake and gene expression but in a slightly different manner. Our analysis reveals a great degree of heterogeneity in the transcriptomic and metabolic profiles of L6, C2C12, or primary human myotubes. Based on these distinct signatures, we provide recommendations for the appropriate use of these models depending on scientific hypotheses and biological relevance.

Train like an athlete: applying exercise interventions to manage type 2 diabetes.

Exercise elicits high energy demands, stimulating cardiorespiratory function and substrate mobilisation and oxidation. Repeated bouts of exercise lead to whole-body adaptations, which improve athletic performance. Distinct exercise modalities and intensities and nutritional conditions pose specific physiological challenges, subsequently inducing different adaptations to training. Athletes often modify these variables to achieve individualised training goals and maximise performance. Exercise training improves glycaemic control in individuals with type 2 diabetes; however, the precise training regimen that confers the most beneficial metabolic adaptations in this population is unknown. In this review, we discuss how modifying exercise type, intensity and modality and nutritional status affects the beneficial effects of exercise on glycaemic control in individuals with type 2 diabetes. Evidence indicates that greater improvements in glycaemic control can be achieved through combined aerobic and resistance training regimens compared with either training type alone. However, the increased frequency of training and a greater number of exercise bouts during combined programmes could be responsible for apparent advantages over a single training modality. The beneficial effects of aerobic exercise on glycaemic control seem to rise with training intensity, with superior adaptations achieved by high-intensity interval training (HIT). In addition, training with low carbohydrate availability ('training low') improves cardiorespiratory function and skeletal muscle oxidative capacity more than conventional training in healthy untrained individuals. Examinations of various training regimens are warranted to assess the safety, efficacy, feasibility and beneficial effects in the type 2 diabetes population. Just like competitive athletes, individuals with type 2 diabetes should be encouraged to adopt training regimens that improve fitness and metabolism. Graphical abstract.

Afternoon exercise is more efficacious than morning exercise at improving blood glucose levels in individuals with type 2 diabetes: a randomised crossover trial.

AIMS/HYPOTHESIS: Exercise is recommended for the treatment and prevention of type 2 diabetes. However, the most effective time of day to achieve beneficial effects on health remains unknown. We aimed to determine whether exercise training at two distinct times of day would have differing effects on 24 h blood glucose levels in men with type 2 diabetes. METHODS: Eleven men with type 2 diabetes underwent a randomised crossover trial. Inclusion criteria were 45-68 years of age and BMI between 23 and 33 kg/m2. Exclusion criteria were insulin treatment and presence of another systemic illness. Researchers were not blinded to the group assignment. The trial involved 2 weeks of either morning or afternoon high-intensity interval training (HIIT) (three sessions/week), followed by a 2 week wash-out period and a subsequent period of the opposite training regimen. Continuous glucose monitor (CGM)-based data were obtained. RESULTS: Morning HIIT increased CGM-based glucose concentration (6.9 ± 0.4 mmol/l; mean ± SEM for the exercise days during week 1) compared with either the pre-training period (6.4 ± 0.3 mmol/l) or afternoon HIIT (6.2 ± 0.3 mmol/l for the exercise days during week 1). Conversely, afternoon HIIT reduced the CGM-based glucose concentration compared with either the pre-training period or morning HIIT. Afternoon HIIT was associated with elevated thyroid-stimulating hormone (TSH; 1.9 ± 0.2 mU/l) and reduced T4 (15.8 ± 0.7 pmol/l) concentrations compared with pre-training (1.4 ± 0.2 mU/l for TSH; 16.8 ± 0.6 pmol/l for T4). TSH was also elevated after morning HIIT (1.7 ± 0.2 mU/l), whereas T4 concentrations were unaltered. CONCLUSIONS/INTERPRETATION: Afternoon HIIT was more efficacious than morning HIIT at improving blood glucose in men with type 2 diabetes. Strikingly, morning HIIT had an acute, deleterious effect, increasing blood glucose. However, studies of longer training regimens are warranted to establish the persistence of this adverse effect. Our data highlight the importance of optimising the timing of exercise when prescribing it as treatment for type 2 diabetes.

Normalization of disrupted clock gene expression in males with tetraplegia: a crossover randomized placebo-controlled trial of melatonin supplementation.

STUDY DESIGN: Crossover double blind, randomized placebo-controlled trial. OBJECTIVES: Circadian oscillators are located both in the brain and in peripheral organs. Melatonin, the main brain-derived hormone governing circadian variations, is highly associated with daylight patterns. However, in subjects with tetraplegia the melatonin levels are blunted. Here we studied peripheral oscillators in peripheral blood mononuclear cells (PBMCs) in males with tetraplegia by examining how exogenous melatonin may influence the expression of clock gene mRNAs. SETTING: Sunnaas Rehabilitation Hospital, Nesoddtangen, Norway. METHODS: Six males with tetraplegia received 2 mg of melatonin or placebo 4 days before the study period. We also included six able-bodied men sleeping or kept awake during the night. Plasma samples were collected four times during a 24-h period. The mRNA expression levels of the clock genes PER1, PER2, BMAL1, and REV-ERBα were quantified in PBMCs using quantitative RT-PCR. RESULTS: The mRNA expression levels of PER-1 and -2 and REV-ERBα were increased at 04:00 h compared with the able-bodied controls (p < 0.05). Melatonin supplementation changed mRNA peak-time toward the time of supplementation. CONCLUSIONS: Several peripheral clock genes displayed distorted expression levels in tetraplegia. Supplementation with melatonin changed the mRNA expression levels of these genes toward those observed among able-bodied. SPONSORSHIP: Financial support was provided from the Throne Holst Foundation, Sunnaas Rehabilitation hospital and the University of Ferrara (FAR2016).

Time-dependent reduction in oxidative capacity among cultured myotubes from spinal cord injured individuals.

BACKGROUND: Skeletal muscle adapts in reaction to contractile activity to efficiently utilize energy substrates, primarily glucose and free fatty acids (FA). Inactivity leads to atrophy and a change in energy utilization in individuals with spinal cord injury (SCI). The present study aimed to characterize possible inactivity-related differences in the energy metabolism between skeletal muscle cells cultured from satellite cells isolated 1- and 12-months post-SCI. METHODS: To characterize inactivity-related disturbances in spinal cord injury, we studied skeletal muscle cells isolated from SCI subjects. Cell cultures were established from biopsy samples from musculus vastus lateralis from subjects with SCI 1 and 12 months after the injury. The myoblasts were proliferated and differentiated into myotubes before fatty acid and glucose metabolism were assessed and gene and protein expressions were measured. RESULTS: The results showed that glucose uptake was increased, while oleic acid oxidation was reduced at 12 months compared to 1 month. mRNA expressions of PPARGC1α, the master regulator of mitochondrial biogenesis, and MYH2, a determinant of muscle fiber type, were significantly reduced at 12 months. Proteomic analysis showed reduced expression of several mitochondrial proteins. CONCLUSION: In conclusion, skeletal muscle cells isolated from immobilized subjects 12 months compared to 1 month after SCI showed reduced fatty acid metabolism and reduced expression of mitochondrial proteins, indicating an increased loss of oxidative capacity with time after injury.

Diacylglycerol kinase delta overexpression improves glucose clearance and protects against the development of obesity.

BACKGROUND AND AIM: Diacylglycerol kinase (DGK) isoforms catalyze an enzymatic reaction that removes diacylglycerol (DAG) and thereby terminates protein kinase C signaling by converting DAG to phosphatidic acid. DGKδ (type II isozyme) downregulation causes insulin resistance, metabolic inflexibility, and obesity. Here we determined whether DGKδ overexpression prevents these metabolic impairments. METHODS: We generated a transgenic mouse model overexpressing human DGKδ2 under the myosin light chain promoter (DGKδ TG). We performed deep metabolic phenotyping of DGKδ TG mice and wild-type littermates fed chow or high-fat diet (HFD). Mice were also provided free access to running wheels to examine the effects of DGKδ overexpression on exercise-induced metabolic outcomes. RESULTS: DGKδ TG mice were leaner than wild-type littermates, with improved glucose tolerance and increased skeletal muscle glycogen content. DGKδ TG mice were protected against HFD-induced glucose intolerance and obesity. DGKδ TG mice had reduced epididymal fat and enhanced lipolysis. Strikingly, DGKδ overexpression recapitulated the beneficial effects of exercise on metabolic outcomes. DGKδ overexpression and exercise had a synergistic effect on body weight reduction. Microarray analysis of skeletal muscle revealed common gene ontology signatures of exercise and DGKδ overexpression that were related to lipid storage, extracellular matrix, and glycerophospholipids biosynthesis pathways. CONCLUSION: Overexpression of DGKδ induces adaptive changes in both skeletal muscle and adipose tissue, resulting in protection against HFD-induced obesity. DGKδ overexpression recapitulates exercise-induced adaptations on energy homeostasis and skeletal muscle gene expression profiles.

Exercise timing influences multi-tissue metabolome and skeletal muscle proteome profiles in type 2 diabetic patients - A randomized crossover trial.

AIMS/HYPOTHESIS: Metabolic effects of exercise may partly depend on the time-of-day when exercise is performed. We tested the hypothesis that exercise timing affects the adaptations in multi-tissue metabolome and skeletal muscle proteome profiles in men with type 2 diabetes. METHODS: Men fitting the inclusion (type 2 diabetes, age 45-68 years and body mass index 23-33 kg/m2) and exclusion criteria (insulin treatment, smoking, concurrent systemic disease, and regular exercise training) were included in a randomized crossover trial (n = 15). Participants included in this metabolomics and proteomics analysis fully completed all exercise sessions (n = 8). The trial consisted of two weeks of high-intensity interval training (HIT) (three sessions/week) either in the morning (08:00, n = 5) or afternoon (16:45, n = 3), a two-week wash-out period, and an additional two weeks of HIT at the opposing time. Participants and researchers were not blinded to group allocation. Blood, skeletal muscle and subcutaneous adipose tissue were obtained before the first, and after each training period. Broad-spectrum, untargeted proteomic analysis was performed on skeletal muscle, and metabolomic analysis was performed on all biosamples. Differential content was assessed by linear regression and pathway set enrichment analyses were performed. Coordinated metabolic changes across tissues were identified by Spearman correlation analysis. RESULTS: Metabolic and proteomic profiles remained stable after two weeks of HIT, and individual metabolites and proteins were not altered, irrespective of the time of day at which the training was performed. However, coordinated changes in relevant metabolic pathways and protein categories were identified. Morning and afternoon HIT similarly increased plasma diacylglycerols, skeletal muscle acyl-carnitines, and subcutaneous adipose tissue sphingomyelins and lysophospholipids. Acyl-carnitines were central to training-induced metabolic cross-talk across tissues. Plasma carbohydrates, via the penthose phosphate pathway, were increased and skeletal muscle lipids were decreased after morning compared to afternoon HIT. Skeletal muscle lipoproteins were higher, and mitochondrial complex III abundance was lower after morning compared to afternoon HIT. CONCLUSIONS/INTERPRETATION: We provide a comprehensive analysis of a multi-tissue metabolomic and skeletal muscle proteomic responses to training at different times of the day in men with type 2 diabetes. Increased circulating lipids and changes in adipose tissue lipid composition were common between morning and afternoon HIT. However, afternoon HIT increased skeletal muscle lipids and mitochondrial content to a greater degree than morning training. Thus, there is a diurnal component in the metabolomic and proteomic response to exercise in men with type 2 diabetes. The clinical relevance of this response warrants further investigation.

Altered oxidative stress and antioxidant defence in skeletal muscle during the first year following spinal cord injury.

Oxidative stress promotes protein degradation and apoptosis in skeletal muscle undergoing atrophy. We aimed to determine whether spinal cord injury leads to changes in oxidative stress, antioxidant capacity, and apoptotic signaling in human skeletal muscle during the first year after spinal cord injury. Vastus lateralis biopsies were obtained from seven individuals 1, 3, and 12 months after spinal cord injury and from seven able-bodied controls. Protein content of enzymes involved in reactive oxygen species production and detoxification, and apoptotic signaling were analyzed by western blot. Protein carbonylation and 4-hydroxynonenal protein adducts were measured as markers of oxidative damage. Glutathione content was determined fluorometrically. Protein content of NADPH oxidase 2, xanthine oxidase, and pro-caspase-3 was increased at 1 and 3 months after spinal cord injury compared to able-bodied controls. Furthermore, total and reduced glutathione content was increased at 1 and 3 months after spinal cord injury. Conversely, mitochondrial complexes and superoxide dismutase 2 protein content were decreased 12 months after spinal cord injury compared to able-bodied controls. In conclusion, we provide indirect evidence of increased reactive oxygen species production and increased apoptotic signaling at 1 and 3 months after spinal cord injury. Concomitant increases in glutathione antioxidant defences may reflect adaptations poised to maintain redox homeostasis in skeletal muscle following spinal cord injury.

Retained differentiation capacity of human skeletal muscle satellite cells from spinal cord-injured individuals.

Despite the well-known role of satellite cells in skeletal muscle plasticity, the effect of spinal cord injury on their function in humans remains unknown. We determined whether spinal cord injury affects the intrinsic ability of satellite cells to differentiate and produce metabolically healthy myotubes. We obtained vastus lateralis biopsies from eight spinal cord-injured and six able-bodied individuals. Satellite cells were isolated, grown and differentiated in vitro. Gene expression was measured by quantitative PCR. Abundance of differentiation markers and regulatory proteins was determined by Western blotting. Protein synthesis and fatty acid oxidation were measured by radioactive tracer-based assays. Activated satellite cells (myoblasts) and differentiated myotubes derived from skeletal muscle of able-bodied and spinal cord-injured individuals expressed similar (P > 0.05) mRNA levels of myogenic regulatory factors. Myogenic differentiation factor 1 expression was higher in myoblasts from spinal cord-injured individuals. Desmin and myogenin protein content was increased upon differentiation in both groups, while myotubes from spinal cord-injured individuals contained more type I and II myosin heavy chain. Phosphorylated and total protein levels of Akt-mechanistic target of rapamycin and forkhead box protein O signalling axes and protein synthesis rate in myotubes were similar (P > 0.05) between groups. Additionally, fatty acid oxidation of myotubes from spinal cord-injured individuals was unchanged (P > 0.05) compared to able-bodied controls. Our results indicate that the intrinsic differentiation capacity of satellite cells and metabolic characteristics of myotubes are preserved following spinal cord injury. This may inform potential interventions targeting satellite cell activation to alleviate skeletal muscle atrophy.