Adiponectin stimulates Sca1+CD34--adipocyte precursor cells associated with hyperplastic expansion and beiging of brown and white adipose tissue.

BACKGROUND: The adipocyte hormone adiponectin improves insulin sensitivity and there is an inverse correlation between adiponectin levels and type-2 diabetes risk. Previous research shows that adiponectin remodels the adipose tissue into a more efficient metabolic sink. For instance, mice that overexpress adiponectin show increased capacity for hyperplastic adipose tissue expansion as evident from smaller and metabolically more active white adipocytes. In contrast, the brown adipose tissue (BAT) of these mice looks "whiter" possibly indicating reduced metabolic activity. Here, we aimed to further establish the effect of adiponectin on adipose tissue expansion and adipocyte mitochondrial function as well as to unravel mechanistic aspects in this area. METHODS: Brown and white adipose tissues from adiponectin overexpressing (APN tg) mice and littermate wildtype controls, housed at room and cold temperature, were studied by histological, gene/protein expression and flow cytometry analyses. Metabolic and mitochondrial functions were studied by radiotracers and Seahorse-based technology. In addition, mitochondrial function was assessed in cultured adiponectin deficient adipocytes from APN knockout and heterozygote mice. RESULTS: APN tg BAT displayed increased proliferation prenatally leading to enlarged BAT. Postnatally, APN tg BAT turned whiter than control BAT, confirming previous reports. Furthermore, elevated adiponectin augmented the sympathetic innervation/activation within adipose tissue. APN tg BAT displayed reduced metabolic activity and reduced mitochondrial oxygen consumption rate (OCR). In contrast, APN tg inguinal white adipose tissue (IWAT) displayed enhanced metabolic activity. These metabolic differences between genotypes were apparent also in cultured adipocytes differentiated from BAT and IWAT stroma vascular fraction, and the OCR was reduced in both brown and white APN heterozygote adipocytes. In both APN tg BAT and IWAT, the mesenchymal stem cell-related genes were upregulated along with an increased abundance of Lineage-Sca1+CD34- "beige-like" adipocyte precursor cells. In vitro, the adiponectin receptor agonist Adiporon increased the expression of the proliferation marker Pcna and decreased the expression of Cd34 in Sca1+ mesenchymal stem cells. CONCLUSIONS: We propose that the seemingly opposite effect of adiponectin on BAT and IWAT is mediated by a common mechanism; while reduced adiponectin levels are linked to lower adipocyte OCR, elevated adiponectin levels stimulate expansion of adipocyte precursor cells that produce adipocytes with intrinsically higher metabolic rate than classical white but lower metabolic rate than classical brown adipocytes. Moreover, adiponectin can modify the adipocytes' metabolic activity directly and by enhancing the sympathetic innervation within a fat depot.

Exercise-induced crosstalk between immune cells and adipocytes in humans: Role of oncostatin-M.

The discovery of exercise-regulated circulatory factors has fueled interest in organ crosstalk, especially between skeletal muscle and adipose tissue, and the role in mediating beneficial effects of exercise. We studied the adipose tissue transcriptome in men and women with normal glucose tolerance or type 2 diabetes following an acute exercise bout, revealing substantial exercise- and time-dependent changes, with sustained increase in inflammatory genes in type 2 diabetes. We identify oncostatin-M as one of the most upregulated adipose-tissue-secreted factors post-exercise. In cultured human adipocytes, oncostatin-M enhances MAPK signaling and regulates lipolysis. Oncostatin-M expression arises predominantly from adipose tissue immune cell fractions, while the corresponding receptors are expressed in adipocytes. Oncostatin-M expression increases in cultured human Thp1 macrophages following exercise-like stimuli. Our results suggest that immune cells, via secreted factors such as oncostatin-M, mediate a crosstalk between skeletal muscle and adipose tissue during exercise to regulate adipocyte metabolism and adaptation.

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.

Palmitate impairs circadian transcriptomics in muscle cells through histone modification of enhancers.

Obesity and elevated circulating lipids may impair metabolism by disrupting the molecular circadian clock. We tested the hypothesis that lipid overload may interact with the circadian clock and alter the rhythmicity of gene expression through epigenomic mechanisms in skeletal muscle. Palmitate reprogrammed the circadian transcriptome in myotubes without altering the rhythmic mRNA expression of core clock genes. Genes with enhanced cycling in response to palmitate were associated with post-translational modification of histones. The cycling of histone 3 lysine 27 acetylation (H3K27ac), a marker of active gene enhancers, was modified by palmitate treatment. Chromatin immunoprecipitation and sequencing confirmed that palmitate exposure altered the cycling of DNA regions associated with H3K27ac. The overlap between mRNA and DNA regions associated with H3K27ac and the pharmacological inhibition of histone acetyltransferases revealed novel cycling genes associated with lipid exposure of primary human myotubes. Palmitate exposure disrupts transcriptomic rhythmicity and modifies enhancers through changes in histone H3K27 acetylation in a circadian manner. Thus, histone acetylation is responsive to lipid overload and may redirect the circadian chromatin landscape, leading to the reprogramming of circadian genes and pathways involved in lipid biosynthesis in skeletal muscle.

Genes Whose Gain or Loss of Function Changes Type 1, 2A, 2X, or 2B Muscle Fibre Proportions in Mice-A Systematic Review.

Adult skeletal muscle fibres are classified as type 1, 2A, 2X, and 2B. These classifications are based on the expression of the dominant myosin heavy chain isoform. Muscle fibre-specific gene expression and proportions of muscle fibre types change during development and in response to exercise, chronic electrical stimulation, or inactivity. To identify genes whose gain or loss-of-function alters type 1, 2A, 2X, or 2B muscle fibre proportions in mice, we conducted a systematic review of transgenic mouse studies. The systematic review was conducted in accordance with the 2009 PRISMA guidelines and the PICO framework. We identified 25 "muscle fibre genes" (Akirin1, Bdkrb2, Bdnf, Camk4, Ccnd3, Cpt1a, Epas1, Esrrg, Foxj3, Foxo1, Il15, Mapk12, Mstn, Myod1, Ncor1, Nfatc1, Nol3, Ppargc1a, Ppargc1b, Sirt1, Sirt3, Thra, Thrb, Trib3, and Vgll2) whose gain or loss-of-function significantly changes type 1, 2A, 2X or 2B muscle fibre proportions in mice. The fact that 15 of the 25 muscle fibre genes are transcriptional regulators suggests that muscle fibre-specific gene expression is primarily regulated transcriptionally. A reanalysis of existing datasets revealed that the expression of Ppargc1a and Vgll2 increases and Mstn decreases after exercise, respectively. This suggests that these genes help to regulate the muscle fibre adaptation to exercise. Finally, there are many known DNA sequence variants of muscle fibre genes. It seems likely that such DNA sequence variants contribute to the large variation of muscle fibre type proportions in the human population.

Distinctive exercise-induced inflammatory response and exerkine induction in skeletal muscle of people with type 2 diabetes.

Mechanistic insights into the molecular events by which exercise enhances the skeletal muscle phenotype are lacking, particularly in the context of type 2 diabetes. Here, we unravel a fundamental role for exercise-responsive cytokines (exerkines) on skeletal muscle development and growth in individuals with normal glucose tolerance or type 2 diabetes. Acute exercise triggered an inflammatory response in skeletal muscle, concomitant with an infiltration of immune cells. These exercise effects were potentiated in type 2 diabetes. In response to contraction or hypoxia, cytokines were mainly produced by endothelial cells and macrophages. The chemokine CXCL12 was induced by hypoxia in endothelial cells, as well as by conditioned medium from contracted myotubes in macrophages. We found that CXCL12 was associated with skeletal muscle remodeling after exercise and differentiation of cultured muscle. Collectively, acute aerobic exercise mounts a noncanonical inflammatory response, with an atypical production of exerkines, which is potentiated in type 2 diabetes.

Genome-wide association analyses of physical activity and sedentary behavior provide insights into underlying mechanisms and roles in disease prevention.

Although physical activity and sedentary behavior are moderately heritable, little is known about the mechanisms that influence these traits. Combining data for up to 703,901 individuals from 51 studies in a multi-ancestry meta-analysis of genome-wide association studies yields 99 loci that associate with self-reported moderate-to-vigorous intensity physical activity during leisure time (MVPA), leisure screen time (LST) and/or sedentary behavior at work. Loci associated with LST are enriched for genes whose expression in skeletal muscle is altered by resistance training. A missense variant in ACTN3 makes the alpha-actinin-3 filaments more flexible, resulting in lower maximal force in isolated type IIA muscle fibers, and possibly protection from exercise-induced muscle damage. Finally, Mendelian randomization analyses show that beneficial effects of lower LST and higher MVPA on several risk factors and diseases are mediated or confounded by body mass index (BMI). Our results provide insights into physical activity mechanisms and its role in disease prevention.

Glutamine Regulates Skeletal Muscle Immunometabolism in Type 2 Diabetes.

Dysregulation of skeletal muscle metabolism influences whole-body insulin sensitivity and glucose homeostasis. We hypothesized that type 2 diabetes-associated alterations in the plasma metabolome directly contribute to skeletal muscle immunometabolism and the subsequent development of insulin resistance. To this end, we analyzed the plasma and skeletal muscle metabolite profile and identified glutamine as a key amino acid that correlates inversely with BMI and insulin resistance index (HOMA-IR) in men with normal glucose tolerance or type 2 diabetes. Using an in vitro model of human myotubes and an in vivo model of diet-induced obesity and insulin resistance in male mice, we provide evidence that glutamine levels directly influence the inflammatory response of skeletal muscle and regulate the expression of the adaptor protein GRB10, an inhibitor of insulin signaling. Moreover, we demonstrate that a systemic increase in glutamine levels in a mouse model of obesity improves insulin sensitivity and restores glucose homeostasis. We conclude that glutamine supplementation may represent a potential therapeutic strategy to prevent or delay the onset of insulin resistance in obesity by reducing inflammatory markers and promoting skeletal muscle insulin sensitivity.

Pannexin 3 deletion reduces fat accumulation and inflammation in a sex-specific manner.

BACKGROUND: Pannexin 3 (PANX3) is a channel-forming glycoprotein that enables nutrient-induced inflammation in vitro, and genetic linkage data suggest that it regulates body mass index. Here, we characterized inflammatory and metabolic parameters in global Panx3 knockout (KO) mice in the context of forced treadmill running (FEX) and high-fat diet (HFD). METHODS: C57BL/6N (WT) and KO mice were randomized to either a FEX running protocol or no running (SED) from 24 until 30 weeks of age. Body weight was measured biweekly, and body composition was measured at 24 and 30 weeks of age. Male WT and KO mice were fed a HFD from 12 to 28 weeks of age. Metabolic organs were analyzed for a panel of inflammatory markers and PANX3 expression. RESULTS: In females there were no significant differences in body composition between genotypes, which could be due to the lack of PANX3 expression in female white adipose tissue, while male KOs fed a chow diet had lower body weight and lower fat mass at 24 and 30 weeks of age, which was reduced to the same extent as 6 weeks of FEX in WT mice. In addition, male KO mice exhibited significantly lower expression of multiple pro-inflammatory genes in white adipose tissue compared to WT mice. While on a HFD body weight differences were insignificant, multiple inflammatory genes were significantly different in quadriceps muscle and white adipose tissue resulting in a more anti-inflammatory phenotype in KO mice compared to WT. The lower fat mass in male KO mice may be due to significantly fewer adipocytes in their subcutaneous fat compared to WT mice. Mechanistically, adipose stromal cells (ASCs) cultured from KO mice grow significantly slower than WT ASCs. CONCLUSION: PANX3 is expressed in male adult mouse adipose tissue and may regulate adipocyte numbers, influencing fat accumulation and inflammation.

Genes controlling skeletal muscle glucose uptake and their regulation by endurance and resistance exercise.

Exercise improves the insulin sensitivity of glucose uptake in skeletal muscle. Due to that, exercise has become a cornerstone treatment for type 2 diabetes mellitus (T2DM). The mechanisms by which exercise improves skeletal muscle insulin sensitivity are, however, incompletely understood. We conducted a systematic review to identify all genes whose gain or loss of function alters skeletal muscle glucose uptake. We subsequently cross-referenced these genes with recently generated data sets on exercise-induced gene expression and signaling. Our search revealed 176 muscle glucose-uptake genes, meaning that their genetic manipulation altered glucose uptake in skeletal muscle. Notably, exercise regulates the expression or phosphorylation of more than 50% of the glucose-uptake genes or their protein products. This included many genes that previously have not been associated with exercise-induced insulin sensitivity. Interestingly, endurance and resistance exercise triggered some common but mostly unique changes in expression and phosphorylation of glucose-uptake genes or their protein products. Collectively, our work provides a resource of potentially new molecular effectors that play a role in the incompletely understood regulation of muscle insulin sensitivity by exercise.

Disrupted circadian oscillations in type 2 diabetes are linked to altered rhythmic mitochondrial metabolism in skeletal muscle.

Circadian rhythms are generated by an autoregulatory feedback loop of transcriptional activators and repressors. Circadian rhythm disruption contributes to type 2 diabetes (T2D) pathogenesis. We elucidated whether altered circadian rhythmicity of clock genes is associated with metabolic dysfunction in T2D. Transcriptional cycling of core-clock genes BMAL1, CLOCK, and PER3 was altered in skeletal muscle from individuals with T2D, and this was coupled with reduced number and amplitude of cycling genes and disturbed circadian oxygen consumption. Inner mitochondria­associated genes were enriched for rhythmic peaks in normal glucose tolerance, but not T2D, and positively correlated with insulin sensitivity. Chromatin immunoprecipitation sequencing identified CLOCK and BMAL1 binding to inner-mitochondrial genes associated with insulin sensitivity, implicating regulation by the core clock. Inner-mitochondria disruption altered core-clock gene expression and free-radical production, phenomena that were restored by resveratrol treatment. We identify bidirectional communication between mitochondrial function and rhythmic gene expression, processes that are disturbed in diabetes.

Epigenetic rewiring of skeletal muscle enhancers after exercise training supports a role in whole-body function and human health.

OBJECTIVES: Regular physical exercise improves health by reducing the risk of a plethora of chronic disorders. We hypothesized that endurance exercise training remodels the activity of gene enhancers in skeletal muscle and that this remodeling contributes to the beneficial effects of exercise on human health. METHODS AND RESULTS: By studying changes in histone modifications, we mapped the genome-wide positions and activities of enhancers in skeletal muscle biopsies collected from young sedentary men before and after 6 weeks of endurance exercise. We identified extensive remodeling of enhancer activities after exercise training, with a large subset of the remodeled enhancers located in the proximity of genes transcriptionally regulated after exercise. By overlapping the position of enhancers with genetic variants, we identified an enrichment of disease-associated genetic variants within the exercise-remodeled enhancers. CONCLUSION: Our data provide evidence of a functional link between epigenetic rewiring of enhancers to control their activity after exercise training and the modulation of disease risk in humans.

The Comparative Methylome and Transcriptome After Change of Direction Compared to Straight Line Running Exercise in Human Skeletal Muscle.

The methylome and transcriptome signatures following exercise that are physiologically and metabolically relevant to sporting contexts such as team sports or health prescription scenarios (e.g., high intensity interval training/HIIT) has not been investigated. To explore this, we performed two different sport/exercise relevant high-intensity running protocols in five male sport team members using a repeated measures design of: (1) change of direction (COD) versus; (2) straight line (ST) running exercise with a wash-out period of at least 2 weeks between trials. Skeletal muscle biopsies collected from the vastus lateralis 30 min and 24 h post exercise, were assayed using 850K methylation arrays and a comparative analysis with recent (subject-unmatched) sprint and acute aerobic exercise meta-analysis transcriptomes was performed. Despite COD and ST exercise being matched for classically defined intensity measures (speed × distance and number of accelerations/decelerations), COD exercise elicited greater movement (GPS-Playerload), physiological (HR), metabolic (lactate) as well as central and peripheral (differential RPE) exertion measures compared with ST exercise, suggesting COD exercise evoked a higher exercise intensity. The exercise response alone across both conditions evoked extensive alterations in the methylome 30 min and 24 h post exercise, particularly in MAPK, AMPK and axon guidance pathways. COD evoked a considerably greater hypomethylated signature across the genome compared with ST exercise, particularly at 30 min post exercise, enriched in: Protein binding, MAPK, AMPK, insulin, and axon guidance pathways. Comparative methylome analysis with sprint running transcriptomes identified considerable overlap, with 49% of genes that were altered at the expression level also differentially methylated after COD exercise. After differential methylated region analysis, we observed that VEGFA and its downstream nuclear transcription factor, NR4A1 had enriched hypomethylation within their promoter regions. VEGFA and NR4A1 were also significantly upregulated in the sprint transcriptome and meta-analysis of exercise transcriptomes. We also confirmed increased gene expression of VEGFA, and considerably larger increases in the expression of canonical metabolic genes PPARGC1A (that encodes PGC1-α) and NR4A3 in COD vs. ST exercise. Overall, we demonstrate that increased physiological/metabolic load via COD exercise in human skeletal muscle evokes considerable epigenetic modifications that are associated with changes in expression of genes responsible for adaptation to exercise.

Metabolic consequences of obesity and type 2 diabetes: Balancing genes and environment for personalized care.

The prevalence of type 2 diabetes and obesity has risen dramatically for decades and is expected to rise further, secondary to the growing aging, sedentary population. The strain on global health care is projected to be colossal. This review explores the latest work and emerging ideas related to genetic and environmental factors influencing metabolism. Translational research and clinical applications, including the impact of the COVID-19 pandemic, are highlighted. Looking forward, strategies to personalize all aspects of prevention, management and care are necessary to improve health outcomes and reduce the impact of these metabolic diseases.

Contraction influences Per2 gene expression in skeletal muscle through a calcium-dependent pathway.

KEY POINTS: Exercising at different times of day elicits different effects on exercise performance and metabolic health. However, the specific signals driving the observed time-of-day specific effects of exercise have not been fully identified. Exercise influences the skeletal muscle circadian clock, although the relative contribution of muscle contraction and extracellular signals is unknown. Here, we show that contraction acutely increases the expression of the core circadian clock gene Period Circadian Regulator 2 (Per2) and phase-shifts Per2 rhythmicity in muscle cells. This contraction effect on core clock genes is mediated through a calcium-dependant mechanism; The results obtained in the present study suggest that a proportion of the ability of exercise to entrain the skeletal muscle clock is driven directly by muscle contraction. Contraction interventions may be used to mimic some time-of-day specific effects of exercise on metabolism and muscle performance. ABSTRACT: Exercise entrains the central and peripheral circadian clocks, although the mechanism by which exercise modulates expression of skeletal muscle clock genes is unclear. The present study aimed to determine whether skeletal muscle contraction alone could directly influence circadian rhythmicity and uncover the underlying mechanism by which contraction modulates clock gene expression. We investigated the expression of core clock genes in human skeletal muscle after acute exercise, as well as following in vitro contraction in mouse soleus muscle and cultured C2C12 skeletal muscle myotubes. Additionally, we interrogated the molecular pathways by which skeletal muscle contraction could influence clock gene expression. Contraction acutely increased the expression of the core circadian clock gene Period Circadian Regulator 2 (Per2) and phase-shifted Per2 rhythmicity in C2C12 myotubes in vitro. Further investigation revealed that pharmacologically increasing cytosolic calcium concentrations by ionomycin treatment mimicked the effect of contraction on Per2 expression. Similarly, treatment with a calcium channel blocker, nifedipine, blocked the effect of electric pulse stimulation-induced contraction on Per2 expression. Increased calcium influx from contraction lead to binding of the phosphorylated form of cAMP response element-binding protein (CREB) to the Per2 promoter, suggesting a role of CREB in contraction-induced Per2 transcription. Thus, by dissociating the effect of muscle contraction alone from the whole effect of exercise, our investigations indicate that a proportion of the ability of exercise to entrain the skeletal muscle clock is driven directly by contraction.

Electroacupuncture Mimics Exercise-Induced Changes in Skeletal Muscle Gene Expression in Women With Polycystic Ovary Syndrome.

CONTEXT: Autonomic nervous system activation mediates the increase in whole-body glucose uptake in response to electroacupuncture but the mechanisms are largely unknown. OBJECTIVE: To identify the molecular mechanisms underlying electroacupuncture-induced glucose uptake in skeletal muscle in insulin-resistant overweight/obese women with and without polycystic ovary syndrome (PCOS). DESIGN/PARTICIPANTS: In a case-control study, skeletal muscle biopsies were collected from 15 women with PCOS and 14 controls before and after electroacupuncture. Gene expression and methylation was analyzed using Illumina BeadChips arrays. RESULTS: A single bout of electroacupuncture restores metabolic and transcriptional alterations and induces epigenetic changes in skeletal muscle. Transcriptomic analysis revealed 180 unique genes (q < 0.05) whose expression was changed by electroacupuncture, with 95% of the changes towards a healthier phenotype. We identified DNA methylation changes at 304 unique sites (q < 0.20), and these changes correlated with altered expression of 101 genes (P < 0.05). Among the 50 most upregulated genes in response to electroacupuncture, 38% were also upregulated in response to exercise. We identified a subset of genes that were selectively altered by electroacupuncture in women with PCOS. For example, MSX1 and SRNX1 were decreased in muscle tissue of women with PCOS and were increased by electroacupuncture and exercise. siRNA-mediated silencing of these 2 genes in cultured myotubes decreased glycogen synthesis, supporting a role for these genes in glucose homeostasis. CONCLUSION: Our findings provide evidence that electroacupuncture normalizes gene expression in skeletal muscle in a manner similar to acute exercise. Electroacupuncture might therefore be a useful way of assisting those who have difficulties performing exercise.

Transcriptomic profiling of skeletal muscle adaptations to exercise and inactivity.

The molecular mechanisms underlying the response to exercise and inactivity are not fully understood. We propose an innovative approach to profile the skeletal muscle transcriptome to exercise and inactivity using 66 published datasets. Data collected from human studies of aerobic and resistance exercise, including acute and chronic exercise training, were integrated using meta-analysis methods (www.metamex.eu). Here we use gene ontology and pathway analyses to reveal selective pathways activated by inactivity, aerobic versus resistance and acute versus chronic exercise training. We identify NR4A3 as one of the most exercise- and inactivity-responsive genes, and establish a role for this nuclear receptor in mediating the metabolic responses to exercise-like stimuli in vitro. The meta-analysis (MetaMEx) also highlights the differential response to exercise in individuals with metabolic impairments. MetaMEx provides the most extensive dataset of skeletal muscle transcriptional responses to different modes of exercise and an online interface to readily interrogate the database.