ABSTRACT
Nutrient composition in obesogenic diets may influence the severity of disorders associated with obesity such as insulin-resistance and chronic inflammation. Here we hypothesized that obesogenic diets rich in fat and varying in fatty acid composition, particularly in omega 6 (ω6) to omega 3 (ω3) ratio, have various effects on energy metabolism, neuroinflammation and behavior. Mice were fed either a control diet or a high fat diet (HFD) containing either low (LO), medium (ME) or high (HI) ω6/ω3 ratio. Mice from the HFD-LO group consumed less calories and exhibited less body weight gain compared to other HFD groups. Both HFD-ME and HFD-HI impaired glucose metabolism while HFD-LO partly prevented insulin intolerance and was associated with normal leptin levels despite higher subcutaneous and perigonadal adiposity. Only HFD-HI increased anxiety and impaired spatial memory, together with increased inflammation in the hypothalamus and hippocampus. Our results show that impaired glucose metabolism and neuroinflammation are uncoupled, and support that diets with a high ω6/ω3 ratio are associated with neuroinflammation and the behavioral deterioration coupled with the consumption of diets rich in fat.
Subject(s)
Insulins , Neuroinflammatory Diseases , Animals , Mice , Obesity/metabolism , Diet, High-Fat/adverse effects , Fatty Acids/metabolism , Inflammation , GlucoseABSTRACT
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.
Subject(s)
Histones , Transcriptome , Humans , Histones/metabolism , Transcriptome/genetics , Palmitates/pharmacology , Palmitates/metabolism , Histone Code/genetics , Protein Processing, Post-Translational , RNA, Messenger/metabolism , Muscle Fibers, Skeletal/metabolism , DNA/metabolismABSTRACT
Aim: To perform a comparative epigenomic analysis of DNA methylation in spermatozoa from humans, mice, rats and mini-pigs. Materials & methods: Genome-wide DNA methylation analysis was used to compare the methylation profiles of orthologous CpG sites. Transcription profiles of early embryo development were analyzed to provide insight into the association between sperm methylation and gene expression programming. Results: We identified DNA methylation variation near genes related to the central nervous system and signal transduction. Gene expression dynamics at different time points of preimplantation stages were modestly associated with spermatozoal DNA methylation at the nearest promoters. Conclusion: Conserved genomic regions subject to epigenetic variation across different species were associated with specific organ functions, suggesting their potential contribution to organ speciation and long-term adaptation to the environment.
Subject(s)
DNA Methylation , Epigenesis, Genetic , Humans , Male , Animals , Mice , Rats , Swine , Epigenomics , Swine, Miniature/genetics , Semen , Spermatozoa/metabolism , CpG IslandsABSTRACT
Polycystic ovary syndrome (PCOS) is characterised by a hormonal imbalance affecting the reproductive and metabolic health of reproductive-aged women. Exercise is recommended as a first-line therapy for women with PCOS to improve their overall health; however, women with PCOS are resistant to the metabolic benefits of exercise training. Here, we aimed to gain insight into the mechanisms responsible for such resistance to exercise in PCOS. We employed an in vitro approach with electrical pulse stimulation (EPS) of cultured skeletal muscle cells to explore whether myotubes from women with PCOS have an altered gene expression signature in response to contraction. Following EPS, 4719 genes were differentially expressed (false discovery rate <0.05) in myotubes from women with PCOS compared to 173 in healthy women. Both groups included genes involved in skeletal muscle contraction. We also determined the effect of two transforming growth factor ß (TGFß) ligands that are elevated in plasma of women with PCOS, TGFß1 and anti-Müllerian hormone (AMH), alone and on the EPS-induced response. While AMH (30 ng/ml) had no effect, TGFß1 (5 ng/ml) induced the expression of extracellular matrix genes and impaired the exercise-like transcriptional signature in myotubes from women with and without PCOS in response to EPS by interfering with key processes related to muscle contraction, calcium transport and actin filament. Our findings suggest that while the fundamental gene expression responses of skeletal muscle to contraction is intact in PCOS, circulating factors like TGFß1 may be responsible for the impaired adaptation to exercise in women with PCOS. KEY POINTS: Gene expression responses to in vitro contraction (electrical pulse stimulation, EPS) are altered in myotubes from women with polycystic ovary syndrome (PCOS) compared to healthy controls, with an increased expression of genes related to pro-inflammatory pathways. Transforming growth factor ß1 (TGFß1) upregulates genes related to extracellular matrix remodelling and reduces the expression of contractile genes in myotubes, regardless of the donor's health status. TGFß1 alters the gene expression response to EPS, providing a possible mechanism for the impaired exercise adaptations in women with PCOS.
Subject(s)
Polycystic Ovary Syndrome , Adult , Anti-Mullerian Hormone/genetics , Anti-Mullerian Hormone/metabolism , Female , Humans , Muscle Fibers, Skeletal/metabolism , Polycystic Ovary Syndrome/genetics , Polycystic Ovary Syndrome/metabolism , Transcriptome , Transforming Growth Factor beta1/metabolismABSTRACT
BACKGROUND: In obesity, adipose tissue dysfunction resulting from excessive fat accumulation leads to systemic insulin resistance (IR), the underlying alteration of Type 2 Diabetes. The specific pathways dysregulated in dysfunctional adipocytes and the extent to which it affects adipose metabolic functions remain incompletely characterized. METHODS: We interrogated the transcriptional adaptation to increased adiposity in association with insulin resistance in visceral white adipose tissue from lean men, or men presenting overweight/obesity (BMI from 19 to 33) and discordant for insulin sensitivity. In human adipocytes in vitro, we investigated the direct contribution of IR in altering metabolic gene programming and glucose utilization using 13C-isotopic glucose tracing. RESULTS: We found that gene expression associated with impaired glucose and lipid metabolism and inflammation represented the strongest association with systemic insulin resistance, independently of BMI. In addition, we showed that inducing IR in mature human white adipocytes was sufficient to reprogram the transcriptional profile of genes involved in important metabolic functions such as glycolysis, the pentose phosphate pathway and de novo lipogenesis. Finally, we found that IR induced a rewiring of glucose metabolism, with higher incorporation of glucose into citrate, but not into downstream metabolites within the TCA cycle. CONCLUSIONS: Collectively, our data highlight the importance of obesity-derived insulin resistance in impacting the expression of key metabolic genes and impairing the metabolic processes of glucose utilization, and reveal a role for metabolic adaptation in adipose dysfunction in humans.
Subject(s)
Diabetes Mellitus, Type 2 , Insulin Resistance , Adipocytes, White/metabolism , Adipose Tissue/metabolism , Diabetes Mellitus, Type 2/complications , Diabetes Mellitus, Type 2/genetics , Glucose/metabolism , Humans , Insulin/metabolism , Insulin Resistance/genetics , Male , Obesity/metabolismABSTRACT
Today, Alzheimer's disease (AD) is one of the most important neurodegenerative disorders that affected millions of people worldwide. Hundreds of academic investigations highlighted the potential roles of natural metabolites in the cornerstone of AD prevention. Nevertheless, alkaloids are only metabolites that successfully showed promising clinical therapeutic effects on the prevention of AD. In this regard, other plant metabolites such as flavonoids are also considered as promising substances in the improvement of AD complications. The lack of data on molecular mode of action of flavonoids inside brain tissues, and their potential to transport across the blood-brain barrier, a physical hindrance between bloodstream and brain tissues, limited the large-scale application of these compounds for AD therapy programs. Herein, a coupled docking and quantum study was applied to determine the binding mode of flavonoids and three protein kinases involved in the pathogenesis of AD. The results suggested that all docked metabolites showed considerable binding affinity to interact with target receptors, but some compounds possessed higher binding energy values. Because docking simulation cannot entirely reveal the potential roles of ligand substructures in the interaction with target residues, quantum chemical analyses (QCAs) were performed to cover this drawback. Accordingly, QCAs determined that distribution of molecular orbitals have a pivotal function in the determination of the type of reaction between ligands and receptors; therefore, using such quantum chemical descriptors may correct the results of virtual docking outcomes to highlight promising backbones for further developments.Communicated by Ramaswamy H. Sarma.
Subject(s)
Alzheimer Disease , Flavonoids , Alzheimer Disease/drug therapy , Flavonoids/pharmacology , Humans , Ligands , Molecular Docking Simulation , Molecular Dynamics SimulationABSTRACT
Polycystic ovary syndrome (PCOS) is a common endocrine disorder associated with insulin resistance and impaired energy metabolism in skeletal muscle, the aetiology of which is currently unclear. Here, we mapped the gene expression profile of skeletal muscle from women with PCOS and determined if cultured primary myotubes retain the gene expression signature of PCOS in vivo. Transcriptomic analysis of vastus lateralis biopsies collected from PCOS women showed lower expression of genes associated with mitochondrial function, while the expression of genes associated with the extracellular matrix was higher compared to controls. Altered skeletal muscle mRNA expression of mitochondrial-associated genes in PCOS was associated with lower protein expression of mitochondrial complex II-V, but not complex I, with no difference in mitochondrial DNA content. Transcriptomic analysis of primary myotube cultures established from biopsies did not display any differentially expressed genes between controls and PCOS. Comparison of gene expression profiles in skeletal muscle biopsies and primary myotube cultures showed lower expression of mitochondrial and energy metabolism-related genes in vitro, irrespective of the group. Together, our results show that the altered mitochondrial-associated gene expression in skeletal muscle in PCOS is not preserved in cultured myotubes, indicating that the in vivo extracellular milieu, rather than genetic or epigenetic factors, may drive this alteration. Dysregulation of mitochondrial-associated genes in skeletal muscle by extracellular factors may contribute to the impaired energy metabolism associated with PCOS.
Subject(s)
Disease Susceptibility , Gene Expression Regulation , Genes, Mitochondrial , Mitochondria/genetics , Mitochondria/metabolism , Polycystic Ovary Syndrome/etiology , Polycystic Ovary Syndrome/metabolism , Biomarkers , Cells, Cultured , Cluster Analysis , Computational Biology/methods , DNA Copy Number Variations , Female , Gene Expression Profiling , Glucose/metabolism , Humans , Muscle Fibers, Skeletal/metabolism , Muscle, Skeletal/metabolism , Polycystic Ovary Syndrome/pathology , TranscriptomeABSTRACT
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.
ABSTRACT
Perinatal smoke/nicotine exposure alters lung development and causes asthma in exposed offspring, transmitted transgenerationally. The mechanism underlying the transgenerational inheritance of perinatal smoke/nicotine-induced asthma remains unknown, but germline epigenetic modulations may play a role. Using a well-established rat model of perinatal nicotine-induced asthma, we determined the DNA methylation pattern of spermatozoa of F1 rats exposed perinatally to nicotine in F0 gestation. To identify differentially methylated regions (DMRs), reduced representation bisulfite sequencing was performed on spermatozoa of F1 litters. The top regulated gene body and promoter DMRs were tested for lung gene expression levels, and key proteins involved in lung development and repair were determined. The overall CpG methylation in F1 sperms across gene bodies, promoters, 5'-UTRs, exons, introns, and 3'-UTRs was not affected by nicotine exposure. However, the methylation levels were different between the different genomic regions. Eighty one CpG sites, 16 gene bodies, and 3 promoter regions were differentially methylated. Gene enrichment analysis of DMRs revealed pathways involved in oxidative stress, nicotine response, alveolar and brain development, and cellular signaling. Among the DMRs, Dio1 and Nmu were the most hypermethylated and hypomethylated genes, respectively. Gene expression analysis showed that the mRNA expression and DNA methylation were incongruous. Key proteins involved in lung development and repair were significantly different (FDR < 0.05) between the nicotine and placebo-treated groups. Our data show that DNA methylation is remodeled in offspring spermatozoa upon perinatal nicotine exposure. These epigenetic alterations may play a role in transgenerational inheritance of perinatal smoke/nicotine induced asthma.
Subject(s)
DNA Methylation , Epigenesis, Genetic , Lung/pathology , Nicotine/toxicity , Nicotinic Agonists/toxicity , Prenatal Exposure Delayed Effects/pathology , Spermatozoa/metabolism , Animals , Animals, Newborn , Female , Gene Expression Regulation, Developmental , Lung/drug effects , Lung/metabolism , Male , Pregnancy , Prenatal Exposure Delayed Effects/chemically induced , Prenatal Exposure Delayed Effects/genetics , Prenatal Exposure Delayed Effects/metabolism , Rats , Rats, Sprague-Dawley , Spermatozoa/pathologyABSTRACT
OBJECTIVE: NAD+ is a co-factor and substrate for enzymes maintaining energy homeostasis. Nicotinamide phosphoribosyltransferase (NAMPT) controls NAD+ synthesis, and in skeletal muscle, NAD+ is essential for muscle integrity. However, the underlying molecular mechanisms by which NAD+ synthesis affects muscle health remain poorly understood. Thus, the objective of the current study was to delineate the role of NAMPT-mediated NAD+ biosynthesis in skeletal muscle development and function. METHODS: To determine the role of Nampt in muscle development and function, we generated skeletal muscle-specific Nampt KO (SMNKO) mice. We performed a comprehensive phenotypic characterization of the SMNKO mice, including metabolic measurements, histological examinations, and RNA sequencing analyses of skeletal muscle from SMNKO mice and WT littermates. RESULTS: SMNKO mice were smaller, with phenotypic changes in skeletal muscle, including reduced fiber area and increased number of centralized nuclei. The majority of SMNKO mice died prematurely. Transcriptomic analysis identified that the gene encoding the mitochondrial permeability transition pore (mPTP) regulator Cyclophilin D (Ppif) was upregulated in skeletal muscle of SMNKO mice from 2 weeks of age, with associated increased sensitivity of mitochondria to the Ca2+-stimulated mPTP opening. Treatment of SMNKO mice with the Cyclophilin D inhibitor, Cyclosporine A, increased membrane integrity, decreased the number of centralized nuclei, and increased survival. CONCLUSIONS: Our study demonstrates that NAMPT is crucial for maintaining cellular Ca2+ homeostasis and skeletal muscle development, which is vital for juvenile survival.
Subject(s)
Calcium/metabolism , Cytokines/metabolism , Homeostasis , Mitochondria/metabolism , Muscle, Skeletal/metabolism , Nicotinamide Phosphoribosyltransferase/metabolism , Animals , Cells, Cultured , Female , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Muscle DevelopmentABSTRACT
The profound energy-expending nature of brown adipose tissue (BAT) thermogenesis makes it an attractive target tissue to combat obesity-associated metabolic disorders. While cold exposure is the strongest inducer of BAT activity, the temporal mechanisms tuning BAT adaptation during this activation process are incompletely understood. Here we show that the scaffold protein Afadin is dynamically regulated by cold in BAT, and participates in cold acclimation. Cold exposure acutely increases Afadin protein levels and its phosphorylation in BAT. Knockdown of Afadin in brown pre-adipocytes does not alter adipogenesis but restricts ß3-adrenegic induction of thermogenic genes expression and HSL phosphorylation in mature brown adipocytes. Consistent with a defect in thermogenesis, an impaired cold tolerance was observed in fat-specific Afadin knockout mice. However, while Afadin depletion led to reduced Ucp1 mRNA induction by cold, stimulation of Ucp1 protein was conserved. Transcriptomic analysis revealed that fat-specific ablation of Afadin led to decreased functional enrichment of gene sets controlling essential metabolic functions at thermoneutrality in BAT, whereas it led to an altered reprogramming in response to cold, with enhanced enrichment of different pathways related to metabolism and remodeling. Collectively, we demonstrate a role for Afadin in supporting the adrenergic response in brown adipocytes and BAT function.
Subject(s)
Adipocytes, Brown/metabolism , Adipose Tissue, Brown/metabolism , Cold Temperature , Gene Expression Regulation , Kinesins/biosynthesis , Myosins/biosynthesis , Thermogenesis , Animals , Kinesins/genetics , Mice , Mice, Knockout , Myosins/geneticsABSTRACT
Parental health influences embryonic development and susceptibility to disease in the offspring. We investigated whether maternal voluntary running during gestation could protect the offspring from the adverse effects of maternal or paternal high-fat diet (HF) in mice. We performed transcriptomic and whole-genome DNA methylation analyses in female offspring skeletal muscle and targeted DNA methylation analysis of the peroxisome proliferator-activated receptor-γ coactivator-1α (Pgc-1α) promoter in both male and female adult offspring. Maternal HF resulted in impaired metabolic homeostasis in male offspring at 9 mo of age, whereas both male and female offspring were negatively impacted by paternal HF. Maternal exercise during gestation completely mitigated these metabolic impairments. Female adult offspring from obese male or female parent had skeletal muscle transcriptional profiles enriched in genes regulating inflammation and immune responses, whereas maternal exercise resulted in a transcriptional profile similar to offspring from normal chow (NC)-fed parents. Maternal HF, but not paternal HF, resulted in hypermethylation of the Pgc-1α promoter at CpG-260, which was abolished by maternal exercise. These findings demonstrate the negative consequences of maternal and paternal HF for the offspring's metabolic outcomes later in life possibly through different epigenetic mechanisms, and maternal exercise during gestation mitigates the negative consequences.NEW & NOTEWORTHY Maternal or paternal obesity causes metabolic impairment in adult offspring in mice. Maternal exercise during gestation can completely mitigate metabolic impairment. Maternal obesity, but not paternal obesity, results in hypermethylation of the Pgc-1α promoter at CpG-260, which can be abolished by maternal exercise.
Subject(s)
Physical Conditioning, Animal , Prenatal Exposure Delayed Effects , Adult Children , Animals , Diet, High-Fat , Female , Humans , Male , Mice , Obesity , Parents , PregnancyABSTRACT
OBJECTIVE: Increasing adaptive thermogenesis by stimulating browning in white adipose tissue is a promising method of improving metabolic health. However, the molecular mechanisms underlying this transition remain elusive. Our study examined the molecular determinants driving the differentiation of precursor cells into thermogenic adipocytes. METHODS: In this study, we conducted temporal high-resolution proteomic analysis of subcutaneous white adipose tissue (scWAT) after cold exposure in mice. This was followed by loss- and gain-of-function experiments using siRNA-mediated knockdown and CRISPRa-mediated induction of gene expression, respectively, to evaluate the function of the transcriptional regulator Y box-binding protein 1 (YBX1) during adipogenesis of brown pre-adipocytes and mesenchymal stem cells. Transcriptomic analysis of mesenchymal stem cells following induction of endogenous Ybx1 expression was conducted to elucidate transcriptomic events controlled by YBX1 during adipogenesis. RESULTS: Our proteomics analysis uncovered 509 proteins differentially regulated by cold in a time-dependent manner. Overall, 44 transcriptional regulators were acutely upregulated following cold exposure, among which included the cold-shock domain containing protein YBX1, peaking after 24 h. Cold-induced upregulation of YBX1 also occurred in brown adipose tissue, but not in visceral white adipose tissue, suggesting a role of YBX1 in thermogenesis. This role was confirmed by Ybx1 knockdown in brown and brite preadipocytes, which significantly impaired their thermogenic potential. Conversely, inducing Ybx1 expression in mesenchymal stem cells during adipogenesis promoted browning concurrent with an increased expression of thermogenic markers and enhanced mitochondrial respiration. At a molecular level, our transcriptomic analysis showed that YBX1 regulates a subset of genes, including the histone H3K9 demethylase Jmjd1c, to promote thermogenic adipocyte differentiation. CONCLUSION: Our study mapped the dynamic proteomic changes of murine scWAT during browning and identified YBX1 as a novel factor coordinating the genomic mechanisms by which preadipocytes commit to brite/beige lineage.
Subject(s)
Adipose Tissue, White/metabolism , Thermogenesis/genetics , Thermogenesis/physiology , Transcription Factors/genetics , Transcription Factors/metabolism , Adipocytes, Brown/metabolism , Adipogenesis , Adipose Tissue, Brown/metabolism , Animals , Cell Differentiation , Cell Line , Cell Proliferation , Gene Expression Regulation , Jumonji Domain-Containing Histone Demethylases/genetics , Jumonji Domain-Containing Histone Demethylases/metabolism , Male , Mesenchymal Stem Cells , Mice , Mice, Inbred C57BL , Obesity/metabolism , Proteomics , Subcutaneous Fat/metabolism , Transcriptome , Up-RegulationABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
Time-restricted feeding (TRF) improves metabolism independent of dietary macronutrient composition or energy restriction. To elucidate mechanisms underpinning the effects of short-term TRF, we investigated skeletal muscle and serum metabolic and transcriptomic profiles from 11 men with overweight/obesity after TRF (8 h day-1) and extended feeding (EXF, 15 h day-1) in a randomised cross-over design (trial registration: ACTRN12617000165381). Here we show that muscle core clock gene expression was similar after both interventions. TRF increases the amplitude of oscillating muscle transcripts, but not muscle or serum metabolites. In muscle, TRF induces rhythmicity of several amino acid transporter genes and metabolites. In serum, lipids are the largest class of periodic metabolites, while the majority of phase-shifted metabolites are amino acid related. In conclusion, short-term TRF in overweight men affects the rhythmicity of serum and muscle metabolites and regulates the rhythmicity of genes controlling amino acid transport, without perturbing core clock gene expression.
Subject(s)
Amino Acids/blood , Circadian Rhythm/genetics , Fasting , Lipids/blood , Muscle, Skeletal/metabolism , Adult , Circadian Clocks/genetics , Cross-Over Studies , Gene Expression , Humans , Lipid Metabolism , Male , Muscle, Skeletal/physiology , Overweight/diet therapy , Overweight/metabolismABSTRACT
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.
Subject(s)
Calcium , Circadian Clocks , Animals , Circadian Clocks/genetics , Circadian Rhythm , Gene Expression , Mice , Muscle, Skeletal/metabolism , Period Circadian Proteins/genetics , Period Circadian Proteins/metabolismABSTRACT
Aim: Innate circadian rhythms are critical for optimal tissue-specific functions, including skeletal muscle, a major insulin-sensitive tissue responsible for glucose homeostasis. We determined whether transcriptional oscillations are associated with CpG methylation changes in skeletal muscle. Materials & methods: We performed rhythmicity analysis on the transcriptome and CpG methylome of circadian synchronized myotubes. Results: We identified several transcripts and CpG-sites displaying oscillatory behavior, which were enriched with Gene Ontology terms related to metabolism and development. Oscillating CpG methylation was associated with rhythmic expression of 31 transcripts. Conclusion: Although circadian oscillations may be regulated by rhythmic DNA methylation, strong rhythmic associations between transcriptome and CpG methylation were not identified. This resource constitutes a transcriptomic/epigenomic atlas of skeletal muscle and regulation of circadian rhythms.
Subject(s)
Circadian Rhythm/physiology , Energy Metabolism , Epigenomics , Gene Expression Profiling , Muscle Fibers, Skeletal/metabolism , Algorithms , Animals , Biomarkers , Cell Line , Circadian Clocks/genetics , Computational Biology/methods , DNA Methylation , Epigenomics/methods , Gene Expression Profiling/methods , High-Throughput Nucleotide Sequencing , Mice , Organ Specificity/genetics , Promoter Regions, Genetic , TranscriptomeABSTRACT
KEY POINTS: This is the first long-term human clinical trial to report on effects of nicotinamide riboside (NR) on skeletal muscle mitochondrial function, content and morphology. NR supplementation decreases nicotinamide phosphoribosyltransferase (NAMPT) protein abundance in skeletal muscle. NR supplementation does not affect NAD metabolite concentrations in skeletal muscle. Respiration, distribution and quantity of muscle mitochondria are unaffected by NR. NAMPT in skeletal muscle correlates positively with oxidative phosphorylation Complex I, sirtuin 3 and succinate dehydrogenase. ABSTRACT: Preclinical evidence suggests that the nicotinamide adenine dinucleotide (NAD+ ) precursor nicotinamide riboside (NR) boosts NAD+ levels and improves diseases associated with mitochondrial dysfunction. We aimed to determine if dietary NR supplementation in middle-aged, obese, insulin-resistant men affects mitochondrial respiration, content and morphology in skeletal muscle. In a randomized, placebo-controlled clinical trial, 40 participants received 1000 mg NR or placebo twice daily for 12 weeks. Skeletal muscle biopsies were collected before and after the intervention. Mitochondrial respiratory capacity was determined by high-resolution respirometry on single muscle fibres. Protein abundance and mRNA expression were measured by Western blot and quantitative PCR analyses, respectively, and in a subset of the participants (placebo n = 8; NR n = 8) we quantified mitochondrial fractional area and mitochondrial morphology by laser scanning confocal microscopy. Protein levels of nicotinamide phosphoribosyltransferase (NAMPT), an essential NAD+ biosynthetic enzyme in skeletal muscle, decreased by 14% with NR. However, steady-state NAD+ levels as well as gene expression and protein abundance of other NAD+ biosynthetic enzymes remained unchanged. Neither respiratory capacity of skeletal muscle mitochondria nor abundance of mitochondrial associated proteins were affected by NR. Moreover, no changes in mitochondrial fractional area or network morphology were observed. Our data do not support the hypothesis that dietary NR supplementation has significant impact on skeletal muscle mitochondria in obese and insulin-resistant men. Future studies on the effects of NR on human skeletal muscle may include both sexes and potentially provide comparisons between young and older people.
Subject(s)
Insulin Resistance , Mitochondria, Muscle/physiology , Muscle, Skeletal/physiology , Niacinamide/analogs & derivatives , Obesity/physiopathology , Humans , Male , Middle Aged , NAD/metabolism , Niacinamide/administration & dosage , Nicotinamide Phosphoribosyltransferase/metabolism , Pyridinium CompoundsABSTRACT
AIM: Hepatic insulin resistance is a hallmark of type 2 diabetes and non-alcoholic fatty liver disease. Dysregulation of microRNA (miRNA) expression in insulin-resistant livers might coordinate impaired hepatic metabolic function. Here, we aimed to discover miRNAs and their downstream targets involved in hepatic insulin resistance. METHODS: We determined miRNA expression profiles by small RNA sequencing of two mouse models of impaired hepatic insulin action: high-fat diet-induced obesity and liver-specific insulin receptor knockout. Conversely, we assessed the hepatic miRNA expression profile after treatment with the antidiabetic hormone, fibroblast growth factor 21 (FGF21). Ontology analysis of predicted miRNA gene targets was performed to identify regulated gene pathways. Target enrichment analysis and miRNA mimic overexpression in vitro were used to identify unified protein targets of nodes of regulated miRNAs. RESULTS: We identified an array of miRNA species regulated by impaired liver insulin action or after fibroblast growth factor 21 treatment. Ontology analysis of predicted miRNA gene targets identified pathways controlling hepatic energy metabolism and insulin sensitivity. We identified a node of two miRNAs downregulated in the livers of liver-specific insulin receptor knockout mice, miR-883b and miR-205, which positively regulate the expression of transcription factor zinc finger E-box-binding homeobox 1 (ZBED1). We found another node of two miRNAs upregulated in the livers of fibroblast growth factor 21-treated mice, miR-155-3p and miR-1968-5p, which canonically downregulates the caveola component, polymerase I and transcript release factor (PTRF), a gene previously implicated in hepatic energy metabolism. CONCLUSIONS: This study identifies two nodes of coregulated miRNAs that might coordinately control hepatic energy metabolism in states of insulin resistance.
ABSTRACT
CONTEXT: Polycystic ovary syndrome (PCOS) is a chronic disease affecting reproductive function and whole-body metabolism. Although the etiology is unclear, emerging evidence indicates that the epigenetics may be a contributing factor. OBJECTIVE: To determine the role of global and genome-wide epigenetic modifications in specific immune cells in PCOS compared with controls and whether these could be related to clinical features of PCOS. DESIGN: Cross-sectional study. PARTICIPANTS: Women with (n = 17) or without PCOS (n = 17). SETTING: Recruited from the general community. MAIN OUTCOME MEASURES: Isolated peripheral blood mononuclear cells were analyzed using multicolor flow cytometry methods to determine global DNA methylation levels in a cell-specific fashion. Transcriptomic and genome-wide DNA methylation analyses were performed on T helper cells using RNA sequencing and reduced representation bisulfite sequencing. RESULTS: Women with PCOS had lower global DNA methylation in monocytes (P = 0.006) and in T helper (P = 0.004), T cytotoxic (P = 0.004), and B cells (P = 0.03). Specific genome-wide DNA methylation analysis of T helper cells from women with PCOS identified 5581 differentially methylated CpG sites. Functional gene ontology enrichment analysis showed that genes located at the proximity of differentially methylated CpG sites belong to pathways related to reproductive function and immune cell function. However, these genes were not altered at the transcriptomic level. CONCLUSIONS: It was shown that PCOS is associated with global and gene-specific DNA methylation remodeling in a cell type-specific manner. Further investigation is warranted to determine whether epigenetic reprogramming of immune cells is important in determining the different phenotypes of PCOS.