Transcription profiling of mouse C1C12 cells cultured in high and low glucose conditions
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ABSTRACT: The integration of positive and negative intra- and extra-cellular signals dictates whether a cell will proliferate or differentiate. While it is intuitive to speculate that nutrients availability may influence this alternative, a comprehensive complement of the molecular determinants involved in this process has not been elucidated yet. In this study, we will investigate how nutrients (glucose) affect skeletal myogenesis. C2C12 cells will be cultured in high glucose and low glucose conditions, and their differenciation will be studied. Experiment Overall Design: We hypothesize that calorie restriction may have an effect on appropriate differentiation of skeletal myoblasts and this effect can be demonstrated by expression profiling.
Project description:The integration of positive and negative intra- and extra-cellular signals dictates whether a cell will proliferate or differentiate. While it is intuitive to speculate that nutrients availability may influence this alternative, a comprehensive complement of the molecular determinants involved in this process has not been elucidated yet. In this study, we will investigate how nutrients (glucose) affect skeletal myogenesis. C2C12 cells will be cultured in high glucose and low glucose conditions, and their differenciation will be studied. Keywords: Differential Design, Differentiation Medium (DM), Growth Medium (GM)
Project description:Metabolic stress and changes in nutrient levels modulate many aspects of skeletal muscle function during aging and disease. Growth factors and cytokines secreted by skeletal muscle, known as myokines, are important signaling factors but it is largely unknown whether they modulate muscle growth and differentiation in response to nutrients. Here, we find that changes in glucose levels increase the activity of the glucose-responsive transcription factor MLX, which promotes and is necessary for myoblast fusion. MLX promotes myogenesis not via an adjustment of glucose metabolism but rather by inducing the expression of several myokines, including insulin like-growth factor-2 (IGF2), whereas RNAi and dominant-negative MLX reduce IGF2 expression and block myogenesis. This phenotype is rescued by conditioned media from control muscle cells and by recombinant IGF2, which activates the myogenic kinase Akt. Importantly, MLX null mice display decreased IGF2 induction and diminished muscle regeneration in response to injury, indicating that the myogenic function of MLX is conserved in vivo. Thus, glucose is a signaling molecule that regulates myogenesis and muscle regeneration via MLX/IGF2/Akt signaling.â??The data pproided are histome H4 acetlation data for MLX DN and MLX wt samples; 3 MLX DN H4 Ac Chip seq samples , 3 Inputs, 3 MLX WT H4 Ac samples and 3 WT inputs
Project description:Metabolic stress and changes in nutrient levels modulate many aspects of skeletal muscle function during aging and disease. Growth factors and cytokines secreted by skeletal muscle, known as myokines, are important signaling factors but it is largely unknown whether they modulate muscle growth and differentiation in response to nutrients. Here, we find that changes in glucose levels increase the activity of the glucose-responsive transcription factor MLX, which promotes and is necessary for myoblast fusion. MLX promotes myogenesis not via an adjustment of glucose metabolism but rather by inducing the expression of several myokines, including insulin like-growth factor-2 (IGF2), whereas RNAi and dominant-negative MLX reduce IGF2 expression and block myogenesis. This phenotype is rescued by conditioned media from control muscle cells and by recombinant IGF2, which activates the myogenic kinase Akt. Importantly, MLX null mice display decreased IGF2 induction and diminished muscle regeneration in response to injury, indicating that the myogenic function of MLX is conserved in vivo. Thus, glucose is a signaling molecule that regulates myogenesis and muscle regeneration via MLX/IGF2/Akt signaling. The data pproided are histome H4 acetlation data for MLX DN and MLX wt samples;
Project description:In utero undernutrition is associated with obesity and insulin resistance, although its effect on skeletal muscle remains poorly defined. We report that, in mice, adult offspring from undernourished dams have decreased energy expenditure, decreased skeletal muscle mitochondrial content, and altered energetics in isolated mitochondria and permeabilized muscle fibers. Strikingly, when these mice are put on a 40% calorie restricted diet they lose half as much weight as calorie restricted controls. Our results reveal for the first time that in utero undernutrition alters metabolic physiology having a profound effect on skeletal muscle energetics and response to calorie restriction in adulthood.
Project description:In utero undernutrition is associated with obesity and insulin resistance, although its effect on skeletal muscle remains poorly defined. We report that, in mice, adult offspring from undernourished dams have decreased energy expenditure, decreased skeletal muscle mitochondrial content, and altered energetics in isolated mitochondria and permeabilized muscle fibers. Strikingly, when these mice are put on a 40% calorie restricted diet they lose half as much weight as calorie restricted controls. Our results reveal for the first time that in utero undernutrition alters metabolic physiology having a profound effect on skeletal muscle energetics and response to calorie restriction in adulthood. We have used a mouse model of low birth weight generated through 50% food restriction of mouse dams during the third week of gestation. We have studied in utero food restricted offspring and control offspring that were not food restricted in utero in both the ad libitum and calorie restricted states. Gene expression profiling was performed on tibialis anterior muscle from 8 mice per group, pooled in pairs.
Project description:We generated transcriptome profiles of skeletal muscles from Largewhite pigs at 35, 55, and 90 days of gestation (dg), using high-throughput deep sequencing methods. A large number of differentially expressed (DE) genes and miRNAs were identified between stages and dietary treatments. Interestingly, reduced maternal calorie supply led to significant changes in mRNA and miRNA expression patterns. Down-regulation of critical genes and miRNAs associated with increased cell growth and myoblast differentiation at certain stages was found to be responsible for repressed myofiber formation in calorie-restricted pigs. Moreover, an integrative analysis of the miRNA and mRNA using sequence-based target prediction and negative correlation of miRNA-mRNA profiles identified a number of novel miRNA-mRNA interactions that are potentially involved in prenatal myogenesis regulation. Importantly, some critical miRNA-mRNA interactions were validated. And we first proposed a miRNA-TF-mRNA regulatory loop, which significantly increased the number of biologically relevant targets of miRNAs and introduced new mechanisms underlying the nutrient-modulated myogenesis.
Project description:Analysis of treatment at gene expression level in aged mice. Results provide important information of the response of drug modifying NAD metabolism which has been implicated in anti-aging effect of calorie restriction in aging process. Total RNA obtained from skeletal muscles and brain (cortex) subjected to calorie restriction or β-lapachone treatment compared to untreated control.
Project description:Tamoxifen, a selective estrogen receptor modulator (SERM), is commonly used in the treatment of hormone-responsive cancers. The effects of tamoxifen in anabolic tissues harboring estrogen-receptors, such as skeletal muscle, are poorly understood. As estrogen and estrogen receptors play an important role in skeletal muscle development and repair, we hypothesize that tamoxifen may have specific effects on myogenesis, the developmental process underlying muscle cells differentiation and repair. Myogenesis is characterized by fine-tuned changes in protein expression as embryonic myoblasts and adult satellite cells transition from pluripotent stem cells to multinucleated, contractile muscle fibers: we undertake a quantitative proteomic analysis of tamoxifen-induced changes in developing skeletal muscle cells which we expect may also shed light on the effect of tamoxifen on muscle repair.
We report a tandem mass-tag (TMT) approach to tamoxifen-treated myogenesis in C2C12 cells, a well-characterized model of in vitro murine skeletal muscle differentiation. A longitudinal analysis of >10,000 proteins identified in C2C12 myogenesis revealed a novel subset of 1,239 myogenically-regulated proteins. This set of regulatory proteins clustered into five distinct longitudinal expression trends which significantly overlap those obtained in similar analyses performed in human myocytes. A longitudinal analysis of myogenesis in the presence of tamoxifen, when contrasted with a similar analysis in untreated myogenesis finds that while the vast majority of myogenically-regulated proteins were unaffected by tamoxifen treatment, specific pathways and networks are affected. We document a specific functional enrichment for adiponectin-signaling, whereby a set of 198 proteins were differentially expressed relative to controls at one or more stages of myogenesis, the majority of which were involved in steroid biosynthesis, lipid storage and/or metal ion homeostasis. Interestingly, the only protein that was differentially expressed in the tamoxifen-treated cells at every stage of myogenesis was metallothionein-1 (MT1). Elevated levels of MT1 have been correlated with tamoxifen resistance and increased patient mortality and relapse in breast cancer, as well as with cachexia and muscle atrophy in rodent models. Increased MT1 expression levels may contribute to the musculoskeletal effects reported by patients undergoing tamoxifen treatment. Finally, we present a powerful, self-validating pipeline for analyzing the total proteomic response to in vitro treatment across every stage of muscle cells development which can be easily adapted to study the effects of other drugs on myogenesis.
Project description:Background: Diet induced weight reduction promotes a decrease in resting energy expenditure that could partly explain the difficulty to maintain reduced body mass. Whether this reduction remains after stabilized weight loss is still controversial. The molecular mechanisms are unknown. Objective: To investigate the effect of a stabilized 10%-weight loss on resting metabolic rate, body composition and skeletal muscle gene expression profile in obese women. Design: Obese women were successively submitted to a 4-w very low-calorie diet, a 3-6-wk low-calorie diet, and a 4-wk weight maintenance program to achieve a 10% weight loss. Resting energy expenditure, body composition, plasma parameters and skeletal muscle transcriptome were compared before weight loss and during stabilized weight reduction. Results: Energy restriction caused an 11% weight loss. Stabilization to the new weight was accompanied by an 11% decrease of the resting metabolic rate normalized to the body cellular mass which was below that of lean subjects. The range of the changes in the skeletal muscle transcriptome was modest. The main regulated genes were that of slow/oxidative fiber markers which were overexpressed and the gene encoding the glucose metabolism inhibitor PDK4 which was down-regulated. The knowledge based approach, gene set enrichment analysis, identified pathways related to insulin and interleukin 6 and long term calorie restriction adaptations during weight loss. Set of arrays that are part of repeated experiments Keywords: Biological Replicate
Project description:Cultivation methods used to investigate microbial calorie restriction often result in carbon and energy starvation. This study aims to dissect cellular responses to calorie restriction and starvation in Saccharomyces cerevisiae by using retentostat cultivation. In retentostats, cells are continuously supplied with a small, constant carbon and energy supply, sufficient for maintenance of cellular viability and integrity but insufficient for growth. When glucose-limited retentostats cultivated under extreme calorie restriction were subjected to glucose starvation, calorie-restricted and glucose-starved cells were found to share characteristics such as increased heat-shock tolerance and expression of quiescence-related genes. However, they also displayed strikingly different features. While calorie-restricted yeast cultures remained metabolically active and viable for prolonged periods of time, glucose starvation resulted in rapid consumption of reserve carbohydrates, population heterogeneity due to appearance of senescent cells and, ultimately, loss of viability. Moreover, during starvation, calculated rates of ATP synthesis from storage carbohydrates were 2-3 orders of magnitude lower than steady-state ATP-turnover rates calculated under extreme calorie restriction in retentostats. Stringent reduction of ATP turnover during glucose starvation was accompanied by a strong down-regulation of genes involved in protein synthesis. These results demonstrate that extreme calorie restriction and carbon starvation represent different physiological states in S. cerevisiae.