Mass spectrometry-based analysis of the TBC1D4 interactome in human skeletal muscle
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive HF
ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)
TISSUE(S): Skeletal Muscle
SUBMITTER: 
atul shahaji deshmukh
LAB HEAD: Atul S Deshmukh
PROVIDER: PXD026225 | Pride | 2022-03-01
REPOSITORIES: Pride
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Project description:Insulin resistance in skeletal muscle is a key phenotype associated with type 2 diabetes (T2D) and is even present in offspring of diabetic parents. However, molecular mediators of insulin resistance remain unclear. We find that the top-ranking gene set in expression analysis of muscle from humans with T2D and normoglycemic insulin resistant subjects with parental family history (FH+) of T2D is increased expression of actin cytoskeleton genes regulated by serum response factor (SRF) and its coactivator MKL1. Furthermore, the SRF activator STARS is upregulated in FH+ and T2D and inversely correlated with insulin sensitivity. These patterns are recapitulated in insulin resistant mice, and linked to alterations in two other regulators of this pathway: reduced G-actin and increased nuclear localization of MKL1. Both genetic and pharmacologic manipulation of STARS/MKL1/SRF pathway significantly alter insulin action: 1) Overexpression of MKL1 or reduction in G-actin decreased insulin-stimulated Akt phosphorylation; 2) reduced STARS expression increased insulin signalling and glucose uptake, and 3) SRF inhibition by CCG-1423 reduced nuclear MKL1, improved glucose uptake, and improved glucose tolerance in insulin resistant mice in vivo. Thus, SRF pathway alterations are a signature of insulin resistance which may also contribute to T2D pathogenesis and be a novel therapeutic target. Skeletal muscle samples were obtained from 10 subjects with type 2 diabetes, 25 subjects with a family history of type 2 diabetes (one or both parents), and 15 subjects with no family history of type 2 diabetes. In this analysis RNA was isolated for cRNA preparation and hybridized to Affymetrix Human Genome U133 Plus 2.0 microarrays.
2011-03-01 | E-GEOD-25462 | biostudies-arrayexpress
Project description:Skeletal muscle insulin resistance is the earliest defect in type 2 diabetes (T2D), preceding and predicting disease development. Whether this represents the underlying primary defect in T2D or effects of changes in hormones or circulating metabolites is unknown. To address this question, we have developed a “disease-in-a-dish” model by differentiating iPS cells from T2D patients and controls into myoblasts (iMyo) and studied their function in vitro. We find that T2D iMyos exhibit multiple defects mirroring human disease including altered insulin signaling through the IRS/AKT pathway, decreased insulin-stimulated glucose uptake, and reduced mitochondrial oxidation. In addition, using global phosphoproteomics we find that T2D alters phosphorylation of a large network of targets of mTOR, S6K, PKC and other kinases including proteins involved in regulation of Rho-GTPases, mRNA splicing/processing, vesicular trafficking, gene transcription and chromatin remodeling. This cell-autonomous dysregulated phosphorylation network reveals a new dimension in the mechanism underlying insulin resistance in T2D.
2020-08-27 | PXD015430 | Pride
Project description:Insulin resistance in skeletal muscle is a key phenotype associated with type 2 diabetes (T2D) and is even present in offspring of diabetic parents. However, molecular mediators of insulin resistance remain unclear. We find that the top-ranking gene set in expression analysis of muscle from humans with T2D and normoglycemic insulin resistant subjects with parental family history (FH+) of T2D is increased expression of actin cytoskeleton genes regulated by serum response factor (SRF) and its coactivator MKL1. Furthermore, the SRF activator STARS is upregulated in FH+ and T2D and inversely correlated with insulin sensitivity. These patterns are recapitulated in insulin resistant mice, and linked to alterations in two other regulators of this pathway: reduced G-actin and increased nuclear localization of MKL1. Both genetic and pharmacologic manipulation of STARS/MKL1/SRF pathway significantly alter insulin action: 1) Overexpression of MKL1 or reduction in G-actin decreased insulin-stimulated Akt phosphorylation; 2) reduced STARS expression increased insulin signalling and glucose uptake, and 3) SRF inhibition by CCG-1423 reduced nuclear MKL1, improved glucose uptake, and improved glucose tolerance in insulin resistant mice in vivo. Thus, SRF pathway alterations are a signature of insulin resistance which may also contribute to T2D pathogenesis and be a novel therapeutic target.
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Project description:Lifestyle intervention including exercise restores glucose homeostasis and pancreatic β-cell function in type 2 diabetes (T2D). However, exercise compliance is a challenge. Novel alternative or adjuvant approaches are necessary. During exercise, the contracting skeletal muscle acts as endocrine organ via the secretion and endocrine signaling of functional proteins. We postulated that contracting skeletal muscle secretes proteins that target pancreatic β-cells and regulate insulin secretion and glucose metabolism. To test this hypothesis, we used an in vitro cell-based skeletal muscle contraction system to uncover proteins released in the muscle secretome. Using an RNAseq screen, we identified growth differentiation factor 15 (GDF15) as a lead candidate. β-cells, human pancreatic islets, and C57BL/6J mice exposed to acute GDF15 treatment exhibited increased glucose-stimulated insulin secretion, and the mechanism involved activation of the insulin release pathway. Chronic GDF15 treatment in db/db mice reduced insulin resistance and preserved pancreatic PDX-1 expression. Consistently, plasma GDF15 increased concurrently with C-peptide prior to the onset of chronic hyperglycemia in humans with pre-diabetes. In addition, in humans with T2D, exercise-induced GDF15 was associated with enhanced β-cell function. These findings support GDF15 as a potential therapeutic target for type 2 diabetes and associated co-morbidities.
2022-10-31 | GSE139872 | GEO
Project description:Type 2 diabetes (T2D) is major cause of mortality and morbidity. The key manifestations of this disease are insulin resistance (IR), hyperglycemia and hyperinsulinemia. Secretion of insulin from the endocrine pancreas is triggered by elevated circulating glucose and acts on skeletal muscle and adipose tissues to remove excess glucose from the blood (1). This is achieved through a series of intracellular events that trigger the translocation of vesicles containing the glucose transporter 4 (GLUT4) to the surface of skeletal muscle cells and adipocytes (2,3). This process is impaired in patients with IR or T2D and insulin fails to promote GLUT4 translocation, thus resulting in inefficient glucose uptake in storage tissue and hyperglycemia (4). Chronic hyperglycemic condition increases the risk of cardiovascular disease, stroke, neuropathy, and death (5). Previously developed insulin sensitizer like thiazolidinediones (TZDs) have proven effective in improving glycemic control, however, they significantly increase the risk of cardiovascular disease due to their agonist activity at PPAR(6-8). Consequently, identifying insulin sensitizers that restore insulin-stimulated GLUT4 translocation in diabetic patients without targeting PPAR is urgently needed. The discovery of such molecules has been challenging due to the lack of a GLUT4 translocation assay amenable to high throughput screening (HTS). For this reason, we have developed a new luminescence based HTS assay that allows real time monitoring of GLUT4 translocation in mammalian cells and further adapted the assay to measure GLUT4 translocation in live mice. Using this assay, we identified a new insulin sensitizer which greatly improves insulin-dependent glucose uptake in storage tissues and glucose tolerance in insulin resistant rodents. This new insulin sensitizers acts through interaction with the Unc119 family of proteins which are known to facilitate the transport of specific cargos (9) but had not yet been implicated in insulin action or GLUT4 translocation. This study identifies new PPAR-independent insulin sensitizers with in-vivo efficacy and identify Unc119 proteins as new targets for the treatment of T2D.
2024-07-03 | PXD034052 | Pride
Project description:Skeletal muscle insulin resistance is a prominent early feature in the pathogenesis of type 2 diabetes (T2D). In attempt to overcome this defect, we generated mice overexpressing insulin receptors (IR) specifically in skeletal muscle (IRMOE). On normal chow, IRMOE mice have similar body weight as controls, but an increase in lean mass and glycolytic muscle fibers and reduced fat mass. IRMOE mice also show higher basal phosphorylation of IR, IRS-1 and Akt in muscle and improved glucose tolerance compared to controls. When challenged with high fat diet (HFD), IRMOE mice are protected from diet-induced obesity. This is associated with reduced inflammation in fat and liver, improved glucose tolerance and improved systemic insulin sensitivity. Surprisingly, however, in both chow and HFD-fed mice, insulin stimulated Akt phosphorylation is significantly reduced in muscle of IRMOE mice, indicating post-receptor insulin resistance. RNA sequencing reveals downregulation of several post-receptor signaling proteins that contribute to this resistance. Thus, enhancing early insulin signaling in muscle by overexpression of the insulin receptor protects mice from diet-induced obesity and its effects on glucose metabolism. However, chronic overstimulation of this pathway leads to post-receptor desensitization, indicating the critical balance between normal signaling and hyperstimulation of the insulin signaling pathway.
2020-09-01 | GSE149662 | GEO
Project description:Physical inactivity and western diet are the main factors leading to skeletal muscle (and whole-body) insulin resistance – a defect in insulin-mediated glucose uptake and metabolism – and associated metabolic diseases, such as obesity and type 2 diabetes mellitus (T2D). The study of the response to a mixed meal (normalized for body mass and/or basal metabolic rate) is of particular interest, as it simulates the physiological response to a typical (daily) single meal that depends on both the secretory function of the beta cells (insulin response) and the sensitivity of skeletal muscle signaling to insulin – variables, which markedly changing during development of insulin resistance and T2D. We aimed to compare the early transcriptomic response to a mixed meal normalized to body mass in skeletal muscle of healthy subjects and obese patients without (Ob) and with T2D (Ob+T2D). To investigate the early transcriptomic response we took biopsy samples from the vastus lateralis muscle prior to and 1 h after a meal. In the Ob+T2D group the average level and increase in C-peptide and insulin in the blood were comparable with healthy subjects that, apparently, is explained by dysfunction of the beta-cells induced by chronic T2D. At the same time, the Ob+T2D group demonstrated dysregulation of mixed meal-induced gene expression in skeletal muscle: morу than two times lower number of DEGs and a small overlapping with the gene response in the control group. On the contrary, the postprandial level of C-peptide and insulin in the Ob group was significantly greater than in healthy control that is related to insulin oversecretion associated with insulin resistance. Despite this, the gene response in skeletal muscle was also significantly dysregulated; moreover, it differed from the response in the Ob+T2D group. It means that defect in regulation of insulin-induced gene expression (insulin resistance in terms of gene expression) appears at the first stage of the development of metabolic disorders.
2023-05-05 | GSE231509 | GEO
Project description:<p>The Finland-United States Investigation of NIDDM Genetics (FUSION) study is a long-term effort to identify genetic variants that predispose to type 2 diabetes (T2D) or that impact the variability of T2D-related quantitative traits (QTs). Skeletal muscle and adipose are major insulin target tissues and play key roles in insulin resistance. We hypothesize that a subset of T2D and related QT variants alter gene expression in skeletal muscle and adipose tissue. For this FUSION Tissue Biopsy Study, we have obtained and are analyzing RNA-Seq, microRNA (miRNA)-Seq, and DNA methylation (methyl)-Seq data on biopsy samples from 331 individuals from across the range of glucose tolerance: 124 normal glucose tolerance (NGT), 77 impaired glucose tolerance (IGT), 44 impaired fasting glucose (IFG), and 86 newly-diagnosed T2Ds. Participants completed two study visits, two weeks apart. First visits comprised most of the clinical phenotyping, including four-point OGTT (fasting, and 30, 60, and 120 minute post-load); BMI, WHR; lipids; blood pressure; and many other variables. Participants also completed FUSION health history, medication, and lifestyle questionnaires. At second visit, we obtained ~250mg <i>vastus lateralis</i> skeletal muscle, ~750mg abdominal subcutaneous adipose, and a ~5x15mm section of abdominal skin. Visits were completed in March 2013. RNA isolation is ongoing in the Collins laboratory at the NIH, RNA and miRNA sequencing at the NIH Intramural Sequencing Center (NISC), and genotyping at the Center for Inherited Disease Research (CIDR). Individual-level data is available here for the 306 individuals who consented to data deposit.</p> <p>To focus on evaluation of gene expression and its regulation in skeletal muscle, we analyzed mRNA extracted from <i>vastus lateralis</i> skeletal muscle obtained from 271 of the 331 individual subjects from Finland, along with genome-wide genotypes. Individual-level data is available here for the 250 subjects who reconsented to the use of their data. Release phs001048.v2.p1 adds muscle data for an additional 42 subjects and data from adipose tissue for 276 subjects. Total RNA was isolated using Trizol extraction in the Collins laboratory at the NIH. The mRNA was poly-A selected, 24-plex libraries were generated using the Illumina TruSeq directional mRNA-seq library protocol and RNA sequencing was performed on HiSeq2000 sequencers using 101bp paired-end reads at NISC. miRNA libraries were prepared from total RNA from 296 muscle and 270 adipose samples, pooled and sequenced 50bp single-end reads on Illumina HiSeq2500. Data for 272 muscle and 251 adipose samples are available here for individuals with consent for data deposit. DNA was extracted from blood in the Collins laboratory, and genotyping on the Illumina Omni2.5M array was performed at CIDR. Genotypes were imputed using the HRC 2016 reference panel. In order to assess regions of open chromatin in skeletal muscle, we obtained muscle tissue from a commercial provider to perform ATAC-seq; these samples were sequenced at the University of Michigan DNA Sequencing Core.</p> <p>Greater than 90% of the approximately 80 loci associated with T2D and the 100s of loci associated with T2D-related traits (glucose and insulin, anthropometrics, lipids) through genome-wide association studies occur in non-coding regions, suggesting a strong regulatory component to disease susceptibility. Regulatory element activity is often tissue-specific, which further complicates discovery of the causal/functional variation. Therefore, there is a critical need to understand the full spectrum of genetic variation and regulatory element usage in T2D-relevant tissues. To that end, this study contains whole genome sequence and whole genome bisulfite sequence, and/or Illumina MethylationEPIC Array data, of two tissues relevant to T2D: skeletal muscle and adipose tissue from individuals with glucose tolerance categories ranging from normal to T2D, providing a comprehensive survey of both individual genetic variation as well as DNA methylation across different tissues from multiple individuals.</p>
| phs001048 | dbGaP
Project description:Skeletal myocytes are metabolically active and susceptible to insulin resistance, thus implicated in type 2 diabetes (T2D). This complex disease involves systemic metabolic changes and their elucidation at the systems level requires genome-wide data and biological networks. Genome-scale metabolic models (GEMs) provide a network-context to integrate high-throughput data. We generated myocyte-specific RNA-seq data and investigated their correlation with proteome data. These data were then used to reconstruct a comprehensive myocyte GEM. Next, we performed a meta-analysis of six studies comparing muscle transcription in T2D versus healthy subjects. Transcriptional changes were mapped on the myocyte GEM, revealing extensive transcriptional regulation in T2D, particularly around pyruvate oxidation, branched-chain amino acid catabolism, and tetrahydrofolate metabolism, connected through the down-regulated dihydrolipoamide dehydrogenase. Strikingly, the gene signature underlying this metabolic regulation successfully classifies the disease state of individual samples, suggesting that regulation of these pathways is a ubiquitous feature of myocytes in response to T2D. Isolated skeletal muscle precursor cells from six normal glucose tolerant and non-obese males and females were differentiated in vitro. RNA from fully differentiated myotubes was sequenced using RNA-seq.
2015-04-07 | E-GEOD-63887 | biostudies-arrayexpress
Project description:Obesity and type 2 diabetes (T2D) are metabolic disorders influenced by lifestyle and genetic factors, and characterized by insulin resistance in skeletal muscle, a prominent site of glucose disposal. Numerous genetic variants have been associated with obesity and T2D, of which the majority is located in non-coding DNA regions. This suggest that most variants mediate their effect by altering the activity of gene-regulatory elements, including enhancers. Here, we map skeletal muscle genomic enhancer elements that are dynamically regulated after exposure to the free fatty acid palmitate or the inflammatory cytokine TNFα. By overlapping enhancer positions with the location of disease-associated genetic variants, and resolving long-range chromatin interactions between enhancers and gene promoters, we identify target genes involved in metabolic dysfunction in skeletal muscle. The majority of these genes also associate with altered whole-body metabolic phenotypes in the murine BXD genetic reference population. Thus, our combined genomic investigations identified genes that are involved in skeletal muscle metabolism and linked to SNPs associated with the development of obesity and T2D.
2020-03-26 | GSE126101 | GEO