Development of a physiological insulin resistance model in human stem cell-derived adipocytes
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE201908 | GEO | 2022/06/17
REPOSITORIES: GEO
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Project description:Background: Systemic glucose homeostasis is affected by adipose cell function. Still, the cellular events preceding the onset of insulin resistance in adipocytes are not yet resolved. To capture the immediate cellular changes during diet-induced expansion of cell volume and number, we characterized the initial changes in mature adipocytes during a short high fat diet (HFD) intervention. Methods: C57BL6/J male mice were fed chow diet, and switched to HFD for 2, 4, 6 or 14 days. Systemic glucose clearance was assessed by glucose tolerance test. Adipose tissue was dissected for RNA seq analysis and cell size distribution analyzed using coulter counter technique. Insulin response in isolated adipocytes was measured by glucose uptake and Western blotting of insulin signaling intermediates. Confocal microscopy was used to assess autophagic flux in cells pre-incubated with rapamycin and chloroquine. Results: Switching to HFD was accompanied by an immediate adipose cell size expansion followed by recruitment of new adipocytes. Despite an increase in both basal and insulin-stimulated glucose uptake in adipocytes, we observed a progressive decrease in insulin-stimulated activation of Protein Kinase B (PKB) Ser473, PKB Thr308 and Akt substrate of 160 (AS160) Tyr642, and onset of systemic insulin-resistance already after two days. Moreover, RNA-seq analysis of adipose tissue revealed marked changes in gene expression at day four, of which the most significant included genes directly involved in autophagy (Trp53Inp2 and Beclin1). Enhanced autophagy was observed in isolated adipocytes from HFD-fed mice, detected as an increased density of LC3-positive puncta by confocal microscopy, and increase of LC3II. Conclusions: HFD rapidly caused impaired insulin signal transduction in adipocytes, while both basal and insulin-stimulated glucose uptake increased, which support the concept of spare signaling. Interestingly, our data suggests autophagy as one of the early cellular events that could contribute to insulin resistance.
2018-10-25 | GSE106174 | GEO
Project description:Whether amino acids act on cellular insulin signaling remains unclear, given that increased circulating amino acid levels are associated with the onset of type 2 diabetes (T2D). Here, we report that phenylalanine modifies insulin receptor beta (IRβ) and inactivates insulin signaling and glucose uptake. Mice fed phenylalanine-rich chow or phenylalanine-producing aspartame or overexpressing human phenylalanyl-tRNA synthetase (hFARS) developed insulin resistance and T2D symptoms. Mechanistically, FARS phenylalanylated lysine 1057/1079 of IRβ (F-K1057/1079), inactivating IRβ and preventing insulin from promoting glucose uptake by cells. SIRT1 reversed F-K1057/1079 and counteracted the insulin-inactivating effects of hFARS and phenylalanine. F-K1057/1079 and SIRT1 levels in white blood cells from T2D patients wereare positively and negatively correlated with T2D onset, respectively. Blocking F-K1057/1079 with phenylalaninol sensitizeds insulin signaling and relieveds T2D symptoms in hFARS-transgenic and db/db mice. These findings shed light on the activation of insulin signaling and T2D progression through inhibition of phenylalanylation.
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Project description:Insulin action in adipocytes affects whole-body insulin sensitivity. Studies of adipose-specific Glut4 knockout mice have established that adipose Glut4 contributes to the control of systemic glucose homeostasis. Presumably, this reflects a role for Glut4-mediated glucose transport in the regulation of secreted adipokines. In cultured 3T3-L1 adipocytes, Rab10 GTPase is required for insulin-stimulated translocation of Glut4 (Sano et al., 2007). The physiological importance of adipose Rab10 and the significance of its role in the control of Glut4 vesicle trafficking in vivo are unknown. Here we report that adipocytes from adipose-specific Rab10 knockout mice have a ~50% reduction in glucose uptake and Glut4 translocation to the cell surface in response to insulin, demonstrating a role for Rab10 in Glut4 trafficking. Moreover, hyperinsulinemic-euglycemic clamp shows decreased whole-body glucose uptake as well as impaired suppression of hepatic glucose production in adipose Rab10 knockout mice. Thus, fully functional Glut4 vesicle trafficking in adipocytes is critical for maintaining insulin sensitivity. Comparative transcriptome analysis of perigonadal adipose tissue demonstrates significant transcriptional similarities between adipose Rab10 knockout mice and adipose Glut4 knockout mice, consistent with the notion that the phenotypic similarities between the two models are mediated by reduced insulin-stimulated glucose transport into adipocytes. Transcriptome sequencing of perigonadal white adipose tissue
2016-06-30 | E-GEOD-70123 | 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. 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: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:Insulin action in adipocytes affects whole-body insulin sensitivity. Studies of adipose-specific Glut4 knockout mice have established that adipose Glut4 contributes to the control of systemic glucose homeostasis. Presumably, this reflects a role for Glut4-mediated glucose transport in the regulation of secreted adipokines. In cultured 3T3-L1 adipocytes, Rab10 GTPase is required for insulin-stimulated translocation of Glut4 (Sano et al., 2007). The physiological importance of adipose Rab10 and the significance of its role in the control of Glut4 vesicle trafficking in vivo are unknown. Here we report that adipocytes from adipose-specific Rab10 knockout mice have a ~50% reduction in glucose uptake and Glut4 translocation to the cell surface in response to insulin, demonstrating a role for Rab10 in Glut4 trafficking. Moreover, hyperinsulinemic-euglycemic clamp shows decreased whole-body glucose uptake as well as impaired suppression of hepatic glucose production in adipose Rab10 knockout mice. Thus, fully functional Glut4 vesicle trafficking in adipocytes is critical for maintaining insulin sensitivity. Comparative transcriptome analysis of perigonadal adipose tissue demonstrates significant transcriptional similarities between adipose Rab10 knockout mice and adipose Glut4 knockout mice, consistent with the notion that the phenotypic similarities between the two models are mediated by reduced insulin-stimulated glucose transport into adipocytes.
2016-06-30 | GSE70123 | GEO
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.
2011-03-01 | GSE25462 | GEO
Project description:The pathogenesis of obesity-related metabolic diseases has been linked to the inflammation of white adipose tissue (WAT), but the molecular interconnections are still not fully understood. MiR-146a controls inflammatory processes by suppressing pro-inflammatory signaling pathways. The aim of this study was to characterize the role of miR-146a in obesity and insulin sensitivityresistance . MiR-146a-/- mice were subjected to a high fat diet followed by metabolic tests and WAT transcriptomics. Gain- and loss-of-function studies were performed using human Simpson-Golabi-Behmel syndrome (SGBS) adipocytes. Compared to controls, miR-146a-/- mice gained significantly more body weight on a high fat diet with increased fat mass and adipocyte hypertrophy. This was accompanied by exacerbated liver steatosis, insulin resistance, and glucose intolerance. Likewise, adipocytes transfected with an inhibitor of miR-146a displayed a decrease in insulin-stimulated glucose uptake, while transfecting miR-146a mimics caused the opposite effect. Natriuretic peptide receptor 3 (NPR3) was identified as a direct target gene of miR-146a in adipocytes and CRISPR/Cas9-mediated knockout of NPR3 increased insulin-stimulated glucose uptake and enhanced de-novo lipogenesis. In summary, miR-146a regulates systemic and adipocyte insulin sensitivity via downregulation of NPR3.
2020-10-12 | GSE151268 | GEO
Project description:Insulin-stimulated muscle glucose uptake is a key process to alleviate hyperglycemia. This process depends on the redistribution of glucose transporters to the muscle surface membrane following phosphorylation of TBC1D1 and TBC1D4. Genetic evidence from a TBC1D4 loss-of-function mutation in human skeletal muscle is associated with an increased risk of type 2 diabetes (T2D). However, little is known about the potential regulating interactors of TBC1D4 in skeletal muscle. Here, we sought to identify interactors of TBC1D4 in human skeletal muscle by an unbiased proteomics approach. We detected 76 proteins as candidate TBC1D4 interactors, whereof 12 were regulated by insulin stimulation including known proteins involved in glucose metabolism (e.g. 14-3-3 proteins and ACTN4). TBC1D1 also co-precipitated with TBC1D4 and vice versa in both human and mouse skeletal muscle. This interaction was not regulated by insulin or exercise in young healthy lean individuals. In contrast, we observed an altered interaction as well as compromised insulin-stimulated phospho-regulation of the TBC1D1-TBC1D4 complex in muscle of obese individuals with T2D. In conclusion, we provide a list of TBC1D4 interactors in human and mouse skeletal muscle. These protein interactors serve as potential regulators of TBC1D4 function and thus insulin-stimulated glucose uptake in skeletal muscle.
2022-03-01 | PXD026225 | Pride