Quantitative proteomics and phosphoproteomics reveal TNF-alpha mediated downstream protein functions in hepatocytes
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion Lumos
ORGANISM(S): Mus Musculus (ncbitaxon:10090)
SUBMITTER: 
Uma Aryal
PROVIDER: MSV000087248 | MassIVE | Tue Apr 20 15:10:00 BST 2021
REPOSITORIES: MassIVE
Similar Datasets
Project description:Insulin resistance is a heterogeneous disorder caused by a range of genetic and environmental factors, and we hypothesise that its aetiology varies considerably between individuals. This heterogeneity provides significant challenges to the development of effective therapeutic regimes for long-term management of type 2 diabetes. We describe a novel strategy, using large-scale gene expression profiling, to develop a gene expression signature (GES) that reflects the overall state of insulin resistance in cells and patients. The GES was developed from 3T3-L1 adipocytes that were made M-bM-^@M-^\insulin resistantM-bM-^@M-^] by treatment with tumour necrosis factor (TNF-alpha and then reversed with aspirin and troglitazone (M-bM-^@M-^\re-sensitizedM-bM-^@M-^]). The GES consisted of 5 genes whose expression levels best discriminated between the insulin resistant and insulin re-sensitized states. We then used this GES to screen a compound library for agents that affected the GES genes in 3T3-L1 adipocytes in a way that most closely resembled the changes seen when insulin resistance was successfully reversed using aspirin and troglitazone. This screen identified both known and new insulin sensitising compounds including non-steroidal anti-inflammatory agents, beta-adrenergic antagonists, beta-lactams and sodium channel blockers. We tested the biological relevance of this GES in participants in the San Antonio Family Heart Study (n = 1,240) and showed that patients with the lowest GES scores were more insulin resistant (according to HOMA_IR and fasting plasma insulin levels, P < 0.001). These findings show that GES technology can be used for both discovery of insulin sensitising compounds and characterisation of patients into subtypes of insulin resistance according to GES scores, opening the possibility of developing a personalised medicine approach to type 2 diabetes. Three-condition experiment, Vehicle, TNF and TNF+ASA+TGZ with biological replicates: 22 Vehicle, 21 TNF and 21 TNF+ASA+TGZ , independently grown and harvested. One replicate per array.
2010-09-10 | E-GEOD-18103 | biostudies-arrayexpress
Project description:Insulin resistance is a sine qua non of Type 2 diabetes (T2D) and a frequent complication of multiple clinical conditions, including obesity, aging, and steroid use, among others. How such a panoply of insults can result in a common phenotype is incompletely understood. Furthermore, very little is known about the transcriptional and epigenetic basis of this disorder, despite evidence that such pathways are likely to play a fundamental role. Here, we compare cell autonomous models of insulin resistance induced by the cytokine tumor necrosis factor-a (TNF) or by the steroid dexamethasone (Dex) to construct detailed transcriptional profiles associated with cellular insulin resistance. Gene expression data from mouse adipocyte, with TNF or Dex treatments at different time points. Murine 3T3-L1 adipocytes were treated separately with dexamethasone (Dex; 20nM) or tumor necrosis factor-alpha. To comprehensively assess gene expression changes caused by Dex and TNF in a time-dependent manner, we profiled cells at early (2 hours), intermediate (24 hours), and late (6 days) points in the development of insulin resistance.
2014-12-23 | E-GEOD-62635 | biostudies-arrayexpress
Project description:Insulin resistance is a sine qua non of Type 2 diabetes (T2D) and a frequent complication of multiple clinical conditions, including obesity, aging, and steroid use, among others. How such a panoply of insults can result in a common phenotype is incompletely understood. Furthermore, very little is known about the transcriptional and epigenetic basis of this disorder, despite evidence that such pathways are likely to play a fundamental role. Here, we compare cell autonomous models of insulin resistance induced by the cytokine tumor necrosis factor-a (TNF) or by the steroid dexamethasone (Dex) to construct detailed epigenomic maps associated with cellular insulin resistance. Murine 3T3-L1 adipocytes were treated separately with dexamethasone (Dex; 20nM) or tumor necrosis factor-alpha. To comprehensively assess epigenomic changes caused by Dex and TNF in a time-dependent manner, we profiled cells at early (2 hours), intermediate (24 hours), and late (6 days) points in the development of insulin resistance.
2014-12-23 | E-GEOD-58491 | biostudies-arrayexpress
Project description:Cytokines such as TNF-alpha and IL-1beta are known for their contribution to inflammatory processes in liver . In contrast, the cytokine IL-17 has not yet been assigned a role in liver diseases. IL-17 can cooperate with TNF-alpha to induce a synergistic response on several target genes in different cell lines, but no data exist for primary hepatocytes. To enhance our knowledge on the impact of IL-17 alone and combined with TNF-alpha in primary murine hepatocytes a comprehensive microarray study was designed. IL-1beta was included as this cytokine is suggested to act in a similar manner as the combination of TNF-alpha and IL-17, especially with respect to its role in mRNA stabilization. Results: The present microarray analysis demonstrates that primary murine hepatocytes responded to IL-17 stimulation by upregulation of chemokines and genes, which are functionally responsible to increase and sustain inflammation. Cxcl2, Nfkbiz and Zc3h12a were strongly induced, whereas the majority of the genes were only very moderately upregulated. Promoter analysis revealed involvement of NF-kappaB in the activation of many genes. Combined stimulation of TNF-alpha/IL-17 resulted in enhanced induction of gene expression, but significantly synergistic effects could be applied only to a few genes, such as Nfkbiz, Cxcl2, Zc3h12 and Steap4. Comparison of the gene expression profile obtained after stimulation of TNF-alpha/IL-17 versus IL-1 proposed a IL-1beta-like effect of the latter cytokine combination. Moreover, evidence was provided that modulation of mRNA stability may be a major mechanism by which IL-17 regulates gene expression in primary hepatocytes. This assumption was exemplarily proven for Nfkbiz mRNA for the first time in hepatocytes. Our studies also suggest that RNA stability can partially be correlated to the existence of AU rich elements, but further mechanisms like the RNase-activity of the upregulated Zc3h12a have to be considered. Conclusions: Our microarray analysis gives new insights in IL-17 induced gene expression in primary hepatocytes highlighting the crosstalk with the NF-kappaB signalling pathway. Gene expression profile suggests IL-17 a role in sustaining liver inflammatory processes most likely by RNA stabilization. Altogether, our results provide evidence that IL-17 alone and in concert with TNF-alpha may play a role in inflammatory liver diseases. Primary murine hepatocytes of three animals stimulated for 1 or 4h by TNF-alpha, IL-1beta, IL-17 or TNF-alpha followed by IL-17 were used for microarray analysis.
2010-03-07 | E-GEOD-19272 | biostudies-arrayexpress
Project description:Obesity is a growing epidemic worldwide and is a major risk factor for several chronic diseases, including diabetes, kidney disease, heart disease, and cancer. The global epidemic of obesity has been accompanied with a dramatic increase in the incidence and prevalence of type 2 diabetes mellitus (T2DM). Obesity often leads to T2D, via the increased production of proinflammatory cytokines such as tumor necrosis factor alpha. Our study combines different proteomic techniques to investigate the changes in the global proteome, secretome and phosphoproteome of adipocytes under chronic inflammation condition, as well as fundamental cross-talks between different cellular pathways regulated by chronic TNF alpha exposure. Our results show that many key regulator proteins of the canonical and non-canonical NF-kB pathways, such as Nfkb2, and its downstream effectors, including Csf-1 and Lgals3bp, directly involved in leukocyte migration and invasion, were significantly upregulated at the intra and extracellular levels, culminating in the progression of inflammation. Our data provides evidence of several key proteins that play a role in the development of insulin resistance.
2020-06-08 | MSV000085562 | MassIVE
Project description:Glycoproteomics, TNF-alpha, membrane proteomics, insulin resistance, adipocyte, SILAC Insulin resistance (IR) is a complex process arising from both environmental and genetic perturbations and leads to a variety of diseases including type-2 diabetes (T2D). The expansion of adipose tissue during obesity results in secretion of proinflammatory cytokines which significantly impairs insulin sensitivity throughout the entire body. Inflammation modulates glycosylation in a variety of cell types however, an investigation of changes in glycosylation following inflammation-induced IR in adipocytes has not been performed. In the present study, we performed a quantitative N-glycomic analysis of TNF-alpha induced IR in adipocytes and identified the regulation of specific N-glycans including an increase in terminal di-galactose- and decrease in di-sialic acid-containing glycans with alpha-2,3 linkages. Quantitative analysis of the membrane-associated proteome in TNF-alpha treated adipocytes identified the regulation of specific glycosyltransferases and glycosidases which correlated with the regulated N-glycans. This included the up- and down-regulation of B4GalT5 and ST3Gal6, respectively at both the protein and mRNA level. To identify the protein and glycosylation sites modified with these regulated N-glycans, we performed a site-specific quantitative glycoproteomic analysis of enriched N-glycopeptides from TNF-alpha treated adipocytes with and without deglycosylation. The combined workflow provided a relative quantification of changes in protein abundance verses N-glycosylation occupancy verses site-specific N-glycans on a proteome-wide level. This revealed the modulation of a subset of N-glycan compositions on specific proteins including those previously associated with insulin-stimulated GLUT4 trafficking to the plasma membrane. Finally, knockdown of B4GalT5 with siRNA and analysis of released N-glycans resulted in the down-regulation of di-galactose containing glycans, confirming the involvement of this enzyme in the TNF-alpha regulated N-glycome.
2015-11-09 | PXD002435 | Pride
Project description:This experiment was conducted to identify target genes of the peroxisome proliferator-activated receptor alpha (PPARa) in skeletal muscle of transgenic mice that overexpressed PPARa. The following abstract from the published manuscript describes the major findings of this work. A potential link between muscle peroxisome proliferator- activated receptor-alpha signaling and obesity-related diabetes.Finck BN, Bernal-Mizrachi C, Han DH, Coleman T, Sambandam N, LaRiviere LL, Holloszy JO, Semenkovich CF, Kelly DP. The role of the peroxisome proliferator-activated receptor-alpha (PPARalpha) in the development of insulin-resistant diabetes was evaluated using gain- and loss-of-function approaches. Transgenic mice overexpressing PPARalpha in muscle (MCK-PPARalpha mice) developed glucose intolerance despite being protected from diet-induced obesity. Conversely, PPARalpha null mice were protected from diet-induced insulin resistance in the context of obesity. In skeletal muscle, MCK-PPARalpha mice exhibited increased fatty acid oxidation rates, diminished AMP-activated protein kinase activity, and reduced insulin-stimulated glucose uptake without alterations in the phosphorylation status of key insulin-signaling proteins. These effects on muscle glucose uptake involved transcriptional repression of the GLUT4 gene. Pharmacologic inhibition of fatty acid oxidation or mitochondrial respiratory coupling prevented the effects of PPARalpha on GLUT4 expression and glucose homeostasis. These results identify PPARalpha-driven alterations in muscle fatty acid oxidation and energetics as a potential link between obesity and the development of glucose intolerance and insulin resistance. Experiment Overall Design: RNA from two wild-type (non-transgenic (NTG)) and two PPARalpha overexpressing (MCK-PPARa) mice was analyzed. Two replicates of each are provided.
2008-06-14 | E-GEOD-5777 | biostudies-arrayexpress
Project description:Liver-specific Knockdown of JNK1 Up-regulates Proliferator-activated Receptor Coactivator 1 and Increases Plasma Triglyceride despite Reduced Glucose and Insulin Levels in Diet-induced Obese Mice. The c-Jun N-terminal kinases (JNKs) have been implicated in the development of insulin resistance, diabetes, and obesity. Genetic disruption of JNK1, but not JNK2, improves insulin sensitivity in diet-induced obese (DIO) mice. We applied RNA interference to investigate the specific role of hepatic JNK1 in contributing to insulin resistance in DIO mice. Adenovirus-mediated delivery of JNK1 short-hairpin RNA (Ad-shJNK1) resulted in almost complete knockdown of hepatic JNK1 protein without affecting JNK1 protein in other tissues. Liver-specific knockdown of JNK1 resulted in significant reductions in circulating insulin and glucose levels, by 57 and 16%, respectively. At the molecular level, JNK1 knockdown mice had sustained and significant increase of hepatic Akt phosphorylation. Furthermore, knockdown of JNK1 enhanced insulin signaling in vitro. Unexpectedly, plasma triglyceride levels were robustly elevated upon hepatic JNK1 knockdown. Concomitantly, expression of proliferator-activated receptor coactivator 1, glucokinase, and microsomal triacylglycerol transfer protein was increased. Further gene expression analysis demonstrated that knockdown of JNK1 up-regulates the hepatic expression of clusters of genes in glycolysis and several genes in triglyceride synthesis pathways. Our results demonstrate that liver-specific knockdown of JNK1 lowers circulating glucose and insulin levels but increases triglyceride levels in DIO mice. Experiment Overall Design: Liver sample from vehicle, GFP Adv-shRNA, or Jnk1 Adv-shRNA treated DIO mice with 5, 4, and 5 replicates, respectively
2008-06-15 | E-GEOD-7648 | biostudies-arrayexpress
Project description:Diet-induced obesity (DIO) predisposes individuals to insulin resistance, and adipose tissue has a major role in the disease. Insulin resistance can be induced in cultured adipocytes by a variety of treatments, but what aspects of the in vivo responses are captured by these models remains unknown. We use global RNA sequencing to investigate changes induced by TNF-a, hypoxia, dexamethasone, high insulin, and a combination of TNF-a and hypoxia, comparing the results to the changes in white adipose tissue from DIO mice. We found that different in vitro models capture distinct features of DIO adipose insulin resistance, and a combined treatment of TNF-a and hypoxia is most able to mimic the in vivo changes. Using genome-wide DNase I hypersensitivity followed by sequencing, we further examined the transcriptional regulation of TNF-a-induced insulin resistance, and we found that C/EPBM-CM-^_ key regulator of adipose insulin resistance. RNA-seq for 6 insulin resistance conditions and 2 control conditions, Dnase hypersensitivity-seq of 4 conditions and 1 control condition, ChIP-seq on p65 after TNFa treatment.
2013-10-18 | E-GEOD-35724 | biostudies-arrayexpress
Project description:Insulin resistance is a heterogeneous disorder caused by a range of genetic and environmental factors, and we hypothesise that its aetiology varies considerably between individuals. This heterogeneity provides significant challenges to the development of effective therapeutic regimes for long-term management of type 2 diabetes. We describe a novel strategy, using large-scale gene expression profiling, to develop a gene expression signature (GES) that reflects the overall state of insulin resistance in cells and patients. The GES was developed from 3T3-L1 adipocytes that were made “insulin resistant” by treatment with tumour necrosis factor (TNF-alpha and then reversed with aspirin and troglitazone (“re-sensitized”). The GES consisted of 5 genes whose expression levels best discriminated between the insulin resistant and insulin re-sensitized states. We then used this GES to screen a compound library for agents that affected the GES genes in 3T3-L1 adipocytes in a way that most closely resembled the changes seen when insulin resistance was successfully reversed using aspirin and troglitazone. This screen identified both known and new insulin sensitising compounds including non-steroidal anti-inflammatory agents, beta-adrenergic antagonists, beta-lactams and sodium channel blockers. We tested the biological relevance of this GES in participants in the San Antonio Family Heart Study (n = 1,240) and showed that patients with the lowest GES scores were more insulin resistant (according to HOMA_IR and fasting plasma insulin levels, P < 0.001). These findings show that GES technology can be used for both discovery of insulin sensitising compounds and characterisation of patients into subtypes of insulin resistance according to GES scores, opening the possibility of developing a personalised medicine approach to type 2 diabetes.
2010-09-10 | GSE18103 | GEO