ABSTRACT: Insulin is a potent regulator of protein metabolism. Here we describe a time-resolved map of insulin-regulated protein turnover in 3T3-L1 adipocytes using metabolic pulse-chase labelling and high-resolution mass spectrometry.
Project description:Insulin resistance is a major risk factor for human metabolic diseases including type 2 diabetes, cardiovascular diseases and some cancers. We analysed the proteome analyses across in vitro and in vivo models of insulin resistance to find common features of insulin resistance and identified lower expression of enzymes within the mevalonate pathway. mitochondrial CoQ was lower in all models of insulin resistance. Specifically, the coenzyme Q (CoQ) biosynthetic proteins Coq 7 and 9 were decreased in adipose tissue from a mouse model of diet-induced obesity, and mitochondrial CoQ was lower in all models of insulin resistance. Moreover, the mitochondrial content of CoQ in adipose tissue correlated positively with insulin sensitivity in obese humans. Inhibition of CoQ biosynthesis induced insulin resistance, while replenishment of CoQ restored insulin sensitivity in cell culture models and in animals, demonstrating that loss of CoQ is both necessary and sufficient for adipocyte insulin resistance. Mechanistically, loss of CoQ increased mitochondrial peroxides. These findings place defective mitochondrial CoQ homeostasis upstream of increased mitochondrial oxidants in the induction of insulin resistance in adipocytes and highlight the CoQ biosynthetic pathway as an appealing therapeutic target to combat insulin resistance.
Project description:To elucidate the signalling network responsible for mediating the effects of FGF21, we quantified the dynamic phosphoproteome of adipocytes following acute exposure to this hormone. A major FGF21-regulated signalling node was mTORC1/S6K. In contrast to insulin, we find that FGF21 activates mTORC1 via MAPK signalling rather than through the canonical PI3K/AKT pathway. This study provides a systems view of FGF21 signalling and reveals a new role for mTORC1 in maintaining nutrient homeostasis.
Project description:Insulin binds the insulin receptor (IR), which in turn has showed to form nanoclusters at the cell membrane. Trying to exploit the nanoscale spatial organization of IR, we developed rod-like insulin-DNA-origami nanostructures carrying different numbers of insulin molecules. These structures (referred to as NanoRods, or NR) were then utilized to investigate receptor activity to spatial distribution of insulin molecules. One part of this investigation was to study the transcriptional response of brown adipocytes treated with NR bound to one insulin (NR-1) and NR bound to seven insulin (NR-7), and compare to untreated controls. Furthermore, free insulin was also used to ensure that similar pathways were activated when using NR-bound insulin.
Project description:There are an estimated 21million diabetics in the United States and 150 million diabetics worldwide. The World Health Organization anticipates that these numbers will double in the next 20 years. Metabolic syndrome is a well recognized set of symptoms that increases a patient’s risk of developing diabetes. Insulin resistance is a factor in both metabolic syndrome and Type 2 diabetes. It is characterized by decreased insulin stimulated glucose uptake in peripheral tissues, decreased adiponectin levels, increased adipocyte FFA and cytokine production, and increased insulin and hepatic glucose output. Prevention or reversal of insulin resistance should serve as an important strategy in addressing the growing health concerns posed by the Diabetes epidemic. While increased adiposity is associated with insulin resistance, the role of the cell types present within adipose (adipocytes, pre-adipocytes, endothelial cells, macrophages, fibroblasts, leukocytes and smooth muscle cells) in insulin resistance is unclear. In an effort to begin dissection of this question, we examined the transcriptional response of the buoyant and non-buoyant fractions isolated from insulin sensitive or TNF induced insulin resistant hMSC derived adipocytes before and after treatment with insulin. hMSC derived adipocytes were treated with 0 or 10ng/ml TNF for 24 hours to induce insulin resistance and subsequently serum starved for 5 h followed by treatment with 0 or 20nM insulin for 2 hours. At the end of the incubation period, cells were harvested as is (mixed) or subjected to fractionation to separate the adipocytes (buoyant fraction) and the stromal cells (non-buoyant fraction). These isolated cells were resuspended in RLT buffer to prepare lysates for total RNA isolation using the Qiagen RNeasy kit. Total RNA was submitted to GeneLogic for cRNA preparation using the Affymetrix GenChip IVT kit and hybridization to Affymetrix U133 Plus 2.0 arrays.
Project description:Obesity is an energy balance disorder in which nutrient intake chronically exceeds energy expenditure, resulting in the accumulation of white adipose tissue. Increased adiposity is due to increases in the number and size of adipocytes, which leads to increased body fat and metabolic consequences. Adipocytes induce insulin resistance by promoting lipotoxicity and modulating adipokine secretion. Therefore, a thorough understanding of the mechanisms that regulate adipogenesis could have clinical relevance in preventing and treating obesity and the metabolic syndrome. In this study, we performed miRNA array to measure miRNA profiles of undifferentiated and differentiated 3T3-L1 adipocytes using Agilent(r) miRNA array. We show that miRNA profile changes during adipogenesis. Thus, this data would be useful to find the distinct role of miRNAs for the regulation of adipogenesis.
Project description:Activation of protein kinase C epsilon (PKCε) in the liver has been widely associated with hepatic insulin resistance. PKCε is proposed to inhibit insulin signalling through phosphorylation of the insulin receptor. We have tested this directly by breeding PKCε floxed mice with mice expressing Cre recombinase under the control of the cytomegalovirus, albumin or adiponectin promoters to generate global, liver- and adipose tissue-specific PKCε knockout (KO) mice. Global deletion of PKCε recapitulated the benefits for diet-induced glucose intolerance that we previously described using conventional PKCε KO mice. However, we did not detect PKCε-dependent alterations in hepatic insulin receptor phosphorylation. Furthermore, liver-specific KO mice were not protected against diet-induced glucose intolerance or insulin resistance determined by euglycemic clamp. In contrast, adipose tissue-specific KO mice exhibited improved glucose tolerance and mildly increased hepatic triglyceride storage, but no change in liver insulin sensitivity. Phosphoproteomic analysis of insulin signalling in PKCε KO adipocytes revealed no defect in the canonical INSR/AKT/mTOR pathways. However, PKCε KO resulted in changes in phosphorylation of several proteins associated with the endosome and cell junctions suggesting regulation in secretory pathways and a potential role of PKCε in endocrine function. Indeed, RNA-seq analysis revealed adipose-tissue PKCε-dependent changes in the hepatic expression of several genes linked to glucose homeostasis and hepatic lipid metabolism. The primary effect of PKCε on glucose homeostasis is therefore not exerted directly in the liver as currently assumed. However, PKCε in adipose tissue modulates glucose tolerance and is involved in crosstalk with the liver that affects gene expression and lipid accumulation.
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.
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.
Project description:The intimate association between obesity and type II diabetes urges for a deeper understanding of adipocyte function. We and others have previously delineated a role for the tumor suppressor p53 in adipocyte biology. Here, we show that mice haploinsufficient for MDM2, a key regulator of p53, in their adipose stores suffer from overt obesity, insulin resistance, and hepatic steatosis. These mice had decreased levels of circulating palmitoleic acid (non-esterified fatty acid (NEFA) 16:1) concomitant with impaired visceral adipose tissue expression of Scd1 and Ffar4. A similar decrease in Scd and Ffar4 expression was found in in vitro differentiated adipocytes with perturbed MDM2 expression. Mechanistically, lowered MDM2 levels led to nuclear exclusion of the transcriptional cofactors, MORC2 and LIPIN1, thus hampering adipocyte function by antagonizing LIPIN1-mediated PPARγ coactivation. Collectively, these data argue for a p53-independent role of MDM2 in controlling adipocyte function through LIPIN1.
Project description:We profiled PPARg dependent gene expression changes during differntiation of 3T3L1 cell using PPARg siRNA 3T3-L1 (Pre-adipocyte) cell line was induced to differentiate using standard adipocyte differentiation media (IBMX, Dex and Insulin) 48hrs post-confluency. RNA was harvested at day -2 (confluent fibroblasts), 48hrs post-induction with IBMX, DEX and Insulin (day=0) and for each subsequent day after rosiglitazone treatment. Illumina beadchip microarrays were used to determine expression profiles of genes differentially regulated in cells transfected with either siRNA targeting PPARgamma or a non-targeting control siRNA. 3T3L1 cell were induced to differentiate into adipocytes using IBMX, DEX and Insulin. RNA from cell treated with PPARg-specific siRNA and non-specific siRNA was isolated at different timepoints. Illumina MouseRef-8 v1.1 Bead chips were used for expression profiling