Evidence that insulin activates casein kinase 2 in rat epididymal fat-cells and that this may result in the increased phosphorylation of an acid-soluble 22 kDa protein.
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ABSTRACT:
SUBMITTER: Diggle TA
PROVIDER: S-EPMC1151638 | biostudies-other | 1991 Oct
REPOSITORIES: biostudies-other
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Project description:1. Earlier studies have shown that exposure of fat-cells to insulin results in the rapid increased phosphorylation of an acid-soluble 22 kDa protein and that increases in phosphorylation were also evident in cells exposed to adrenaline [Belsham & Denton (1980) Biochem. Soc. Trans. 8, 382-383; Belsham, Brownsey, Hughes & Denton (1980) Diabetologia 18, 307-312]. 2. The effects of adrenaline are shown to be brought about through beta-adrenergic receptors and to be mimicked by other agents which increase cell cyclic AMP concentrations. The maximum extent of phosphorylation is about 60% of that observed with insulin. Increased phosphorylation is also observed in fat-cells exposed to vasopressin, oxytocin and phorbol esters, but not to alpha-adrenergic agonists. 3. No changes in the phosphorylation of the protein are evident in epididymal fat-pads from fat-fed, starved or starved/refed animals, despite the large changes in protein composition of fat-cells which accompany these nutritional alterations. This suggests that the protein is not closely involved in lipogenesis or associated metabolic pathways, but rather that it may play a more general regulatory role. 4. The 22 kDa protein migrates as a doublet on SDS/PAGE even after purification to apparent homogeneity by sequential use of Mono Q chromatography, SDS/PAGE and h.p.l.c. The amino acid compositions of the two components are very similar and share features in common with a number of proteins, including inhibitor-1, inhibitor-2, dopamine- and cyclic-AMP-regulated phosphoprotein (DARPP-32), and G-substrate, which may be involved in the regulation of protein phosphatase activity. 5. Phosphopeptide mapping and phosphoamino acid analysis reveals that insulin increases the phosphorylation of two distinct peptides within the protein (in one peptide insulin increases the amount of phosphothreonine, whereas in the other the hormone increases the amounts of phosphothreonine and phosphoserine). Both components of the doublet exhibit similar changes in phosphorylation, and hence the differences in migration are not the result of differences in phosphorylation, as suggested previously [Blackshear, Nemenoff & Avruch (1983) Biochem. J. 214, 11-19]. The pattern of phosphorylation observed with the beta-adrenergic agonist isoprenaline was similar to that observed with insulin. 6. The possible role and regulation of the 22 kDa protein are discussed.
| S-EPMC1130848 | biostudies-other
Project description:1. A new rapid method for the purification of fat-cell acetyl-CoA carboxylase is described; the key step is sedimentation after specific polymerization by citrate. 2. Incubation of epididymal fat-pads or isolated fat-cells with insulin or adrenaline leads to a rapid increase or decrease respectively in the activity of acetyl-CoA carboxylase measured in fresh tissue extracts. The persistence of the effect of insulin through high dilution of tissue extracts and through purification involving precipitation with (NH4)2SO4 suggests that the enzyme undergoes a covalent modification after exposure of intact tissue to the hormone. The opposed effects of insulin and adrenaline are not adequately explained through modification of a common site on acetyl-CoA carboxylase, since these hormones bring about qualitatively different alterations in the kinetic properties of the enzyme measured in tissue extracts. 3. The state of phosphorylation of acetyl-CoA carboxylase within intact fat-cells exposed to insulin was determined, and results indicate a small but consistent rise in overall phosphorylation of the Mr-230000 subunit after insulin treatment. 4. Acetyl-CoA carboxylase from fat-cells previously incubated in medium containing [32P]phosphate was purified by immunoprecipitation and then digested with performic acid and trypsin before separation of the released phosphopeptides by two-dimensional analysis. Results obtained show that the exposure of fat-cells to insulin leads to a 5-fold increase in incorporation of 32P into a peptide which is different from those most markedly affected after exposure of fat-cells to adrenaline. 5. These studies indicate that the activation of acetyl-CoA carboxylase in cells incubated with insulin is brought about by the increased phosphorylation of a specific site on the enzyme, possibly catalysed by the membrane-associated cyclic AMP-independent protein kinase described by Brownsey, Belsham & Denton [(1981) FEBS Lett. 124, 145-150].
| S-EPMC1158076 | biostudies-other
Project description:The first steps in insulin action are binding of insulin to its receptor and activation of the insulin receptor kinase. As there is indirect evidence that further signal transduction might involve a guanine-nucleotide-binding protein (G-protein), we studied whether insulin modulates GTP binding to plasma membrane proteins of fat cells and skeletal muscle. We found that insulin rapidly increased (30 s) binding of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) in a dose dependent manner (0.03-2.0 nM). This effect was not altered by pertussis toxin, but it was abolished by cholera toxin treatment of fat cells. Scatchard analysis of the binding data showed that the increased GTP[S] binding is due to a decrease in the Kd for GTP from 100 nM to 50 nM. Furthermore, binding of GTP to these plasma membranes inhibited both the binding of 125I-insulin to the insulin receptor and the stimulation of the insulin receptor kinase, suggesting a feedback interaction between the insulin-stimulated GTP-binding site and the insulin receptor. In order to identify this insulin-stimulated GTP-binding site, plasma membranes were labelled with the photoreactive GTP analogue [alpha-32P]GTP gamma-azidoanilide. We found that insulin selectively stimulated GTP binding to a 40 kDa protein. In conclusion, in plasma membranes of fat cells and skeletal muscle, the insulin receptor interacts with a 40 kDa GTP-binding site. We speculate that this 40 kDa GTP-binding site might be a G-protein which is involved in insulin signal transmission.
| S-EPMC1151149 | biostudies-other
Project description:Intact rat epididymal fat-cells were incubated with 32Pi and the intracellular proteins separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. One of the phosphorylated proteins has the same RF value as [14C]biotin-labelled acetyl-CoA carboxylase purified from fat-cells and is specifically precipitated after incubation with antiserum raised against acetyl-CoA carboxylase. No significant changes in the extent of phosphorylation of acetyl-CoA carboxylase were detected after exposure of the cells to insulin.
| S-EPMC1183791 | biostudies-other
Project description:1. Earlier studies have shown that exposure of fat-cells to insulin results in the rapid increased phosphorylation of an acid-soluble protein which migrates as a doublet on SDS/PAGE with an apparent molecular mass of close to 22 kDa; agents such as isoprenaline, which increase cell concentrations of cyclic AMP, also increase phosphorylation, but to a lesser extent [Belsham, Brownsey, Hughes and Denton (1980) Diabetologia 18, 307-312; Diggle and Denton (1992) Biochem. J. 282, 729-736]. 2. The protein has been purified from rat epididymal adipose tissue, and the sequences of six tryptic peptides were determined. All six peptides are present in the deduced sequence of a protein of similar properties, designated PHAS-I by Hu, Pang, Kong, Velleca and Lawrence [(1994) Proc. Natl. Acad. Sci. U.S.A. 91, 3730-3734]. Hence the proteins are the same or extremely similar. 3. A rabbit anti-peptide antibody has been raised against one of the peptides (AGGDESQFEMD). The antibody was found to be highly specific for the phosphorylated and non-phosphorylated forms of the acid-soluble 22 kDa protein in Western blots and by immunoprecipitation. Studies with the antibody preparation have shown that both phosphorylated and non-phosphorylated forms of the protein appear to be exclusively located in the cytoplasm, and that exposure of cells to isoprenaline causes increased phosphorylation of the same acid-soluble 22 kDa protein as does insulin treatment. 4. Western blots carried out with the antibody preparation indicate that the protein is also present in other insulin-sensitive tissues, including liver, skeletal muscle, heart and brown adipose tissue. The protein was also detected in lung and spleen, but not brain and kidney. It is concluded that the protein may play an important role in some of the actions of insulin.
| S-EPMC1136492 | biostudies-other
Project description:Objective: Long-term dysregulation of energy balance is the key component of the obesity epidemic. Given the harm of central obesity and the discovery that beige cells appear within white adipose tissue (WAT), enhancing the energy-expending or "browning" ability of visceral adipose tissue (VAT) has become of therapeutic interest. In this study, we focused on the regulating role of microRNA (miRNA)-27b-3p in mice epididymal white adipose tissue (eWAT) browning. Methods: High-fat diet (HFD) induced obese mice model was constructed. Expression of miR-27b-3p and Ucp1 in eWAT was measured during the course of HFD. Through tail vein injection of antimiR-27b-3p, miR-27b-3p expression was inhibited to analyze the potential role of miR-27b-3p in fat browning and metabolism. Results: miR-27b-3p was predominantly expressed in eWAT and browning ability of eWAT in HFD induced obese mice was impaired. Inhibition of miR-27b-3p enhanced browning capacity of eWAT in mice fed an HFD and led to weight loss and insulin sensitivity improvement. Conclusions: High expression of miR-27b-3p in eWAT inhibits browning ability and leads to visceral fat accumulation. It is suggested miR-27b-3p may become a potential therapeutic option for visceral obesity and its associated diseases.
| S-EPMC6369196 | biostudies-literature
Project description:Aims/hypothesisDespite a similar fat storing function, visceral (intra-abdominal) white adipose tissue (WAT) is detrimental, whereas subcutaneous WAT is considered to protect against metabolic disease. Recent findings indicate that thermogenic genes, expressed in brown adipose tissue (BAT), can be induced primarily in subcutaneous WAT. Here, we investigate the hypothesis that the Wilms tumour gene product (WT1), which is expressed in intra-abdominal WAT but not in subcutaneous WAT and BAT, suppresses a thermogenic program in white fat cells.MethodsHeterozygous Wt1 knockout mice and their wild-type littermates were examined in terms of thermogenic and adipocyte-selective gene expression. Glucose tolerance and hepatic lipid accumulation in these mice were assessed under normal chow and high-fat diet conditions. Pre-adipocytes isolated from the stromal vascular fraction of BAT were transduced with Wt1-expressing retrovirus, induced to differentiate and analysed for the expression of thermogenic and adipocyte-selective genes.ResultsExpression of the thermogenic genes Cpt1b and Tmem26 was enhanced and transcript levels of Ucp1 were on average more than tenfold higher in epididymal WAT of heterozygous Wt1 knockout mice compared with wild-type mice. Wt1 heterozygosity reduced epididymal WAT mass, improved whole-body glucose tolerance and alleviated severe hepatic steatosis upon diet-induced obesity in mice. Retroviral expression of WT1 in brown pre-adipocytes, which lack endogenous WT1, reduced mRNA levels of Ucp1, Ppargc1a, Cidea, Prdm16 and Cpt1b upon in vitro differentiation by 60-90%. WT1 knockdown in epididymal pre-adipocytes significantly lowered Aldh1a1 and Zfp423 transcripts, two key suppressors of the thermogenic program. Conversely, Aldh1a1 and Zfp423 mRNA levels were increased approximately five- and threefold, respectively, by retroviral expression of WT1 in brown pre-adipocytes.Conclusion/interpretationWT1 functions as a white adipocyte determination factor in epididymal WAT by suppressing thermogenic genes. Reducing Wt1 expression in this and other intra-abdominal fat depots may represent a novel treatment strategy in metabolic disease.
| S-EPMC8803700 | biostudies-literature
Project description:The heart requires a continuous supply of energy but has little capacity for energy storage and thus relies on exogenous metabolic sources. We previously showed that cardiac MED13 modulates systemic energy homeostasis in mice. Here we sought to define the extra-cardiac tissue(s) that respond to cardiac MED13 signaling. We show that cardiac over-expression of MED13 in transgenic (MED13cTg) mice confers a lean phenotype that is associated with increased lipid uptake, beta-oxidation and mitochondrial content in white adipose tissue (WAT) and liver. Cardiac expression of MED13 decreases metabolic gene expression and metabolite levels in heart and liver but enhances them in WAT. Although exhibiting increased energy expenditure in the fed state, MED13cTg mice metabolically adapt to fasting. Furthermore, MED13cTg hearts oxidize fuel that is readily available, rendering them more efficient in the fed state. Parabiosis experiments in which circulations of wild-type and MED13cTg mice are joined, reveal that circulating factor(s) in MED13cTg mice promote enhanced metabolism and leanness. These findings demonstrate that MED13 acts within the heart to promote systemic energy expenditure in extra-cardiac energy depots and point to an unexplored metabolic communication system between the heart and other tissues.
2014-12-01 | GSE62450 | GEO
Project description:The epididymal fat pad is a male gonadal adipocyte tissue and is histochemically separated into distal and proximal parts. The development of epididymal fat during postnatal period has not been examined in detail. A previous research showed that expression of adipocyte-associated molecules in the distal epididymal fat of mouse is generally increased as postnatally aged. In the present study, expressional patterns of same adipocyte-associated molecules in the mouse proximal epididymal fat at 2, 5, 8, and 12 months of age were studied by quantitative real-time PCR analysis and were compared with those in the distal epididymal fat. The expressional levels of peroxisome proliferator-activated receptor gamma (Pparg), lipoprotein lipase (Lpl), and fatty acid synthase (Fasn) at 5 months of age were significantly lower than those at 2 months of age, while transcript level of leptin (Lep) at 5 months was higher than that at 2 months of age. The transcript levels of all molecules at 8 months of age were significantly increased, compared with those at 2 and 5 months of age. At 12 months of age, expression of delta like non-canonical Notch ligand 1 (Dlk1) was further significantly increased, while there was no change on the transcript level of Pparg and significant decreases of Fabp4, Retn, Lpl, Lep, Fasn, and adiponectin (Adipoq) transcript levels. The current findings show that expressional patterns of molecules associated with adipocyte in the proximal epididymal fat is somewhat different with those of the distal epididymal fat, suggesting the existence of regional variance in the epididymal fat.
| S-EPMC6985298 | biostudies-literature
Project description:In the multicellular tissue, cell-cell interaction is important for a precise control of its function. The exchange of signaling molecules between adjacent cells via connexon allows the functional harmony of cells in the tissue. The present research was to determine the presence and expressional patterns of connexin (Cx) isoforms in the rat epididymal fat during postnatal development using quantitative real-time polymerase chain reaction (PCR) analysis. Of 13 Cx isoforms examined, expression of 11 Cx isoforms in the epididymal fat during postnatal development was detected. These Cx isoforms include Cx26, Cx31, Cx31.1, Cx32, Cx33, Cx36, Cx37, Cx40, Cx43, Cx45, and Cx50. Expressional levels of all Cx isoforms at 1 and 2 years of age were significantly higher than those at the early postnatal ages, such as 7 days, 14 days, and 24 days of ages. Except Cx33 and Cx43, the transcript levels of rest Cx isoforms at 1 year of age were significantly lower than that at 2 years of age. In addition, expressional patterns of Cx isoforms between 7 days and 5 months of ages generally varied according to the isoform. The existence of various Cx isoforms in the rat epididymal fat has been identified and expression of each Cx isoform in the epididymal fat during postnatal development has shown a particular pattern, distinguishable from the others. To our knowledges, this is the first report showing expressional patterns of Cx isoforms at transcript level in the epididymal fat at various postnatal ages.
| S-EPMC5915765 | biostudies-literature