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Comparison of the effects of insulin and adrenergic agonists on the phosphorylation of an acid-soluble 22 kDa protein in rat epididymal fat-pads and isolated fat-cells.

ABSTRACT: 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.

SUBMITTER: Diggle TA

PROVIDER: S-EPMC1130848 | biostudies-other | 1992 Mar

REPOSITORIES: biostudies-other

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Project description:1. Dose-dependent effects of adrenaline on PDHa activity were investigated with both incubated rat epidiymal fat-pads and isolated adipocytes. 2. Adrenaline (10nM- 5 micrometer) decreased PDHa activity in fat-pads incubated with 5 mM-[U-14C]glucose + insulin (20 munits/ml). Changes in [U-14C]glucose incorporation into fatty acids in these tissues correlated only loosely with changes in PDHa activity. There was a good inverse relationship between adrenaline-induced changes in PDHa activity and increases in lipolysis (glycerol release). 3. Adrenaline (10nM - 0.5 micrometer) decreased PDHa activity in fat-pads incubated with 5 mM-[U-14C]pyruvate + insulin (20 munits/ml), whereas 1 micrometer- and 5 micrometer-adrenaline slightly increased PDHa activity. All concentrations of adrenaline tested decreased [U-14C]pyruvate incorporation into fatty acids. Between 10nM- and 0.5 micrometer-adrenaline percentage decreases in PDHa activity paralleled decreases in faty acid synthesis. 4. Effects of adrenaline on PDHa activity and fatty acid synthesis in fat-pads incubated with 5mM-[U-14C]pyruvate + insulin (20 munits/ml) could not be mimicked by addition of albumin-bound palmitate. 5. The response of PDHa activity to adrenaline (0.1 nM - 1 micrometer) in isolated adipocytes differed with the carbohydrate substrate used in the incubations. With 5 mM-glucose + insulin (20 munits/ml), PDHa activity was significantly increased by 10 nM-adrenaline, but not by 1 micrometer-adrenaline, the response to adrenaline being biphasic. There was some correlation between PDHa activity and accumulation of non-esterified fatty acids. With 5 mM-glucose alone adrenaline (0.1 nM - 1 micrometer) had no effect on PDHa activity even though lipolysis was increased by adrenaline (0.1 micrometer - 1 micrometer). With 5mM-fructose in the presence and absence of insulin, lipolytic doses of adrenaline decreased PDHa activity. No tested concentrations of adrenaline increased PDHa with this substrate. 6. In the presence of 5 mM-fructose, palmitate was significantly more effective than adrenaline with respect to the maximum decrease in PDHa activity that could be elicited. 4. The relationship of changes in PDHa activity to changes in lipogenesis and the likelihood of adrenaline-induced changes in PDHa activity being secondary to changes in non-esterified fatty acid metabolism are discussed.

Project description:Fat-cells were prepared from rat and guinea-pig epididymal adipose tissue and compared on the basis of the intracellular distributions and activities of enzymes and with respect to their utilization of various U-(14)C-labelled substrates for lipogenesis. 1. Compared with the rat, guinea-pig extramitochondrial enzyme activities differed in that aconitate hydratase, alanine aminotransferase, ATP-citrate lyase, lactate dehydrogenase, NAD-malate dehydrogenase, NADP-malate dehydrogenase and phosphoenolpyruvate carboxykinase activities were appreciably lower, whereas aspartate aminotransferase, glucose 6-phosphate dehydrogenase, NADP-isocitrate dehydrogenase and 6-phosphogluconate dehydrogenase activities were appreciably higher. Mitochondrial activities of citrate synthase, NADP-isocitrate dehydrogenase and pyruvate carboxylase were appreciably lower, whereas mitochondrial activities of aspartate aminotransferase, glutamate dehydrogenase, NAD-malate dehydrogenase and phosphoenolpyruvate carboxykinase were higher in the guinea pig compared with the rat. 2. In general guinea-pig fat-cells incorporated acetate and lactate into fatty acids more readily than rat fat-cells, whereas rat fat-cells incorporated glucose and pyruvate more readily than guinea-pig fat-cells. 3. Acetate stimulated the incorporation of glucose into fatty acids in rat fat-cells, but had no appreciable effect upon this process in guinea-pig fat-cells. Acetate greatly decreased the incorporation of lactate into fatty acids in cells from both species. 4. Lactate/pyruvate ratios produced by incubation of guinea-pig cells with glucose+insulin were very low compared with those found with rat cells under the same conditions. 5. With glucose (+insulin) or with glucose+acetate (+insulin) as substrates guinea-pig cells produced enough NADPH by the hexose monophosphate pathway to satisfy the NADPH requirements of lipogenesis. In rat fat-cells under the same conditions, hexose monophosphate-pathway NADPH provision was not sufficient to meet the requirements of lipogenesis. 6. These results are discussed, particularly in relationship to the disposition of cytosolic reducing equivalents in the cells.

Project description:1. The effects of the protein phosphatase inhibitors okadaic acid and microcystin LR on the regulation by insulin of pyruvate dehydrogenase and acetyl-CoA carboxylase have been studied in rat epididymal fat-pads and isolated cells. These inhibitors both completely blocked the phosphatase activity (against phosphorylase a) present in extracts of epididymal fat-pads, with half-maximal effects in the nanomolar range. 2. Okadaic acid treatment of pads and cells lowered the activity of acetyl-CoA carboxylase assayed in tissue extracts, both before and after treatment of the extracts with the activator, citrate. Further, okadaic acid treatment abolished the 2-3-fold difference in activity observed between extracts from control and insulin-treated tissues, assayed without prior treatment with citrate. 3. Incubation of pads with [32P]Pi, sufficient to label the intracellular pool of ATP, demonstrated that okadaic acid increased the overall phosphorylation of acetyl-CoA carboxylase on a number of distinct sites, as judged by two-dimensional mapping of tryptic peptides. These included the 'I-peptide' [Brownsey & Denton (1982) Biochem. J. 202, 77-86], the phosphorylation of which may be associated with the stimulation of the activity of the enzyme by insulin, as well as inhibitory phosphorylation sites. 4. Incubation with 1 microM-okadaic acid had no effect on the basal level of active pyruvate dehydrogenase apparent after tissue extraction, but abolished the 2-3-fold increase in this parameter which was elicited by insulin in the absence of okadaic acid. However, okadaic acid treatment did not affect the persistent increase in active pyruvate dehydrogenase levels which was apparent in mitochondria subsequently isolated from insulin-treated pads and re-incubated with an oxidizable substrate. It is concluded that the effects of okadaic acid are exerted through changes in metabolite concentrations rather than some direct action on the signalling pathway whereby insulin stimulates pyruvate dehydrogenase. 5. Microcystin LR did not mimic the effects of okadaic acid on intact cells and pads described above.

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Comparison of the effects of insulin and adrenergic agonists on the phosphorylation of an acid-soluble 22 kDa protein in rat epididymal fat-pads and isolated fat-cells.

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