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Sphingosine, an Inhibitor of Protein Kinase C, Suppresses the Insulin

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Sphingosine, an Inhibitor of Protein Kinase C, Suppresses the Insulin Proc. Nati. Acad. Sci. USA Vol. 86, pp. 4705-4709, June 1989 Medical Sciences Sphingosine, an inhibitor of protein kinase C, suppresses the insulin-like effects of growth hormone in rat adipocytes (insulin action/lipogenesis/glucose uptake/phorbol esters) JEAN SMAL AND PIERRE DE MEYTS Department of Diabetes, Endocrinology and Metabolism, City of Hope National Medical Center, Duarte, CA 91010 Communicated by Rachmiel Levine, February 27, 1989 (received for review July 18, 1988) ABSTRACT Insulin, human growth hormone (hGH), and the GH receptors of 3T3-F442A fibroblasts and adipocytes phorbol 12-myristate 13-acetate all stimulate lipogenesis in rat are phosphorylated when occupied by GH, but the biological adipocytes preincubated without hGH for 4 hr. As previous significance of this phosphorylation has still to be addressed. data suggested that protein kinase C plays an important role in In contrast, the mechanisms of action of insulin have been the action ofinsulin and in the insulin-like effects of hGH in rat thoroughly investigated over the past decade. The advances in adipocytes, we tested the effects of sphingosine, a potent the molecular biology of the insulin receptor (10, 11), including inhibitor of protein kinase C, on the lipogenic activity of both site-directed mutagenesis, have helped build extensive circum- hormones. At 50 IAM, sphingosine had no effect on basal stantial evidence for an important role ofthe receptor ,-subunit lipogenesis but completely abolished the action of phorbol tyrosine kinase (12), although specific intracellular substrates 12-myristate 13-acetate and decreased by 65% and 89%, have yet to be identified. The insulin receptor has also recently respectively, the effects of hGH and insulin. At higher concen- been proposed to activate a phospholipase C that specifically trations (100 ,uM), sphingosine abolished both basal and hor- hydrolyzes aglycosylphosphatidylinositol in the cell membrane mone-stimulated lipogenesis; this effect was partially reversible (13). This glycosylphosphatidylinositol breakdown generates an after washing the cells. Similar effects of sphingosine on basal inositol phosphate glycan (13) that mimics some intracellular and stimulated glucose uptake were seen in parallel, suggesting effects of insulin and diacylglycerols (14), which are potential inhibits lipogenesis at the glucose-uptake step in activators of protein kinase C. This kinase, an important inter- that sphingosine mediate in various pathways of signal transduction, hormone rat adipocytes. N-Acetylsphingosine and sphingomyelin, two to analogs ofsphingosine that are inactive on protein kinase C, did action, and cell regulation (15), has also been suggested or phorbol participate in insulin action. Indeed, insulin has been reported not inhibit lipogenesis induced by hGH, insulin, to provoke rapid increases in the synthesis and intracellular 12-myristate 13-acetate. Sphingosine did not inhibit insulin contents of phosphatidic acid, inositol phospholipids, and di- binding to rat adipocytes at concentrations up to 200 ,M but acylglycerols in BC3H1 myocytes and adipose tissue (13, 14, decreased hGH binding to its receptors by 44% at 50 ,uM. These 16-25), although one recent work showed no effects of insulin data suggest a direct link between the inhibition ofprotein kinase on the breakdown of inositolphospholipids in rat adipocytes C and that of lipogenesis and provide new evidence for the (26). Moreover, insulin stimulates the activity ofprotein kinase involvement of protein kinase C in the mechanism of action of C in both cytosolic and membrane fraction ofBC3H1 myocytes growth hormone and insulin in rat adipocytes. and in rat diaphragm membranes (27). Finally, protein kinase C activators such as phorbol esters mimic some of the insulin Human growth hormone (hGH) exerts a variety of effects on effects in different cell types (20, 25, 28-45). somatic growth, development, and metabolism (1-3). While Because phorbol esters stimulate lipogenesis in rat adipo- most of growth hormone (GH) effects on skeletal growth are cytes (36-38), we evaluated the possible involvement ofprotein thought to be indirect and mediated through somatomedins kinase C in our postulated common pathway shared by insulin such as insulin-like growth factor I (2, 4), GH actions on and hGH in these cells. We showed that phorbol 12-myristate carbohydrate and lipid metabolism are probably direct (5). 13-acetate (PMA), an activator of protein kinase C, stimulated GH is classically an antagonist of insulin, with lipolytic and lipogenesis and that its effect was nonadditive to the effects of diabetogenic effects in vivo-but paradoxically GH also insulin or GH (43). Furthermore, downregulation of protein exerts acute and transient insulin-like effects in vivo and in kinase C markedly inhibited the effects of both hormones (43). vitro in models where endogenous GH has been suppressed In the present work, we looked for a specific inhibitor ofprotein (1, 2, 5). We recently showed that hGH, like insulin, stimu- kinase C to further evaluate the potential role of protein kinase lates lipogenesis in rat adipocytes preincubated without hor- C in the actions of insulin and GH. Sphingosine, a sphingoid mones during 4 hr, and the nonadditivity of the two maximal long-chain base, was recently shown to be a potent inhibitor of responses strongly suggested that the two hormones share a purified protein kinase C-and ofphorbol dibutyrate binding in common subset of activated pathways (6k. vitro and in vivo (46-48). We report here that sphingosine Despite advances in our knowledge of the mechanisms of strongly inhibits GH-, insulin-, and PMA-stimulated lipogenesis action of GH and insulin, we still do not understand at the and glucose uptake in rat adipocytes. These results are con- molecular level how these hormones transduce their signals sistent with an involvement of protein kinase C in the mecha- from their specific membrane receptors into the cell. The nisms of action of GH and insulin. recent cloning of a putative GH receptor (7) has provided no clue as to a possible mechanism of action. The GH receptor sequence deduced from the cDNA is not related to known MATERIALS AND METHODS tyrosine kinases or to any other known protein except the Reagents and Hormones. hGH (NIDDK hGH bulk at 2.4 prolactin receptor (8). Foster et al. (9) reported recently that international units per mg) was obtained from the National The publication costs of this article were defrayed in part by page charge Abbreviations: GH, growth hormone; hGH, human GH; PMA, payment. This article must therefore be hereby marked "advertisement" phorbol 12-myristate 13-acetate; KRH buffer, Krebs-Ringer-Hepes in accordance with 18 U.S.C. §1734 solely to indicate this fact. buffer; BSA, bovine serum albumin. 4705 4706 Medical Sciences: Smal and De Meyts Proc. Natl. Acad. Sci. USA 86 (1989) Hormone and Pituitary Program, National Institute of Ar- Glucose Uptake. Adipocytes were isolated and then prein- thritis, Diabetes, and Digestive and Kidney Diseases cubated 4 hr at 37°C in KRH buffer/BSA (10 mg/ml)/0.27 (NIDDK) (S. Raiti, University of Maryland School of Med- mM glucose as described in the lipogenesis assay procedure. icine). Porcine insulin was purchased from Sigma; D- For glucose-uptake measurements, isolated adipocytes (16 x [3-3H]glucose was from DuPont/NEN; collagenase (Cls II, 104 cells per ml) were incubated according to the lipogenesis batch 67157M) was from Cooper Biomedical; bovine serum procedure with increased concentrations of sphingosine (0- albumin (BSA) fraction V (batch 17F-0149), PMA, sphin- 100 ,uM) during 10 min at 37°C before the addition of a gosine, and sphingomyelin were from Sigma; and both 2,5- maximally effective dose of insulin (100 ng/ml), hGH (1 diphenyloxazole (PPO) and 1,4-bis-2(5-phenyloxazolyl)ben- Ag/ml), or PMA (100 ng/ml) and 9 nM D-[3-3H]glucose for 2 zene (POPOP) were purchased from Eastman Kodak. N- hr at 37°C. Incubation was interrupted by adding successively Acetylsphingosine was prepared as reported (49). Other 3 ml of ice-cold 0.9o NaCl and 1 ml of dinonylphthalate per chemicals were ofreagent grade and were obtained from J. T. tube. The tubes were immediately centrifuged at 4°C, 3000 x Baker. The tubes used for lipogenesis assays were 7-ml g for 5 min. Floating cells were recovered with a 500-,ul pipet high-density polyethylene S/P scintillation mini-vials from fitted with a truncated tip, transferred in a scintillation vial American Scientific Products (McGaw Park, IL). containing 10 ml of universal scintillating fluid (Aquasol-2, Animals. Male Wistar rats (120-140 g) were obtained from NEN Research Product, Boston), and thoroughly shaken to Charles River Breeding Laboratories and were maintained in break the cells before counting. light/dark cycles of 12 hr and fed ad libitum. Hormone Binding. The binding of insulin and GH to their Lipogenesis. The adipocyte isolation and lipogenesis assay receptors was studied as described (6). We prepared 25I- were conducted as described (6). Briefly, dissected epididymal labeled insulin (labeled on tyrosine A14) and 1251I-labeled hGH and retroperitoneal fat pads were digested by vigorous shaking as described (51, 52). Isolated adipocytes (8 x 105 cells per at 37TC for 30 min with collagenase (1.0 mg/ml) in Krebs- ml for insulin binding; 16 x 105 cells per ml for hGH binding) Ringer-Hepes (KRH) buffer, pH 7.4/BSA (35 mg/ml)/0.27 were preincubated in duplicate (final volume, 0.5 ml per tube) mM glucose. After filtration
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