Association of (c)AMP-degrading glycosylphosphatidylinositol-anchored proteins with lipid droplets is induced by palmitate, H2O2 and the sulfonylurea drug, glimepiride, in rat adipocytes

Inhibition of lipolysis in rat adipocytes by palmitate, H2O2 and the antidiabetic sulfonylurea drug, glimepiride, has been demonstrated to rely on the upregulated conversion of cAMP to adenosine by enzymes associated with lipid droplets (LD) rather than on cAMP degradation by the insulin-stimulated microsomal phosphodiesterase 3B (Müller, G., Wied, S., Over, S., and Frick, W. (2008) Biochemistry 47, 1259-1273). Here these two enzymes were identified as the glycosylphosphatidylinositol (GPI)-anchored phosphodiesterase, Gce1, and the 5'-nucleotidase, CD73, on basis of the following findings: (i) Photoaffinity labeling with 8-N3-[32P]cAMP and [14C]5'-FSBA of LD from palmitate-, glucose oxidase- and glimepiride-treated, but not insulin-treated and basal, adipocytes led to the identification of 54-kDA cAMP- and 62-kDa AMP-binding proteins. (ii) The amphiphilic proteins were converted into hydrophilic versions and released from the LD by chemical or enzymic treatments specifically cleaving GPI anchors, but resistant toward carbonate extraction. (iii) The cAMP-to-adenosine conversion activity was depleted from the LD by adsorption to (c)AMP-Sepharose. (iv) cAMP-binding to LD was increased upon challenge of the adipocytes with palmitate, glimepiride or glucose oxidase and abrogated by phospholipase C digestion. (v) The 62-kDa AMP-binding protein was labeled with typical GPI anchor constituents and reacted with anti-CD73 antibodies. (vi) Inhibition of the bacterial phosphatidylinitosol-specific phospholipase C or GPI anchor biosynthesis blocked both agent-dependent upregulation and subsequent loss of cAMP-to-adenosine conversion associated with LD and inhibition of lipolysis. (vii) Gce1 and CD73 can be reconstituted into and exchanged between LD in vitro. These data suggest a novel insulin-independent antilipolytic mechanism engaged by palmitate, glimepiride and H2O2 in adipocytes which involves the upregulated expression of a GPI-anchored PDE and 5'-nucleotidase at LD. Their concerted action may ensure degradation of cAMP and inactivation of hormone-sensitive lipase in the vicinity of LD.     

Insulin-mimetic signaling by the sulfonylurea glimepiride and phosphoinositolglycans involves distinct mechanisms for redistribution of lipid raft components.

The insulin signal transduction cascade provides a number of sites downstream of the insulin receptor (IR) for cross-talk from other signaling pathways. Tyrosine phosphorylation of the IR substrates IRS-1/2 and metabolic insulin-mimetic activity in insulin-responsive cells can be provoked by soluble phosphoinositolglycans (PIG), which trigger redistribution from detergent-insoluble glycolipid-enriched raft domains (DIGs) to other areas of the plasma membrane and thereby activation of nonreceptor tyrosine kinases (NRTK) [Müller, G., Jung, C., Wied, S., Welte, S., Jordan, H., and Frick, W. (2001) Mol. Cell. Biol. 21, 4553-4567]. Here we describe that stimulation of glucose transport in isolated rat adipocytes by a different stimulus, the sulfonylurea glimepiride, is also based on IRS-1/2 tyrosine phosphorylation and downstream insulin-mimetic signaling involving activation of the NRTK, pp59(Lyn), and pp125(Fak), as well as tyrosine phosphoryation of the DIGs component caveolin. As is the case for PIG 41, glimepiride causes the concentration-dependent dissociation of pp59(Lyn) from caveolin and release of this NRTK and the glycosyl-phosphatidylinositol-anchored (GPI) proteins, Gce1 and 5'-nucleotidase, from total and anti-caveolin-immunoisolated DIGs. This results in their movement to detergent-insoluble raft domains of higher buoyant density (non-DIGs areas). IRS-1/2 tyrosine phosphorylation and glucose transport activation by both glimepiride and PIG are blocked by introduction into adipocytes of the caveolin scaffolding domain peptide which mimicks the negative effect of caveolin on pp59(Lyn) activity. Tyrosine phosphorylation of the NRTK, IRS-1/2, and caveolin as well as release of the NRTK and GPI proteins from DIGs and their redistribution into non-DIGs areas in response to PIG is also inhibited by treatment of intact adipocytes with either trypsin plus salt or N-ethylmaleimide (NEM). In contrast, the putative trypsin/salt/NEM-sensitive cell surface component (CIR) is not required for glimepiride-induced glucose transport, IRS-1/2 tyrosine phosphorylation, and redistribution of GPI proteins and NRTK. The data suggest that CIR is involved in concentrating signaling molecules at DIGs vs detergent-insoluble non-DIGs areas. These inhibitory interactions are relieved in response to putative physiological (PIG) or pharmacological (sulfonylurea) stimuli via different molecular mechanisms (dependent on or independent of CIR, respectively) thereby inducing IR-independent positive cross-talk to metabolic insulin signaling.     

Induced release of membrane vesicles from rat adipocytes containing glycosylphosphatidylinositol-anchored microdomain and lipid droplet signalling proteins

Synthesis and degradation of lipids in mammalian adipocytes are tightly and coordinatedly regulated by insulin, fatty acids, reactive oxygen species and drugs. Conversely, the lipogenic or lipolytic state of adipocytes is communicated to other tissues by the secretion of soluble adipocytokines. Here we report that insulin, palmitate, H₂O₂ and the antidiabetic sulfonylurea drug glimepiride induce the release of the typical lipid droplet (LD) protein, perilipin-A, as well as typical plasma membrane microdomain (DIGs) proteins, such as caveolin-1 and the glycosylphosphatidylinositol (GPI)-anchored proteins, Gce1 and CD73 from rat adipocytes. According to biochemical and morphological criteria these LD and GPI-proteins are embedded within two different types of phospholipid-containing membrane vesicles, collectively called adiposomes. Adiposome release was not found to be causally related to cell lysis or apoptosis. The interaction of Gce1 and CD73 with the adiposomes apparently depends on their intact GPI anchor. Pull-down of caveolin-1, perilipin-A and CD73 together with phospholipids (via binding to annexin-V) as well as mutually of caveolin-1 with CD73 or perilipin-A (via coimmunoprecipitation) argues for their colocalization within the same adiposome vesicle.Taken together, certain lipogenic and anti-lipolytic agents induce the specific release of a subset of LD and DIGs proteins, including certain GPI-proteins, in adiposomes from primary rat adipocytes. Given the (c)AMP-degrading activities of Gce1 and CD73 and LD-forming function of perilipin-A and caveolin-1, the physiological relevance of the release of adiposomes from adipocytes may rely on the intercellular transfer of lipogenic and anti-lipolytic information.     

Convergence and divergence of the signaling pathways for insulin and phosphoinositolglycans.

Phosphoinositolglycan molecules isolated from insulin-sensitive mammalian tissues have been demonstrated in numerous in vitro studies to exert partial insulin-mimetic activity on glucose and lipid metabolism in insulin-sensitive cells. However, their ill-defined structures, heterogeneous nature, and limited availability have prohibited the analysis of the underlying molecular mechanism. Phosphoinositolglycan-peptide (PIG-P) of defined and homogeneous structure prepared in large scale from the core glycan of a glycosyl-phosphatidylinositol-anchored membrane protein from Saccharomyces cerevisiae has recently been shown to stimulate glucose transport as well as a number of glucose-metabolizing enzymes and pathways to up to 90% (at 2 to 10 microns) of the maximal insulin effect in isolated rat adipocytes, cardiomyocytes, and diaphragms (G. Müller et al., 1997, Endocrinology 138: 3459-3476). Consequently, we used this PIG-P for the present study in which we compare its intracellular signaling with that of insulin. The activation of glucose transport by both PIG-P and insulin in isolated rat adipocytes and diaphragms was found to require stimulation of phosphatidylinositol (PI) 3-kinase but to be independent of functional p70S6kinase and mitogen-activated protein kinase. The increase in glycerol-3-phosphate acyltransferase activity in rat adipocytes in response to PIG-P and insulin was dependent on both PI 3-kinase and p70S6kinase. This suggest that the signaling pathways for PIG-P and insulin to glucose transport and metabolism converage at the level of PI 3-kinase. A component of the PIG-P signaling pathway located up-stream of PI 3-kinase was identified by desensitization of isolated rat adipocytes for PIG-P action by combined treatment with trypsin and NaCl under conditions that preserved cell viability and the insulin-mimetic activity of sodium vanadate but completely blunted the insulin response. Incubation of the cells with either trypsin or NaCl alone was ineffective. The desensitized adipocytes were reconstituted for stimulation of lipogenesis by PIG-P by addition of the concentrated trypsin/salt extract. The reconstituted adipocytes exhibited 65-75% of the maximal PIG-P response and similar EC50 values for PIG-P (2 to 5 microns) compared with control cells. A proteinaceous N-ethylmaleimide (NEM)-sensitive component contained in the trypsin/salt extract was demonstrated to bind in a functional manner to the adipocyte plasma membrane of desensitized adipocytes via bipolar interactions. An excess of trypsin/salt extract inhibited PIG-P action in untreated adipocytes in a competitive fashion compatible with a receptor function for PIG-P of this protein. The presence of the putative PIG-P receptor protein in detergent-insoluble complexes prepared from isolated rat adipocytes suggests that caveolae/detergent-insoluble complexes of the plasma membrane may play a role in insulin-mimetic signaling by PIG-P. Furthermore, treatment of isolated rat diaphragms and adipocytes with PIG-P as well as with other agents exerting partially insulin-mimetic activity, such as PI-specific phospholipase C (PLC) and the sulfonylurea glimepiride, triggered tyrosine phosphorylation of the caveolar marker protein caveolin, which was apparently correlated with stimulation of lipogenesis. Strikingly, in adipocytes subjected to combined trypsin/salt treatment, PIG-P, PI-specific PLC, and glimepiride failed completely to provoke insulin-mimetic effects. A working model is presented for a signaling pathway in insulin-sensitive cells used by PIG(-P) molecules which involves GPI structures, the trypsin/salt- and NEM-sensitive receptor protein for PIG-P, and additional proteins located in caveolae/detergent-insoluble complexes.     

Cholesterol depletion blocks redistribution of lipid raft components and insulin-mimetic signaling by glimepiride and phosphoinositolglycans in rat adipocytes

Glycosylphosphatidylinositol-anchored plasma membrane (GPI) proteins, such as Gce1, the dually acylated nonreceptor tyrosine kinases (NRTKs), such as pp59(Lyn), and the membrane protein, caveolin, together with cholesterol are typical components of detergent/carbonate-insoluble glycolipid-enriched raft domains (DIGs) in the plasma membrane of most eucaryotes. Previous studies demonstrated the dissociation from caveolin and concomitant redistribution from DIGs of Gce1 and pp59(Lyn) in rat adipocytes in response to four different insulin-mimetic stimuli, glimepiride, phosphoinositolglycans, caveolin-binding domain peptide, and trypsin/NaCl-treatment. We now characterized the structural basis for this dynamic of DIG components. MATERIALS AND METHODS: Carbonate extracts from purified plasma membranes of basal and stimulated adipocytes were analyzed by high-resolution sucrose gradient centrifugation. RESULTS: This process revealed the existence of two distinct species of detergent/carbonate-insoluble complexes floating at higher buoyant density and harboring lower amounts of cholesterol, caveolin, GPI proteins, and NRTKs (lcDIGs) compared to typical DIGs of high cholesterol content (hcDIGs). The four insulin-mimetic stimuli decreased by 40-70% and increased by 2.5- to 5-fold the amounts of GPI proteins and NRTKs at hcDIGs and lcDIGs, respectively. Cholesterol depletion of adipocytes per se by incubation with methyl-beta-cyclodextrin or cholesterol oxidase also caused translocation of GPI proteins and NRTKs from hcDIGs to lcDIGs and their release from caveolin in reversible fashion without concomitant induction of insulin-mimetic signaling. Cholesterol depletion, however, reduced by 50-60% the stimulus-induced translocation as well as dissociation from hcDIGs-associated caveolin of GPI proteins and NRTKs, activation of NRTKs as well as insulin-mimetic signaling and metabolic action. In contrast, insulin-mimetic signaling induced by vanadium compounds was not significantly diminished by cholesterol depletion. CONCLUSIONS: The data provide evidence that insulin-mimetic signaling in rat adipocytes provoked by glimepiride, phosphoinositolglycans, caveolin-binding domain peptide, and trypsin/NaCl-treatment, but not vanadium compounds, relies on the dynamics of DIGs-the translocation of certain GPI proteins and NRTKs from hcDIGs to lcDIGs mediated by a trypsin/NaCl-sensitive cell surface component. The resultant stimulation of pp59(Lyn) in course of its dissociation from caveolin and incorporation into lcDIGs in combination with an lcDIGs-independent signal seems to substitute for activation of the insulin receptor tyrosine kinase.     

Interaction of phosphatidylinositolglycan(-peptides) with plasma membrane lipid rafts triggers insulin-mimetic signaling in rat adipocytes

The phosphoinositolglycan(-peptide) (PIG-P) portion of glycosylphosphatidylinositol-anchored plasma membrane (GPI) proteins or synthetic PIG(-P) molecules interact with proteinaceous binding sites which are located in high-cholesterol-containing detergent/carbonate-insoluble glycolipid-enriched raft domains (hcDIGs) of the plasma membrane. In isolated rat adipocytes, PIG(-P) induce the redistribution of GPI proteins from hcDIGs to low-cholesterol-containing DIGs (lcDIGs) and concomitantly provoke insulin-mimetic signaling and metabolic action. Using a set of synthetic PIG(-P) derivatives we demonstrate here that their specific binding to hcDIGs and their insulin-mimetic signaling/metabolic activity strictly correlate with respect to (i) translocation of the GPI proteins, Gce1 and 5(')-nucleotidase, from hcDIGs to lcDIGs, (ii) dissociation of the nonreceptor tyrosine kinase, pp59(Lyn), from caveolin residing at hcDIGs, (iii) translocation of pp59(Lyn) from hcDIGs to lcDIGs, (iv) activation of pp59(Lyn), (v) tyrosine phosphorylation of insulin receptor substrate proteins-1/2, and finally (vi) stimulation of glucose transport. The natural PIG(-P) derived from the carboxy-terminal tripeptide of Gce1, YCN-PIG, exhibits the highest potency followed by a combination of the separate peptidylethanolamidyl and PIG constituents. We conclude that efficient positive cross-talk of PIG(-P) to the insulin signaling cascade requires their interaction with hcDIGs. We suggest that PIG(-P) thereby displace GPI proteins from binding to hcDIGs leading to their release from hcDIGs for lateral movement to lcDIGs which initiates signal transduction from DIGs via caveolin and pp59(Lyn) to the insulin receptor substrate proteins of the insulin signaling pathway.     

Mechanism of insulin resistance in the post receptor events in sheep: 3-O-methylglucose transport in ovine adipocytes

A severe resistance to the stimulatory action of insulin on glucose metabolism has been shown in ruminant adipose tissue or isolated adipocytes as compared to that of rats. To elucidate the mechanism of insulin resistance in ruminants, we measured the stimulatory effect of insulin on 3-O-methylgulose transport and on intracellular glucose metabolism in isolated adipocytes from sheep and rats. At a glucose concentration (0.1 mM) where transport is thought to be rate-limiting for metabolism, lipogenesis from [U-14C]glucose by ovine adipocytes was markedly less than by rat adipocytes in both the basal state and at all insulin concentrations. The responsiveness to insulin assessed by percent increase above basal was reduced to about 15% of that in rat adipocytes, but the insulin sensitivity was similar, because the insulin concentration giving half-maximal stimulation, ED50, did not differ significantly between ovine and rat adipocytes. The maximal insulin-stimulated 3-O-methylglucose transport in ovine adipocytes per cell was less than 20% of that in rat adipocytes, with a significant lowering in basal rates of transport. However, when data was expressed per 3-O-methylglucose equilibrium space no significant differences were found between ovine and rat in the basal transport rates, but a lowered ability of insulin to stimulate glucose transport was still seen in ovine adipocytes. The dose-response curve for glucose transport was slightly shifted to the right in ovine adipocytes compared to rat adipocytes, indicating a small decrease in insulin sensitivity. The decrease in glucose transport was due to 60% reduction in the maximum velocity in the insulin--stimulated state, with no change in the Km.     

Glucose-induced sequential processing of a glycosyl-phosphatidylinositol-anchored ectoprotein in Saccharomyces cerevisiae.

Transfer of spheroplasts from the yeast Saccharomyces cerevisiae to glucose leads to the activation of an endogenous (glycosyl)-phosphatidylinositol-specific phospholipase C ([G]PI-PLC), which cleaves the anchor of at least one glycosyl-phosphatidylinositol (GPI)-anchored protein, the cyclic AMP (cAMP)-binding ectoprotein Gce1p (G. Müller and W. Bandlow, J. Cell Biol. 122:325-336, 1993). Analyses of the turnover of two constituents of the anchor, myo-inositol and ethanolamine, relative to the protein label as well as separation of the two differently processed versions of Gce1p by isoelectric focusing in spheroplasts demonstrate the glucose-induced conversion of amphiphilic Gce1p first into a lipolytically cleaved hydrophilic intermediate, which is then processed into another hydrophilic version lacking both myo-inositol and ethanolamine. When incubated with unlabeled spheroplasts, the lipolytically cleaved intermediate prepared in vitro is converted into the version lacking all anchor constituents, whereby the anchor glycan is apparently removed as a whole. The secondary cleavage ensues independently of the carbon source, attributing the key role in glucose-induced anchor processing to the endogenous (G)PI-PLC. The secondary processing of the lipolytically cleaved intermediate of Gce1p at the plasma membrane is correlated with the emergence of a covalently linked high-molecular-weight form of a cAMP-binding protein at the cell wall. This protein lacks anchor components, and its protein moiety appears to be identical with double-processed Gce1p detectable at the plasma membrane in spheroplasts. The data suggest that glucose-induced double processing of GPI anchors represents part of a mechanism of regulated cell wall expression of proteins in yeast cells.     

Control of lipid storage and cell size between adipocytes by vesicle-associated glycosylphosphatidylinositol-anchored proteins

Adipose tissue mass in mammals is expanding by increasing the average cell volume as well as the total number of the adipocytes. Up-regulation of lipid storage in fully differentiated adipocytes resulting in their enlargement is well documented and thought to be a critical mechanism for the expansion of adipose tissue depots during the growth of both lean and obese animals and human beings. A novel molecular mechanism for the regulation of lipid storage and cell size in rat adipocytes has recently been elucidated for the physiological stimuli, palmitate and hydrogen peroxide, the anti-diabetic sulfonylurea drug, glimepiride, and insulin-mimetic phosphoinositolglycans. It encompasses (i) the release of small vesicles, so-called adiposomes, harbouring the glycosylphosphatidylinositol-anchored (c)AMP-degrading phosphodiesterase Gce1 and 5'-nuceotidase CD73 from large donor adipocytes, (ii) the transfer of the adiposomes and their interaction with detergent-insoluble glycolipid-enriched microdomains of the plasma membrane of small acceptor adipocytes, (iii) the translocation of Gce1 and CD73 from the adiposomes to the intracellular lipid droplets of the acceptor adipocytes and (iv) the degradation of (c)AMP at the lipid droplet surface zone by Gce1 and CD73 in the acceptor adipocytes. In concert, this sequence of events leads to up-regulation of esterification of fatty acids into triacylglycerol and down-regulation of their release from triacylglycerol. This apparent mechanism for shifting the triacylglycerol burden from large to small adipocytes may provide novel strategies for the therapy of metabolic diseases, such as type 2 diabetes and obesity.     

Redistribution of Glycolipid Raft Domain Components Induces Insulin-Mimetic Signaling in Rat Adipocytes

Caveolae and caveolin-containing detergent-insoluble glycolipid-enriched rafts (DIG) have been implicated to function as plasma membrane microcompartments or domains for the preassembly of signaling complexes, keeping them in the basal inactive state. So far, only limited in vivo evidence is available for the regulation of the interaction between caveolae-DIG and signaling components in response to extracellular stimuli. Here, we demonstrate that in isolated rat adipocytes, synthetic intracellular caveolin binding domain (CBD) peptide derived from caveolin-associated pp59(Lyn) (10 to 100 microM) or exogenous phosphoinositolglycan derived from glycosyl-phosphatidylinositol (GPI) membrane protein anchor (PIG; 1 to 10 microM) triggers the concentration-dependent release of caveolar components and the GPI-anchored protein Gce1, as well as the nonreceptor tyrosine kinases pp59(Lyn) and pp125(Fak), from interaction with caveolin (up to 45 to 85%). This dissociation, which parallels redistribution of the components from DIG to non-DIG areas of the adipocyte plasma membrane (up to 30 to 75%), is accompanied by tyrosine phosphorylation and activation of pp59(Lyn) and pp125(Fak) (up to 8- and 11-fold) but not of the insulin receptor. This correlates well to increased tyrosine phosphorylation of caveolin and the insulin receptor substrate protein 1 (up to 6- and 15-fold), as well as elevated phosphatidylinositol-3' kinase activity and glucose transport (to up to 7- and 13-fold). Insulin-mimetic signaling by both CBD peptide and PIG as well as redistribution induced by CBD peptide, but not by PIG, was blocked by synthetic intracellular caveolin scaffolding domain (CSD) peptide. These data suggest that in adipocytes a subset of signaling components is concentrated at caveolae-DIG via the interaction between their CBD and the CSD of caveolin. These inhibitory interactions are relieved by PIG. Thus, caveolae-DIG may operate as signalosomes for insulin-independent positive cross talk to metabolic insulin signaling downstream of the insulin receptor based on redistribution and accompanying activation of nonreceptor tyrosine kinases.     

Stimulation of glucose transport in rat adipocytes by insulin, adenosine, nicotinic acid and hydrogen peroxide. Role of adenosine 3′:5′-cyclic monophosphate

Glucose transport into adipocytes of the rat was measured by monitoring the conversion of [1-(14)C]glucose into (14)CO(2). Glucose transport was made rate-limiting by increasing the flux through the pentose phosphate pathway with phenazine methosulphate, an agent that rapidly reoxidizes NADPH. Under these conditions, the observed rate of glucose disappearance from the incubation medium was about 20% higher than the rate of conversion of the C-1 of glucose into (14)CO(2). Apparent rates of glucose transport were significantly increased by insulin, H(2)O(2), adenosine and nicotinic acid. Stimulation of the apparent rate of glucose transport by insulin was dependent on adipocyte concentration, the hormone being most effective at relatively high cell concentrations. Adenosine and nicotinic acid further enhanced the maximum stimulation of glucose transport by insulin. Potentiation of insulin action by adenosine was more pronounced at lower cell concentrations. At relatively high cell concentrations the stimulatory action of insulin was markedly decreased by adenosine deaminase. Stimulation of apparent rates of glucose transport by the compounds noted above were antagonized by agents that increased intracellular cyclic AMP concentrations (theophylline and isoprenaline) and by dibutyryl cyclic AMP. Intracellular concentrations of cyclic AMP were significantly lowered when adipocytes were incubated with insulin, H(2)O(2), adenosine or nicotinic acid. These effects were observed under basal conditions or when intracellular cyclic AMP concentrations were elevated by theophylline or isoprenaline. On the basis of the above data, we suggest that insulin, H(2)O(2), adenosine and nicotinic acid may all stimulate glucose transport in rat adipocytes by lowering the intracellular cyclic AMP concentration. These data therefore support the hypothesis that cyclic AMP inhibits glucose transport in rat adipocytes.     

Inhibition of lipolysis by palmitate, H2O2 and the sulfonylurea drug, glimepiride, in rat adipocytes depends on cAMP degradation by lipid droplets

The release of fatty acids and glycerol from lipid droplets (LD) of mammalian adipose cells is tightly regulated by a number of counterregulatory signals and negative feedback mechanisms. In humans unrestrained lipolysis contributes to the pathogenesis of obesity and type II diabetes. In order to identify novel targets for the pharmacological interference with lipolysis, the molecular mechanisms of four antilipolytic agents were compared in isolated rat adipocytes. Incubation of the adipocytes with insulin, palmitate, glucose oxidase (for the generation of H2O2) and the antidiabetic sulfonylurea drug, glimepiride, reduced adenylyl cyclase-dependent, but not dibutyryl-cAMP-induced lipolysis as well as the translocation of hormone-sensitive lipase and the LD-associated protein, perilipin-A, to and from LD, respectively. The antilipolytic activity of palmitate, H2O2 and glimepiride rather than that of insulin was dependent on rolipram-sensitive but cilostamide-insensitive phosphodiesterase (PDE) but was not associated with detectable downregulation of total cytosolic cAMP and insulin signaling via phosphatidylinositol-3 kinase and protein kinase B. LD from adipocytes treated with palmitate, H2O2 and glimepiride were capable of converting cAMP to adenosine in vitro, which was hardly observed with those from basal cells. Conversion of cAMP to adenosine was blocked by rolipram and the 5'-nucleotidase inhibitor, AMPCP. Immunoblotting analysis revealed a limited salt-sensitive association with LD of some of the PDE isoforms currently known to be expressed in rat adipocytes. In contrast, the cAMP-to-adenosine converting activity was stripped off the LD by bacterial phosphatidylinositol-specific phospholipase C. These findings emphasize the importance of the compartmentalization of cAMP signaling for the regulation of lipolysis in adipocytes, in general, and of the involvement of LD-associated proteins for cAMP degradation, in particular.     

Effects of insulin, triiodothyronine and fat soluble vitamins on adipocyte differentiation and LPL gene expression in the stromal-vascular cells of red sea bream, Pagrus major

Various kinds of hormones including insulin, triiodothyronine (T(3)) and fat-soluble vitamins have been proposed as mediators of adipocyte differentiation in mammals. To investigate the factors which are responsible for fish adipocyte differentiation, we developed a serum-free culture system of stromal-vascular cells of red sea bream adipose tissue and examined the effects of bovine insulin, T(3), and fat-soluble vitamins (all-trans retinoic acid, retinyl acetate and 1,25-dihydroxyvitamin D(3)) on the differentiation-linked expression of the lipoprotein lipase (LPL) gene. As assessed by the increase in LPL gene expression after 3 day cultivation, like in mammalian adipocytes, insulin enhanced the adipocyte differentiation in a concentration-dependent manner. During 2 week cultivation, bovine insulin promoted lipid accumulation in differentiating adipocytes concentration-dependently until the terminal differentiation. These results indicate that the differentiation of fish adipocytes is inducible by insulin alone. T(3) alone had no effect but enhanced the differentiation-linked LPL gene expression in the presence of insulin. Fat-soluble vitamins, unlike in mammalian adipocytes, did not show any significant effects. The method developed in this study should be of interest for the characterization of factors involved in fish adipocyte differentiation.     

Inositol glycan mimics the action of insulin on glucose utilization in rat adipocytes

Some of the actions of insulin may be mediated by the intracellular generation of an inositol phosphate glycan that modulates the activities of certain metabolic enzymes. The actions of this molecule were evaluated on glucose utilization in intact rat adipocytes. The inositol glycan led to the dose-dependent stimulation of glucose oxidation and lipogenesis. The extent of stimulation was similar to that observed for insulin. The stimulation of lipogenesis was seen only at high concentrations of glucose, suggesting regulation of processes distal to glucose uptake. The effects of the inositol glycan on intact adipocytes were specifically attenuated with inositol monophosphate in a dose dependent manner. These results further support a role for this substance as a second messenger for some of the actions of insulin, and indicate that the cellular uptake of the inositol glycan may occur by a specific transport system.     

Insulin-like signaling in yeast: modulation of protein phosphatase 2A, protein kinase A, cAMP-specific phosphodiesterase, and glycosyl-phosphatidylinositol-specific phospholipase C activities

Previously, we have described significant effects of human insulin on glucose metabolism in the yeast Saccharomyces cerevisiae under conditions of growth limitation. These regulations apparently rely on a transmembrane receptor capable of binding human insulin and responding by tyrosine/serine phosphorylation of a specific set of polypeptides [Müller, G., Rouveyre, N., Crecelius, A., and Bandlow, W. (1998) Biochemistry 37, 8683-8695; Müller, G., Rouveyre, N., Upshon, C., Gross, E., and Bandlow, W. (1998) Biochemistry 37, 8696-8704; Müller, G., Rouveyre, N., Upshon, C., and Bandlow, W. (1998) Biochemistry 37, 8705-8713]. To characterize the molecular link between the initial steps in insulin-like signaling in yeast and the changes in the activities of glycogen synthase and glycogen phosphorylase, we examined here the effects of human insulin on a set of key regulatory enzymes of glycogen metabolism, protein phosphatase 2A (PP2A), cAMP-specific phosphodiesterase (cAMP-PDE), and protein kinase A (PKA). PP2A was activated about 2-fold by insulin in spheroplasts and in intact cells, whereas the fraction of active PKA was significantly reduced in a cAMP-independent manner as well as through a subsequent up to 3-fold increase in particulate cAMP-PDE activity accompanied by a 50% decrease in cytosolic cAMP levels. In addition, glycosyl-phosphatidylinositol-specific phospholipase C (GPI-PLC), which in isolated rat adipocytes is activated by insulin, was stimulated to up to 5-fold by glucose and 10-fold by glucose plus insulin in both yeast spheroplasts and intact cells leading to a concentration-dependent leftward shift of the glucose-response curve for activation of the GPI-PLC. GPI-PLC was most pronouncedly stimulated by authentic human insulin compared to various insulin analogues and insulin-like growth factor I. In addition to lipolytic cleavage by GPI-PLC, the GPI anchor of the cAMP-binding ectoprotein, Gce1p, was secondarily processed by a rapid proteolytic event. As the GPI-PLC reaction is rate limiting, the efficiency of the two-step anchor cleavage was significantly increased when insulin was present together with glucose as compared to glucose alone. The insulin concentrations effective in modulating PP2A, PKA, cAMP-PDE, and GPI-PLC activities correlate well with those required for half-saturation of the specific binding sites as well as for stimulation of protein phosphorylation and glycogen accumulation. The data suggest that mammalian insulin-sensitive cells and yeast share (part of) the key regulatory mechanism (consisting of PP2A, PKA, cAMP-PDE, and GPI-PLC) involved in the transduction of the insulin signal from the respective receptor systems to glycogen synthase and phosphorylase.     

Phospholipase C mimics insulin action on pyruvate dehydrogenase and insulin mediator generation but not glucose transport or utilization

This study investigated the extent to which a purified phosphatidylinositol-specific and a commercial non-specific phospholipase C mimicked acute insulin action in rat adipocytes. The enzymes mimicked insulin stimulation of pyruvate dehydrogenase (PDH) and breakdown of a glycophospholipid proposed as a precursor for an intracellular mediator of insulin action, but were much less effective in stimulating glucose transport and utilization. These observations corroborate recent suggestions that insulin may activate a phospholipase C to generate a mediator that can account for insulin activation of PDH from a mediator precursor with a phosphatidylinositol anchor. This mediator precursor is probably an outer membrane component since effects were obtained with intact cells. It is unlikely that this mechanism accounts fully for insulin action since phosphatidylinositol-specific and commercial phospholipase C stimulation of glucose transport was significantly less than that elicited by insulin.     

Insulin increases the synthetic rate and messenger RNA level of lipoprotein lipase in isolated rat adipocytes

Lipoprotein lipase (LPL) is the enzyme responsible for hydrolysis of circulating triglyceride-rich lipoproteins and is important for storage of adipocyte lipid. To study the regulation of LPL synthetic rate in adipose tissue, primary cultures of isolated rat adipocytes were pulse-labeled with [35S]methionine, and LPL was immunoprecipitated with an LPL-specific antibody. A pulse-chase experiment identified the cellular and secreted forms of LPL as a 55-57-kDa protein. In the presence of heparin, there was a large increase in secretion of newly synthesized LPL from the cells, although heparin did not stimulate cellular LPL synthetic rate. When cells were exposed to insulin for 2 h, pulse-labeling revealed that insulin stimulated a maximal dose-related increase in LPL synthetic rate of 300% of control. This increase in LPL synthetic rate was observed after an exposure to insulin for as little as 60 min and was accompanied by only a 10-25% increase in total protein synthesis. In addition, insulin had no effect on the turnover of intracellular LPL. Using a cDNA probe for LPL, insulin induced a 2-fold increase in the LPL mRNA. Thus, insulin stimulated an increase in specific LPL mRNA in isolated rat adipocytes. This increase in LPL mRNA then leads to an increase in the synthetic rate of the LPL protein.     

Okadaic acid inhibits insulin-induced glucose transport in fetal brown adipocytes in an Akt-independent and protein kinase C zeta-dependent manner

In the present study we have investigated the effect of increased serine/threonine phosphorylation of insulin receptor substrates-1 and -2 (IRS-1 and IRS-2) by okadaic acid pretreatment on brown adipocyte insulin signalling leading to glucose transport, an important metabolic effect of insulin in brown adipose tissue. Okadaic acid pretreatment before insulin stimulation decreased IRS-1 and IRS-2 tyrosine phosphorylation in parallel to a decrease in their sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility. IRS-1/IRS-2-associated p85alpha and phosphatidylinositol (PI) 3-kinase enzymatic activity were partly reduced in brown adipocytes pretreated with okadaic acid upon stimulation with insulin. Furthermore, insulin-induced glucose uptake was totally abolished by the inhibitor in parallel with a total inhibition of insulin-induced protein kinase C (PKC) zeta activity. However, activation of Akt/PKB or p70 S6 kinase (p70(s6k)) by insulin remained unaltered. Our results suggest that downstream of PI 3-kinase, insulin signalling diverges into at least two independent pathways through Akt/PKB and PKC zeta, the PKC zeta pathway contributing to glucose transport induced by insulin in fetal brown adipocytes.