CB1 Receptor-Dependent and Independent Induction of Lipolysis in Primary Rat Adipocytes by the Inverse Agonist Rimonabant (SR141716A).

(1) Background: Acute administration of the cannabinoid receptor 1 (CB1R) inverse agonist Rimonabant (SR141716A) to fed Wistar rats was shown to elicit a rapid and short-lasting elevation of serum free fatty acids. (2) Methods: The effect of Rimonabant on lipolysis in isolated primary rat adipocytes was studied to raise the possibility for direct mechanisms not involving the (hypothalamic) CB1R. (3) Results: Incubation of these cells with Rimonabant-stimulated lipolysis to up to 25% of the maximal isoproterenol effect, which was based on both CB1R-dependent and independent mechanisms. The CB1R-dependent one was already effective at Rimonabant concentrations of less than 1 µM and after short-term incubation, partially additive to ß-adrenergic agonists and blocked by insulin and, in part, by adenosine deaminase, but not by propranolol. It was accompanied by protein kinase A (PKA)-mediated association of hormone-sensitive lipase (HSL) with lipid droplets (LD) and dissociation of perilipin-1 from LD. The CB1R-independent stimulation of lipolysis was observed only at Rimonabant concentrations above 1 µM and after long-term incubation and was not affected by insulin. It was recapitulated by a cell-free system reconstituted with rat adipocyte LD and HSL. Rimonabant-induced cell-free lipolysis was not affected by PKA-mediated phosphorylation of LD and HSL, but abrogated by phospholipase digestion or emulsification of the LD. Furthermore, LD isolated from adipocytes and then treated with Rimonabant (>1 µM) were more efficient substrates for exogenously added HSL compared to control LD. The CB1R-independent lipolysis was also demonstrated in primary adipocytes from fed rats which had been treated with a single dose of Rimonabant (30 mg/kg). (4) Conclusions: These data argue for interaction of Rimonabant (at high concentrations) with both the LD surface and the CB1R of primary rat adipocytes, each leading to increased access of HSL to LD in phosphorylation-independent and dependent fashion, respectively. Both mechanisms may lead to direct and acute stimulation of lipolysis at peripheral tissues upon Rimonabant administration and represent targets for future obesity therapy which do not encompass the hypothalamic CB1R.

Upregulation of lipid synthesis in small rat adipocytes by microvesicle-associated CD73 from large adipocytes.

Filling-up lipid stores is critical for size increase of mammalian adipocytes. The glycosylphosphatidylinositol (GPI)-anchored protein, CD73, is released from adipocytes into microvesicles in response to the lipogenic stimuli, palmitate, the antidiabetic sulfonylurea drug glimepiride, phosphoinositolglycans (PIG), and H(2)O(2). Upon incubation of microvesicles with adipocytes, CD73 is translocated to cytoplasmic lipid droplets (LD) and esterification is upregulated. The role of CD73-harboring microvesicles in coordinating esterification between differently sized adipocytes was studied here. Populations consisting of either small or large or of both small and large isolated rat adipocytes as well as native adipose tissue pieces from young and old rats were incubated with or depleted of endogenous microvesicles and analyzed for translocation of CD73 and esterification in response to the lipogenic stimuli. Large adipocytes exhibited higher and lower efficacy in releasing CD73 into microvesicles and in translocating CD73 to LD, respectively, compared to small adipocytes. Populations consisting of both small and large adipocytes were more active in esterification in response to the lipogenic stimuli than either small or large adipocytes. With both adipocytes and adipose tissue pieces from young rats esterification stimulation by the lipogenic stimuli was abrogated by depletion of CD73-harboring microvesicles from the incubation medium and interstitial spaces, respectively. In conclusion, stimulus-induced lipid synthesis between differently sized adipocytes is controlled by the release of microvesicle-associated CD73 from large cells and its subsequent translocation to LD of small cells. This information transfer via microvesicles harboring GPI-anchored proteins may shift the burden of triacylglycerol storage from large to small adipocytes.

Microvesicles released from rat adipocytes and harboring glycosylphosphatidylinositol-anchored proteins transfer RNA stimulating lipid synthesis.

Small microvesicles, such as microparticles and exosomes, have been demonstrated to transfer proteins and nucleic acids from a variety of donor to acceptor cells with corresponding (patho)physiological consequences. Recently the in vitro transfer of glycosylphosphatidylinositol (GPI)-anchored proteins from microvesicles released from large rat adipocytes to intracellular lipid droplets (LDs) of small adipocytes has been shown to be upregulated by physiological (palmitate, H(2)O(2)) and pharmacological (anti-diabetic sulfonylurea drug glimepiride) stimuli and to increase the esterification into as well as to reduce the release of fatty acids from triacylglycerol. Here microvesicles derived from (preferentially large) rat adipocytes or plasma and harboring the GPI-anchored proteins, Gce1 and CD73, were demonstrated to contain specific transcripts and microRNAs that are both transferred into and expressed in acceptor adipocytes and are involved in the upregulation of lipogenesis and cell size. The transferred transcripts were specific for fatty acid esterification (glycerol-3-phosphate acyltransferase-3, diacylglycerol acyltransferase-2), lipid droplet biogenesis (FSP27, caveolin-1) and adipokines (leptin, adiponectin). The transfer and lipogenic activity were more efficient for small rather than large acceptor adipocytes and significantly upregulated by palmitate, glimepiride and H(2)O(2). Together the data suggest that microvesicles released from large adipocytes stimulate lipid storage in small adipocytes by mediating horizontal transfer of lipogenic information which is encoded by relevant (micro)RNA and GPI-anchored protein species. Paracrine and endocrine regulation of lipid storage and, in parallel, cell size of white adipocytes by specific (micro)RNAs in GPI-anchored protein-harboring microvesicles may represent a novel target for interference with metabolic diseases, such as obesity and metabolic syndrome.

Glycosylphosphatidylinositol-anchored proteins coordinate lipolysis inhibition between large and small adipocytes.

In response to palmitate, the antidiabetic sulfonylurea drug glimepiride, phosphoinositoglycans, or H(2)O(2), the release of the glycosylphosphatidylinositol-anchored and cyclic adenosine monophosphate-degrading phosphodiesterase Gce1 from adipocytes into small vesicles (adiposomes) and its translocation from adiposomes to cytoplasmic lipid droplets (LD) of adipocytes have been reported. Here the role of Gce1-harboring adiposomes in coordinating lipolysis between differently sized adipocytes was studied. Separate or mixed populations of isolated epididymal rat adipocytes of small and large size and native adipose tissue pieces from young and old rats were incubated with exogenous adiposomes or depleted of endogenous adiposomes and then analyzed for translocation of Gce1 and lipolysis in response to above antilipolytic stimuli. Large compared with small adipocytes are more efficient in releasing Gce1 into adiposomes but less efficient in translocating Gce1 from adiposomes to LDs. Maximal lipolysis inhibition by above antilipolytic stimuli, but not by insulin, was observed with mixed populations of small and large adipocytes (1:1 to 1:2) rather than with separate populations. In mixed adipocyte populations and adipose tissue pieces from young, but not old, rats, lipolysis inhibition by above antilipolytic stimuli, but not by insulin, was dependent on the function of Gce1-harboring adiposomes. Inhibition of lipolysis in rat adipose tissue in response to palmitate, glimepiride, and H(2)O(2) is coordinated via the release of adiposome-associated and glycosylphosphatidylinositol-anchored Gce1 from large "donor" adipocytes and their subsequent translocation to the LDs of small "acceptor" adipocytes. This transfer of antilipolytic information may be of pathophysiologic relevance.

Lipid storage in large and small rat adipocytes by vesicle-associated glycosylphosphatidylinositol-anchored proteins.

Adipose tissue mass in mammals expands by increasing the average cell volume and/or total number of the adipocytes. Upregulated 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 was recently elucidated for the physiological stimuli, palmitate and H(2)O(2), and the antidiabetic sulfonylurea drug, glimepiride. It encompasses (1) the release of small vesicles, so-called adiposomes, harboring the glycosylphosphatidylinositol -anchored (c)AMP-degrading phosphodiesterase Gce1 and 5'-nucleotidase CD73 from donor adipocytes, (2) the transfer of the adiposomes and their interaction with detergent-insoluble glycolipid-enriched microdomains of the plasma membrane of acceptor adipocytes, (3) the translocation of Gce1 and CD73 from the adiposomes to the intracellular lipid droplets of the acceptor adipocytes, and (4) the degradation of (c)AMP at the lipid droplet surface zone by Gce1 and CD73 in the acceptor adipocytes, leading to the upregulation of the esterification of fatty acids into triacylglycerol s and the downregulation of their release from triacylglycerols. This mechanism may provide novel strategies for the therapy of metabolic diseases, such as type 2 diabetes and obesity.

Synthetic phosphoinositolglycans regulate lipid metabolism between rat adipocytes via release of GPI-protein-harbouring adiposomes.

A novel molecular mechanism for the regulation of lipid metabolism by palmitate, H2O2 and the anti-diabetic sulfonylurea drug, glimepiride, in rat adipocytes was recently elucidated. It encompasses the translocation of the glycosylphosphatidylinositol-anchored (GPI-) and (c)AMP degrading enzymes Gce1 and CD73 from detergent-insoluble glycolipid-enriched microdomains of the plasma membrane (DIGs) to intracellular lipid droplets (LD), the incorporation of Gce1 and CD73 into vesicles (adiposomes) which are then released from donor adipocytes and finally the transfer of Gce1 and CD73 from the adiposomes to acceptor adipocytes, where they degrade (c)AMP at the LD surface. Here the stimulation of esterification and inhibition of lipolysis by synthetic phosphoinositolglycans (PIGs), such as PIG37, which represents the glycan component of the GPI anchor, are shown to be correlated to translocation from DIGs to LD and release into adiposomes of Gce1 and CD73. PIG37 actions were blocked upon disruption of DIGs, inactivation of PIG receptor and removal of adiposomes from the incubation medium as was true for those induced by palmitate, H2O2 or glimepiride. In contrast, only the latter actions were dependent on the GPI-specific phospholipase C (GPI-PLC), which may generate PIGs, or on exogenous PIG37 in case of inhibited GPI-PLC. At submaximal concentrations PIG37 and palmitate, H2O2 or glimepiride acted in synergistic fashion. These data suggest that PIGs provoke the transfer of GPI-proteins from DIGs via LD and adiposomes of donor adipocytes to acceptor adipocytes and thereby mediate the regulation of lipid metabolism by palmitate, H2O2 and glimepiride between adipocytes.

Novel glimepiride derivatives with potential as double-edged swords against type II diabetes.

Sulphonylurea drugs have been widely used in the safe and efficacous therapy of type II diabetes during the past five decades. They lower blood glucose predominantly via the stimulation of insulin release from pancreatic beta-cells. However, a moderate insulin-independent regulation of fatty acid esterification and release in adipose tissue cells has been reported for certain sulphonylureas, in particular for glimepiride. On basis of the known pleiotropic pathogenesis of type II diabetes with a combination of beta-cell failure and peripheral, including adipocyte, insulin resistance, anti-diabetic drugs exerting both insulin releasing- and fatty acid-metabolizing activities in a more balanced and potent fashion may be of advantage. However, the completely different molecular mechanisms underlying the insulin-releasing and fatty acid-metabolizing activities, as have been delineated so far for glimepiride, may hamper their optimization within a single sulphonylurea molecule. By analyzing conventional sulphonylureas and novel glimepiride derivatives for their activities at the primary targets and downstream steps in both beta-cells and adipocytes in vitro we demonstrate here that the insulin-releasing and fatty acid-metabolizing activities are critically dependent on both overlapping and independent structural determinants. These were unravelled by the parallel losses of these two activities in a subset of glimepiride derivatives and the impairment in the insulin-releasing activity in parallel with elevation in the fatty acid-metabolizing activity in a different subset. Together these findings may provide a basis for the design of novel sulphonylureas with blood glucose-lowering activity relying on less pronounced stimulation of insulin release from pancreatic beta-cells and more pronounced insulin-independent stimulation of esterification as well as inhibition of release of fatty acids by adipocytes than provoked by the sulphonylureas currently used in therapy.

Inhibition of lipolysis by adiposomes containing glycosylphosphatidylinositol-anchored Gce1 protein in rat adipocytes.

Small membrane vesicles released from large adipocytes and harbouring the glycosylphosphatidylinositol-anchored (GPI-) AMP-degrading protein CD73 have previously been demonstrated to stimulate the signal-induced esterification of free fatty acids into neutral lipids suggesting a role of these so-called adiposomes (ADIP) in the paracrine regulation of lipid metabolism in the adipose tissue. Here the involvement of another constituent GPI-protein of ADIP, the cAMP-degrading protein Gce1 in the signal-induced inhibition of lipolysis was investigated in primary rat adipocytes. Incubation of small, and to a lower degree, large adipocytes with ADIP inhibited lipolysis and increased its sensitivity toward inhibition by H(2)O(2), the anti-diabetic drug glimepiride and palmitate. This was accompanied by the transfer of Gce1 from the ADIP to detergent-insoluble glycolipid-enriched plasma membrane microdomains (DIGs) and its subsequent translocation to cytoplasmic lipid droplets (LD) of the acceptor adipocytes. The translocation from DIGs to LD rather than the transfer from ADIP to DIGs of Gce1 was stimulated by H(2)O(2) > glimepiride > palmitate. Both transfer and translocation led to salt- and carbonate-resistant association of Gce1 with DIGs and LD, respectively, and relied on the structural integrity of the DIGs and GPI anchor of Gce1. In conclusion, the trafficking of GPI-proteins from ADIP of donor adipocytes via DIGs to LD of acceptor adipocytes mediates paracrine regulation of lipolysis within adipose tissue.

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, H(2)O(2) 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.

Coordinated regulation of esterification and lipolysis by palmitate, H2O2 and the anti-diabetic sulfonylurea drug, glimepiride, in rat adipocytes.

Inhibition of lipolysis by palmitate, H2O2 and the anti-diabetic sulfonylurea drug, glimepiride, in isolated rat adipocytes has previously been shown to rely on the degradation of cyclic adenosine monophosphate by the phosphodiesterase, Gce1, and the 5'-nucleotidase, CD73. These glycosylphosphatidylinositol (GPI)-anchored proteins are translocated from plasma membrane lipid rafts to intracellular lipid droplets upon H2O2-induced activation of a GPI-specific phospholipase C (GPI-PLC) in response to palmitate and glimepiride in intact adipocytes and, as demonstrated here, in cell-free systems as well. The same agents are also known to stimulate the incorporation of fatty acids into triacylglycerol. Here the involvement of H2O2 production, GPI-PLC activation and translocation of Gce1 and CD73 in the agent-induced esterification and accompanying lipid droplet formation was tested in rat adipocytes using relevant inhibitors. The results demonstrate that upregulation of the esterification and accumulation of triacylglycerol by glimepiride depends on the sequential H2O2-induced GPI-PLC activation and GPI-protein translocation as does inhibition of lipolysis. In contrast, stimulation of the esterification and triacylglycerol accumulation by palmitate relies on insulin-independent tyrosine phosphorylation and thus differs from its anti-lipolytic mechanism. As expected, insulin regulates lipid metabolism via typical insulin signalling independent of H2O2 production, GPI-PLC activation and GPI-protein translocation, albeit these processes are moderately stimulated by insulin. In conclusion, triacylglycerol and lipid droplet formation in response to glimepiride and H2O2 may involve the hydrolysis of cyclic adenosine monophosphate by lipid droplet-associated Gce1 and CD73 which may regulate lipid droplet-associated triacylglycerol-synthesizing and hydrolyzing enzymes in coordinated and inverse fashion.

Translocation of glycosylphosphatidylinositol-anchored proteins from plasma membrane microdomains to lipid droplets in rat adipocytes is induced by palmitate, H2O2, and the sulfonylurea drug glimepiride.

Inhibition of lipolysis by palmitate, H(2)O(2), and the antidiabetic sulfonylurea drug, glimepiride, in rat adipocytes has been shown previously to rely on the concerted degradation of cAMP by the glycosylphosphatidylinositol (GPI)-anchored phosphodiesterase Gce1 and 5'-nucleotidase CD73, which both gain access to the lipid droplets (LDs). The present report demonstrates the translocation of Gce1 and CD73, harboring the intact GPI anchor, from detergent-insoluble glycolipid-enriched plasma membrane domains (DIGs) to the LDs in response to palmitate, H(2)O(2), and glimepiride by analysis of their steady-state distribution using photoaffinity labeling and activity determination as well as of their redistribution after pulse or equilibrium metabolic labeling. We were surprised to find that palmitate, H(2)O(2), and glimepiride induced the activation of the GPI-specific phospholipase C (GPI-PLC) at DIGs of rat adipocytes, leading to anchorless Gce1 and CD73. Inhibition of the GPI-PLC or the presence of nonhydrolyzable substrate analogs of Gce1 and CD73 interfered with the palmitate-, H(2)O(2)-, and glimepiride-induced 1) lipolytic cleavage of Gce1 and CD73, 2) translocation of their GPI-anchored versions from DIGs to LDs, 3) up-regulation of cAMP degradation, and 4) inhibition of lipolysis. These data suggest a novel insulin-independent antilipolytic mechanism in rat adipocytes, which relies on the palmitate-, H(2)O(2)-, and glimepiride-induced and GPI-PLC-dependent translocation of (c)AMP-degrading GPI-anchored proteins from the adipocyte plasma membrane to LDs. The findings may shed new light on the biogenesis and degradation of LDs in response to physiological and pharmacological stimuli.

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.

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.

Regulation of lipid raft proteins by glimepiride- and insulin-induced glycosylphosphatidylinositol-specific phospholipase C in rat adipocytes.

The insulin receptor-independent insulin-mimetic signalling provoked by the antidiabetic sulfonylurea drug, glimepiride, is accompanied by the redistribution and concomitant activation of lipid raft-associated signalling components, such as the acylated tyrosine kinase, pp59(Lyn), and some glycosylphosphatidylinositol-anchored proteins (GPI-proteins). We now found that impairment of glimepiride-induced lipolytic cleavage of GPI-proteins in rat adipocytes by the novel inhibitor of glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC), GPI-2350, caused almost complete blockade of (i) dissociation from caveolin-1 of pp59(Lyn) and GPI-proteins, (ii) their redistribution from high cholesterol- (hcDIGs) to low cholesterol-containing (lcDIGs) lipid rafts, (iii) tyrosine phosphorylation of pp59(Lyn) and insulin receptor substrate-1 protein (IRS-1) and (iv) stimulation of glucose transport as well as (v) inhibition of isoproterenol-induced lipolysis in response to glimepiride. In contrast, blockade of the moderate insulin activation of the GPI-PLC and of lipid raft protein redistribution by GPI-2350 slightly reduced insulin signalling and metabolic action, only. Importantly, in response to both insulin and glimepiride, lipolytically cleaved hydrophilic GPI-proteins remain associated with hcDIGs rather than redistribute to lcDIGs as do their uncleaved amphiphilic versions. In conclusion, GPI-PLC controls the localization within lipid rafts and thereby the activity of certain GPI-anchored and acylated signalling proteins. Its stimulation is required and may even be sufficient for insulin-mimetic cross-talking to IRS-1 in response to glimepiride via redistributed and activated pp59(Lyn).

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

UNLABELLED: 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.