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Publications (23)67.96 Total impact

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    ABSTRACT: 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.
    Archives of Physiology and Biochemistry 07/2010; 116(3):97-115.
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    ABSTRACT: In addition to predominant localization at detergent-insoluble, glycolipid-enriched plasma membrane microdomains (DIGs), glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-proteins) have been found associated with lipid droplets (LDs) and adiposomes. Adiposomes are vesicles that are released from adipocytes in response to anti-lipolytic and lipogenic signals, such as H(2)O(2), palmitate and the antidiabetic sulfonylurea drug, glimepiride, and harbour (c)AMP-degrading GPI-proteins, among them the 5-nucleotidase CD73. Here the role of adiposomes in GPI-protein-mediated information transfer was studied. Adiposomes were incubated with isolated rat adipocytes under various conditions. Trafficking of CD73 and lipid synthesis were analysed. Upon blockade of GPI-protein trafficking, CD73 specifically associated with DIGs of small, and to a lower degree, large, adipocytes. On reversal of the blockade, CD73 appeared at cytosolic LD in time- adiposome concentration- and signal (H(2)O(2) > glimepiride > palmitate)-dependent fashion. The salt- and carbonate-resistant association of CD73 with structurally intact DIGs and LD was dependent on its intact GPI anchor. Upon incubation with small and to a lower degree, large adipocytes, adiposomes increased lipid synthesis in the absence or presence of H(2)O(2), glimepiride and palmitate and improved the sensitivity toward these signals. Upregulation of lipid synthesis by adiposomes was dependent on the translocation of CD73 with intact GPI anchors from DIGs to LD. The signal-induced transfer of GPI-anchored CD73 from adiposomes via DIGs to LD of adipocytes mediates paracrine upregulation of lipid synthesis within the adipose tissue.
    British Journal of Pharmacology 06/2010; 160(4):878-91. · 5.07 Impact Factor
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    ABSTRACT: 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.
    Archives of Physiology and Biochemistry 02/2010; 116(1):28-41.
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    ABSTRACT: 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.
    Archives of Physiology and Biochemistry 02/2010; 116(1):3-20.
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    ABSTRACT: 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.
    Biochemistry 03/2008; 47(5):1259-73. · 3.38 Impact Factor
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    ABSTRACT: 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.
    Biochemistry 03/2008; 47(5):1274-87. · 3.38 Impact Factor
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    ABSTRACT: AVE2268, a substituted glycopyranoside, is an orally active and selective inhibitor of sodium-dependent glucose transporter 2 (SGLT2; IC50 = 13 nmol/L). Investigation of the pharmacological profile of AVE2268 on urinary glucose excretion (UGE) and blood glucose after glucose challenge (po or Intraperitoneal) was performed in mice and rats. AVE2268 caused a dose-dependent increase of UGE in mice (ID30 = 79 +/- 8.1 mg/kg p.o.) and rats (ID30 = 39.8 +/- 4.0 mg/kg p.o.). AVE2268 in mice was more potent to decrease blood glucose ascent when glucose was given intraperitoneally (ID50 = 13.2 +/- 3.9 mg/ kg), compared to orally administered glucose (ID50 = 26.1 +/- 3.9 mg/kg), showing that AVE2268 has no effects on SGLT 1 in the gut in vivo, which is in accordance with ist very low affinity to the SGLT 1 in vitro (IC50 >10,000 nmol/L). During an oral glucose tolerance test, AVE2268 dose-dependently increased UGE, with subsequent decreases of AUC and blood glucose. A highly significant inverse correlation between AUC and UGE was found (p < 0.001). The increase in UGE is linked to the inhibition of SGLT2 only. This profile renders AVE2268 as a new antidiabetic drug for the treatment of type 2 diabetes.
    Arzneimittel-Forschung 02/2008; 58(11):574-80. · 0.56 Impact Factor
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    ABSTRACT: 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).
    Biochemical Pharmacology 04/2005; 69(5):761-80. · 4.58 Impact Factor
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    ABSTRACT: Intestinal cholesterol absorption is an important regulator of serum cholesterol levels. Ezetimibe is a specific inhibitor of intestinal cholesterol absorption recently introduced into medical practice; its mechanism of action, however, is still unknown. Ezetimibe neither influences the release of cholesterol from mixed micelles in the gut lumen nor the transfer of cholesterol to the enterocyte brush border membrane. With membrane-impermeable Ezetimibe analogues we could demonstrate that binding of cholesterol absorption inhibitors to the brush border membrane of small intestinal enterocytes from the gut lumen is sufficient for inhibition of cholesterol absorption. A 145-kDa integral membrane protein was identified as the molecular target for cholesterol absorption inhibitors in the enterocyte brush border membrane by photoaffinity labeling with photoreactive Ezetimibe analogues (Kramer, W., Glombik, H., Petry, S., Heuer, H., Schafer, H. L., Wendler, W., Corsiero, D., Girbig, F., and Weyland, C. (2000) FEBS Lett. 487, 293-297). The 145-kDa Ezetimibe-binding protein was purified by three different methods and sequencing revealed its identity with the membrane-bound ectoenzyme aminopeptidase N ((alanyl)aminopeptidase; EC 3.4.11.2; APN; leukemia antigen CD13). The enzymatic activity of APN was not influenced by Ezetimibe (analogues). The uptake of cholesterol delivered by mixed micelles by confluent CaCo-2 cells was partially inhibited by Ezetimibe and nonabsorbable Ezetimibe analogues. Preincubation of confluent CaCo-2 cells with Ezetimibe led to a strong decrease of fluorescent APN staining with a monoclonal antibody in the plasma membrane. Independent on its enzymatic activity, aminopeptidase N is involved in endocytotic processes like the uptake of viruses. Our findings suggest that binding of Ezetimibe to APN from the lumen of the small intestine blocks endocytosis of cholesterol-rich membrane microdomains, thereby limiting intestinal cholesterol absorption.
    Journal of Biological Chemistry 02/2005; 280(2):1306-20. · 4.65 Impact Factor
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    ABSTRACT: The design and synthesis of a biotin-tagged photoreactive analogue C-4 of the cholesterol absorption inhibitor Ezetimibe is described. Photoaffinity labeling of intestinal brush border membrane vesicles with C-4 and subsequent streptavidin-biotin chromatography leads to selective extraction of a 145 kDa integral membrane protein as the molecular target for cholesterol absorption inhibitors.
    Bioorganic & Medicinal Chemistry 05/2003; 11(8):1639-42. · 2.90 Impact Factor
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    ABSTRACT: Insulin receptor-independent activation of the insulin signal transduction cascade in insulin-responsive target cells by phosphoinositolglycans (PIG) and PIG-peptides (PIG-P) is accompanied by redistribution of glycosylphosphatidylinositol (GPI)-anchored plasma membrane proteins (GPI proteins) and dually acylated nonreceptor tyrosine kinases from detergent/carbonate-resistant glycolipid-enriched plasma membrane raft domains of high-cholesterol content (hcDIGs) to rafts of lower cholesterol content (lcDIGs). Here we studied the nature and localization of the primary target of PIG(-P) in isolated rat adipocytes. Radiolabeled PIG-P (Tyr-Cys-Asn-NH-(CH(2))(2)-O-PO(OH)O-6Manalpha1(Manalpha1-2)-2Manalpha1-6Manalpha1-4GluN1-6Ino-1,2-(cyclic)-phosphate) prepared by chemical synthesis or a radiolabeled lipolytically cleaved GPI protein from Saccharomyces cerevisiae, which harbors the PIG-P moiety, bind to isolated hcDIGs but not to lcDIGs. Binding is saturable and abolished by pretreatment of intact adipocytes with trypsin followed by NaCl or with N-ethylmaleimide, indicating specific interaction of PIG-P with a cell surface protein. A 115-kDa polypeptide released from the cell surface by the trypsin/NaCl-treatment is labeled by [(14)C]N-ethylmaleimide. The labeling is diminished upon incubation of adipocytes with PIG-P which can be explained by direct binding of PIG-P to the 115-kDa protein and concomitant loss of its accessibility to N-ethylmaleimide. Binding of PIG-P to hcDIGs is considerably increased after pretreatment of adipocytes with (glycosyl)phosphatidylinositol-specific phospholipases compatible with lipolytic removal of endogenous ligands, such as GPI proteins/lipids. These data demonstrate that in rat adipocytes synthetic PIG(-P) as well as lipolytically cleaved GPI proteins interact specifically with hcDIGs. The interaction depends on the presence of a trypsin/NaCl/NEM-sensitive 115-kDa protein located at hcDIGs which thus represents a candidate for a binding protein for exogenous insulin-mimetic PIG(-P) and possibly endogenous GPI proteins/lipids.
    Archives of Biochemistry and Biophysics 01/2003; 408(1):17-32. · 3.37 Impact Factor
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    ABSTRACT: 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.
    Archives of Biochemistry and Biophysics 01/2003; 408(1):7-16. · 3.37 Impact Factor
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    ABSTRACT: 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. Carbonate extracts from purified plasma membranes of basal and stimulated adipocytes were analyzed by high-resolution sucrose gradient centrifugation. 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. 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.
    Molecular Medicine 04/2002; 8(3):120-36. · 4.47 Impact Factor
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    ABSTRACT: 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.
    Biochemistry 01/2002; 40(48):14603-20. · 3.38 Impact Factor
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    ABSTRACT: 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.
    Molecular and Cellular Biology 08/2001; 21(14):4553-67. · 5.37 Impact Factor
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    G Müller, S Wied, W Frick
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    ABSTRACT: Signaling molecules downstream from the insulin receptor, such as the insulin receptor substrate protein 1 (IRS-1), are also activated by other receptor tyrosine kinases. Here we demonstrate that the non-receptor tyrosine kinases, focal adhesion kinase pp125(FAK) and Src-class kinase pp59(Lyn), after insulin-independent activation by phosphoinositolglycans (PIG), can cross talk to metabolic insulin signaling in rat and 3T3-L1 adipocytes. Introduction by electroporation of neutralizing antibodies against pp59(Lyn) and pp125(FAK) into isolated rat adipocytes blocked IRS-1 tyrosine phosphorylation in response to PIG but not insulin. Introduction of peptides encompassing either the major autophosphorylation site of pp125(FAK), tyrosine 397, or its regulatory loop with the twin tyrosines 576 and 577 inhibited PIG-induced IRS-1 tyrosine phosphorylation and glucose transport. PIG-induced pp59(Lyn) kinase activation and pp125(FAK) tyrosine phosphorylation were impaired by the former and latter peptide, respectively. Up-regulation of pp125(FAK) by integrin clustering diminished PIG-induced IRS-1 tyrosine phosphorylation and glucose transport in nonadherent but not adherent adipocytes. In conclusion, PIG induced IRS-1 tyrosine phosphorylation by causing (integrin antagonized) recruitment of IRS-1 and pp59(Lyn) to the common signaling platform molecule pp125(FAK), where cross talk of PIG-like structures and extracellular matrix proteins to metabolic insulin signaling may converge, possibly for the integration of the demands of glucose metabolism and cell architecture.
    Molecular and Cellular Biology 08/2000; 20(13):4708-23. · 5.37 Impact Factor
  • G Müller, W Frick
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    ABSTRACT: In recent years, a number of cross-talk systems have been identified which feed into the insulin signalling cascade at the level of insulin receptor substrate (IRS) tyrosine phosphorylation, e.g., receptor and non-receptor tyrosine kinases and G-protein-coupled receptors. At the molecular level, a number of negative modulator and feedback systems somehow interacting with the beta-subunit (catecholamine-, phorbolester-, or tumor necrosis factor-alpha-induced serine/threonine phosphorylation, carboxy-terminal trimming by a thiol-dependent protease, association of inhibitory/regulatory proteins such as RAD, PC1, PED, alpha2-HS-glycoprotein) have been identified as candidate mechanisms for the impairment of insulin receptor function by elevations in the activity and/or amount of the corresponding modification enzymes/inhibitors. Both decreased responsiveness and sensitivity of the insulin receptor beta-subunit for insulin-induced tyrosine autophosphorylation have been demonstrated in several cellular and animal models of metabolic insulin resistance as well as in the adipose tissue and skeletal muscle of diabetic patients and obese Pima Indians compared to non-obese subjects. Therefore, induction of the insulin signalling cascade by bypassing the defective insulin receptor kinase may be useful for the therapy of non-insulin dependent diabetes mellitus. During the past two decades, phosphoinositolglycans (PIGs) of various origin have been demonstrated to exert potent insulin-mimetic metabolic effects upon incubation with cultured or isolated muscle and adipose cells. However, it remained to be elucidated whether these compounds actually manage to trigger insulin signalling and if so at which level of hierarchy within the signalling cascade the site of interference is located. Recent studies using isolated rat adipocytes and chemically synthesized PIG compounds point to IRS1/3 tyrosine phosphorylation by p59Lyn kinase as the site of cross-talk, the negative regulation of which by interaction with caveolin is apparently abrogated by PIG. This putative mechanism is thus compatible with the recently formulated caveolin signalling hypothesis, the supporting data for which are reviewed here. Though we have not obtained experimental evidence for the involvement of PIG in physiological insulin action, the potential cross-talk between insulin and PIG signalling, including the caveolae/detergent-insoluble glycolipid-enriched rafts as the compartments where the corresponding signalling components are concentrated, thus represent novel targets for signal transduction therapy.
    Cellular and Molecular Life Sciences CMLS 01/2000; 56(11-12):945-70. · 5.62 Impact Factor
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    ABSTRACT: A set of synthetic phosphoinositolglycan (PIG) compounds has been demonstrated to exert insulin-mimetic activity on glucose and lipid metabolism in rat adipocytes differing considerably in potency [compound 41>37>45>7>1; W. Frick, A. Bauer, J. Bauer, S. Wied and G. Müller, G. (1998) Biochemistry 37, 13421-13436]. In the present study we examine whether these differences are based on the capability of the PIG compounds to stimulate signalling components which are thought to mediate metabolic insulin action. Studies using a tyrosine kinase inhibitor and introduction into adipocytes of anti-phosphotyrosine or inhibitory anti-insulin receptor beta-subunit antibodies demonstrated dependence on tyrosine phosphorylation but independence of insulin receptor kinase activation of the insulin-mimetic signalling and metabolic activity of the PIG compounds. The five compounds elicited in rat adipocytes a significant increase in tyrosine phosphorylation of both insulin receptor substrate 1 (IRS-1) and IRS-3 and, to a minor degree, IRS-2, in IRS-1/3-associated phosphatidylinositol 3-kinase (PI 3-K) protein as well as activity, and in protein kinase B (PKB) activity as well as phosphorylation. This was most pronounced for compound 41, approaching 65-95% of the maximal insulin response (MIR) at 20 microM, and declined in the order of compounds 37, 45, 7 and 1. The same ranking was true for the maximal inhibition of glycogen synthase kinase 3 activity (GSK-3) (41, 75% of MIR; compound 37, 65%; compound 7, 25%; compound 1, 10%) and GSK-3 autophosphorylation. The half-maximal concentrations effective for signalling (compound 41, 2-5 microM; compound 37, 10-20 microM) corresponded well to those stimulating glucose and lipid metabolism. Interestingly, compounds 37 and 41 stimulated mitogen-activated protein kinase (MAPK) and protein synthesis in rat adipocytes to only about 20-30% (at 50 microM) of MIR. We conclude that in rat adipocytes: (i) the potency of PIG compounds to regulate glucose/lipid metabolism depends on the activation of PI 3-K and PKB and inhibition of GSK-3; (ii) initiation of tyrosine phosphorylation of IRS-1/3 is sufficient and activation of the PI 3-K cascade is required for insulin-mimetic metabolic signalling; and (iii) PIG compounds are quite selective for the PI 3-K compared to the MAPK cascade, (iv) PIG compounds seem to use the same signalling components downstream of PI 3-K (including Rab4) for stimulation of glucose transport as does insulin. Thus the early signalling step(s) used by PIG, but not by insulin, may represent a target for the treatment of insulin-resistant states.
    Biochemical Journal 11/1998; 336 ( Pt 1):163-81. · 4.65 Impact Factor
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    ABSTRACT: Phosphoinositolglycan (PIG) molecules have been implicated to stimulate glucose and lipid metabolism in insulin-sensitive cells and tissues in vitro and in vivo. The structural requirements for this partial insulin-mimetic activity remained unclear so far. For establishment of a first structure-activity relationship, a number of PIG compounds were synthesized consisting of the complete or shortened/mutated glycan moiety derived from the structure of the glycosylphosphatidylinositol (GPI) anchor of the GPI-anchored protein, Gce1p, from yeast. The PIG compounds were divided into four classes according to their insulin-mimetic activity in vitro with the typical representatives: compound 41, HO-SO2-O-6Manalpha1(Manalpha1-2)-2Manalpha1 (6-HSO3)- -6Manalpha1-4GluNb eta1-6(D)inositol-1,2-(cyclic)-phosphate; compound 37, HO-PO(H)O-6Manalpha1(Manalpha1-2)-2Manalpha1-6Manal pha1-4GluNbeta1-6( D)inositol-1,2-(cyclic)-phosphate; compound 7, HO-PO(H)O-6Manalpha1-4GluN(1-6(L)inositol-1,2-(cyclic)-ph osp hate; and compound 1, HO-PO(H)O-6Manalpha1-4GluN(1-6(L)inositol. Compounds 41 and 37 stimulated lipogenesis up to 90% (at 20 microM) of the maximal insulin response but with differing concentrations required for 50% activation (EC50 values 2.5 +/- 0.9 vs 4.9 +/- 1.7 microM) as well as glycogen synthase (4.7 +/- 1 vs 9.5 +/- 1.5 microM) and glycerol-3-phosphate acyltransferase (3.5 +/- 0.8 vs 8.0 +/- 1.1 microM). Compound 7 was clearly less potent (20% of the maximal insulin response at 100 microM), whereas compound 1 was almost inactive. This relative ranking in the insulin-mimetic potency between members of the PIG classes (e.g., 41 > 37 > 7 > 1) was also observed for the (i) activation of glucose transport and glucose transporter isoform 4 translocation in isolated normal and insulin-resistant adipocytes, (ii) inhibition of lipolysis in adipocytes, (iii) stimulation of glucose transport and glycogen synthesis in isolated normal and insulin-resistant diaphragms, and (iv) induction of tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) in diaphragms. The complete glycan core structure (Man3-GluN) of typical GPI anchors including a mannose side chain and the inositolphosphate moiety was required for maximal insulin-mimetic activity of the PIG compounds with some variations possible with respect to the type of residues coupled to the terminal mannose/inositol as well as the type of linkages involved. These data argue for the potency and specificity of the interaction of PIG molecules with putative signaling component(s) (presumably at the level of the IRS proteins) in adipose and muscle cells which finally lead to insulin-mimetic metabolic activity even in insulin-resistant states.
    Biochemistry 10/1998; 37(38):13421-36. · 3.38 Impact Factor
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    ABSTRACT: 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.
    Molecular Medicine 06/1998; 4(5):299-323. · 4.47 Impact Factor