Antihypertensive and laxative effects by pharmacological inhibition of sodium-proton-exchanger subtype 3-mediated sodium absorption in the gut.

High intestinal sodium absorption is one mechanism of hypertension and constipation. The sodium-proton-exchanger subtype 3 (NHE3) is an important mediator of sodium absorption in the gut. SAR218034 (SAR) is an orally nonabsorbable specific NHE3 inhibitor. The effect of SAR (1 mg/kg per day in chow) on feces sodium excretion, systolic blood pressure via tail cuff, and gene expression of NHE3 in the gut were studied in senescent lean hypertensive rats (spontaneously hypertensive rats-lean, loaded with NaCl 0.7% in drinking water) and in hypertensive, obese, and hyperinsulinemic rats (spontaneously hypertensive rats-obese, not loaded with NaCl). In spontaneously hypertensive rats-lean, inhibition of intestinal NHE3 by SAR increased feces sodium excretion and reduced urinary sodium excretion, whereas absolute sodium balance and serum sodium concentration were not changed. This suggests reduced intestinal sodium absorption in SAR-treated animals and was associated with increased feces water content (58% versus 42% in placebo treated animals; P=0.0001) and reduction in systolic blood pressure from 222 ± 7 to 198 ± 2 mm Hg (P=0.0001). Angiotensin-converting enzyme inhibition by ramipril plus NHE3 inhibition resulted in an additive blood pressure-lowering effect. In spontaneously hypertensive rats-obese, SAR lowered systolic blood pressure but did not modify serum insulin or cholesterol levels. Gene expression of NHE3 was upregulated in the ileum and colon but not in the jejunum of SAR-treated rats. Reduction of intestinal sodium absorption by selective NHE3 inhibition in the gut reduces high blood pressure and increases feces water excretion. Intestinal NHE3 blockade could be a new treatment strategy for elderly patients suffering from high blood pressure and constipation.

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.

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.

Effects of AVE2268, a substituted glycopyranoside, on urinary glucose excretion and blood glucose in mice and rats.

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.

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).

Aminopeptidase N (CD13) is a molecular target of the cholesterol absorption inhibitor ezetimibe in the enterocyte brush border membrane.

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.

Synthesis of a biotin-tagged photoaffinity probe of 2-azetidinone cholesterol absorption inhibitors.

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.

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.

Interaction of phosphoinositolglycan(-peptides) with plasma membrane lipid rafts of rat adipocytes.

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.

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.