Sulfonylureas (SUs) are still among the mostly prescribed antidiabetic drugs with an established mode of action: release of insulin from pancreatic β-cells. In addition, effects of SUs on adipocytes by activation of the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) have been described, which might explain their insulin-sensitizing potential observed in patients. However, there is a discrepancy between the impact of SUs on antidiabetic action and their rather moderate in vitro effect on PPARγ transcriptional activity. Recent studies have shown that some PPARγ ligands can improve insulin sensitivity by blocking PPARγ Ser-273 phosphorylation without having full agonist activity. It is unknown if SUs elicit their antidiabetic effects on adipocytes by inhibition of PPARγ phosphorylation. Here, we investigated if binding of SUs to PPARγ can interfere with PPARγ Ser-273 phosphorylation and determined their antidiabetic actions in vitro in primary human white adipocytes and in vivo in high-fat diet (HFD) obese mice.

Primary human white preadipocytes were differentiated in the presence of glibenclamide, glimepiride and PPARγ ligands rosiglitazone and SR1664 to compare PPARγ Ser-273 phosphorylation, glucose uptake and adipokine expression. Transcriptional activity at PPARγ was determined by luciferase assays, quantification of PPARγ Ser-273 phosphorylation was determined by Western blotting and CDK5 kinase assays. In silico modelling was performed to gain insight into the binding characteristics of SUs to PPARγ. HFD mice were administered SUs and rosiglitazone for 6 days. PPARγ Ser-273 phosphorylation in white adipose tissue (WAT), body composition, glucose tolerance, adipocyte morphology and expression levels of genes involved in PPARγ activity in WAT and brown adipose tissue (BAT) were evaluated.

SUs inhibit phosphorylation of PPARγ at Ser-273 in primary human white adipocytes and exhibit a positive antidiabetic expression profile, which is characterized by up regulation of insulin-sensitizing and down regulation of insulin resistance-inducing adipokines. We demonstrate that SUs directly bind to PPARγ by in silico modelling and inhibit phosphorylation in kinase assays to a similar extend as rosiglitazone and SR1664. In HFD mice SUs reduce PPARγ phosphorylation in WAT and have comparable effects on gene expression to rosiglitazone. In BAT SUs increase UCP1 expression and reduce lipid droplets sizes.

Our findings indicate that a part of SUs extra-pancreatic effects on adipocytes in vitro and in vivo is probably mediated via their interference with PPARγ phosphorylation rather than via classical agonistic activity at clinical concentrations.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are anchored at the outer leaflet of eukaryotic plasma membranes (PMs) only by carboxy-terminal covalently coupled GPI. GPI-APs are known to be released from the surface of donor cells in response to insulin and antidiabetic sulfonylureas (SUs) by lipolytic cleavage of the GPI or upon metabolic derangement as full-length GPI-APs with the complete GPI attached. Full-length GPI-APs become removed from extracellular compartments by binding to serum proteins, such as GPI-specific phospholipase D (GPLD1), or insertion into the PMs of acceptor cells. Here, the interplay between the lipolytic release and intercellular transfer of GPI-APs and its potential functional impact was studied using transwell co-culture with human adipocytes as insulin-/SU-responsive donor cells and GPI-deficient erythroleukemia as acceptor cells (ELCs). Measurement of the transfer as the expression of full-length GPI-APs at the ELC PMs by their microfluidic chip-based sensing with GPI-binding α-toxin and GPI-APs antibodies and of the ELC anabolic state as glycogen synthesis upon incubation with insulin, SUs and serum yielded the following results: (i) Loss of GPI-APs from the PM upon termination of their transfer and decline of glycogen synthesis in ELCs, as well as prolongation of the PM expression of transferred GPI-APs upon inhibition of their endocytosis and upregulated glycogen synthesis follow similar time courses. (ii) Insulin and SUs inhibit both GPI-AP transfer and glycogen synthesis upregulation in a concentration-dependent fashion, with the efficacies of the SUs increasing with their blood glucose-lowering activity. (iii) Serum from rats eliminates insulin- and SU-inhibition of both GPI-APs' transfer and glycogen synthesis in a volume-dependent fashion, with the potency increasing with their metabolic derangement. (iv) In rat serum, full-length GPI-APs bind to proteins, among them (inhibited) GPLD1, with the efficacy increasing with the metabolic derangement. (v) GPI-APs are displaced from serum proteins by synthetic phosphoinositolglycans and then transferred to ELCs with accompanying stimulation of glycogen synthesis, each with efficacies increasing with their structural similarity to the GPI glycan core. Thus, both insulin and SUs either block or foster transfer when serum proteins are depleted of or loaded with full-length GPI-APs, respectively, i.e., in the normal or metabolically deranged state. The transfer of the anabolic state from somatic to blood cells over long distance and its "indirect" complex control by insulin, SUs and serum proteins support the (patho)physiological relevance of the intercellular transfer of GPI-APs.

Insulin resistance is associated with obesity and can lead to several metabolic disorders including type II diabetes, nonalcoholic fatty liver disease and cardiovascular problems. Search for the small molecules which can either induce or mimic the insulin action are of great interest and can be utilized to manage insulin resistance. There are several dietary phytochemicals which can potentially have insulinomimetic action. Nevertheless, high throughput screening methods to test efficiency of small molecules to act as an insulinomimetic are not fully established. In this paper we have performed chemical screen analysis based on GLUT4 translocation using a cell line CHO-HIRC-myc-GLUT4 eGFP that expresses GLUT4-GFP in association with human Insulin receptor. We have established a high content screening-based method which can track and quantify the GLUT4 translocation from perinuclear area to the cell membrane. The assay involves measuring fluorescence intensity in a defined perinuclear area and a defined area along the cell membrane; and the results are expressed as the ratio of fluorescence intensity in the perinuclear to membrane area. The assay could collect real time data of GLUT4 translocation from thousand of cells/ sample and from many such samples in one experiment. We validated the assay using Insulin, insulin mimics/sensitizers and insulin inhibitors. The agonist or antagonists were analyzed for their ability to enhance or block the GLUT4 translocation independent of insulin. The outcome of the assay was correlated by performing glucose uptake assay using differentiated 3T3L1 cells. Using this platform we further identified several plant extracts which had the insulin mimetic action. We confirmed that these plant extracts were non-toxic to the beta cells using RIN mf5cells and 3T3L1 cells. We have identified plant extracts with the potential insulinomimetic action using novel high-content screening approach; these can be further tested for their efficiency in-vivo in pre-clinical trials.

Both physicians and patients in Egypt often express concern as to the clinical efficacy of locally manufactured glimepiride tablet generics whenever adequate control of blood sugar is not achieved with these products. The present study addresses this issue. The pharmaceutical quality of four glimepiride 3 mg tablet generics purchased in Egypt from local pharmacies was assessed relative to the innovator product (Amaryl^®), 3 mg tablets. Uniformity of Content, dissolution rate, disintegration time and hardness were determined. Products were subjected to a 6-month stability study under stress condition (40 °c/75%RH). The same brands were evaluated in vivo in a clinical study conducted in the Main Alexandria University Hospital involving 100 patients (20 patients per brand including innovator). Patients recruited were newly diagnosed type II diabetics. Glimepiride tablets were used as a monotherapy. Fasting blood glucose (FBG) and glycosylated hemoglobin (HbA1C) were measured over a period of 3 months. The results indicated differences (p ≤ .05) in the in vitro and in vivo performance of the tested products; innovator and tested generics substitution was not evident. The stability study indicated that the tablets were prone to deterioration in their physical characteristics, particularly dissolution profiles, upon storage of blisters in a hot humid climate. In vitro/in vivo correlations were investigated seeking to identify an in vitro test to serve as a performance indicator for glimepiride tablets in the post-marketing period. The similarity factor (f₂) of the dissolution data proved to be a good indicator of in vivo performance of the tablets.

Glimepiride is a sulfonylurea known to have unique insulin mimetic and sensitizing effects. We aimed to study the efficacy of glimepiride in a patient with type A insulin resistance syndrome.

A 15-year-old girl with type A insulin resistance syndrome was treated with glimpiride for 6 months. Self-monitoring of blood glucose was recorded, and oral glucose tolerance tests on glucose and insulin were measured during the treatment. Hyperinsulinemic euglycemic clamp was used to evaluate whole-body insulin sensitivity before and after the treatment.

A novel heterozygous missense mutation at exon 19 (c.3427A>T) in the tyrosine kinase domain of the INSR gene was identified, causing an amino acid replacement of phenylalanine for isoleucine at codon 1143 (Ile1143Phe). Before the treatment, the patient's glycated hemoglobin was 7.0%, plasma glucose during oral glucose tolerance test was 6.7, 12.8 and 17.3 mmol/L, and simultaneous serum insulin was 80.7, 137.5 and >300 μU/mL. There were no significant differences between self-monitored blood glucose measured at each time-point among different glimepiride dosages, or during the 14 weeks when glimepiride was used at its maximal dosage (6 mg/day). Oral glucose tolerance test showed little change in plasma glucose and serum insulin. Glycated hemoglobin decreased by 0.8% after the treatment. However, a euglycemic clamp study showed that the M value decreased from 5.25 to 2.90 mg/kg/min, showing increased insulin resistance.

Treatment with glimepiride did not improve insulin sensitivity in a patient with type A insulin resistance syndrome carrying Ile1143Phe heterozygous mutation in the INSR gene. Large-scale long-term studies assembled worldwide are required to optimize treatment algorithms for patients with type A insulin resistance syndrome.

The aim of the present study was to investigate the mechanism of action of a sulfonamide derivative on glucose uptake in adipose tissue, as well as to characterize the effects of this compound on intestinal disaccharidases and advanced glycation end-products (AGEs) formation. Camphoryl-benzene sulfonamide (CS) was able to stimulate glucose uptake in isolated adipocytes, adipose tissue, and in soleus muscle. The stimulatory effect of the compound (10 μM) on glucose uptake on adipose tissue was blocked by diazoxide, wortmannin, U73122, colchicine, and N-ethylmaleimide. On the other hand, the effects of CS were not blocked by glibenclamide, an inhibitor of the K⁺ -ATP channel, or even by the inhibitor of protein p38 MAPK, SB 203580. In vivo, this compound reduced intestinal disaccharidase activity, while, in vitro, CS reduced the formation of AGEs at 7, 14, and 28 days of incubation. The stimulatory effect of CS on glucose uptake requires the activation of the K⁺ -ATP channel, translocation, and fusion of GLUT4 vesicles to the plasma membrane on adipocytes for glucose homeostasis. In addition, the inhibition of disaccharidase activity contributes to the glucose homeostasis in a short-term as well as the remarkable reduction in AGE formation indicates that the CS may prevent of complications of late diabetes.

Liu, Xiang-Wen, Jie Yin, Qi-Sheng Ma, Chu-Chu Qi, Ji-Ying Mu, Lang Zhang, Li-Ping Gao, and Yu-Hong Jing. Role of arcuate nucleus in the regulation of feeding behavior in the process of altitude acclimatization in rats. High Alt Med Biol. 18:234-241, 2017.-Highly efficient energy utilization and metabolic homeostasis maintenance rely on neuromodulation. Altitude exposure is known to stimulate neuroendocrine systems to respond to acute hypoxia and adaptive acclimatization. However, limited data on how the adaptive regulation of the arcuate nucleus performs in the process of altitude acclimatization are available. In the present study, male Sprague Dawley rats were transported to Huashixia, Qinghai (with an altitude of 4400 m) from Xian (with an altitude of 300 m) by air; rats were consistently raised in Xian as control. Food uptake and body weight were measured consecutively after being subjected to high-altitude condition. Contents of plasma leptin and ghrelin were analyzed by the Enzyme Linked Immunosorbent Assay (ELISA) Kits. Brain coronal sections were obtained, and neuropeptide Y (NPY), proopiomelanocotin (POMC), and c-fos immunoreactivity in arcuate nucleus were observed. Arcuate nucleus was isolated from the hypothalamus, and the mRNA of NPY and POMC were measured by quantitative real-time polymerase chain reaction. Our results showed both food consumption and body weight decreased in the high plateau compared with rats raised in the low-altitude condition. Plasma leptin increased at the early stage, and ghrelin decreased at a later stage after reaching the high plateau. The peak of c-fos immunoreactivity in the arcuate nucleus was at day 3 after reaching the high plateau. The expression level of NPY increased, and POMC decreased in the arcuate nucleus at day 7 after reaching the high plateau compared with the plain control group. These results indicate that the arcuate nucleus of hypothalamus performs an important function in regulating feeding behavior during altitude acclimatization. Our study suggested that altitude acclimation is regulated by the hypothalamus that received leptin and ghrelin signals to response by its microcircuit, including NPY- and POMC-neurons in the arcuate nucleus.

Autonomic nervous system imbalance is associated with metabolic diseases, including diabetes. Glibenclamide is an antidiabetic drug that acts by stimulating insulin secretion from pancreatic beta cells and is widely used in the treatment of type 2 diabetes. Since there is scarce data concerning autonomic nervous system activity and diabetes, the aim of this work was to test whether glibenclamide can improve autonomic nervous system activity and muscarinic acetylcholine receptor function in pre-diabetic obese male rats.

Pre-diabetes was induced by treatment with monosodium L-glutamate in neonatal rats. The monosodium L-glutamate group was treated with glibenclamide (2 mg/kg body weight /day) from weaning to 100 days of age, and the control group was treated with water. Body weight, food intake, Lee index, fasting glucose, insulin levels, homeostasis model assessment of insulin resistance, omeostasis model assessment of β-cell function, and fat tissue accumulation were measured. The vagus and sympathetic nerve electrical activity were recorded. Insulin secretion was measured in isolated islets challenged with glucose, acetylcholine, and the selective muscarinic acetylcholine receptor antagonists by radioimmunoassay technique.

Glibenclamide treatment prevented the onset of obesity and diminished the retroperitoneal (18%) and epididymal (25%) fat pad tissues. In addition, the glibenclamide treatment also reduced the parasympathetic activity by 28% and glycemia by 20% in monosodium L-glutamate-treated rats. The insulinotropic effect and unaltered cholinergic actions in islets from monosodium L-glutamate groups were increased.

Early glibenclamide treatment prevents monosodium L-glutamate-induced obesity onset by balancing autonomic nervous system activity.

Previous studies suggested that dyslipidemia was potentially associated with anti-diabetic medications of sulfonylureas (SUs). The results were, however, inconsistent. Therefore, we conducted a meta-analysis of randomized controlled trials (RCTs) to assess the effects of SUs on the level of lipids in patients with type 2 diabetes mellitus (T2DM).

We searched PubMed, EMBASE, and CENTRAL databases for RCTs that addressed the effects of second- and/or third-generation SUs used in T2DM patients on lipids profiles with study duration of at least 12 weeks. Two reviewers independently screened literature, collected data, and assessed methodological quality of included studies. The meta-analysis was performed by using the RevMan5.1 software.

A total of 59 RCTs were included, of which 52 were included for final meta-analysis. The results suggested that SUs statistically increased the levels of FFA (SMD = 0.24, 95%CI 0.06 to 0.42) and TG (MD = 0.06, 95%CI 0.02 to 0.10), but decreased HDL-C (MD = -0.07, 95%CI -0.11 to -0.04) and LDL-C (MD = -0.11, 95%CI -0.17 to -0.04); but the SUs had no effect on TC (MD = 0.01, 95%CI -0.05 to 0.08), ApoA1 (MD = 0.01, 95%CI -0.03 to 0.04), and Apo B (MD = -0.01, 95%CI -0.05 to 0.03). When compared to metformin, SUs could increase TC and LDL-C; compared to glinides, SUs increased TC and lowered HDL-C; compared to thiazolidinediones, SUs reduced TC, LDL-C, HDL-C, and increase TG.

SUs have a small effect on lipids, although they may statistically increase the level of FFA and TG, and decrease LDL-C and HDL-C.

Diabetes mellitus (DM) affects over 10% of the world's population. Hyperglycemia is the main feature for the diagnosis of this disease. The zebrafish (Danio rerio) is an established model organism for the study of various metabolic diseases. In this paper, hyperglycemic zebrafish, when immersed in a 111 mM glucose solution for 14 days, developed increased glycation of proteins from the eyes, decreased mRNA levels of insulin receptors in the muscle, and a reversion of high blood glucose level after treatment with anti-diabetic drugs (glimepiride and metformin) even after 7 days of glucose withdrawal. Additionally, hyperglycemic zebrafish developed an impaired response to exogenous insulin, which was recovered after 7 days of glucose withdrawal. These data suggest that the exposure of adult zebrafish to high glucose concentration is able to induce persistent metabolic changes probably underlined by a hyperinsulinemic state and impaired peripheral glucose metabolism.

Gene expression changes have been associated with type 2 diabetes mellitus (T2DM); however, the alterations are not fully understood. We investigated the effects of anti-diabetic drugs on gene expression in Zucker diabetic fatty (ZDF) rats using oligonucleotide microarray technology to identify gene expression changes occurring in T2DM. Global gene expression in the pancreas, adipose tissue, skeletal muscle, and liver was profiled from Zucker lean control (ZLC) and anti-diabetic drug treated ZDF rats compared with those in ZDF rats. We showed that anti-diabetic drugs regulate the expression of a large number of genes. We provided a more integrated view of the diabetic changes by examining the gene expression networks. The resulting sub-networks allowed us to identify several biological processes that were significantly enriched by the anti-diabetic drug treatment, including oxidative phosphorylation (OXPHOS), systemic lupus erythematous, and the chemokine signaling pathway. Among them, we found that white adipose tissue from ZDF rats showed decreased expression of a set of OXPHOS genes that were normalized by rosiglitazone treatment accompanied by rescued blood glucose levels. In conclusion, we suggest that alterations in OXPHOS gene expression in white adipose tissue may play a role in the pathogenesis and drug mediated recovery of T2DM through a comprehensive gene expression network study after multi-drug treatment of ZDF rats.

Glimepiride is a third-generation sulfonylurea agent and is widely used in the treatment of type 2 diabetes mellitus. In addition to the stimulatory effects on pancreatic insulin secretion, glimepiride has also been reported to have extrapancreatic functions including activation of PI3 kinase (PI3K) and Akt in rat adipocytes and skeletal muscle. PI3-kinase and Akt are important signaling molecules in the regulation of proliferation and differentiation in various cells. This study investigated the actions of glimepiride in rat osteoblasts and the role of PI3K/Akt pathway. Cell proliferation was determined by measuring absorbance at 550 nm. Supernatant assay was used for measuring alkaline phosphatase activity. Western blot analysis was used for determining collagen I, insulin receptor substrate-1/2, PI3K/Akt, and endothelial nitric oxide synthase expression. We found that glimepiride significantly enhanced proliferation and differentiation of osteoblasts and led to activation of several key signaling molecules including insulin receptor substrate-1/2, PI3K/Akt, and endothelial nitric oxide synthase. Furthermore, a specific inhibitor of PI3K abolished the stimulatory effects of glimepiride on proliferation and differentiation. Taken together, these observations provide concrete evidence that glimepiride activates the PI3K/Akt pathway; and this activation is likely required for glimepiride to stimulate proliferation and differentiation of rat osteoblasts.

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.

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).

An improved liquid chromatographic tandem mass spectrometric method for the determination of glimepiride in human plasma has been developed and fully validated. The article describes in detail the bioanalytical procedure and summarizes the validation results obtained. The samples were extracted using liquid--liquid extraction with a mixture of 1-chlorobutane-isopropanol-ethyl acetate (88:2:10, v/v/v). The chromatographic separation was performed on a reversed-phase Hypersil ODScolumn (250 x 4.6 mm i.d.; 5 microm particle size) using a mobile phase consisting of formic acid 0.05 M-acetonitrile (28:72, v/v), pumped at a flow rate of 0.3 ml min(-1) heated to 25 degrees C. The analytes were detected using an API 3000 triple quadrupole mass spectrometer with positive electrospray ionization in multiple reaction monitoring mode. Tandem mass spectrometric detection enabled the quantitation of glimepiride down to 0.50 ng mL(-1). Calibration graphs were linear (r better than 0.998, n=1), in concentration range 0.50--1000 ng mL(-1), and the intra- and inter- day RSD values were less than 10.37 and 11.55% for glimepiride. The method was successfully applied to a kinetic study in order to assess the main pharmacokinetic parameters of glimepiride.

The exact mechanism of the efficacy of glimepiride in the achievement of glycemic control has not yet been clearly defined.

This study was conducted to examine the influence of glimepiride on insulin secretion and sensitivity in patients with type 2 diabetes mellitus (DM) of recent onset.

This 24-week, open-label, controlled trial was conducted at the Division of Endocrinology and Metabolism, Veterans Affairs Medical Center (Phoenix, Arizona). Study participants were aged 32 to 75 years and had recent-onset (established by a short duration of symptoms 6 weeks to 6 months prior to the study) type 2 DM, or were age-matched healthy volunteers (control group). In the diabetic patients, glimepiride tablets were administered orally, initially at 2 mg once daily in the morning, with the dosage increased by 1 mg every 2 weeks until fasting plasma glucose (FPG) decreased to 6.7 mmol/L; the dosage was then maintained for the remainder of the 24-week study period. Oral glucose tolerance tests (OGTTs) were conducted in the control group and before treatment and at 24 weeks after the achievement and maintenance of glycemic control (glycosylated hemoglobin <7.0%) in the diabetic group. For OGTT, plasma insulin and glucose levels were determined after the subjects fasted overnight and then at every 15 minutes for 2 hours after glucose challenge.

Fourteen diabetic men (mean [SEM] age, 50 [6] years; range, 32-75 years) and 10 male healthy controls (mean [SD] age, 48 [5] years; range, 30-68 years) were enrolled. In the DM group, FPG decreased significantly after treatment ( P<0.001); fasting plasma insulin was markedly elevated before treatment (P<0.001 vs controls) and decreased after treatment ( P<0.01) but did not normalize; first-phase insulin secretion was markedly inhibited before treatment ( P<0.001 vs controls) and normalized after treatment ( P<0.001) total insulin secretion significantly improved after treatment ( P<0.01) but did not normalize. Finally, the pretreatment insulin sensitivity index decreased significantly (P<0.01) after treatment and normalized in 6 of 14 patients (42.9%) with type 2 DM.

In this study, glimepiride achieved desirable glycemic control in patients with recent-onset type 2 DM through improvement in insulin secretion and sensitivity.

Glimepiride has the lowest ratio of insulin release to glucose decrease compared with other sulphonylureas. This prompted us to study in vitro and in vivo in a placebo-controlled study the effect of glimepiride on the redox-sensitive transcription factor nuclear factor-kappa B (NF-kappaB).

Fifteen patients with type 2 diabetes on glibenclamide with a stable HbA1c over the last 6 months were included. After sampling for determination of baseline values, 10 patients were changed to an equivalent dose of glimepiride, while the placebo group was maintained at glibenclamide plus placebo. The glimepiride dose in these patients was adjusted so that no change in glucose control occurred, allowing for direct comparison. The others were kept on glibenclamide and received additional placebo. After 4 weeks of glimepiride or glibenclamide plus placebo, a second blood sample was taken. Mononuclear cells were isolated and assayed in a tissue-culture-independent electrophoretic mobility shift assay (EMSA)-based detection system for NF-kappaB binding activity, and by Western Blot for nuclear localization of NF-kappaB-p65, the cytoplasmic content of IkappaBalpha and the NF-kappaB-controlled haemoxygenase-1. Glimepiride dose-dependent inhibition of carboxymethyllysin (CML) albumin or tumour necrosis factor alpha (TNFalpha)- and H2O2-induced activation of NF-kappaB binding were determined, using isolated peripheral blood mononuclear cells from healthy volunteers, and transcriptional activity of bovine aortic endothelial cells either left untreated or induced with CML albumin incubated with or without glimepiride. Furthermore, in-vitro studies were implemented to demonstrate radical quenching properties of glimepiride in the cell-free 2,2'-azo-bis(2-aminopropane)-dihydrochloride system.

Baseline glucose and HbA1c remained stable in the patients switched from glibenclamide to a corresponding dose of glimepiride or kept on glibenclamide plus placebo. While in the group of patients only taking glibenclamide plus placebo the NF-kappaB binding activity did not change significantly (p = 0.58), the NF-kappaB binding activity in the group of patients taking glimepiride was reduced from 19.3 relative NF-kappaB-p65-equivalents to 15.5 relative NF-kappaB-p65-equivalents (p = 0.04). The nuclear translocation of NF-kappaB-p65 was reduced from 100% at baseline to 58% after 4 weeks (p = 0.04); the cytoplasmic localization of NF-kappaB-p65 increased from 100% to 129% (p = 0.03) and the cytoplasmic content of IkappaBalpha increased from 100% to 109% (p = 0.06). The redox-sensitive haemoxygenase-1 antigen was reduced from 100% to 82% (p = 0.04). To prove directly that glimepiride reduces NF-kappaB activation, we isolated peripheral blood mononuclear cells (PBMC) from healthy volunteers. In vitro, glimepiride reduced TNFalpha-(1 nmol/l) and CML albumin (800 nmol/l)-induced NF-kappaB activation dose dependently, being half maximal at 120 micromol/l. H2O2-mediated NF-kappaB activation was only partially reduced. In addition, glimepiride reduced NF-kappaB-dependent gene expression using a NF-kappaB-driven luciferase reporter system. Finally, a cell-free detection system showed that glimepiride has radical quenching properties.

Glimepiride can affect the activation of the redox-sensitive transcription factor NF-kappaB in vitro and in vivo.

In the last few years, there has been increasing interest in the physiological role of acylation-stimulating protein (ASP). Recent studies in rats and mice, in particular in C3 (-/-) mice that are ASP deficient, have advanced our understanding of the role of ASP. Of note, the background strain of the mice influences the phenotype of delayed postprandial triglyceride clearance in ASP-deficient mice. Administration of ASP in all types of lean and obese mice studied to date, however, enhances postprandial triglyceride clearance. On the other hand, regardless of the background strain, ASP-deficient mice demonstrate reduced body weight, reduced leptin and reduced adipose tissue mass, suggesting that ASP deficiency results in protection against development of obesity. In humans, a number of studies have examined the relationship between ASP, obesity, diabetes and dyslipidemia as well as the influence of diet, exercise and pharmacological therapy. While many of these studies have small subject numbers, interesting observations may help us to better understand the parameters that may influence ASP production and ASP action. The aim of the present review is to provide a comprehensive overview of the recent literature on ASP, with particular emphasis on those studies carried out in rodents and humans.

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.

To examine the effect of glimepiride on insulin-stimulated glycogen synthesis in cultured human skeletal muscle cells in comparison with glibenclamide.

Myotubes derived from glucose-tolerant subjects were incubated with glimepiride or glibenclamide (0-100 micro mol/l) for 4 h and with or without insulin (100 nmol/l) for 2 h, and subsequently glycogen synthesis was determined.

Glimepiride had no significant effect on basal glycogen synthesis; in contrast, glimepiride caused a dose-dependent increase of insulin-stimulated glycogen synthesis, with a maximal effect of 39.97 +/- 8.4% (mean +/- SEM, n = 4, P < 0,02). The time course of this glimepiride effect on insulin-stimulated glycogen synthesis showed a peak after 12 h incubation with a half maximal effect after 4 h. Preincubation of the myotubes with wortmannin (100 nmol/l), an inhibitor of phosphatidylinositol (PI)- 3 kinase, caused an inhibition of this glimepiride effect on insulin-stimulated glycogen synthesis. In contrast to glimepiride, incubation of myotubes with glibenclamide (0-100nmol/l), a second generation sulfonylurea, had no significant effect on basal or insulin-stimulated glycogen synthesis.

Incubation of cultured human skeletal muscle cells derived from glucose-tolerant subjects with glimepiride caused a dose-dependent increase of insulin-stimulated glycogen synthesis using therapeutic glimepiride concentrations. This glimepiride effect seems to be mediated via the PI3 kinase pathway. In contrast to glimepiride, glibenclamide had no significant effect on basal or insulin-stimulated glycogen synthesis. These results suggest that glimepiride, beside its well-known effect to stimulate insulin secretion, possess an insulin-sensitizing action in cultured human skeletal muscle cells in support of the concept of an extrapancreatic action of glimepiride.

The critical role of the heterogeneous nature of cellular plasma membranes in transmembrane signal transduction has become increasingly appreciated during the past decade. Areas of relatively disordered, loosely packed phospholipids are disrupted by hydrophobic detergent/carbonate-insoluble glycolipid-enriched raft microdomains (DIGs) of highly ordered (glyco)sphingolipids and cholesterol. DIGs exhibit low buoyant density and are often enriched in glycosylphosphatidylinositol-anchored plasma membrane proteins (GPI proteins), dually acylated signalling proteins, such as non-receptor tyrosine kinases (NRTKs), and caveolin. At least two types of DIGs, hcDIGs and lcDIGs, can be discriminated on basis of higher and lower content, respectively, of these typical DIGs components. In quiescent differentiated cells, GPI proteins and NRTKs are mainly associated with hcDIGs, however, in adipose cells certain insulin-mimetic stimuli trigger redistribution of subsets of GPI proteins and NRTKs from hcDIGs to lcDIGs. Presumably, these stimuli induce displacement of GPI proteins from a GPI receptor located at hcDIGs whereas simultaneously NRTKs dissociate from a complex with caveolin located at hcDIGs, too. NRTKs are thereby activated and, in turn, modulate intracellular signalling pathways, such as stimulation of metabolic insulin signalling in insulin-sensitive cells. The apparent dynamics of DIGs may provide a target mechanism for regulating the activity of lipid-modified signalling proteins by small drug molecules, as exemplified by the sulfonylurea, glimepiride, which lowers blood glucose in an insulin-independent fashion, in part.

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.

Glimepiride (Amaryl), which is a new oral antidiabetic drug in the sulfonylurea class, was analysed by using second order derivative UV spectrophotometry. The quantification of glimepiride in dimethylformamide was performed in the wavelength range of 245-290 nm at N = 6, ?lambda = 21. The second order derivative spectra was calculated using peak to peak (lambdaDMF = 263.3-268.2 nm), peak to zero (lambdaDMF = 268.2 nm) and tangent (lambdaDMF = 263.3-271.8 nm) method for calibration curves, the linearity range of 1.00-500.00 microg ml(-1) by using the second order derivative UV spectrophotometric method. The developed method was applied to directly and easily to the analysis of the pharmaceutical tablet preparations. R.S.D. were found to be 4.18% (Amaryl tablet; 1 mg) and 2.21% (Amaryl tablet; 2 mg). The method was completely validated and proven to be rugged. The limit of quantitation and the limit of detection were found as 1.00 and 0.4 microg ml(-1), respectively. This validated derivative UV spectrophotometric method is potentially useful for a routine laboratory because of its simplicity, rapidity, sensitivity, precision and accuracy.

Diabetes mellitus type 2 is a world-wide growing health problem affecting more than 150 million people at the beginning of the new millennium. It is believed that this number will double in the next 25 yr. The pathophysiological hallmarks of type 2 diabetes mellitus consist of insulin resistance, pancreatic beta-cell dysfunction, and increased endogenous glucose production. To reduce the marked increase of cardiovascular mortality of type 2 diabetic subjects, optimal treatment aims at normalization of body weight, glycemia, blood pressure, and lipidemia. This review focuses on the pathophysiology and molecular pathogenesis of insulin resistance and on the capability of antihyperglycemic pharmacological agents to treat insulin resistance, i.e., a-glucosidase inhibitors, biguanides, thiazolidinediones, sulfonylureas, and insulin. Finally, a rational treatment approach is proposed based on the dynamic pathophysiological abnormalities of this highly heterogeneous and progressive disease.

K(+) channel effectors are widely used in the treatment of various diseases, including diabetes mellitus type II, hypertension, and cardiac arrhythmia. In addition, a constantly growing body of literature reveals that some of these substances, despite their direct effect on K(+) channels, may influence cellular lipid metabolism. As a result, membrane lipid content and cellular concentrations of lipid messengers are changed. Due to the dependence of K(+) channel activity on membrane lipids, these observations seem to be of particular importance not only to characterize secondary effects of K(+) channel effectors but also to understand the long-term effects of these agents on K(+) channel activity.

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.

Caveolin has been shown to play an important role in signal transduction and nitric oxide synthase production. The purpose of this study was to investigate whether caveolin was tyrosine phosphorylated or activated by shear stress or cyclic strain in bovine aortic endothelial cells (BAECs).

BAECs were subjected to an average of 10% strain at a rate of 60 cycles/min or a laminar shear stress of 10 dyn/cm(2) for up to 4 h. Immunoblotting with anticaveolin antibody was performed to assess activation of caveolin. Coimmunoprecipitation of anticaveolin antibody with anti-tyrosine phosphorylation antibody was performed to detect the tyrosine phosphorylation of caveolin.

Neither cyclic strain nor shear stress at physiologic levels altered the level of caveolin protein. Tyrosine phosphorylation of caveolin could not be observed at any time under either cyclic strain or shear stress condition.

Although hemodynamic forces alter nitric oxide synthase production and activate signal transduction, caveolin levels or activity is not altered in endothelial cells exposed to shear stress or cyclic strain.

Caveolin-1, a scaffolding protein of caveolae, is known to be tyrosine-phosphorylated by Src kinases. Recently we generated a specific antibody to caveolin-1 phosphorylated at tyrosine-14 (PY14) (R. Nomura and T. Fujimoto, 1999, Mol. Biol. Cell 10, 975-986). In the present study, by applying PY14 to sections of normal rat tissues, we found that tyrosine phosphorylation of caveolin-1 occurred in limited locations, including the endothelium of the continuous capillaries and small venules. Cultured endothelial cells were not labeled by PY14 under a standard culture condition, but became positively labeled when exposed to oxidative stresses and/or tyrosine phosphatase inhibitors. The reaction was prohibited by pretreating the cells with herbimycin A or genistein. Vasoactive reagents or physical stimuli did not cause the phosphorylation. Concomitant with the tyrosine phosphorylation, the number of invaginated caveolae decreased drastically, and vesicles labeled intensely for caveolin-1 appeared in the cytoplasm; the average diameter of the vesicles was larger than that of caveolae. The result implies that tyrosine phosphorylation of caveolin-1 occurs at tyrosine-14 in the normal rat endothelium in vivo and may induce caveolar vesiculation and/or fusion.

A regulatory role for intracellular Ca2+ ([Ca2+]i) in adipocyte lipogenesis, lipolysis and triglyceride accumulation has been demonstrated. Compounds acting on the pancreatic sulfonylurea receptor (SUR) to increase (e.g., glibenclamide) or decrease (e.g., diazoxide) [Ca2+]i cause corresponding increases and decreases in weight gain. However, these weight gain and loss effects have been attributed to insulin release rather than to the primary effects of these compounds on the adipocyte SUR and its associated K(ATP) channel. Accordingly, we have evaluated the direct role of the human adipocyte SUR in regulating adipocyte metabolism. We used RT-PCR with primers for a highly conserved region of SUR1 to demonstrate that human adipocytes express SUR1. The PCR product was confirmed by sequence analysis and used as a probe to demonstrate adipocyte SUR1 expression by Northern blot analysis. Adipocytes exhibited glibenclamide dose-responsive (0-20 microM) increases in [Ca2+]i (P<0.05). Similarly, glibenclamide (10 microM) caused a 67% increase in adipocyte fatty acid synthase activity (P<0.001), a 48% increase in glycerol-3-phosphate dehydrogenase activity (P<0.01) and a 68% inhibition in lipolysis (P<0.01), whereas diazoxide (10 microM) completely prevented each of these effects. These data demonstrate that human adipocytes express a SUR that regulates [Ca2+]i and, consequently, exerts coordinate control over lipogenesis and lipolysis. Accordingly, the adipocyte SUR1 may represent an important target for the development of therapeutic interventions in obesity.

Caveolae are specialized plasmalemmal microdomains originally studied in numerous cell types for their involvement in the transcytosis of macromolecules. They are enriched in glycosphingolipids, cholesterol, sphingomyelin, and lipid-anchored membrane proteins, and they are characterized by a light buoyant density and resistance to solubilization by Triton X-100 at 4 degreesC. Once the identification of the marker protein caveolin made it possible to purify this specialized membrane domain, it was discovered that caveolae also contain a variety of signal transduction molecules. This includes G protein-coupled receptors, G proteins and adenylyl cyclase, molecules involved in the regulation of intracellular calcium homeostasis, and their effectors including the endothelial isoform of nitric oxide synthase, multiple components of the tyrosine kinase-mitogen-activated protein kinase pathway, and numerous lipid signaling molecules. More recent work has indicated that caveolae further serve to compartmentalize, modulate, and integrate signaling events at the cell surface. This specialized plasmalemmal domain warrants direct consideration in future investigations of both normal and pathological signal transduction in pulmonary cell types.

The cell biology of caveolae is a rapidly growing area of biomedical research. Caveolae are known primarily for their ability to transport molecules across endothelial cells, but modern cellular techniques have dramatically extended our view of caveolae. They form a unique endocytic and exocytic compartment at the surface of most cells and are capable of importing molecules and delivering them to specific locations within the cell, exporting molecules to extracellular space, and compartmentalizing a variety of signaling activities. They are not simply an endocytic device with a peculiar membrane shape but constitute an entire membrane system with multiple functions essential for the cell. Specific diseases attack this system: Pathogens have been identified that use it as a means of gaining entrance to the cell. Trying to understand the full range of functions of caveolae challenges our basic instincts about the cell.

To review the clinical pharmacology data regarding the sulfonylurea glimepiride, and to summarize the clinical trials of glimepiride efficacy and safety alone and in combination with insulin for the treatment of type 2 diabetes mellitus.

A MEDLINE database search (English language, January 1985-April 1997) was performed to identify relevant published articles, including reviews and abstracts; the manufacturer (Hoechst Marion Roussel, Kansas City, MO) provided unpublished data.

Pharmacology information was taken from representative original research articles. Eight clinical studies were selected for analysis on the basis of large enrollment, appropriate study design, and publication of results.

All clinical trials, published and unpublished, were reviewed.

Glimepiride is a sulfonylurea that is pharmacologically distinct from other sulfonylureas because of differences in receptor-binding properties and potentially selective effects on ATP-sensitive K+ channels. The pharmacokinetic and pharmacodynamic profile of glimepiride makes it suitable for once-daily dosing. The safety and efficacy of glimepiride have been confirmed in studies involving more than 5000 patients with type 2 diabetes. In one study, once-daily doses of 1-8 mg reduced fasting plasma glucose from baseline by 43-74 mg/dL more than did placebo (p < 0.001), and hemoglobin (Hb) A1C values decreased by 1.2-1.9% more than with placebo (p < 0.001). Two-thirds of patients achieved tight control (i.e., HbA1C < or = 7.2%). Glimepiride was as effective as second-generation sulfonylureas. The most common adverse events were dizziness and headache, but no single adverse event occurred in more than 2% of patients.

Glimepiride appears to be a useful option for patients with type 2 diabetes not controlled by diet and exercise and who want to achieve tight glucose control. Glimepiride can be used alone, in combination with other antihyperglycemic agents, or in patients with secondary sulfonylurea failure, as an adjunct to insulin therapy.

Glimepiride is a sulphonylurea agent that stimulates insulin release from pancreatic beta-cells and may act via extrapancreatic mechanisms. It is administered once daily to patients with type 2 (non-insulin-dependent) diabetes mellitus in whom glycaemia is not controlled by diet and exercise alone, and may be combined with insulin in patients with secondary sulphonylurea failure. The greatest blood glucose lowering effects of glimepiride occur in the first 4 hours after the dose. Glimepiride has fewer and less severe effects on cardiovascular variables than glibenclamide (glyburide). Pharmacokinetics are mainly unaltered in elderly patients or those with renal or liver disease. Few drug interactions with glimepiride have been documented. In patients with type 2 diabetes, glimepiride has an effective dosage range of 0.5 to 8 mg/day, although there is little difference in efficacy between dosages of 4 and 8 mg/day. Glimepiride was similar in efficacy to glibenclamide and glipizide in 1-year studies. However, glimepiride appears to reduce blood glucose more rapidly than glipizide over the first few weeks of treatment. Glimepiride and gliclazide were compared in patients with good glycaemic control at baseline in a 14-week study that noted no differences between their effects. Glimepiride plus insulin was as effective as insulin plus placebo in helping patients with secondary sulphonylurea failure to reach a fasting blood glucose target level of < or = 7.8 mmol/L, although lower insulin dosages and more rapid effects on glycaemia were seen with glimepiride. Although glimepiride monotherapy was generally well tolerated, hypoglycaemia occurred in 10 to 20% of patients treated for < or = 1 year and > or = 50% of patients receiving concomitant insulin for 6 months. Pooled clinical trial data suggest that glimepiride may have a lower incidence of hypoglycaemia than glibenclamide, particularly in the first month of treatment. Dosage is usually started at 1 mg/day, titrated to glycaemic control at 1- to 2-week intervals to a usual dosage range of 1 to 4 mg/day (maximum 6 mg/day in the UK or 8 mg/day in the US).

Glimepiride is a conveniently administered alternative to other sulphonylureas in patients with type 2 diabetes mellitus not well controlled by diet alone. Its possible tolerability advantages and use in combination with other oral antidiabetic drugs require further study. Glimepiride is also reported to reduce exogenous insulin requirements in patients with secondary sulphonylurea failure when administered in combination with insulin.

A novel phosphoinositolglycan-peptide (PIG-P) from the yeast Saccharomyces cerevisiae potently mimicks insulin action on glucose transport and metabolism in rat muscle and adipose tissue. The aim of the present study was to elucidate the cellular signalling pathways of this insulin-mimetic compound. Rapid onset and reversibility of PIG-P action on glucose transport were observed in isolated adipocytes with a half-time of transport stimulation of 6-8 min (insulin less than 5 min). Combined treatment with PIG-P and insulin indicated additive stimulation of glucose transport at submaximal concentrations and non-additive action of both agents at maximal doses. The tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) was markedly increased in response to PIG-P in rat cardiomyocytes without any effect on the tyrosine phosphorylation of the insulin receptor beta-subunit. PIG-P action in these cells was accompanied by phosphorylation/dephosphorylation of several proteins with molecular masses of 15-30 kDa, a response not detected with insulin. Downstream signalling of IRS-1 was then analysed by monitoring IRS-1-associated phosphatidylinositol 3-kinase (PI 3-kinase) activity in cardiomyocytes. A stable (2 and 15 min incubation with PIG-P) 7-fold stimulation corresponding to about 50% of insulin action could be detected. Increased tyrosine phosphorylation of IRS-1 and enhanced PI 3-kinase activity in response to PIG-P independent of the insulin receptor was also observed in isolated adipocytes. Involvement of PI 3-kinase in PIG-P action was subsequently confirmed by the dose-dependent inhibition of PIG-P-activated glucose transport in rat diaphragm and adipocytes by the PI 3-kinase inhibitors wortmannin and LY294002. These data suggest divergent upstream signalling by insulin and PIG-P involving phosphoproteins not affected by insulin. However, PIG-P and insulin action converge at the level of IRS-1 inducing insulin-independent PI 3-kinase-mediated signalling to glucose transport.

Polar headgroups of free glycosyl-phosphatidylinositol (GPI) lipids or protein-bound GPI membrane anchors have been shown to exhibit insulin-mimetic activity in different cell types. However, elucidation of the molecular mode of action of these phospho-inositolglycan (PIG) molecules has been hampered by 1) lack of knowledge of their exact structure; 2) variable action profiles; and 3) rather modest effects. In the present study, these problems were circumvented by preparation of PIG-peptides (PIG-P) in sufficient quantity by sequential proteolytic (V8 protease) and lipolytic (phosphatidylinositol-specific phospholipase C) cleavage of the GPI-anchored plasma membrane protein, Gce1p, from the yeast Saccharomyces cerevisiae. The structure of the resulting PIG-P, NH2-Tyr-Cys-Asn-ethanolamine-PO4-6(Man1-2)Man1-2Man1-+ ++6Man1-4GlcNH(2)1-6myo-inositol-1,2-cyclicPO4, was revealed by amino acid analysis and Dionex exchange chromatography of fragments generated enzymatically or chemically from the neutral glycan core and is in accordance with the known consensus structures of yeast GPI anchors. PIG-P stimulated glucose transport and lipogenesis in normal, desensitized and receptor-depleted isolated rat adipocytes, increased glycerol-3-phosphate acyltransferase activity and translocation of the glucose transporter isoform 4, and inhibited isoproterenol-induced lipolysis and protein kinase A activation in adipocytes. Furthermore, PIG-P was found to stimulate glucose transport in isolated rat cardiomyocytes and glycogenesis and glycogen synthase in isolated rat diaphragms. The concentration-dependent effects of the PIG-P reached 70-90% of the maximal insulin activity with EC50-values of 0.5-5 microM. Chemical or enzymic cleavages within the glycan or peptide portion of the PIG-P led to decrease or loss of activity. The data demonstrate that PIG-P exhibits a potent insulin-mimetic activity which covers a broad spectrum of metabolic insulin actions on glucose transport and metabolism.