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Diabetes Research and Clinical Practice 28 Suppl. (1995) S115-S137 DIABETES RESEARCH AND CLINICAL PRACTICE Extrapancreatic effects of sulfonylureas — a comparison between glimepiride and conventional sulfonylureas Gunter Muller*a, Yusuke Satohb, Karl Geisena a Hoechst AG Frankfurt, Pharmaceutical Research Divison, SBU Metabolic Diseases, H 825, 65926 Frankfurt, Germany b Hoechst Japan Limited, Pharma Research and Development Divison, Kawagoe, Saitama 350-11 Japan Abstract The contribution of extrapancreatic effects of sulfonylureas to the blood glucose-decreasing activity was reevaluated in vivo and in vitro with several conventional sulfonylureas and with the new one glimepiride. In vivo, in dogs, after single approximately equipotent blood glucose-decreasing doses, the sulfonylureas were tested for a ranking in the ratios of mean plasma insulin-increasing and blood glucose-decreasing activity. Studies were also performed in hyperglycemic hyperinsulinemic KK-Ay mice under once daily treatment for 8 weeks. In vitro, glimepiride and glibenclamide were tested for the ranking of their extrapancreatic activity with respect to the stimulation of glucose transport and glucose metabolizing processes in normal and insulin-resistant fat cells as well as in the isolated diaphragm. Furthermore, in vitro studies were performed, especially with glimepiride, in order to characterize the molecular mechanism for the extrapancreatic activity. The dog studies revealed a marked ranking in the ratios of plasma insulin-increasing and blood glucose-decreasing activity between the different sulfonyiureas (glimepiride < glipizide < gliclazide < glibenclamide). In the hyperglycemic hyperinsulinemic KK -Ay mice, glimepiride reduced blood glucose by 40%, plasma insulin by 50% and HBAlc by 33%, whereas glibenclamide and gliclazide had no effect on these parameters. In vitro, glimepiride and glibenclamide had extrapancreatic effects within the lower uM range, with glimepiride exhibiting 2-3-fold lower ED50 values than glibenclamide. In the absence of insulin, both stimulated glucose transport — up to 60% of the maximum insulin response in the rat diaphragm and up to 35% in 3T3 adipocytes. Glycogenesis was stimulated in the rat diaphragm — up to 55% of the maximum insulin effect; lipogenesis in 3T3 adipocytes — up to 40%. The studies on the molecular mechanism of extrapancreatic activity with rat adipocytes and diaphragm suggest that these direct insulin -mimetic effects rely on the induction of GLUT4 translocation from internal stores to the plasma membrane and on the activation of the key metabolic enzymes, glycogen synthase and glycerol-3-phosphate acyltransferase. These processes occur within the same drug concentration range and with the same ranking between glimepiride and glibenclamide as observed for glucose utilization and transport. The direct effects of sulfonylureas may ultimately be regulated by a glycosyl-phosphatidylinositol-specific phospholipase C, shown to be activated by glimepiride in rat adipocytes. Lipolytic cleavage products thereby generated from glycolipidic structures may in turn stimulate specific protein phosphatases which activate key regulatory proteins/enzymes of glucose and lipid metabolism. Conclusion: From the ranking of the sulfonylureas in the ratio of mean insulin-releasing and mean blood glucose decreasing-activity at single doses in vivo, it is deduced that sulfonylureas have a variable degree of blood glucose decreasing-acivity which is independent of their insulin secretion-stimulating activity. The different degree of this extrapancreatic activity in vivo is confirmed * Corresponding author, Tel.: +49 069 3054271; Fax: +49 069 311454. 0168-8227/95/$09.50 © 1995 Elsevier Science Ireland Ltd. All rights reserved. SSDI 0168-8227(95)01089-U G. Müller et al. /Diabetes Research and Clinical Practice 28 Suppl. (1995) S115-S137 S116 by a corresponding ranking in stimulation of glucose utilization in vitro. Dephosphorylation and activation of key enzymes of glucose transport and metabolism induced by a glycosyl phosphatidylinositol-specific phospholipase C might be the molecular basis for the extrapancreatic activity of sulfonylureas. Keywords: cAMP Glibenclamide; Glimepiride; Insulin action; Adipocytes; Diaphragm; Glucose transport; Metabolism; 1. Introduction Since the introduction of sulfonylureas into the treatment of non-insulin-dependent diabetes mellitus (NIDDM) about four decades ago, there has been some debate as to whether their blood glucose-decreasing activity is based solely on the stimulation of insulin release—or—is-also -due to extrapancreatic activity (for reviews see Refs. 1 and 2). The observation that, after long-term treatment of NIDDM patients with sulfonylurea compounds, plasma insulin levels often return to pre-treatment levels without loss of improved blood glucose control, raised suspicion of extrapancreatic effects [3-5], especially since, in in vitro studies with isolated or cultured muscle and fat cells, direct (as assayed in the absence of insulin) stimulation of glucose transport and metabolism was demonstrated [6-14]. The interpretation of this long-term effect of sulfonylureas in patients as extrapancreatic activity [15-17] has, however, been questioned. Blood glucose control without adequately elevated mean plasma insulin levels has rather been explained as a drug-induced postprandial kick of insulin release resulting in lower mean blood glucose values and thereby also reduced basal insulin levels [18-20]. Furthermore, long-term treatment with sulfonylureas has been suspected of reducing insulin resistance by normalizing blood glucose and/or basal insulin levels since permanently increased blood glucose and/or basal plasma insulin levels are said to induce insulin resistance in peripheral tissues of Type II diabetics [21-23] and of normal [24] and diabetic rats [25]. The molecular mechanism for desensitization due to increased mean glucose or basal insulin concentrations has been considered to reside within receptor and post-receptor steps of the insulin signaling cascade [26-28] (e.g. phosphorylation and inactivation of IRS-129). With different sulfonylureas, we have tried in vivo and in vitro to re-evaluate the extrapancreatic activity. In addition, we have attempted to elucidate the underlying molecular mechanism. We hypothetised that the ratio of mean plasma insulin increase and mean blood glucose decrease should be the same for all sulfonylureas if time response curves for blood glucose decrease and plasma insulin increase had a similar shape and blood glucose were solely decreased by insulin release. Ranking of plasma insulin/blood glucose ratios, however, would be an indication of extrapancreatic blood glucose decreasing activity which does not rely on additional insulin release. Furthermore, we postulated that in vivo ranking in plasma insulin/blood glucose ratios should also be reflected in extrapancreatic activity in vitro of the sulfonylureas tested. Identical ranking in vivo and in vitro would indicate that the cellular sites of action of sulfonylureas, as identified in the in vitro studies, are responsible for the extrapancreatic activity in vivo. In vivo, to exclude the postulated indirect effect of sulfonylureas via metabolic improvement during long-term treatment, we performed the studies at single approximately equipotent blood glucose-decreasing doses. Study animals were normal fasted dogs. Study parameters were the mean plasma insulin-increasing and mean blood glucose-decreasing activity during the whole study period. The sulfonylureas tested were glibenclamide, gliclazide, glipizide and glimepiride. Glimepiride is a new sulfonylurea. Its pharmacological characterization is presented elsewhere [30]. Glimepiride, glibenclamide and gliclazide were additionally studied subchronically in diabetic hyperinsulinemic KK-Ay-mice for their ef- G. Müller et al /Diabetes Research and Clinical Practice 28 Supp. (1995)S115-S137 feet on plasma insulin, blood glucose and HBAlc. In vitro, glimepiride and glibenclamide were examined in normal and insulin-resistant fat cells and in the isolated diaphragm. Potency ratios and EC50 values of dose response curves were used as ranking parameters for the extrapancreatic activity in vitro. The in vitro studies on the molecular mechanism of the extrapancreatic activity were performed with isolated and cultured fat cells predominantly using glimepiride. 2. Experimental procedures 2. 1 Biological Methods which have already been published were used for the determination of blood glucose, plasma insulin and glycated hemoglobin [30,31]; for preparation of rat diaphragms and adipocytes [32]; for differentiation of 3T3 adipocytes [33]; for primary culture [34], metabolic labeling with 32Pi or myo-[14C]inositol and cellular fractination of rat adipocytes [35,36]; for immunoblotting and immunoprecipitation of GLUT4 [35]; for assays of 2-deoxyglucose or 3-omethylglucose transport [35], lipogenesis, glycogenesis, glycogen synthase, glycerol-3-phosphate acyltransferase, protein kinase A, low Km cAMP-specific phosphodiesterase [32] and glycosyl-phosphatidylinositol-specific phospholipase C [33,3.6]. S117 with confidence limits of two drugs were determined as the ratio of equieffective concentrations of each drug from the respective graded dose-response relations [37]. The level of significance for all test procedures was 5%. 3. Results 3.1 Extrapancreatic activity in vivo After intravenous administration of 60 ug/kg to normal fasted dogs, glimepiride lowered the blood glucose markedly less than glibenclamide up to 16 h, and thereafter up to 51 h, twice as much as glibenclamide (Fig. 1). Over the whole study period, glimepiride had a total blood glucose-decreasing activity of 18,4 ±1,8% (mean S.E.M.), that of glibenclamide being 11.7 + 2.6%. Both compounds showed a plasma insulin peak after 30 min, the peak value after glibenclamide being twice as high as that after glimepiride. After glibenclamide, insulin gradually declined up to the end of the study, whereas, 2 h after 2.2. Statistical Raw data were used for the evaluation of plasma insulin, and raw data or raw data expressed as a percent of the initial values were used to evaluate the blood glucose. Mean plasma insulin and mean blood glucose changes over the whole study period were calculated by the trapezoidal rule. In case of dose-response curves for plasma insulin and blood glucose, the equipotent mean blood glucose decrease and corresponding mean plasma insulin change over the whole study period were each taken from the respective regression lines. For comparison of means of several groups, analysis of variance was performed and subsequently Duncan's multiple range test [37]. EC50 values were determined by a fourparameter logistic function fit [38]. Potency ratios Fig. 1. Time course for blood glucose and plasma insulin levels in fasted male beagle dogs after a single intravenous dose of glimepiride or glibenclamide as clear aqueous sodium salt solutions (mean values), cf. Fig. 2, Exp. 1. S118 G. Muller et al, /Diabetes Research and Clinical Practice 28 Suppl.(1995) S115-S137 Fig. 2. Ratios of mean plasma insulin increase/mean percentage blood glucose decrease (PI/BG ratio) over the whole study period in fasted male beagle dogs after a single oral dose of different sulfonylureas at approximately equipotent blood glucose-decreasing doses. administration of glimepiride, insulin did not differ markedly from the control. The mean plasma insulin concentration after glimepiride was decreased by about 1.06 ± 1.84 U/ml, and after glibenclamide it was increased by about 1.34 ± 1.62 U/ml. The ratio for plasma insulin change and blood glucose decrease after glimepiride was < 0, and after glibenclamide 0.12 (Fig. 2, Exp. 1). After oral administration of 90 g/kg to normal fasted dogs, the blood glucose decrease induced by glimepiride was initially faster than that induced by glibenclamide; subsequently, up to 16 h it was lower, and thereafter up to 48 h, it was two times greater than that induced by glibenclamide (Fig. 3). Over the whole study period, glimepiride had a total blood glucose-decreasing activity of 15.3 ± 1.2%, that of glibenclamide being 15.7 ± 2.4%. Both compounds showed a plasma insulin peak after 60 min, this being 37.7 ± 7.4 U/ml for glibenclamide and G. Muller et al. / Diabetes Research and Clinical Practice 28 Suppl. (1995) S115S137 Fig. 3. Time course for blood glucose and plasma insulin levels in fasted male beagle dogs after a single oral dose of glimepiride or glibenclamide as clear aqueous sodium salt solutions (mean values), cf. Fig. 2, Exp. 2. 31.1 ± 4.3 U/ml for glimepiride. After glimepiride, the plasma insulin values declined within 3 h to those of the controls; after glibenclamide they gradually declined up to the end of study. The mean total plasma insulin increase after glimepiride was about 0.41 ± 0.47 U/ml, that after glibenclamide being about 2.70 ± 1.44. The ratio for plasma insulin change and blood glucose decrease after glimepiride was 0.03 and after glibenclamide 0.17 (Fig. 2, Exp. 2). After oral administration to dogs, glimepiride, at single doses of 0.01, 0.03 or 0.1 mg/kg, and gliclazide, at single doses of 0.4, 1.2 or 4.0 mg/kg, both decreased the blood glucose dose-dependently almost to the same degree (Fig. 4). However, the corresponding plasma insulin levels with glimepiride at all doses were significantly lower than those with gliclazide. The regression lines through the mean percentage blood glucose decrease over 8 h yielded an ED 25 of 0.056 mg/kg for glimepiride, and one of 1.5 mg/kg for gliclazide. The corresponding mean plasma insulin increase was 0.88 U/ml for glimepiride and 3.70 S119 U/ml for gliclazide, resulting in a total plasma insulin increase/total blood glucose decrease ratio of 0.04 for glimepiride and 0.15 for gliclazide (Fig. 2, Exp. 4). After oral administration of glimepiride or glibenclamide at doses of 90 g/kg, or of gliclazide or glipizide at doses of 1.8 and 180 g/kg, respectively, to dogs, glimepiride had the highest total blood glucose-lowering activity and lowest insulin-releasing activity; glipizide had the lowest total blood glucose-lowering activity and highest insulin-releasing activity (Fig. 5). The ranking in maximal blood glucose decrease was: glibenclamide > gliclazide = glipizide > glimepiride, the ranking in mean total blood glucose decrease, however, being: glimepiride (-20.4 ± 1.5%) > gibenclamide (-19.0 ± 1.0%) > gliclazide (16.8 ± 1.2%) > glipizide (-13.0 ± 2.2%). The ranking in plasma insulin peak height was: glipizide > gliclazide > glibenclamide > glimepiride, the ranking in mean total plasma insulin increase being gliclazide (12.2 ± 0.7 U/ml) < glimepiride (12.4 ± 1.0 U/ml) < glibenclamide (13.6 ± 1.0 U/ml) < glipizide (14.7 ± 0.7 U/ml). The total plasma insulin increase/total blood glucose decrease ratio for glimepiride was 0.03, for gliclazide 0.07, for glipizide 0.11 and for glibenclamide 0.16 (Fig. 2, Exp. 3). After a single oral dose of 90 g/kg glibenclamide or glimepiride to fasted dogs which, 30 min after administration, had been repeatedly loaded orally with glucose (2 g/kg) at 5-h intervals up to 50 h, the plasma insulin peaks and the peaks and nadirs of blood glucose were lower than those in the control animals (Fig. 6). The mean blood glucose concentration over 52 h in the glimepiride animals was 5.7 ± 0.13 mmol/l and in the glibenclamide animals 5.6 ± 0.10 mmol/l. Both means were statistically significantly lower than that in the controls (6.2 ± 0.14 mmol/l), however, they did not differ from each other. The mean plasma insulin concentration over 52 h in the glimepiride animals was 46.9 ± 4.1 U/ml and in the glibenclamide treated animals 53.5 ± 4.4 U/ml. Both means were also statistically significantly lower than that in the S120 G. Muller et al. /Diabetes Research and Clinical Practice 28 Suppl. (1995) S115-S137 Time in hrs after administration Fig. 4. Time course for blood glucose and plasma insulin levels in fasted male beagle dogs after a single oral dose of glimepiride or gliclazide (mean values), cf. Fig. 2, Exp. 4. Fig. 5. Time course for blood glucose and plasma insulin levels in fasted male beagle dogs after a single oral dose of glimepiride, glibenclamide, gliclazide or glipizide as clear aqueous sodium salt solutions (mean ± S.E.M.). cf. Fig. 2, Exp. 3. G. Muller et al. /Diabetes Research and Clinical Practice 28 Suppl. (1995) S115-S137 controls (60.6 ± U/ml).However, in contrast to the mean blood glucose concentrations, they also differed statistically from each other. After 8 weeks' oral treatment of hyperglycemic hyperinsulinemic KK-Ay mice once daily with 0.5 mg/kg glimepiride, 1.25 mg/kg glibenclamide or 20 mg/kg gliclazide (approximately equipotent blood glucose-decreasing doses in rabbits), glimepiride reduced blood glucose by 40%, plasma insulin levels by 50% and HBAlc by 33% (Fig:7). Glimepiride treatment had provoked changes in these parameters already after 3 weeks of treatment. In contrast, even after 8 weeks of treatment, glibenclamide and gliclazide had no effect on blood glucose, plasma insulin and HBA lc levels. Thepresented animal studies suggest that sulfonylureas also manage to decrease blood glucose independently of their capacity to stimulate insulin release and that sulfonylureas of different structure exhibit this extrapancreatic activity to a variable degree. Since short-term studies were performed; a secondary sulfonylurea effect, i.e. an Blood glucose in mmol/I Plasma insulin in U/ml S121 S122 G. Muller et al /Diabetes Research and Clinical Practice 28 Suppl. (1995) S215-S137 Fig. 7. Time course for blood glucose, plasma insulin and HBAlc levels in insulin-resistant diabetic KK-Ay mice treated once daily for 8 weeks with glimepiride, glibenclamide or gliclazide (mean values). *Significant from the control values with P < 0.05; ** significant from the control values with P < 0.01. tivity of sulfonylureas might be direct stimulation of peripheral glucose disposal. Evidence of direct so-called insulin-mimetic activity — the stimulation of glucose utilization — of sulfonylureas, in particular of glibenclamide, has accumulated during the past two decades in the literature stemming from in vitro studies with isolated and cultured muscle [6-10] and fat cells [11-14]. Effect of sulfonylureas on glucose transport and metabolism. The two major pathways for nonoxidative glucose metabolism in muscle and fat tissue are glycogen and lipid synthesis which are mainly involved in the overall peripheral glucose Fig. 8. Time course for blood glucose levels in fasted male beagle dogs after a single oral dose of glimepiride or glibenclamide as clear aqueous sodium salt solutions (mean values). utilization. We therefore studied glimepiride and glibenclamide for their effects on lipogenesis and glycogenesis in vitro. For lipogenesis, incorporation of [3H]glucose into toluene-extractable acylglycerides in cultured 3T3 adipocytes and, for glycogenesis, incorporation of [14C]glucose into ethanol-precipitable glycogen in the isolated rat diaphragm were determined (Fig. 9). Both drugs stimulated glycogenesis (Panel A) to up to 2.5 times the basal state (corresponding to 65-70% of the maximal insulin effect) and lipogenesis (Panel B) to up to four times the basal state (corresponding to 40-45% of the maximal insulin effect), the stimulation being concentration dependent. There was no difference in the maximal response between the two drugs. However, both dose-response curves of glimepiride were statistically significantly shifted to the left of those of glibenclamide, resulting in a glimepiride/ glibenclamide potency ratio of about two. The EG50 values for lipogenesis were 1.7 M for glimepiride and 2.9 M for glibenclamide; the EC50 values for glycogenesis were 0.4 M for glimepiride and 0.8 M for glibenciamide. The rate-limiting step of lipogenesis and glycogenesis under physiological conditions is glucose transport [39]. To compare glimepiride and glibenclamide for their effect on glucose transport, the uptake of the non-metabolizable glucose ana- G. Muller et al/Diabetes Research and Clinical Practice 28 Suppl. (1995) S115-S137 Panel A S123 Panel B Fig. 9. (according to Ref. 32). Panel A: Effect of sulfonylureas on glycogenesis in isolated rat diaphragms was assayed after incubation with the indicated concentrations of glimepiride and glibenclamide for 4 h or insulin for 30 min. The 14C-incorporation into ethanol-precipitated glycogen measured for the-basal state was set at 100%(mean ± sem, n = 6). Panel B: Effect of sulfonylureas on lipogenesis in 3T3 adipocytes was tested after incubation with differentiated and confluent cells with the indicated concentrations of glimepiride and glibenclamide for 20 h or insulin for 15 min. The 3H-incorporation into toluene-extractable lipids measured_ for the basal state and corrected for a blank value (incubation mixture lacking cells) was set at at 100% <mean ± sem, n = 6). logue, 2-deoxyglucose, in the isolated rat diaphragm and in 3T3 adipocytes was determined (Fig. 10). Both drugs increased the 2-deoxyglucose uptake in the diaphragm (Panel A) by up to 1.8-fold (corresponding to 70-75% of the maximal insulin effect) and in 3T3 cells (Panel B) by up to 7-fold (corresponding to 40% of the maxi- Panel A mal insulin effect), the increase being concentration dependent, The maximal transport velocities for glimepiride and glibenclamide did not differ. Again, the dose-response curves for glimepiride were statistically significantly shifted to the left of those for glibenclamide. The EC50 values in 3T3 cells were 3.2 M for glimepiride and 5.2 M for Panel B Fig. 10. (according to Ref. 32). Panel A: Effect of sulfonylureas on 2-deoxyglucose transport in isolated rat diaphragms was assayed after incubation with the indicated concentrations of glimepiride and glibenclamide or 300 nM insulin for 4 h. The diaphragm-associated 3H-radioactivity measured for the basal state and corrected for non-specific trapping (presence of 25 M cytochalasin B) was set at 100% (mean ± S.E.M., n =4). Panel B: Effect of sulfonylureas on 2-deoxyglucose transport in 3T3 adipocytes was assayed after incubation for 30 min with the indicated drug concentrations or 30 nM insulin. The cell-associated 3H-radioactivity measured for the basal state and corrected for non-specific trapping (presence of 25 M cytochalasin B) was set at 100% (mean ± S.E.M., n = 8). S124 G. Muller et al. /Diabetes Research and Clinical Practice 28 Suppl. (1995) S115-S137 glibenclamide. The EC50 values in rat diaphragm were 0.8 M for glimepiride and 1.4 M for glibenclamide. NIDDM is associated with desensitization of the glucose transport system towards stimulation by insulin in muscle and fat tissue, resulting in lower glucose disposal rates in diabetic patients as revealed by euglycemic hyperinsuimemic clamp studies [40,41]. A primary culture of isolated rat adipocytes desensitized in vitro for insulin-stimulated glucose transport by long-term incubation with high concentrations of insulin, glucose and glutamine is regarded as a corresponding in vitro— model [42,43]. Compared to non-desensitized cells, the maximal insulin response of 3-o-methylglucose transport in these adipocytes was reduced to about one third, and the insulin dose response curve (data not shown) was shifted to the right (EC50 = 1.9 vs. 0.5 nM). With glimepiride, however, there was only a 15% reduction in the maximal response, and only a marginal shift to the right in the dose response curve was observed (EC50 = 22.5 vs. 16.5 M), this shift not being significant statistically. This indicates that the ex-trapancreatic activity of glimepiride is hardly affected in the insulin-resistant state. Effect of sulfonylureas on GLUT4 translocation / dephosphorylation. The molecular mechanism of glucose transport stimulation by insulin in muscle and fat cells is based primarily on the translocation of the glucose transporter isoform GLUT4 [44] from intracellular stores — the low density microsomes — to the plasma membrane [45,46]. The increase in the number of functional glucose transporter molecules in the plasma membrane in response to insulin is dramatically impaired in adipocytes isolated from insulin-resistant hyperinsulinemic Zucker fatty rats, providing a molecular explanation of the defective insulin-stimulated glucose transport in these cells and reduced glucose clearance in these animals [47,48]. The effect of glimepiride (10 M) on the cell surface expression of GLUT4 in normal and desensitized rat adipocytes was studied in comparison to insulin (10 nM). GLUT4 was determined in total membranes, low density microsomes and plasma membranes using SDS-PAGE of the proteins with subsequent immunoblotting with anti-GLUT4 antibodies and [125I]protein A, followed by autoradiography (Fig. 11). Densitometric quantita-tion of the GLUT4 band in the autoradiogram revealed that, in normal cells (upper panel), glimepiride increased the amount of immunore-active GLUT4 in the plasma membrane 3-3.5-fold, while insulin increased it 7-8-fold. This increase was correlated with a decrease in GLUT4 in the low-density microsomes. The observed GLUT4 translocation induced by glimepiride and insulin was in reasonable agreement with the glucose transport stimulation in 3T3 adipocytes (Fig. 10, Panel B). In the insulin-resistant cells (middle-panel), insulinstimulated GLUT4 trans-location was reduced to about 2-fold. The glimepiride-induced translocation was about 3-fold, and thus hardly diminished compared to normal cells, as was the case for glucose transport activation by glimepiride. Obviously, glimepiride causes a redistribution of GLUT4 from low-density microsomes to plasma membranes in rat adipocytes, which is not significantly impaired in insulin-resistant cells compared to normal cells and is presumably responsible for the stimulation of glucose transport by glimepiride. The distribution of GLUT4 between low-density microsomes and the plasma membrane is believed to be regulated by its phosphorylation state. Serine/threonine phosphorylation at the carboxyl terminus may function as an internalization signal for GLUT4 [49,50]. In comparison to insulin, the effect of glimepiride on the phosphorylation state of GLUT4 was determined in normal and desensitized rat adipocytes in vitro. Rat adipocytes were metabolically labeled with 32Pi until equilibrium, prior to short-term incubation with 10 nM insulin or 10 M glimepiride. Subsequently, total membranes were isolated and GLUT4 immunoprecipitated with anti-GLUT4 antibodies and analyzed by SDS-PAGE and fluorography (Fig. 12). In normal cells, in the absence of insulin or glimepiride, there was considerable phosphate incorporation into GLUT4. Densitometric quantitation of the GLUT4 band in the fluorogram revealed that both insulin and glimepiride reduced the phosphorylation state of GLUT4 in total membranes by about 25-35%. In desensitized adipocytes, in the absence of insulin G. Muller et al. /Diabetes Research and Clinical Practice 28 Suppl. (1995) S115-S137 S125 Fig 11 (according to ref. 35). Effect of insulin and glimepiride on GLUT4 translocation was assayed in cultured normal (upper panel) and desensitized rat adipocytes (middle panel) after full deactivation of the glucose transport system and incubation of the cells in the absence (CON) or presence of 10 nM insulin (INS) or 10 M glimepiride (GLI) for 20 min. The amount of GLUT4 was determined in isolated total membranes (T), low-density microsomes (L) and plasma membranes (P) after SDS-PAGE of equivalent amounts of protein by immunoblotting with anti-GLUT4 antibodies and [ I25I]protein A and subsequent autoradiography. A representative autoradiogram of a typical experiment repeated three times with similar results is shown. S126 G. Mller et al. /Diabetes Research and Clinical Practice 28 Suppl. (1995) S115-S137 Fig. 12. (according to Ref. 35). Effect of insulin and glimepiride on the phosphorylation of GLUT4 was assayed in cultured normal and desensitized rat adipocytes after metabolic labeling with 32 P1 for 2 h. After full deactivation of the glucose transport system, the washed cells were incubated in the absence or presence of 10 nM insulin (INS) and 10 M glimepiride (GLI) for 20 min. again washed and then homogenized in the presence of 1 mM sodium phosphate. GLUT4 was immunoprecipitated from equivalent amounts of isolated total membranes with anti-GLUT4 antibodies and protein A-Sepharose and then analyzed by SDS-PAGE and autoradiography. A representative autoradiogram of a typical experiment repeated twice with similar results is shown. or glimepiride, the GLUT4 phosphorylation was increased about 5-fold compared to that in normal adipocytes. Insulin caused only a 20-25% reduction in the amount of phosphorylated GLUT4. In contrast, the phosphorylation state of GLUT4 after incubation with glimepiride was de- G. Muller et al. /Diabetes Research and Clinical Practice 28 Suppl. (1995) S115-S137 creased to 40-35%, approaching a2-fold level of GLUT4 phosphoprotein as observed in normal cells (Fig. 12). Obviously, glimepiride causes dephosphorylation of GLUT4, this desphosphorylation being more efficient in desensitized adipocytes than in normal cells. The dephpsphorylation of GLUT4 in response to sulfonylureas may provide-a molecular mechanism for the stimulation of GLUT4 translocation and glucose transport by glimepiride in normal and especially in insulin-resistant adipocytes. Effect of sulfonylureas on glucose-metabolizing enzymes. The key metabolic enzymes of lipogenesis, glycerol-3-phosphate acyltransferase, and of glycogenesis, glycogen synthase, are both activated_by_ insulin via dephosphorylation (for reviews see Refs. 51 and 32). Since glimepiride— like insulin, as already shown for GLUT4 — seems to regulate certain dephosphorylation processes in adipocytes, the effect of glimepiride and glibenclamide on the activity of glycogen synthase and glycerol-3-phosphate acyltransferase was studied. Glycogen synthase activity was measured in the homogenate from drug-incubated rat diaphragms and expressed as the ratio of activity in the presence of 0.1 mM and 10 mM glucose-6phosphate. At 0.1 mM glucose-6-phosphate, only the dephospho-form of glycogen synthase is active; at 10 mM, the total enzyme activity is measured. Both glibenclamide and glimepiride increased the activity ratio of glycogen synthase up to 45-50% of the maximal insulin effect (Fig. 13), this increase being concentration dependent. Fig. 13. (according to Ref. 32). Effect of sulfonylureas on glycogen synthase activity ratio (in the presence of 0.1 mM and 10 mM glucose-6-phosphate, respectively) in the homogenate of isolated rat diaphragm was assayed after 4 h incubation with the indicated concentrations of glimepiride and glibenclamide or 300 nM insulin (mean ± S.E.M., n = 3). S127 Thus, sulfonylureas lead to a considerable increase in the dephospho-form of glycogen synthase in adipocytes, which may indicate a sulfonylurea-induced dephosphorylation of this enzyme. Glycerol-3-phosphate acyltransferase activity was determined in the homogenate from drug-incubated rat adipoeytes. The activity of glycerol-3phosphate acyltransferase rose up to 35-40% of the maximal insulin effect in response to both drugs (Fig. 14), this rise being concentration dependent. For both enzymes, the glimepiride dose response curves showed a statistically significant shift to the left compared to those of glibenclamide. For glycogen synthase, the EC50 values were 0.8 M for glimepiride and 2.0 M for glibenclamide; for glycerol-3-phosphate acyltransferase the EC50 values were 19 M and 3.7 M, respectively. The sulfonylurea-induced simultaneous stimulation of glucose transport (via dephosphorylation of GLUT4) and of enzymes of the non-oxidative glucose metabolism (via dephosphorylation of key metabolic enzymes) suggests that the activation of specific protein phosphatases in muscle and fat cells is the molecular mechanism for the increase of glucose utilization in peripheral tissues provoked by sulfonylureas. Effect of sulfonylureas on the glycosyl-phosphatidylinositol-specific phospholipase C. The apparent coordinated activation of the multitude of protein phosphatases involved in the regulation Fig. 14. (according to Ref. 32). Effect of sulfonylureas on glycerol-3-phosphate acyltransferase activity in the homogenate from isolated rat adipocytes was assayed after incubation with the indicated concentrations of glimepiride and glibenclamide for 4 h or 20 h, or with 30 nM insulin for 30 min. The 3H-incorporation into butanol-extractable lipids measured for the basal state and corrected for a blank value (incubation mixture lacking protein) was set at 100% (mean ± S.E.M., n = 5). S128 G. Muller et al. /Diabetes Research and Clinical Practice 28 Suppl (1995) S115-S137 of—glucose transport and metabolism by glimepiride raises the question about putative upstream regulatory steps involved in glimepiride signaling. Cleavage products of a certain subtype of free glycolipids and glycolipid membrane anchors, glycosyl-phbsphatidylinositol, generated by a specific phospholipase C can alter the total phosphorylation patterns in rat adipocytea in an insulin-like manner [53-55]. These: putative protein phosphatase-modulating effector molecules, phosphoinositol-glycans of yet unknown structure, stimulate glucose transport [56], lipogenesis [57], glycogenesis [58], glycogen synthase [59] and glyc-erol-3-phosphate acyltransferase [60] in intact adipocytes and muscle cells in vitro. In isolated normal and desensitized rat adipocytes, the effect of glimepiride on the activity of the glycosyl phosphatidylinositol-specific phospholipase C was tested. The assay is based on the cleavage of the covalently attached glycosyl-phosphatidylinositol plasma membrane anchor (for a review see Cross [61]) of an isoform of lipoprotein lipase in rat adipocytes [62]. After metabolic labeling of the cells with myo-[14C]inositol and subsequent incubation with insulin or glimepiride, plasma membranes were isolated. For detection of anchor cleavage, the membrane proteins were partitioned between a Triton X-114 and an aqueous phase [63] to separate the hydrophilic form of lipoprotein lipase (with the degraded anchor structure) from the amphiphilic version (linked to the intact anchor structure). Subsequently, hydrophilic lipoprotein lipase was immunoprecipitated from the aqueous phase and analyzed by SDS-PAGE and fluorography for l4 C-radioactivity (Fig. 15), The retention of the radiolabeled myo-inositol within the hydrophilic lipoprotein lipase indicates cleavage of the glycosyl-phosphatidylinositol membrane anchor by a phospholipase. Densitometric quantitation of the lipoprotein lipase band in the fluorogram revealed that, in both normal and desensitized cells, insulin and glimepiride caused a concentration-dependent increase in hydrophilic lipoprotein lipase. In normal cells, the maximal effect of glimepiride (10 M) approached 25-30% of the maximal insulin effect (30 nM), the EC50 value for glimepiride being 3.1 M. In desensitized adipocytes, the maximal sti- mulation of the-phospholipase by glimepiride was slightly reduced compared to that in normal cells (5.5-fold vs. 6.3-fold), but markedly exceeded the maximal insulin effect (2.7-fold in desensitized vs. 12.5-fold in normal cells). The EC50 value for insulin increased from 1.9 nM in normal cells to 5.1 nM in desensitized cells, whereas the EC50 value for glimepiride remained constant (3.1 vs. 3.5 M). Obviously, the desensitization toward insulin and the maintenance of sensitivity toward glimepiride for stimulation of glucose transport and GLUT4 translocation/dephosphorylation is also reflected in the stimulation of the phospholipase C. Furthermore, the time response curves of activation of the phospholipase and of glucose transport by glimepiride in desensitized as well as in normal adipocytes were similar. These data suggest a mechanistic coupling between the sulfonylurea-mducible phospholipase activity and GLUT4 translocation/dephosphorylation. The maintenance of extrapancreatic activity of glimepiride in the desensitized adipocytes might be responsible for its extrapancreatic activity in the insulin-resistant KK-Ay mice. Taken together with the demonstration of a defect in releasing functional phosphoinositol-glycan structures in insulin-resistant Type II diabetic Goto-Kakizaki rats [64], the data provide evidence that sulfonylureas bypass the site of insulin resistance at the level of the generation of phosphoinositol-glycan structures via direct activation of the glycosylphosphatidylinositol-specific phospholipase C. Effect of sulfonylureas on the cAMP regulatory cascade. Sulfonylureas inhibit concentration-dependently, isoproterenol-stimulated lipolysis in rat adipocytes in vitro [65], presumably via interference with the cAMP-dependent activation or direct inhibition of protein kinase A [66,67]. To test for these possibilities and for differences between glimepiride and glibenclamide, the activity of protein kinase A was assayed in the cytosol from drugtreated rat adipocytes which had been preincubated with isoproterenol to increase cytosolic cAMP levels. Protein kinase A was tested in the absence (for determination of the amount of active enzyme) and presence (for determination of the total amount of enzyme) of saturating concentrations of exogenous cAMP. Glimepiride and G. Muller et al /Diabetes Research and Clinical Practice 28 Suppl. (1995) S115-S137 S129 Fig. 15. (according to Ref. 36). Effect of insulin and glimepiride on the glycosyl-phosphatidylinositol-specific phospholipase C was assayed in isolated rat adipocytes which had been incubated in primary culture in the presence of 0.5 mM glucose (normal) or 20 mM glucose, 16 mM glutamine, 10 nM insulin (desensitized) for 20 h. The washed cells were metabolically labeled with myo-[14 C]inositol and then incubated for 30 min with the indicated concentrations of insulin or glimepiride. Isolated plasma membranes were subjected to TX-114 partitioning. Hydrophilic lipoprotein lipase was purified from the aqueous phase by immunoprecipitation and analyzed by SDS-PAGE and fluorography. A representative fluorogram of a typical experiment repeated three times with similar results is shown. glibenclamide reduced the protein kinase A activity ratio, i.e. the portion of active enzyme (Fig. 16), this reduction being concentration dependent. There was no difference in the maximal inhibition between glimepiride and glibenclamide. However, the dose response curve for glimepiride was shifted to the left, this shift being statistically significant. The EC 50-value was 0.7 M for glimepiride and 1.1 M. for glibenclamide. The protein kinase A activity ratio is assumed mainly to reflect the intracellular cAMP concentration at the time of homogenization. Thus, sulfonylureas seem to lower protein kinase A activity and consequently lipolysis via causing a decrease in cytosolic cAMP levels. One potential mechanism for the cAMP-lowering capacity of sulfonylureas is the stimulation of degradation of cAMP. Consequently, with glimepiride and glibenclamide, the effect on the activity of the particulate low Km cAMP-specific phosphodiesterase in microsomes from drug-incubated rat adipocytes was tested. Both drugs increased the phosphodiesterase activity up to 60% of the maximal insulin effect (Fig. 17). The dose response curve for glimepiride showed a shift to the left of that of glibenclamide (EC50 = 0.8 vs. 2.2 M), this shift being statisti- S130 G. Muller et al. /Diabetes Research and Clinical Practice 28 Suppl. (1995) S115-S137 cAMP-specific phosphodiesterase [72]. These glycolipids were isolated from insulin-sensitive tissues and are structurally related to the lipolytic cleavage product from the lipoprotein lipase glycosyl-phosphatidylinositol membrane anchor. 4. Discussion Fig. 16. (according to Ref. 32). Effect of sulfonylureas on protein kinase A activity ratio (in the absence or presence of 1 M cAMP) in the cytosol of isolated rat adipocytes was assayed after incubation of adipocytes with 1 M isopro-terenol for 15 min and then with the indicated concentrations of glimepiride and glibenclamide or 5 nM insulin for 4 h (mean ± S.E.M., n = 4). cally significant. These data confirm previous reports on the activation of phosphodiesterase by sulfonylureas [68,69]. The consistently lower EC50 values of glimepiride for inhibition of protein kinase A, for activation of cAMP-specific phosphodiesterase, and for activation of glycosyl-phosphatidylinositol specific phospholipase C compared to those of glibenciamide suggest a mechanistic linkage between the cAMP regulatory cascade and the generation of phosphoinositol-glycan molecules in fat cells. In agreement with this putative causal relationship, phosphoinositol-glycans prepared from free glycolipids have been shown to inhibit catecholamine-induced lipolysis [70] and protein kinase A activation [71] as well as to stimulate Fig. 17. (according to Ref. 32). Effect of sulfonylureas on low Km cAMP-specific phosphodiesterase activity in microsomes from isolated rat adipocytes was assayed after 2 h incubation of adipocytes with the drug concentrations indicated or 5 nM insulin (mean S.E.M., n = 5). The ranking in the plasma insulin/blood glucose ratio in vivo suggests that sulfonylureas have extrapancreatic activity and that they differ in their extrapancreatic activity. The dogs treated once with glimepiride or glibenciamide and then repeatedly glucose-loaded showed a smaller blood glucose increase despite a smaller plasma insulin increase than the controls. Although both drugs inhibited blood glucose increase to the same degree, the plasma insulin increase with glimepiride was smaller than that with glibenciamide. The dogs treated with glimepiride, glibenclamide, gliclazide or glipizide showed at a similar total blood glucose decrease a ranking in insulin release, glibenclamide having the highest insulin-releasing activity and glimepiride the lowest. Of the dogs treated with glimepiride or glibenciamide, those given glimepiride showed, 24-48 h after treatment, lower plasma insulin values than the glibenclamide-treated animals, despite their blood glucose values also being significantly lower. In the diabetic hyperinsulinemic KK-Ay mice treated for 8 weeks with glimepiride, glibenclamide or gliclazide, only glimepiride induced a significant blood glucose decrease, this being accompanied by a decrease in plasma insulin. Of the sulfonylureas tested, glimepiride obviously has the most pronounced extrapancreatic activity in vivo. The demonstration of extrapancreatic activity of sulfonylureas and of a similar ranking between glimepiride and glibenclamide in vitro supports the in vivo findings, in the isolated or cultured fat cells and the muscle diaphragm, the EC50 values for the stimulation of lipogenesis, glycogenesis, glucose transport, cAMP-specific phosphodiesterase and glycerol-3-phosphate acyltransferase, as well as for the inhibition of protein kinase A were two to three times lower for glimepiride than for glibenclamide. This argues in favour of G. Muller et al / Diabetes Research and Clinical Practice-2S-Suppl (1995) S115-S137 The physiological relevance of the direct stimulation of glucose utilization in peripheral cells observed in vitro for sulfonylureas in the present and also in previous studies [6-14]. It may result in an increase of glucose disposal in muscle and fat tissues in vivo, which does not depend on insulin release. The present studies on the molecular mechanism of sulfonylurea action in vitro provide an explanation as to how sulfonylureas manage to affect the multiple steps required for increased glucose utilization, i.e. (i) activation of glucose transport and glucose-metabolizing enzymes leading to stimulation of glycogen and lipid synthesis; and (ii) downregulation of the cAMP regulatory cascade, leading to inhibition of lipolysis. The simultaneous and coordinated regulation of these activities resembles the action of insulin. According to our data, glimepiride seems to exert these pleiotropic effects via independent mecha nisms: (a) the stimulation of specific protein phosphatases responsible for regulation of GLUT4 translocation and of glycogen synthase/glycerol3-phosphate acyltransferase activity; and (b) the stimulation of the cAMP-specific phosphodiesterase. At the ultimate hierarchy of regulation, glimepiride, however, also causes rapid and efficient activation of the glycosyl-phosphatidylinositol-specific phospholipase C, as shown in rat adipocytes. The soluble phosphoinositol-glycan molecules thereby generated are known to regulate phospho-/dephosphorylation processes, and the cAMP regulatory cascade (for reviews see Refs. 73 and 74). It is therefore tempting to speculate about the glycosyl-phosphatidylinositol-specific phospholipase C as the primary target for sulfonylurea action on protein phosphorylation and cAMP metabolism in peripheral cells. Insulin has also recently been shown to increase the activity of this enzyme in adipocytes [33,36,75] and myocytes [76]. Thus, sulfonylureas may partly mimic the insulin action by interfering with the metabolic branch of the insulin signaling cascade at the level of the glycosyl-phosphatidylinositolspecific phospholipase. Besides the argument that the effect of sulfonylureas under chronic treatment might be an indirect effect due to an improved metabolic control S131 resulting in increased insulin sensitivity, there are, however, four more concerns about the physiological relevance of extrapancreatic effects of sulfonylureas: i) potential differences between peripheral and portal vein insulin concentrations ii) discrepancy between protein binding in vivo and in vitro, iii) discrepancy between total in vivo and in vitro concentrations, iv) missing effect on blood glucose in totally insulin-deficient animals and diabetic patients. (i) At the first pass, liver removes up to 50% insulin front the portal blood [77, 78]. One might therefore suspect that sulfonylureas may have induced a small plasma insulin increase in the portal vein not detectable in the peripheral plasma, which would have resulted in greater inhibition of the hepatic glucose output by glimepiride. This agrument, however, cannot be sustained. Although the blood glucose in the dogs 24 h after the administration of glimepiride was lower than that after glibenclamide, the insulin was lower too. (ii) The free drug concentration of glimepiride and glibenclamide in medium containing bovine serum albumin is, according to Panten [79, 80], 10-15% in human serum, according to Lehr [81] it is less than 1%. Since only the drug not bound to protein is supposed to be effective [82], the higher protein binding in vivo raises doubts as to whether, even under the assumption of similar total drug concentrations in serum and incubation medium, the effects observed in vitro might become effective in vivo [83]. However, according to Hornke and Jantz (personal communication), the protein binding of glimepiride in vitro is more than 99.5% with bovine serum albumin. (iii) The total drug concentrations required in vitro for half-maximal effects (1-5 /xM) are higher than even the cmax plasma concentrations under the therapeutical oral doses administered to the dogs. The plasma halflife of glimepiride in dogs is 12 h [84]. After administration of 100 g/kg glimepiride orally to dogs, the c max plasma concentra- S132 G. Muller et al. /Diabetes Research and Clinical Practice 28 Suppl. (1995) S115-S137 tion was 2.2 M (at 2 h); after 24 h, the_ plasma concentralion was 6.4 M [85]. It seems unlikely that the short-lasting, early cmax phase of glimepiride would have been sufficient to induce the long-lasting blood glucose decrease without increased plasma insulin in our animals. Activation of the glucose transport by insulin in rat adipocytes in vitro persists only for 10-20 min after removal of insulin [86]. Duration of extrapancreatic activity of sulfonylureas in vitro after drug removal is not known. This is difficult to determine due to the undefined efficiency of a washout procedure. Alternatively, the confict between total drug concentrations effective in vivo and in vitro might be overcome by accepting that the total amount of molecules which interact with the target cell in a functional manner will be dependent on the product of the concentration and exposure time. In the present in vitro studies, the extrapancreatic effects were determined immediately after incubation, with drug, of fat cells for 30 min to 20 h or of diaphragm for 4 h. The short incubation time might have been compensated by a higher drug concentration. The importance of the exposure time is underlined by preliminary experiments which indicate that, in vitro, the maximal cytochalasin B-inhibitable glucose transport velocity in 3T3 adipocytes increases with the incubation time in the presence of 20 M glimepiride from about 6.8-fold over the basal state at 30 min to 9.3-fold at 20 h. At submaximal concentrations, glimepiride was also more efficient with longer exposure times. Furthermore, our studies on the activation of glycerol-3-phosphate acyltransferase by glimepiride and glibenclamide in rat adipocytes revealed an even more striking dependency on the incubation time rising from an approximate 35% increase at 4 h to 90% at 20 h with 100 M drug (Fig. 14). The maximal enzyme activity after the longterm exposure was 2.5-3 times higher throughout the concentration range tested and the do dose response curve was shifted to the left. Time-dependent penetration of the sulfony-lurea molecules into the plasma membrane of the target cell might be the mechanism for the increasing activity. There is strong evidence for a high lipophilicity of sulfonylureas [87], especially of glimepiride and glibenclamide, based on their distributionbetween an octanol and a water phase. The partition coefficients of the unionized forms at pH 7.4 are 2.8 for glipizide, 4.1 for gliclazide, 94 for glibenclamide and 50 for glimepiride [80, 88, 89]. The amount of protein-bound drug may represent the pool for molecules which time-dependently insert into the lipid-bilayer of the membrane either via passage across the aqueous environment or via close physical contact of serum proteins loaded with sulfonylurea with the surface of the target cell. The altered physical state of the lipid bilayer induced by the intercalated sulfonylurea may affect protein components of the signal transduction cascade located in the plasma membrane of fat and muscle cells. Possible candidates for those components are the glycosylphosphatidylinositol-specific phospholipase C in the fat cell and the membrane-associated activated protein kinase C isoforms in the muscle cell Activation of protein kinase C by sulfonylureas has been demonstrated for BC3H1 myocytes [9, 10] and correlated with the increase of glucose transport induced by these drugs. The suggested accumulation of sulfonylurea molecules in the membrane of the target cell and its functional relevance is in conflict with a typical receptor-ligand interaction which solely depends on the affinity of the receptor and the free ligand concentration. It is, however, compatible with the findings that in ventricular myocytes isolated from guinea pig hearts sulfonylurea-induced inhibition of ATP-dependent K+ channels depends on the rates of drug absorption into and release from the membrane lipids [90] and on the presence of the unionized G. Muller et al. / Diabetea Research and Clinical Practice 28 Suppl. (1995) S115-S137 form of the drug which is-charaeterized by-high lipophilicity [91]. Furthermore no high affinity binding sites for sulfonylureas have been described so far in plasma membranes of isolated and cultured adipocytes [92]. Low affinity binding sites recently identified in membranes of isolated rat adipocytes [93] are presumably not involved in mediating the extrapancreatic effects. Their high Kd values exceed the EC 50 values for extrapancreatic effects. With the proposed "partitioning" mechanism, the specificity of extra-pancreatic sulfonylurea action would be defined by the presence of protein components of the signaling cascade regulating glucose transport and metabolism in the membrane of the target cell instead of by a receptorligand interaction. The lower partitioning coefficient of glimepiride compared to that of glibenclamide suggests that lipophilicity is not the main determinant for the strength of the extrapancreatic activity iv) There are several explanations of the missing effect on blood glucose [30] and rate of glucose disposal in totally insulin-deficient animals [94] and type I diabetic patients [95, 96]: a) Insulin may control the activity or protein/gene expression of certain components of the signal transduction cascade mediating the direct insulin-mimetic effects of sulfonylureas in fat and muscle cells (e.g. glycosyl-phosphatidylinositolspecific phospholipase C, protein kinase C). Consequently, sulfonylurea signaling will require a certain basal insulin level, b) The drastically altered overall metabolic state of completely insulin-deficient animals may abolish the direct insulin-mimetic action of sulfonylureas. c) When insulin is deficient, glucose is not only less utilized from the periphery but also excessively produced by the liver due to absence of inhibition of gluconeogenesis. This results in hyperglycemia which exceeds the renal glucose threshold and leads to glucosuria. In vitro, sulfonylureas have been shown to inhibit gluconeogenesis in hepatocytes by stimulation of fructose-2,6-bisphosphate formation S133 [97, 98, 99,l00,l01] and possibly via inhibi-tion of protein kinase A [66, 67]. The sulfonylurea concentrations, however, required for inhibition of hepatic gluconeogenesis in vitro are 10-100 times higher than those for the extrapancreatic effects in peripheral cells in vitro. This discrepancy in the effective concentrations might be due to the mechanism of protein kinase A inhibition in hepatic and adipocyte tissue being fundamentally different. Protein kinase A inhibition was demonstrated with hepatocytes as well as with the isolated rat liver enzyme [102]. However, the sulfonylurea concentration required for inhibition of protein kinase A was also higher for the isolated liver enzyme compared to that of the adipocyte enzyme for incubation of intact cells in agreement with the observed discrepancy between the EC50 for inhibition of hepatic gluconeogenesis and of lipolysis in fat cells. Phosphoinositol-glycans may mediate the inhibition of protein kinase A in fat cells, whereas, in the liver, direct inhibition by sulfonylureas seems to be effective [67]. It is thus possible that, in the totally insulin-deficient animals and diabetic patients, the peripheral extrapancreatic effect was balanced by insufficient inhibition of the hepatic glucose output. The increased glimepiride-induced glucose flux into the peripheral tissues might have led to reduced glucosuria but did not lower blood glucose. Consequently, for manifestation of direct stimulation of glucose disposal in peripheral tissues by sulfonylureas, a certain amount of insulin may be necessary for inhibition of hepatic glucose output. The direct stimulation of glucose utilization in peripheral tissues might be one explanation tor the extrapancreatic activity of sulfonylureas as observed in the above animal studies. Particular attention has been paid to this mode of action in the present and previous studies over the past 2 decades. However, other mechanisms, such as modulation of glucagon release from the pancreas [30, 103], alteration of insulin clearance by S134 G. Muller et al. /Diabetes Research and Clinical Practice 28 Suppl. 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