Triphenyltin impairs insulin secretion by decreasing glucose-induced NADP(H) and ATP production in hamster pancreatic β-cells

Oral administration of triphenyltin chloride (TPT) (6 mg/100g body weight) inhibits insulin secretion by decreasing glucose-induced cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) in pancreatic β-cells of the hamster. To test the possibility that the abnormal level of the [Ca(2+)](i) induced by TPT administration could be due to a defect in the metabolic signal of glucose in the β-cells, we tested the effects of TPT administration on the glucose-induced NAD(P)H and ATP production, and on the changes of membrane potential and [Ca(2+)](i) by glucose and high K(+) in the β-cells. The [Ca(2+)](i) was measured in islet cells loaded with fura-2. TPT administration significantly reduced the NAD(P)H and ATP production, the depolarization of plasma membrane, and insulin secretion by 15 mM glucose in islet cells. TPT administration also reduced the insulin secretion by 10mM dihydroxyacetone and glyceraldehyde. However, TPT administration did not affect the increase of [Ca(2+)](i) and the insulin secretion by 30 mMK(+) or 100 μM tolbutamide, and the membrane potential by 30 mMK(+), and the insulin secretion by 10mM α-ketoisocaproic acid and 0.5mM formycin A, an analog of ATP in the presence of 15 mM glucose. These results suggested that the pathogenesis of TPT-induced hyperglycemia in hamster involves the reduction of [Ca(2+)](i) and insulin secretion in response to K(ATP) channel-dependent depolarization, which is related to the decrease of NAD(P)H and ATP production in pancreatic islet cells after glucose metabolism.     

Effects of tolbutamide and N-benzoyl-D-phenylalanine (NBDP) on the regulation of [Ca2+]i oscillations in mouse pancreatic islets.

The sulfonylurea derivative, tolbutamide, and the phenylalanine derivative, N-benzoyl-D-phenylalanine (NBDP), both of which stimulate insulin secretion through interaction with the sulfonylurea receptor (SUR1), were studied for their ability to increase the [Ca(2+)](i) and to interact with the glucose-induced slow large amplitude [Ca(2+)](i) oscillations in isolated mouse pancreatic islets. Tolbutamide as well as NBDP induced [Ca(2+)](i) oscillations of extremely slow frequency. Both compounds also lowered the threshold for the glucose-induced slow large amplitude [Ca(2+)](i) oscillations and significantly reduced their frequency in intact islets as well as in single pancreatic beta cells. These [Ca(2+)](i) oscillations apparently require a glucokinase-mediated glycolytic flux. This conclusion is supported by the observations that KIC, a mitochondrial fuel, cannot replace glucose in this synergism and that mannoheptulose, an inhibitor of glucokinase and glucose-induced insulin secretion, abolishes these slow [Ca(2+)](i) oscillations. In conclusion, these compounds potentiate the effect of glucose. This additive effect is the likely result of a synergistic closing action upon the ATP-sensitive K(+) (K(ATP)) channel, mediated in the case of glucose through an action upon the channel protein itself of ATP generated in glucose catabolism and in the case of tolbutamide and NBDP upon the sulfonylurea receptor (SUR1) associated with this channel.     

Alterations of insulin secretion following long-term manipulation of ATP-sensitive potassium channels by diazoxide and nateglinide

Previous studies have shown that prolonged exposure to drugs, which act via blocking KATP channels, can desensitize the insulinotropic effects of drugs and nutrients acting via KATP channels. In this study, effects of prolonged exposure to diazoxide, a KATP channel opener, on beta cell function were examined using clonal BRIN-BD11 cells. The findings were compared to the long-term effects of KATP channel blockers nateglinide and tolbutamide. Following 18 h exposure to 200 microM diazoxide, the amounts of insulin secreted in response to glucose, amino acids and insulinotropic drugs were increased. Secretory responsiveness to a variety of agents acting via KATP channels was retained following prolonged diazoxide exposure. In contrast, 18 h exposure to 100 microM nateglinide significantly attenuated the insulin secretory responses to tolbutamide, nateglinide and BTS 67 582. Glucose- and L-alanine-stimulated insulin release were unaffected by prolonged nateglinide exposure, however responsiveness to L-leucine and L-arginine was diminished. Prolonged exposure to nateglinide had no effect on forskolin- and PMA-stimulated insulin release, and the overall pattern of desensitization was similar to that induced by 100 microM tolbutamide. We conclude that in contrast to chronic long-term KATP channel blockade, long-term diazoxide treatment is not harmful to KATP channel mediated insulin secretion and may have beneficial protective effects on beta cell function.     

Control of insulin secretion by sulfonylureas, meglitinide and diazoxide in relation to their binding to the sulfonylurea receptor in pancreatic islets

Sulfonylureas inhibit an ATP-dependent K+ channel in the B-cell plasma membrane and thereby initiate insulin release. Diazoxide opens this channel and inhibits insulin release. In mouse pancreatic islets, we have explored whether other targets for these drugs must be postulated to explain their hypo- or hyperglycaemic properties. At non-saturating drug concentrations the rates of increase in insulin secretion declined in the order tolbutamide = meglitinide greater than glipizide greater than glibenclamide. The same rank order was observed when comparing the rates of disappearance of insulin-releasing and K+ channel-blocking effects. The different kinetics of response depend on the lipid solubility of the drugs, which controls their penetration into the intracellular space. Allowing for the different kinetics, the same maximum secretory rates were caused by saturating concentrations of tolbutamide, meglitinide, glipizide and glibenclamide. A close correlation between insulin-releasing and K+ channel-blocking potencies of these drugs was observed. The relative potencies of tolbutamide, meglitinide, glipizide and glibenclamide corresponded well to their relative affinities for binding to islet-cell membranes, suggesting that the binding site represents the sulfonylurea receptor. The biphasic time-course of dissociation of glibenclamide binding indicates a complex receptor-drug interaction. For diazoxide there was no correlation between affinity of binding to the sulfonylurea receptor and potency of inhibition of insulin secretion. Thus, opening or closing of the ATP-dependent K+ channel by diazoxide or sulfonylureas, respectively, appears to be due to interaction with different binding sites in the B-cell plasma membrane. The free concentrations of tolbutamide, glipizide, glibenclamide and diazoxide which are effective on B-cells are in the range of therapeutic plasma concentrations of the free drugs. It is concluded that the hypo- and hyperglycaemic effects of these drugs result from changing the permeability of the ATP-dependent K+ channel in the B-cell plasma membrane.     

Stimulation of insulin secretion by glucose in the absence of diminished potassium (86Rb+) permeability.

Two inhibitors of the nucleotide-sensitive K+ (KATP) channel, tolbutamide and quinine, were utilized in order to assess the role of this channel in glucose-stimulated insulin release from perifused rat islets. In the absence of these drugs, the addition of 15 mM glucose elicited a marked biphasic stimulation of insulin secretion concomitant with a reduction in the rate of 86Rb+ efflux. In the presence of either 500 microM tolbutamide or 100 microM quinine, a reduced rate of efflux of 86Rb+ was observed together with an elevated rate of insulin release. Under such conditions, the addition of 15 mM glucose retained the ability to stimulate insulin secretion though this was associated with a marked increase in 86Rb+ efflux. It is concluded that a net reduction in beta-cell K+ permeability is not an obligatory step in glucose-stimulated insulin release. Thus, glucose is likely to exert depolarizing actions on the beta-cell in addition to the closure of K+ channels.     

Desensitization of insulin secretory response to imidazolines, tolbutamide, and quinine. I. Secretory and morphological studies.

The desensitization of pancreatic B-cells against stimulation by insulin secretagogues that inhibit ATP-dependent K(+) channels (K(ATP) channels) was investigated by measuring insulin secretion of perifused pancreatic islets. Additionally, the islet insulin content and the number of secretory granules per B-cell were determined. Prior to the measurement of secretion, islets were cultured for 18 h in the presence or absence of the test agents in a cell-culture medium containing 5 mM glucose. The effects of three imidazolines, phentolamine, alinidine, and idazoxan (100 microM each) were compared with those of the well-characterized sulfonylurea, tolbutamide (500 microM), and those of the ion channel-blocking alkaloid, quinine (100 microM). Insulin secretion was strongly reduced upon re-exposure to phentolamine, alinidine, tolbutamide, and quinine, whereas idazoxan, which stimulated secretion only weakly, had no significant effect. The imidazoline secretagogues phentolamine and alinidine induced a cross-desensitization against the stimulatory effect of tolbutamide and quinine. A long-term depolarization with 40 mM KCl was also able to induce a significant reduction of the secretory response to all of the above secretagogues. The insulin content of cultured islets was moderately, but significantly reduced by alinidine, whereas the reduction by phentolamine, tolbutamide, and quinine was not significant. In contrast to these observations, the ultrastructural examination revealed that tolbutamide-treated B-cells had a high degree of degranulation, whereas the other test agents and 40 mM KCl produced only a partial degranulation, except for phentolamine, which produced no significant degranulation at all. These results suggest that the desensitization of insulin secretion is a common property of all agents that stimulate insulin secretion by depolarisation of the plasma membrane. Depending on the specific secretagogue, additional mechanisms, proximal and distal to Ca(2+) influx, appear to contribute to the desensitization (see Rustenbeck et al., pages 1695-1703, this issue).     

3-Methylcholanthrene increases phorbol 12-myristate 13-acetate-induced respiratory burst activity and intracellular calcium levels in common carp (Cyprinus carpio L) macrophages

Oral administration of triphenyltin chloride (TPT) (60 mg/kg body wt) inhibits insulin secretion by perturbing the cytoplasmic Ca(2+) concentration ([Ca(2+)]i) in pancreatic beta-cells of the hamster. To test the possibility that the abnormal levels of [Ca(2+)]i induced by TPT administration could be due to a defect in the cytoplasmic Na(+) concentration ([Na(+)]i) in the beta-cells, we investigated the effects of TPT administration on the changes of [Na(+)]i and [Ca(2+)]i induced by glucose or acetylcholine (ACh) and on the [Na(+)]i induced by ouabain, a potent inhibitor of Na(+),K(+)-ATPase. The changes of [Na(+)]i and [Ca(2+)]i were measured in islet cells loaded with sodium-binding benzofuran isophthalate and fura 2, respectively. TPT administration strongly reduced the rise in [Ca(2+)]i induced by 15 mM glucose with and without extracellular 135 mM Na(+). TPT administration also significantly reduced the rise of [Ca(2+)]i by 100 microM ACh in the presence of 5.5 or 15 mM glucose but not the amplitude of [Ca(2+)]i by 100 microM ACh in Na(+)-free medium. TPT administration attenuated the rise in [Na(+)]i induced by 100 microM ACh in the presence of either 3 or 5.5 mM glucose. However, TPT administration did not impair the [Na(+)]i in the presence of glucose (3, 5.5, and 15 mM) or of 100 microM ouabain with 3 mM glucose. TPT administration significantly suppressed the insulin secretion induced by 15 and 27.8 mM glucose or 100 microM ACh in the presence of 5.5 mM glucose. Our study suggests that triphenyltin has inhibitory effects on the cellular Ca(2+) response due to a reduction of [Ca(2+)]i after Na(+)-dependent and Na(+)-independent depolarization in islet cells of the hamster.     

Insulin release by tolbutamide and glibenclamide

Summary Glibenclamide, a second generation sulfonylurea, produced the same pattern of insulin release from the perfused rat pancreas as did tolbutamide. The stimulatory effect was closely dependent on the glucose concentration present. Both agents enhanced insulin secretion at 5–10 mM glucose, whereas no additional insulin was released when maximally stimulating levels of glucose (20 and 30 mM) were present. The concentrations of glibenclamide stimulating insulin release were 100–400 times lower than equieffective levels of tolbutamide. At glucose levels of 3 or 8 mM, however, glibenclamide did not liberate significantly more insulin from the pancreas than did tolbutamide. Thus the differences of tolbutamide and glibenclamide were quantitative rather than qualitative. Although the active concentrations differed the effects produced were comparable.     

Inhibition of glucose-induced electrical activity in rat pancreatic beta-cells by DCPIB, a selective inhibitor of volume-sensitive anion currents

We have investigated the effects of the ethacrynic acid derivative 4-(2-butyl-6,7-dichloro-2-cyclopentyl-indan-1-on-5-yl) oxobutyric acid (DCPIB), an inhibitor of the volume-sensitive anion channel (VSAC), on electrical activity and insulin secretion in rat pancreatic beta-cells. DCPIB inhibited whole-cell VSAC currents in beta-cells with IC50 values of 2.2 and 1.7 microM for inhibition of outward and inward currents, respectively. DCPIB also inhibited the VSAC at the single channel level in cells activated by glucose. In intact cells, DCPIB caused a net increase in beta-cell input conductance and evoked an outward current that was sensitive to inhibition by tolbutamide, suggesting KATP channel activation. However, no KATP channel activation was evident under conventional whole-cell conditions, suggesting that the drug might activate the channel in intact cells via an indirect mechanism, possibly involving nutrient metabolism. DCPIB suppressed glucose-induced electrical activity in beta-cells, hyperpolarised the cell membrane potential at a substimulatory glucose concentration and prevented depolarisation when the glucose concentration was raised to stimulatory levels. The suppression of electrical activity by DCPIB was associated with a marked inhibition of glucose-stimulated insulin release from intact islets. It is concluded that DCPIB inhibits electrical and secretory activity in the beta-cell as a combined result of a reciprocal inhibition of VSAC and activation of KATP channel activities, thus producing a marked hyperpolarisation of the beta-cell membrane potential.     

Mechanism of the insulin-releasing action of alpha-ketoisocaproate and related alpha-keto acid anions

Alpha-ketoisocaproate directly inhibits the ATP-sensitive K(+) channel (K(ATP) channel) in pancreatic beta-cells, but it is unknown whether direct K(ATP) channel inhibition contributes to insulin release by alpha-ketoisocaproate and related alpha-keto acid anions, which are generally believed to act via beta-cell metabolism. In membranes from HIT-T15 beta-cells and COS-1 cells expressing sulfonylurea receptor 1, alpha-keto acid anions bound to the sulfonylurea receptor site of the K(ATP) channel with affinities increasing in the order alpha-ketoisovalerate < alpha-ketovalerate < alpha-ketoisocaproate < alpha-ketocaproate < beta-phenylpyruvate. Patch-clamp experiments revealed a similar order for the K(ATP) channel-inhibitory potencies of the compounds (applied at the cytoplasmic side of inside-out patches from mouse beta-cells). These findings were compared with the insulin secretion stimulated in isolated mouse islets by alpha-keto acid anions (10 mM). When all K(ATP) channels were closed by the sulfonylurea glipizide, alpha-keto acid anions amplified the insulin release in the order beta-phenylpyruvate < alpha-ketoisovalerate < alpha-ketovalerate approximately alpha-ketocaproate < alpha-ketoisocaproate. The differences in amplification apparently reflected special features of the metabolism of the individual alpha-keto acid anions. In islets with active K(ATP) channels, the first peak of insulin secretion triggered by alpha-keto acid anions was similar for alpha-ketoisocaproate, alpha-ketocaproate, and beta-phenylpyruvate but lower for alpha-ketovalerate and insignificant for alpha-ketoisovalerate. This difference from the above orders indicates that direct K(ATP) channel inhibition is not involved in the secretory responses to alpha-ketoisovalerate and alpha-ketovalerate, moderately contributes to initiation of insulin secretion by alpha-ketoisocaproate and alpha-ketocaproate, and is a major component of the insulin-releasing property of beta-phenylpyruvate.     

Calcium antagonists and blood glucose in normal rabbits

The effects of the calcium antagonists verapamil and nifedipine on blood glucose levels, glucose tolerance, insulin secretion during glucose tolerance and hypoglycaemic effect of tolbutamide were studied in normal nondiabetic rabbits. Daily dosage of 40 mg/kg verapamil and 5 mg/kg nifedipine given orally up to 7 days did not affect blood glucose level, glucose tolerance, insulin secretion during glucose tolerance and hypoglycaemic activity of tolbutamide 250 mg/kg p.o.     

Effects of sulphonylureas and diazoxide on insulin secretion and nucleotide-sensitive channels in an insulin-secreting cell line.

1. The effects of various sulphonylureas and diazoxide on insulin secretion and the activity of various channels have been studied using tissue culture and patch-clamp methods in an insulin-secreting cell line derived from a rat islet cell tumour. 2. Tolbutamide, glibenclamide and HB699 increased the rate of insulin release by 2-5 fold. The concentrations of tolbutamide and glibenclamide giving half-maximum effects on insulin secretion were approximately 40 microM and 0.2 microM, respectively. 3. Diazoxide (0.6-1.0 mM) per se, had either no effect or produced a small increase in insulin secretion, whereas when secretion was maximally stimulated by the combination of glucose (3 mM) and leucine (20 mM), it produced inhibition. Tolbutamide-induced release was also inhibited by diazoxide. 4. Tolbutamide, glibenclamide, HB699 and HB985 reduced the open-state probability of the ATP-K+ channel in a dose-dependent manner. Tolbutamide and glibenclamide were shown to be effective regardless of which side of the membrane they were applied. 5. In whole cell recording, in which the total ATP-sensitive K+ conductance of the cell could be measured, dose-inhibition curves for tolbutamide and glibenclamide were constructed, resulting in Ki values of 17 microM and 27 nM, respectively. The value of Ki for tolbutamide was unchanged when ATP (0.1 mM) was present in the electrode. 6. Diazoxide (0.6 mM) activated the ATP-K+ channels only when they had first been inhibited by intracellular ATP (0.1 mM) or bath applied tolbutamide (3-30 microM). The inhibition produced by glibenclamide could not be reversed by diazoxide. 7. Neither tolbutamide (1.0 mM) nor glibenclamide (10 microM) altered the open-state probability of the Ca2+-activated K+ channel or the Ca2+-activated non-selective cation channel which are present in this cell line. 8. It is concluded that the sulphonylureas and related hypoglycaemic drugs and diazoxide regulate insulin secretion by direct effects on the ATP-K+ channel or a protein closely associated with this channel.     

Inhibition of voltage-gated Ca2+ channels and insulin secretion in HIT cells by the Ca2+/calmodulin-dependent protein kinase II inhibitor KN-62: comparison with antagonists of calmodulin and L-type Ca2+ channels.

To probe for the involvement of Ca2+/calmodulin-dependent protein kinase II in the regulation of insulin secretion, the effects of a specific inhibitor of this enzyme, KN-62, on secretagogue-stimulated insulin secretion, cytosolic Ca2+ concentration ([Ca2+]i) rise, membrane depolarization, and nutrient metabolism were examined in HIT-T15 cells. KN-62 dose-dependently inhibited insulin secretion induced by a nutrient mixture (10 mM glucose, 5 mM leucine, and 5 mM glutamine) alone or combined with either the Ca(2+)-mobilizing receptor agonist bombesin or the cAMP-raising agent forskolin in intact cells. KN-62 did not affect Ca(2+)- or GTP analogue-induced insulin secretion from permeabilized cells, indicating an action at a step before exocytosis. The stimulating effects of nutrients on insulin secretion, [Ca2+]i, and membrane depolarization were potentiated by bombesin. Similarly, bombesin promoted a larger depolarization and [Ca2+]i rise in the presence of nutrients. This was associated with enhanced Ca2+ mobilization and the appearance of sustained [Ca2+]i elevation. The bombesin-induced membrane depolarization, like the nutrient effect, was inhibited by diazoxide, suggesting that this is due to closure of ATP-sensitive K+ channels. Bombesin elicited Ca2+ influx by both membrane potential-sensitive and -insensitive conductance pathways. KN-62 did not affect Ca2+ mobilization and only partially reduced Ca2+ entry during the sustained [Ca2+]i rise in bombesin-stimulated cells. When added before or during the stimulation, KN-62 dose-dependently inhibited nutrient- and KCl-stimulated [Ca2+]i elevation and Mn2+ influx (reflecting Ca2+ entry). The calmodulin antagonist CGS 9343B and the L-type Ca2+ channel blocker SR-7037 mimicked the inhibitory effect of KN-62 on stimulated insulin secretion and [Ca2+]i elevation. Membrane depolarization and nutrient metabolism (reduction of a tetrazolium derivative), however, were not altered by KN-62 treatment, indicating that the early coupling events from nutrient metabolism to closure of ATP-sensitive K+ channels remain operative. These results suggest that KN-62 and the calmodulin antagonist CGS 9343B inhibit Ca2+ influx by means of direct interaction with L-type Ca2+ channels, which, in turn, causes inhibition of stimulated insulin secretion. Thus, it appears that Ca2+/calmodulin-dependent protein kinase II is not involved in the regulation of insulin secretion.     

Identification and pharmacological characterization of sarcolemmal ATP-sensitive potassium channels in the murine atrial HL-1 cell line

Activation of ATP-sensitive potassium (KATP) channels is known to have cardioprotective effects during periods of ischemia and reperfusion, making these channels important targets for clinical drug discovery. Using electrophysiological techniques we identify KATP channels in a mouse atrial cell line (HL-1). HL-1 KATP channels exhibited a concentration-dependent inhibition by ATP (IC50 = 23.3 +/- 3.2 microM), a unitary single-channel conductance of 55 pS, and sensitivity to the isoform-specific KATP channel opener P1075 and inhibitor HMR1098. Adenoviral infection of a dominant-negative Kir6.2 subunit significantly reduced the P1075-sensitive sarcKATP current. Taken together, the data indicate that HL-1 KATP channels are composed of sulfonylurea receptor isoform SUR2A coupled to the pore-forming Kir6.2 subunit--the molecular makeup of sarcKATP channels found in native cardiac myocytes. Pharmacological activation of HL-1 cell KATP channels also resulted in action potential shortening. Using the membrane potential-sensitive dye DiBac4(3), we demonstrated that the sarcKATP channel opener P1075 (20 microM) produced a concentration-dependent hyperpolarization of a monolayer of HL-1 cells that could be reversed by channel inhibition with HMR1098 (20 microM).We conclude that the HL-1 cells are an excellent cell line for studying cardiac sarcKATP channels, and these cells may also provide an important tool for the testing of novel pharmacological modulators of KATP channels in fluorescence-based assays.     

The signalling role of action potential depolarization in insulin secretion: Metabolism-dependent dissociation between action potential increase and secretion increase by TEA

The K⁺ channel blocker, TEA is known to increase action potential amplitude and insulin secretion of mouse β-cells when added to a nutrient secretagogue. In the presence of a maximally effective sulfonylurea concentration (2.7 μM glipizide) the nutrient secretagogue α-ketoisocaproic acid (KIC, 10 mM) strongly increased insulin secretion (about elevenfold). Instead of enhancing the effect of KIC, TEA reduced the KIC-induced secretion by more than 50%. Also, the secretion rate produced by 2.7 μM glipizide alone was significantly reduced by TEA. In contrast, TEA enhanced the insulinotropic effect of glipizide when a basal glucose concentration (5 mM) was present. In the presence as well as in the absence of glucose glipizide produced a plateau depolarization with superimposed action potentials. Under both conditions, TEA increased the glipizide-induced action potential amplitude and further elevated the cytosolic free calcium concentration ([Ca²⁺]_i) with an oscillatory characteristic. These effects depended on the activity of L-type Ca²⁺ channels, even though the effect of TEA differed from that of 1 μM of the Ca²⁺ channel opener, Bay K8644, which primarily increased action potential duration. TEA did not negatively affect parameters of β-cell energy metabolism (NAD(P)H fluorescence and ATP/ADP ratio), rather, it slightly increased NAD(P)H fluorescence. Apparently, TEA inhibits insulin secretion in the absence of glucose in spite of a persistent ability to block K⁺ ion conductance. Thus, the signalling role of action potential depolarization in insulin secretion may require reconsideration and ion conductance-independent actions of K⁺ channels may be involved in this paradox effect of TEA.     

Stoichiometry of sulfonylurea-induced ATP-sensitive potassium channel closure.

Hypoglycemic sulfonylureas (e.g., glibenclamide, glipizide, and tolbutamide) exert their stimulatory effect on excitatory cells by closure of ATP-sensitive potassium (KATP) channels. These channels are heteromultimers composed with a 4:4 stoichiometry of an inwardly rectifying K+ channel (KIR) subunit 6.x plus a sulfonylurea receptor (SUR). SUR1/KIR6.2 reconstitutes the neuronal/pancreatic beta-cell channel, whereas SUR2A/KIR6.2 and SUR2B/KIR6.1 (or KIR6.2) are proposed to reconstitute the cardiac and the vascular smooth muscle-type KATP channels, respectively. SUR2A and SUR2B are splice variants of a single gene differing only in their C-terminal 42 amino acids. Affinities of sulfonylureas for rat SUR2A, rat or human SUR2B, and a SUR2 chimera containing the C-terminal 42 amino acids of SUR1 did not differ significantly, implying that the C terminus does not form part of the binding pocket. Consistent with these findings, reconstituted SUR2A/KIR6.2 and SUR2B/KIR6.2 channels revealed similar sensitivities for glibenclamide and tolbutamide. Dissociation constants of sulfonylureas for SUR2A and SUR2B were 10- to 400-fold higher than for SUR1, however, amazingly the benzoic acid derivative meglitinide did not show lower affinity for SUR2 isoforms. Potencies of glibenclamide, glipizide, tolbutamide, and meglitinide to inhibit activity of SUR1/KIR6.2 and SUR2B/KIR6.2 channels were 3- to 6-fold higher than binding affinities of these drugs with concentration-inhibition relations being significantly steeper (Hill coefficients 1.23-1.32) than binding curves (Hill coefficients 0.93-1.06). The data establish that the C terminus of SURs does not affect sulfonylurea affinity and sensitivity. We conclude that occupation of one of the four SUR sites per channel complex is sufficient to induce KATP channel closure.     

Alterations of insulin secretion from mouse islets treated with sulphonylureas: perturbations of Ca2+ regulation prevail over changes in insulin content.

1. To determine how pretreatment with sulphonylureas alters the beta cell function, mouse islets were cultured (18 - 20 h) without (controls) or with (test) 0.01 microM glibenclamide. Acute responses to glucose were then determined in the absence of glibenclamide. 2. Test islets were insensitive to drugs (sulphonylureas and diazoxide) acting on K+-ATP channels, and their [Ca2+]i was already elevated in the absence of stimulation. 3. Insulin secretion was increased in the absence of glucose, and mainly stimulated between 0 - 10 instead of 7 - 20 mM glucose in controls. The maximum response was halved, but this difference disappeared after correction for the 45% decrease in the islet insulin content. 4. The first phase of glucose-induced insulin secretion was abrogated because of a paradoxical decrease of the high basal [Ca2+]i in beta cells. The second phase was preserved but occurred with little rise of [Ca2+]i. These abnormalities did not result from alterations of glucose metabolism (NADPH fluorescence). 5. In islets cultured with 50 microM tolbutamide, glucose induced biphasic increases in [Ca2+]i and insulin secretion. The decrease in the secretory response was matched by the decrease in insulin content (45%) except at maximal glucose concentrations. Islets pretreated with tolbutamide, however, behaved like those cultured with glibenclamide if tolbutamide was also present during the acute functional tests. 6. In conclusion, treatment with a low glibenclamide concentration causes long-lasting blockade of K+-ATP channels and rise of [Ca2+]i in beta cells. Glucose-induced insulin secretion occurs at lower concentrations, is delayed and is largely mediated by a modulation of Ca2+ action on exocytosis. It is suggested that glucose regulation of insulin secretion mainly depends on a K+-ATP channel-independent pathway during in vivo sulphonylurea treatment.     

Antihyperlipidemic effect of Garlip, a polyherbal formulation in streptozotocin induced diabetic rats

This study was undertaken to investigate the effect of Garlip, a polyherbal drug composed of aqueous extract of six medicinal plants on blood glucose, plasma insulin, tissue lipid profile, and lipidperoxidation in streptozotocin induced diabetic rats. Aqueous extract of Garlip a, polyherbal drug was administered orally (200 mg/kg body weight) for 30 days. The different doses of Garlip on blood glucose and plasma insulin in diabetic rats were studied and the levels of lipid peroxides (TBARS and Hydroperoxide) and tissue lipids (cholesterol, triglyceride, phospholipids and free fatty acids) were also estimated in streptozotocin induced diabetic rats. The effects were compared with tolbutamide. Treatment with Garlip and tolbutamide resulted in a significant reduction of blood glucose and increase in plasma insulin. Garlip also resulted in a significant decrease in tissue lipids and lipid peroxide formation. The effect produced by Garlip was comparable with that of tolbutamide. The decreased lipid peroxides and tissue lipids clearly showed the antihyperlipidemic and antiperoxidative effect of Garlip apart from its antidiabetic effect.     

Heterogeneous characteristics of imidazoline-induced insulin secretion

Imidazolines are regarded as a pharmacological group of insulin secretagogues with one uniform mechanism of action, namely closure of ATP-dependent K⁺ channels (K_ATP channels) and, in consequence, depolarization of the plasma membrane, Ca²⁺ influx and stimulation of secretion. This assumption was investigated by measuring insulin secretion from perifused pancreatic islets in response to three imidazoline compounds and comparing the characteristics of secretion with changes in membrane potential and cytosolic Ca²⁺ concentration [Ca²⁺]_i of single β-cells. Phentolamine (32 μM) stimulated insulin secretion from perifused mouse islets in the presence of stimulatory (10 mM and 30 mM) and substimulatory (5 mM) glucose concentrations and even in the absence of glucose. Idazoxan in concentrations up to 500 μM was virtually ineffective in the presence of 5 mM glucose. At 10 mM glucose, there was a moderate but significant increase of secretion by idazoxan, 20 μM being nearly as effective as 100 μM. The effect of phentolamine was of slow onset and irreversible in the time frame of the experiments, while the effect of idazoxan was of fast onset and reversible. Alinidine also stimulated secretion in the presence of 10 mM glucose with fast and reversible kinetics, but in contrast to idazoxan, 100 μM was clearly more effective than 20 μM. These heterogeneous characteristics of secretion were reflected by changes of [Ca²⁺]_i: the increase of [Ca²⁺]_i by phentolamine was slow and only partially reversible, whereas idazoxan led to a smaller, but faster and reversible response. The increase of [Ca²⁺]_i by phentolamine and idazoxan was abolished by the Ca²⁺ channel blocker D 600. Surprisingly, all three compounds depolarized the β-cell plasma membrane from a resting potential of –71 mV to about –36 mV. Again, the effect of phentolamine was slow and that of idazoxan and alinidine fast. Thus, the characteristics of phentolamine-induced secretion appear to be attributable to the consequences of K_ATP channel closure. It is unclear, however, why all three test compounds achieved the same degree of depolarization in spite of their known different efficiency to close K_ATP channels. Apparently, there are additional mechanisms involved in the action of idazoxan and alinidine, which may contribute to the obvious differences in the characteristics of secretion. Received: 2 October 1998 / Accepted: 21 December 1998     

The non-sulfonylurea moiety of gliquidone mimics the effects of the parent molecule on pancreatic B-cells

Compound UL-DF 9 corresponds to the non-sulfonylurea moiety of gliquidone, a hypoglycaemic sulfonylurea of the second generation. Its effects on the B-cell function were studied in vitro with mouse islets. In the presence of a non-stimulatory concentration of glucose (3 mM), UL-DF 9 decreased 86Rb+ efflux and accelerated 45Ca2+ efflux from islet cells, depolarized the B-cell membrane and induced an electrical activity similar to that triggered by stimulatory concentrations of glucose, and increased insulin release. The changes in 45Ca2+ efflux and insulin release, but not the inhibition of 86Rb+ efflux, were abolished in the absence of Ca2+. In the presence of 10 mM glucose, UL-DF 9 increased 86Rb+ and 45Ca2+ efflux from the islets, augmented the electrical activity in B-cells, and potentiated insulin release. These changes were suppressed by omission of extracellular Ca2+. Qualitatively similar effects were produced by lower concentrations of gliquidone itself. The data suggest that UL-DF 9 and gliquidone decrease the K+ permeability of the B-cell membrane, thereby causing a depolarization which leads to activation of voltage-dependent Ca channels and Ca2+ influx, and thus eventually increase insulin release. Hypoglycaemic sulfonylureas of the second generation therefore seem to contain a second chemical group that interacts with K channels of B-cells as does the sulfonylurea group itself.