U. Panten

Concentration-dependent effects of tolbutamide, meglitinide, glipizide, glibenclamide and diazoxide on ATP-regulated K+ currents in pancreatic B-cells

SummaryThe influence of the hypoglycemic drugs tolbutamide, meglitinide, glipizide and glibenclamide on ATP-dependent K+ currents of mouse pancreatic B-cells was studied using the whole-cell configuration of the patch-clamp technique. In the absence of albumin, tolbutamide blocked the currents half maximally at 4.1 μmol/l. In the presence of 2 mg/ml albumin half maximal inhibition of the currents was observed at 2.1 μmol/l meglitinide, 6.4 nmol/l glipizide and 4.0 nmol/1 glibenclamide. The hyperglycemic sulfonamide diazoxide opened ATP-dependent K+channels. Half maximally effective concentrations of diazoxide were 20 μmol/l with 0.3 mmol/1 ATPand102 μmol/l with 1 mmol/1 ATP in the recording pipette. Thus, the action of diazoxide was dependent on the presence of ATP in the recording pipette. The free concentrations of the drugs which influenced ATP-dependent K+ currents were comparable with the free plasma concentrations in humans and the free concentrations which affected insulin secretion in vitro. The results support the view that the target for the actions of sulfonylureas and of diazoxide is the ATP-dependent K+ channel of the pancreatic B-cell or a structure closely related to this channel.

Cytosolic ADP enhances the sensitivity to tolbutamide of ATP-dependent K+ channels from pancreatic B-cells.

The effects of intracellular purine nucleotides on tolbutamide‐induced block of ATP‐dependent K+ channels from mouse pancreatic B‐cells were studied using the patch‐clamp technique. When applied to the inside of excised patches tolbutamide alone blocked channel activity half‐maximally at 55μM and the concentration‐response curve for the inhibition of K+ channels by tolbutamide was flat. ADP (1 mM), but not other nucleotides (AMP, GTP or GDP) increased the steepness of the concentration‐response curve and decreased the half‐maximally effective tolbutamide concentration to 4.2 μM. It is suggested that the ATP‐dependent K+ channel or a closely related structure contains a receptor which is accessible for cytosolic ADP and controls the sensitivity to tolbutamide.

Effects of L-leucine and α-ketoisocaproic acid upon insulin secretion and metabolism of isolated pancreatic islets

LLeucine has been shown to s~uIate insulin release in vivo [ 1,2] and in vitro [3,4] . It is unknown whether Lleucine must be metabolized to elicit this effect. From in vivo experiments Knopf et al. [2] suggested that o-ketoisocaproic acid (o-KHZ) has no hypoglycemic action of its own but must be transaminated to leucine in order to stimulate insulin secretion. Recently the nonmetabolized, but transported, leucine analogue 2-aminobicyclo(2,2,l)heptane-2-carboxylic acid was found to induce insulin release in vivo [5] and in vitro [6] . From these experiments it was concluded that the receptor sites for the stimulation of ins~in release by Lleucine may be transport sites. Since the above-mentioned experiments did not rule out that Lleucine or a-KIC trigger insulin release by metabolic events in the pcell the present studies were performed. (w-KIC induced a marked stimulation of insulin release by isolated pancreatic islets. The accompanying increase of fluorescence of reduced pyridine nucleotides demonstrated prompt changes of islet cell metabolism. In pancreatic islets cr-KIC enhanced the tissue level and the production of leucine. All other tested leucine metabolites did not stimulate insulin release si~~cantly,

Effect of carbohydrates upon fluorescence of reduced pyridine nucleotides from perifused isolated pancreatic islets

SummaryIn perifused pancreatic islets, the fluorescence of reduced pyridine nucleotides (NAD(P)H) was measured continuously. Elevation of glucose concentration in the medium from 0 – 5 mM to 20 mM led to an increase in NAD(P)H-fluorescence beginning 10–20 sec after change of medium. Perifusion with calcium-free media had no influence on this effect. It was, however, partially or completely blocked by 2-deoxy D-glucose, D-glucosamine, or D-mannoheptulose. D-mannose, but not D-fructose and L-lactate, enhanced NAD(P)H-fluorescence from pancreatic islets. Pyruvate caused but a small fluorescence increase. From these observations it is concluded that D-glucose leads to the increase of NAD(P)H-fluorescence by mediation of the phosphoglyceraldehyde dehydrogenase reaction.

Phosphate and thiophosphate group donating adenine and guanine nucleotides inhibit glibenclamide binding to membranes from pancreatic islets.

SummaryIn microsomes obtained from mouse pancreatic islets, the Mg complex of adenosine 5′-triphosphate (MgATP) increased the dissociation constant (KD) for binding of [3H]glibenclamide by sixfold. In the presence of Mg2+, not only ATP but also adenosine 5′-0-(3-thiotriphosphate) (ATPγS), adenosine 5′-diphosphate (ADP), guanosine 5′-triphosphate (GTP), guanosine 5′-diphosphate (GDP), guanosine 5′-0-(3-thiotriphosphate) (GTPγTS) and guanosine 5′-0-(2-thiodiphosphate) (GDPβ S) inhibited binding of [3H]glibenclamide. These effects were not observed in the absence of Mg2+. Half maximally effective concentrations of the Mg complexes of ATP, ADP, ATPγS and GDP were 11.6, 19.0, 62.3 and 90.1 μmol/l, respectively. The non-hydrolyzable analogues adenosine 5′-(β,γ-imidotriphosphate) (AMP-PNP) and guanosine 5′-(β,γ-imidotriphosphate) (GMP-PNP) did not alter [3H]glibenclamide binding in the presence of Mg2+. MgADP acted much more slowly than MgATP and both MgADP and MgADP did not inhibit [3H]glibenclamide binding when the concentrations of MgATP and MgATP were kept low by the hexokinase reaction. Development of MgATP-induced inhibition of [3H]glibenclamide binding and dissociation of [3H]-glibenclamide binding occurred at similar rates. However, the reversal of MgATP-induced inhibition of [3H]glibenclamide binding was slower than the association of [3H]glibenclamide with its binding site. Exogenous alkaline phosphatase accelerated the reversal of MgATP-induced inhibition of [3H]glibenclamide binding. MgATP enhanced displacement of [3H]glibenclamide binding by diazoxide. The data suggest that sulfonylureas and diazoxide exert their effects by interaction with the same binding site at the sulfonylurea receptor and that protein phosphorylation modulates the affinity of the receptor.

How do sulfonylureas approach their receptor in the B-cell plasma membrane?

SummarySince it was unknown whether the uncharged or the anionic form of hypoglycemic sulfonylureas and meglitinide is the effective modulator of ATP-dependent K+ channels and insulin secretion, we studied the inhibitory effects of tolbutamide and meglitinide on the ATP-dependent K+ current at different external pH. The whole-cell configuration of the patch-clamp technique was used in mouse pancreatic B-cells. When the concentrations of the undissociated forms of these drugs were kept constant at increasing pH of the bath solution (6.4 to 8.4), the rate of development and the degree of K+ channel block varied only slightly. Raising the pH-value in the bath solution at constant total concentration of tolbutamide diminished both the rate of development and the degree of K+ channel block. It is concluded that the undissociated forms of tolbutamide and related compounds are the effective forms.Examination of the K+ current records during the application and removal of different concentrations of tolbutamide, meglitinide, glipizide and glibenclamide at pH 7.4 indicated that the kinetics of the current records reflected not only association and dissociation of the drug-receptor complex but perhaps also the kinetic of drug distribution between bath and the lipid phase of the plasma membrane. As there is evidence against an interaction between sulfonylureas and their receptor via a binding site freely accessible from the cytoplasm, the drugs probably get access to their binding site on the receptor from the lipid phase of the B-cell plasma membrane.

Regulation of energy metabolism in pancreatic islets by glucose and tolbutamide

SummaryThe kinetics of insulin secretion and oxygen uptake in response to D-glucose and tolbutamide were compared in mouse pancreatic islets. In addition, the role of decreased ATP as a driving force for secretagogue-induced oxygen consumption was examined. D-glucose (10–30 mmol/1) triggered a biphasic insulin release which always coincided with a monophasic increase in islet oxygen uptake. In the presence of D-glucose (5–30 mmol/1), tolbutamide (3–500 g,mol/1) consistently elicited an initial peak of insulin secretion which was followed by a continued decline. Tolbutamide-induced secretory profiles were accompanied by similar respiratory profiles. Oxygen consumption per ng of insulin released during the test phase was higher after elevation of the glucose concentration than after addition of tolbutamide. In conjunction with 5 or 10 mmol/l D-glucose, but not with 15 or 30 mmol/1 D-glucose, tolbutamide (30–100 μmol/1) lowered islet ATP content significantly (p < 0.02). Phosphocreatine was not found in isolated islets, although they contained substantial creatine kinase activity. It is concluded that the driving force for Tobutamide-induced oxygen uptake is a decrease in the phosphorylation potential caused by the work load imposed by stimulation of the secretion process. However, a major proportion of the respiratory response to glucose also results from enhancement of biosynthesis.

Glucose both inhibits and stimulates insulin secretion from isolated pancreatic islets exposed to maximally effective concentrations of sulfonylureas

SummaryIsolated pancreatic islets from mice were perifused with media containing maximally effective concentrations of glibenclamide (0.1–10 μmol/l) or glipizide (1 μmol/l). In these islets an increase of the glucose concentration from 10 mmol/l to 40 mmol/l or addition of d-glyc-eraldehyde (20 mmol/1) caused a temporary decrease in insulin release which was followed by a sustained enhancement of release. α-Ketoisocaproate (3 or 20 mmol/1) did not inhibit insulin release; at high concentration it was an even stronger secretagogue than d-glucose or d-glyceraldehyde. It is concluded that high energy phosphates couple B-cell fuel metabolism and insulin release by acting both on the ATP-dependent K+ channel and on other targets not yet identified.

Studies on the mechanism of l-leucine- and alpha-ketoisocaproic acid- -induced insulin release from perifused isolated pancreatic islets.

SummaryThe insulinotropic effects of L-leucine and α-ketoisocaproic acid have been compared in perifused isolated pancreatic islets. In contrast to α-ketoisocaproic acid (10 mM), L-leucine (10 mM) released less insulin in the presence than in the absence of glucose (5 mM). Changes of islet cell metabolism accompanying insulin release were studied by recording the fluorescence of reduced pyridine nucleotides. The traces of L-leucine-or α-ketoisocaproic acid-induced fluorescence increase differed both in the absence and in the presence of glucose (5 mM). When the medium perif using the islets contained 30 mM L-leucine, α-ketoisocaproic acid (10 mM) still triggered a significant insulin release. These results argue against an indirect action of α-ketoisocaproic acid via transformation to L-leucine. Isocaproic acid (10 mM), L-α-hydroxyisocaproic acid (10 mM) or α-ketoisovaleric acid stimulated no remarkable insulin release, demonstrating that the strong insulinotropic effect of α-ketoisocaproic acid is coupled both to its α-ketogroup and to the length of its carbon chain.

Thyroxine treatment and insulin secretion in the rat

SummaryThyroxine treatment increases blood glucose and plasma insulin levels in the rat. The hypoglycemic effect of tolbutamide is more pronounced in treated animals. The immediate insulin secretory response of the isolated perfused pancreas to maximal, but not to submaximal, glucose stimuli was increased after thyroxine treatment, especially in the lower dose range. However, as thyroxine treatment reduces insulin release during the prolonged late phase, the total amount of insulin released from the pancreas is reduced. Both the early response to tolbutamide and the subsequent basal secretion were increased after thyroxine treatment. When the pancreas of treated rats was exposed to glucose plus pyruvate the inhibition of the late phase was reversed. Isoprenaline did not overcome the inhibitory effect of thyroxine treatment on the late phase of glucose-induced insulin release. Thyroxine induces a selective inhibition of glucose induced insulin release which is reversed by pyruvate; this indicates that thyroxine interferes with the glycolysis in the beta cell.