Stephen J. H. Ashcroft

The ATP-sensitivity of K+ channels in rat pancreatic B-cells is modulated by ADP

ATP‐sensitive K+ channels in inside‐out membrane patches from dispersed rat pancreatic B‐cells were studied using patch‐clamp methods. The dose‐response curve for ATP‐induced channel inhibition was shifted to higher concentrations in the presence of ADP (2 mM). In glucose‐free solution, the total intracellular concentration of ATP was 3.8 mM and of ADP 1.5 mM; glucose (20 mM) increased ATP and decreased ADP by approx. 40%. These results suggest that both ADP and ATP may be involved in regulating the activity of the glucose‐sensitive K+ channel in intact B‐cells.

The Insulin Gene Promoter: A Simplified Nomenclature

The tools of molecular biology have rapidly expanded our knowledge of how β-cells regulate insulin gene expression. As this work has progressed in parallel in different laboratories, alternate nomenclature systems have been developed to describe the functionally important elements of the insulin gene. This jumble of names is confusing to those outside the field and intimidating to neophytes. Therefore, we have agreed to a simple, uniform set of names for the major insulin gene promoter elements.

Association studies of variants in promoter and coding regions of beta‐cell ATP‐sensitive K‐channel genes SUR1 and Kir6.2 with Type 2 diabetes mellitus (UKPDS 53)

SUMMARY

Properties of single potassium channels modulated by glucose in rat pancreatic beta-cells.

1. The patch clamp method has been used to examine the effect of glucose on single K+ channel currents recorded from cell‐attached patches on dissociated rat pancreatic beta‐cells. Patch pipettes contained a 140 mM‐K+ solution. 2. In glucose‐free solution three types of K+ channels were observed. Two of these, having conductances of around 50 pS (G‐channel) and 20 pS when the external K+ concentration, [K+]0, was 140 mM, were active at the resting potential of the cell. The G‐channel was observed in more patches and showed higher activity; it therefore appears to contribute the major fraction of the resting K+ permeability of the beta‐cell. At membrane potentials positive to about +20 mV a third type of K+ channel, having a mean conductance of 120 pS, was activated. The open probability of this channel was strongly voltage dependent and increased with depolarization. 3. The reversal potential of the G‐channel current was shifted 59 mV by a 10‐fold change in external K+ (Na+ substitution) indicating the channel is highly K+ selective. The single‐channel conductance varied with [K+]o as predicted from the Goldman‐Hodgkin‐Katz equation; at physiological [K+]o (5 mM‐K+) an inward conductance of around 10 pS is predicted. The amplitude of the single‐channel current showed a tendency to saturate with increasing [K+]o. 4. Single G‐channel currents show burst kinetics indicating at least two closed states. The open and closed (gap) times within the bursts were distributed exponentially with time constants of 2.5 ms (tau o) and 0.5 ms (tau c1) respectively at the resting potential of the cell. There was little change in tau c1 over the voltage range ‐40 to 60 mV (pipette potential) but tau o increased slightly with membrane depolarization. 5. The addition of glucose to the bath solution produced a reversible, dose‐dependent decrease in G‐channel activity. This decrease results principally from a reduction in the frequency and duration of the bursts of openings with increasing glucose. In addition, the mean open time decreases. The short gaps during the bursts were little affected by glucose. 6. At glucose concentrations of .10 mM and above the decrease in G‐channel activity is accompanied by an increase in the input resistance of the cell and by the initiation of action potentials. 7. It is concluded that glucose metabolism results in a reduction of G‐channel open probability and thereby produces depolarization of the beta‐cell.

Metabotropic glutamate and GABAB receptors contribute to the modulation of glucose-stimulated insulin secretion in pancreatic beta cells

Aims/hypothesisThe neurotransmitters glutamate and γ-aminobutyric acid (GABA) could participate in the regulation of the endocrine functions of islets of Langerhans. We investigated the role of the metabotropic glutamate (mGluRs) and GABAB (GABABRs) receptors in this process. MethodsWe studied the expression of mGluRs and GABABRs in rat and human islets of Langerhans and in pancreatic α-cell and beta-cell lines using RT-PCR and immunoblot analysis. Effects of mGluR and GABABR agonists on insulin secretion were determined by radioimmunoassays and enzyme-linked immunoadsorbent assays (ELISAs). ResultsWe detected mGluR3 and mGluR5 (but not mGluR1, 6 and 7) mRNAs in all of the samples examined. Trace amount of mGluR2 was found in MIN6 beta cells; mGluR4 was identified in rat islets; and mGluR8 expression was detected in rat islets, RINm5F and MIN6 cells. GABABR1 a/b and 2 mRNAs were identified in islets of Langerhans and MIN6 cells. The expression of mGluR3, mGluR5, GABABR1 a/b and GABABR2 proteins was confirmed using specific antibodies. Group I (mGluR1/5) and group II (mGluR2/3) specific mGluR agonists increased the release of insulin in the presence of 3 to 10 mmol/l or 3 to 25 mmol/l glucose, respectively, whereas a group III (mGluR4/6–8) specific agonist inhibited insulin release at high (10–25 mmol/l) glucose concentrations. Baclofen, a GABABR agonist, also inhibited the release of insulin but only in the presence of 25 mmol/l glucose. Conclusion/interpretationThese data suggest that mGluRs and GABABRs play a role in the regulation of the endocrine pancreas with mechanisms probably involving direct activation or inhibition of voltage dependent Ca2+-channels, cAMP generation and G-protein-mediated modulation of KATP channels. [Diabetologia (2002) 45: 242–252]

Molecular structure of the glibenclamide binding site of the β-cell KATP channel

We have investigated the structure of the glibenclamide binding site of pancreatic β‐cell ATP‐sensitive potassium (KATP) channels. KATP channels are a complex of four pore‐forming Kir6.2 subunits and four sulfonylurea receptor (SUR1) subunits. SUR1 (ABCC8) belongs to the ATP binding cassette family of proteins and has two nucleotide binding domains (NBD1 and NBD2) and 17 putative transmembrane (TM) sequences. Co‐expression in a baculovirus expression system of two parts of SUR1 between NBD1 and TM12 leads to restoration of glibenclamide binding activity, whereas expression of either individual N‐ or C‐terminal part alone gave no glibenclamide binding activity, confirming a bivalent structure of the glibenclamide binding site. By using N‐terminally truncated recombinant proteins we have shown that CL3 – the cytosolic loop between TM5 and TM6 – plays a key role in formation of the N‐terminal component of the glibenclamide binding site. Analysis of deletion variants of the C‐terminal part of SUR1 showed that CL8 – the cytosolic loop between TM15 and TM16 – is the only determinant for the C‐terminal component of the glibenclamide binding site. We suggest that in SUR1 in the native KATP channel close proximity of CL3 and CL8 leads to formation of the glibenclamide binding site.

Identification of functional ionotropic glutamate receptor proteins in pancreatic beta-cells and in islets of Langerhans.

The presence of ionotropic glutamate receptor proteins in islets of Langerhans and pancreatic β‐cell lines (MIN6, HIT T15, RINm5F) was investigated. For this purpose immunoblot analysis of β‐cell membranes was performed with subunit‐specific antibodies. We identified NMDAR1 subunits of the NMDA and KA‐2 subunits of the kainate receptors, but did not detect GluR1 subunits of the AMPA receptor. The receptor subunits present were shown to be glycosylated. β‐cell membranes contained specific binding sites for glutamate receptor ligands, and NMDA increased insulin secretion. These results demonstrate that ionotropic glutamate receptor proteins, similar to those in the central nervous system, are expressed in rat pancreatic β‐cells.

Protein kinase C in beta-cells: expression of multiple isoforms and involvement in cholinergic stimulation of insulin secretion

The mammalian protein kinase C (PKC) family consists of at least 11 distinct isotypes with marked differences in tissue distribution, localization, cofactor dependence and substrate specificity. Evidence exists for the expression of some of the PKC isoforms in pancreatic beta-cells but no comprehensive analysis of all the known PKC types has been accomplished. To assess the functional relevance of phosphorylation by PKC in the mechanism of insulin secretion we firstly investigated the expression of PKC isoforms in pancreatic beta-cells. The combination of reverse transcription-polymerase chain reaction (RT-PCR), Northern analysis and immunoblotting demonstrated the expression of PKC-alpha, beta II, epsilon, zeta, lambda and mu in MIN6 beta-cells. PKC-mu has not previously been detected in beta-cells. Expression of PKC-delta was also observed at the mRNA level; however, the protein could not be detected by Western blotting in MIN6 cells but was readily observed in RINm5F beta-cells. In short-term incubations, insulin release from MIN6 cells was augmented by 12-0-tetradecanoyl-phorbol-13-acetate (TPA), by carbachol, and by 40 mM K+. Culture of MIN6 cells overnight with TPA resulted in down-regulation of PKC-alpha (totally) and epsilon (partially), without significant change in the other isoforms. In such TPA-treated cells, the secretory response to TPA and to carbachol was abolished but not that elicited by high K+. It is suggested that PKC-alpha and/or epsilon may play a role in cholinergic potentiation of insulin secretion.

Protein phosphorylation in the pancreatic B-cell

92 Valverde, I,, and Malaisse, W.J., Calmodulin and pancreatic B-cell function. Experientia 40 (1984) 1061-1068. 93 Valverde, I., Sener, A., Herchuelz, A., and Malaisse, W.J., The stimulus-secretion coupling of glucose-induced insulin release. XLVII. The possible role of calmodulin. Endocrinology 108 (1981) 1305-1312. 94 Valverde, I., Vandermeers, A., Anjancyulu, R., and Malaisse, W.J., Calmodulin activation of adenylate cyclase in pancreatic islets. Science 206 (1979) 225-227. 95 Wollheim, C.B., Blondel, B., and Sharp, G.W.G., Effect of cholera toxin on insulin release in monolayer cultures of the endocrine pancreas. Diabetologia 10 (1974) 783-787. 1075

ATP-sensitive K-channels in HIT T15 beta-cells studied by patch-clamp methods, 86Rb efflux and glibenclamide binding.

ATP-sensitive K-channels in the cloned β-cell line HIT T15 were studied by patch-clamp methods; by measurement of 86Rb efflux; and by [3H]glibenclamide binding to isolated membrane preparations. In inside-out patches a 50 pS K-channel was found which was blocked by ATP or tolbutamide applied to the intracellular membrane surface. A minimum estimate of about 500 channels per β-cell was obtained by combining whole-cell and single-channel data. The rate of efflux of 86Rb from 86RbCl-loaded HIT cells was markedly increased by intracellular ATP-depletion; 86Rb-efflux was progressively inhibited by increasing concentrations of glibenclamide or tolbutamide. In non-ATP-depleted cells, diazoxide elicited a concentration-dependent stimulation of 86Rb-efflux which was completely blocked by 1 μM glibenclamide. Isolated membranes showed dose-dependent saturable binding of [3H]glibenclamide to both high (Kd=1.12 nM) and low (Kd=136 nM) affinity binding sites. We estimate about 5000 high-affinity binding sites per cell. [3H]-glibenclamide binding was inhibited by tolbutamide (IC50=125 μM) but was not affected by diazoxide. ADP (0.5 or 1.0 mM) markedly reduced binding; other nucleotides tested were ineffective.