Peter Proks

A genetic and physiological study of impaired glucose homeostasis control in C57BL/6J mice

Aims/hypothesisC57BL/6J mice exhibit impaired glucose tolerance. The aims of this study were to map the genetic loci underlying this phenotype, to further characterise the physiological defects and to identify candidate genes.MethodsGlucose tolerance was measured in an intraperitoneal glucose tolerance test and genetic determinants mapped in an F2 intercross. Insulin sensitivity was measured by injecting insulin and following glucose disposal from the plasma. To measure beta cell function, insulin secretion and electrophysiological studies were carried out on isolated islets. Candidate genes were investigated by sequencing and quantitative RNA analysis.ResultsC57BL/6J mice showed normal insulin sensitivity and impaired insulin secretion. In beta cells, glucose did not stimulate a rise in intracellular calcium and its ability to close KATP channels was impaired. We identified three genetic loci responsible for the impaired glucose tolerance. Nicotinamide nucleotide transhydrogenase (Nnt) lies within one locus and is a nuclear-encoded mitochondrial proton pump. Expression of Nnt is more than sevenfold and fivefold lower respectively in C57BL/6J liver and islets. There is a missense mutation in exon 1 and a multi-exon deletion in the C57BL/6J gene. Glucokinase lies within the Gluchos2 locus and shows reduced enzyme activity in liver.Conclusions/interpretationThe C57BL/6J mouse strain exhibits plasma glucose intolerance reminiscent of human type 2 diabetes. Our data suggest a defect in beta cell glucose metabolism that results in reduced electrical activity and insulin secretion. We have identified three loci that are responsible for the inherited impaired plasma glucose tolerance and identified a novel candidate gene for contribution to glucose intolerance through reduced beta cell activity.

Molecular determinants of KATP channel inhibition by ATP

ATP‐sensitive K+ (KATP) channels are both inhibited and activated by intracellular nucleotides, such as ATP and ADP. The inhibitory effects of nucleotides are mediated via the pore‐forming subunit, Kir6.2, whereas the potentiatory effects are conferred by the sulfonylurea receptor subunit, SUR. The stimulatory action of Mg‐nucleotides complicates analysis of nucleotide inhibition of Kir6.2/SUR1 channels. We therefore used a truncated isoform of Kir6.2, that expresses ATP‐sensitive channels in the absence of SUR1, to explore the mechanism of nucleotide inhibition. We found that Kir6.2 is highly selective for ATP, and that both the adenine moiety and the β‐phosphate contribute to specificity. We also identified several mutations that significantly reduce ATP inhibition. These are located in two distinct regions of Kir6.2: the N‐terminus preceding, and the C‐terminus immediately following, the transmembrane domains. Some mutations in the C‐terminus also markedly increased the channel open probability, which may account for the decrease in apparent ATP sensitivity. Other mutations did not affect the single‐channel kinetics, and may reduce ATP inhibition by interfering with ATP binding and/or the link between ATP binding and pore closure. Our results also implicate the proximal C‐terminus in KATP channel gating.

Rapid ATP-Dependent Priming of Secretory Granules Precedes Ca2+ -Induced Exocytosis in Mouse Pancreatic B-Cells

1 The glucose and ATP dependence of exocytosis were investigated in single mouse pancreatic B‐cells by monitoring changes in cell capacitance evoked by voltage‐clamp depolarizations, infusion of high‐[Ca2+]i buffers or photorelease of caged Ca2+ or ATP. 2 In intact B‐cells, using the perforated patch whole‐cell technique, glucose (5 mM) increased exocytotic responses evoked by membrane depolarization 5–fold over that observed in the absence of the sugar. Increasing the glucose concentration to 20 mM produced a further doubling of exocytosis. The stimulatory action of glucose was attributable to glucose metabolism and abolished by mannoheptulose, an inhibitor of glucose phosphorylation. 3 Exocytosis triggered by infusion of high [Ca2+]i and ATP was reduced by 80 % when ATP was replaced by its non‐hydrolysable analogue adenosine 5′‐[β,γ‐methylene]triphosphate (AMP‐PCP) in standard whole‐cell experiments. Exocytosis elicited by GTPγS was similarly affected by replacement of ATP with the stable analogue. 4 Photoreleasing ATP in the presence of 170 nm [Ca2+]i following the complete wash‐out of endogenous ATP, produced a prompt (latency, < 400 ms) and biphasic stimulation of exocytosis. 5 Elevation of [Ca2+]i to exocytotic levels by photorelease from Ca2+‐nitrophenyl EGTA preloaded into the cell evoked a biphasic stimulation in the presence of Mg‐ATP. The response consisted of an initial rapid (completed in < 200 ms) phase followed by a slower (lasting ≥ 10 s) sustained component. Replacement of ATP with AMP‐PCP abolished the late component but did not affect the initial phase. The latency between elevation of [Ca2+]i and exocytosis was determined as < 45 ms. Inclusion of N‐ethylmaleimide (NEM; 0.5 mM for 3 min) in the intracellular solution exerted effects similar to those obtained by substituting AMP‐PCP for ATP. 6 We conclude that the B‐cell contains a small pool (40 granules) of primed granules which are immediately available for release and which are capable of undergoing exocytosis in an ATP‐independent fashion. We propose that this pool of granules is preferentially released during first phase glucose‐stimulated insulin secretion. The short latency between the application of ATP and the onset of exocytosis finally suggests that priming takes place with sufficient speed to participate in the rapid adjustment of the secretory capacity of the B‐cell.

Functional analysis of a structural model of the ATP‐binding site of the KATP channel Kir6.2 subunit

ATP‐sensitive potassium (KATP) channels couple cell metabolism to electrical activity by regulating K+ flux across the plasma membrane. Channel closure is mediated by ATP, which binds to the pore‐forming subunit (Kir6.2). Here we use homology modelling and ligand docking to construct a model of the Kir6.2 tetramer and identify the ATP‐binding site. The model is consistent with a large amount of functional data and was further tested by mutagenesis. Ligand binding occurs at the interface between two subunits. The phosphate tail of ATP interacts with R201 and K185 in the C‐terminus of one subunit, and with R50 in the N‐terminus of another; the N6 atom of the adenine ring interacts with E179 and R301 in the same subunit. Mutation of residues lining the binding pocket reduced ATP‐dependent channel inhibition. The model also suggests that interactions between the C‐terminus of one subunit and the ‘slide helix’ of the adjacent subunit may be involved in ATP‐dependent gating. Consistent with a role in gating, mutations in the slide helix bias the intrinsic channel conformation towards the open state.

Mechanism of Cloned ATP-sensitive Potassium Channel Activation by Oleoyl-CoA

Insulin secretion from pancreatic beta cells is coupled to cell metabolism through closure of ATP-sensitive potassium (KATP) channels, which comprise Kir6.2 and sulfonylurea receptor (SUR1) subunits. Although metabolic regulation of KATP channel activity is believed to be mediated principally by the adenine nucleotides, other metabolic intermediates, including long chain acyl-CoA esters, may also be involved. We recorded macroscopic and single-channel currents from Xenopusoocytes expressing either Kir6.2/SUR1 or Kir6.2ΔC36 (which forms channels in the absence of SUR1). Oleoyl-CoA (1 μm) activated both wild-type Kir6.2/SUR1 and Kir6.2ΔC36 macroscopic currents, ∼2-fold, by increasing the number and open probability of Kir6.2/SUR1 and Kir6.2ΔC36 channels. It was ineffective on the related Kir subunit Kir1.1a. Oleoyl-CoA also impaired channel inhibition by ATP, increasing the Ki values for both Kir6.2/SUR1 and Kir6.2ΔC36 currents by ∼3-fold. Our results indicate that activation of KATP channels by oleoyl-CoA results from an interaction with the Kir6.2 subunit, unlike the stimulatory effects of MgADP and diazoxide which are mediated through SUR1. The increased activity and reduced ATP sensitivity of KATP channels by oleoyl-CoA might contribute to the impaired insulin secretion observed in non-insulin-dependent diabetes mellitus.

Permanent neonatal diabetes caused by dominant, recessive, or compound heterozygous SUR1 mutations with opposite functional effects.

Heterozygous activating mutations in the KCNJ11 gene encoding the pore-forming Kir6.2 subunit of the pancreatic beta cell K(ATP) channel are the most common cause of permanent neonatal diabetes (PNDM). Patients with PNDM due to a heterozygous activating mutation in the ABCC8 gene encoding the SUR1 regulatory subunit of the K(ATP) channel have recently been reported. We studied a cohort of 59 patients with permanent diabetes who received a diagnosis before 6 mo of age and who did not have a KCNJ11 mutation. ABCC8 gene mutations were identified in 16 of 59 patients and included 8 patients with heterozygous de novo mutations. A recessive mode of inheritance was observed in eight patients with homozygous, mosaic, or compound heterozygous mutations. Functional studies of selected mutations showed a reduced response to ATP consistent with an activating mutation that results in reduced insulin secretion. A novel mutational mechanism was observed in which a heterozygous activating mutation resulted in PNDM only when a second, loss-of-function mutation was also present.

Electrogenic arginine transport mediates stimulus-secretion coupling in mouse pancreatic beta-cells.

1. We have investigated the mechanism by which L‐arginine stimulates membrane depolarization, an increase of intracellular calcium ([Ca2+]i) and insulin secretion in pancreatic beta‐cells. 2. L‐Arginine failed to affect beta‐cell metabolism, as monitored by NAD(P)H autofluorescence. 3. L‐Arginine produced a dose‐dependent increase in [Ca2+]i, which was dependent on membrane depolarization and extracellular calcium. 4. The cationic amino acids L‐ornithine, L‐lysine, L‐homoarginine (which is not metabolized) and NG‐monomethyl‐L‐arginine (L‐NMMA, a nitric oxide synthase inhibitor) produced [Ca2+]i responses similar to that produced by L‐arginine. The neutral nitric oxide synthase inhibitors NG‐nitro‐L‐arginine (L‐NNA) and N omega‐monomethyl‐L‐arginine (L‐NAME) also increased [Ca2+]i. D‐Arginine was ineffective. 5. L‐Arginine did not affect whole‐cell Ca2+ currents or ATP‐sensitive K+ currents, but produced an inward current that was carried by the amino acid. 6. The reverse transcriptase‐polymerase chain reaction demonstrated the presence of messenger RNA for the murine cationic amino acid transporters mCAT2A and mCAT2B within the beta‐cell. 7. L‐Arginine did not affect beta‐cell exocytosis as assayed by changes in cell capacitance. 8. Our data suggest that L‐arginine elevates [Ca2+]i and stimulates insulin secretion as a consequence of its electrogenic transport into the beta‐cell. This uptake is mediated by the mCAT2A transporter.

A gating mutation at the internal mouth of the Kir6.2 pore is associated with DEND syndrome

Inwardly rectifying potassium (Kir) channels control cell membrane K+ fluxes and electrical signalling in diverse cell types. Heterozygous mutations in the human Kir6.2 gene (KCNJ11), the pore‐forming subunit of the ATP‐sensitive (KATP) channel, cause permanent neonatal diabetes mellitus. However, the I296L mutation also results in developmental delay, muscle weakness and epilepsy. We investigated the functional effects of the I296L mutation by expressing wild‐type or mutant Kir6.2/SUR1 channels in Xenopus oocytes. The mutation caused a marked increase in resting whole‐cell KATP currents by reducing channel inhibition by ATP, in both homomeric and simulated heterozygous states. Kinetic analysis showed that the mutation impaired ATP sensitivity indirectly, by stabilizing the open state of the channel and possibly also by means of an allosteric effect on ATP binding and/or transduction. The results implicate a new region in Kir‐channel gating and suggest that disease severity is correlated with the extent of reduction in ATP sensitivity.

Review. SUR1: a unique ATP-binding cassette protein that functions as an ion channel regulator.

SUR1 is an ATP-binding cassette (ABC) transporter with a novel function. In contrast to other ABC proteins, it serves as the regulatory subunit of an ion channel. The ATP-sensitive (KATP) channel is an octameric complex of four pore-forming Kir6.2 subunits and four regulatory SUR1 subunits, and it links cell metabolism to electrical activity in many cell types. ATPase activity at the nucleotide-binding domains of SUR results in an increase in KATP channel open probability. Conversely, ATP binding to Kir6.2 closes the channel. Metabolic regulation is achieved by the balance between these two opposing effects. Precisely how SUR1 talks to Kir6.2 remains unclear, but recent studies have identified some residues and domains that are involved in both physical and functional interactions between the two proteins. The importance of these interactions is exemplified by the fact that impaired regulation of Kir6.2 by SUR1 results in human disease, with loss-of-function SUR1 mutations causing congenital hyperinsulinism and gain-of-function SUR1 mutations leading to neonatal diabetes. This paper reviews recent data on the regulation of Kir6.2 by SUR1 and considers the molecular mechanisms by which SUR1 mutations produce disease.

Expression of an activating mutation in the gene encoding the KATP channel subunit Kir6.2 in mouse pancreatic beta cells recapitulates neonatal diabetes.

Neonatal diabetes is a rare monogenic form of diabetes that usually presents within the first six months of life. It is commonly caused by gain-of-function mutations in the genes encoding the Kir6.2 and SUR1 subunits of the plasmalemmal ATP-sensitive K+ (KATP) channel. To better understand this disease, we generated a mouse expressing a Kir6.2 mutation (V59M) that causes neonatal diabetes in humans and we used Cre-lox technology to express the mutation specifically in pancreatic beta cells. These beta-V59M mice developed severe diabetes soon after birth, and by 5 weeks of age, blood glucose levels were markedly increased and insulin was undetectable. Islets isolated from beta-V59M mice secreted substantially less insulin and showed a smaller increase in intracellular calcium in response to glucose. This was due to a reduced sensitivity of KATP channels in pancreatic beta cells to inhibition by ATP or glucose. In contrast, the sulfonylurea tolbutamide, a specific blocker of KATP channels, closed KATP channels, elevated intracellular calcium levels, and stimulated insulin release in beta-V59M beta cells, indicating that events downstream of KATP channel closure remained intact. Expression of the V59M Kir6.2 mutation in pancreatic beta cells alone is thus sufficient to recapitulate the neonatal diabetes observed in humans. beta-V59M islets also displayed a reduced percentage of beta cells, abnormal morphology, lower insulin content, and decreased expression of Kir6.2, SUR1, and insulin mRNA. All these changes are expected to contribute to the diabetes of beta-V59M mice. Their cause requires further investigation.