Hyperinsulinism induced by targeted suppression of beta cell K _ATP channels

Abstract

ATP-sensitive K ؉ (KATP) channels couple cell metabolism to electrical activity. To probe the role of KATP in glucose-induced insulin secretion, we have generated transgenic mice expressing a dominant-negative, GFP-tagged KATP channel subunit in which residues 132-134 (Gly-Tyr-Gly) in the selectivity filter were replaced by Ala-Ala-Ala, under control of the insulin promoter. Transgene expression was confirmed by both beta cell-specific green fluorescence and complete suppression of channel activity in those cells (Ϸ70%) that did fluoresce. Transgenic mice developed normally with no increased mortality and displayed normal body weight, blood glucose levels, and islet architecture. However, hyperinsulinism was evident in adult mice as (i) a disproportionately high level of circulating serum insulin for a given glucose concentration (Ϸ2-fold increase in blood insulin), (ii) enhanced glucose-induced insulin release from isolated islets, and (iii) mild yet significant enhancement in glucose tolerance. Enhanced glucose-induced insulin secretion results from both increased glucose sensitivity and increased release at saturating glucose concentration. The results suggest that incomplete suppression of KATP channel activity can give rise to a maintained hyperinsulinism.T he ATP-sensitive K ϩ channel (K ATP ) has long been proposed as a critical link in glucose-induced insulin release from pancreatic beta cells ( Fig. 1A; refs. 1 and 2). According to this paradigm, a high intracellular [ATP]͞[ADP] ratio in the fed state inhibits K ATP channels, causing membrane depolarization, leading to Ca 2ϩ entry through voltage-dependent Ca 2ϩ channels and insulin exocytosis. A rise in circulating insulin, in turn, leads to an increased peripheral glucose uptake and compensatory drop in blood glucose. Conversely, a falling intracellular [ATP]͞ [ADP] ratio during the fasting state is presumed to relieve inhibition of K ATP channels, resulting in membrane hyperpolarization and cessation of Ca 2ϩ -induced insulin release (3).Direct evidence for this paradigm is provided by the stimulatory and inhibitory effects of the K ATP -specific drugs, sulfonylureas and diazoxide, respectively, on insulin secretion in vivo (4) and the generation of transgenic and knockout mouse models with expression of altered or deficient pancreatic K ATP (5-7) that exhibit beta cell dysfunction. Profound neonatal diabetes due to permanent suppression of insulin release is observed in transgenic mice expressing overactive beta cell K ATP channels, dramatically illustrating the ability of K ATP channels to inhibit secretion (8). Conversely, the recent discovery of numerous mutations in pancreatic K ATP channel subunits (both the poreforming Kir6.2 and SUR1) in human patients with persistent hyperinsulinemic hypoglycemia of infancy (PHHI) (3) establishes a causative link between suppressed K ATP activity, and the corollary metabolic disorder of hyperinsulinism (3). PHHIassociated K ATP mutations can be classified into two major categories: those that suppress channe...

Hyperinsulinism in mice with heterozygous loss of KATP channels

“…Mice were typed using primers for the wild-type Kir6.2 or SUR1 genes and primers against the neomycin-resistance gene that was incorporated in the Kir6.2 or SUR1 gene disruption [12,14]. Kir6.2[AAA] were generated and maintained on the C57/Bl6 background [15].…”

“…Islets were manually isolated under a dissecting microscope, and maintained overnight in CMRL medium (Gibco BRL) containing 5.6 mmol/l glucose in a humidified 37°C incubator. For electrophysiological measurements, islets were washed three times in Minimal Essential Medium (without L-glutamine), followed by one wash in DMEM supplemented with trypsin/EDTA (0.01/0.002%), and a final wash in DMEM [15]. Trypsin-treated islets were dispersed into isolated cells by resuspending gently in complete CMRL medium (supplemented with FCS, 10%), penicillin (100 U/ml) and streptomycin (100 μg/ml).…”

“…Electrophysiological measurements Macroscopic currents from dispersed islet cells were recorded using a standard whole-cell voltage-clamp configuration [15]. Electrodes were filled with K-INT (140 mmol/l KCl, 10 mmol/l K-HEPES, 1 mmol/l K-EGTA, pH 7.4), plus 1 mmol/l MgCl 2 and 1 mmol/l ATP (potassium salt).…”

“…Mice completely lacking Kir6.2 or SUR1 [12][13][14], as well as mice expressing a dominantnegative Kir6.2 transgene in beta cells (Kir6. 2[AAA] [15] or Kir6.2[G132S] [16]) have been generated. Kir6.2 [AAA] mice (which exhibit complete loss of K ATP channels in ∼70% of beta cells, but normal channel density in the remainder) show hyperinsulinaemia, enhanced glucose tolerance and increased glucose-stimulated insulin secretion (GSIS) [15].…”

“…2[AAA] [15] or Kir6.2[G132S] [16]) have been generated. Kir6.2 [AAA] mice (which exhibit complete loss of K ATP channels in ∼70% of beta cells, but normal channel density in the remainder) show hyperinsulinaemia, enhanced glucose tolerance and increased glucose-stimulated insulin secretion (GSIS) [15]. However, mice completely lacking K ATP activity (Kir6.2 −/− ) show transient hyperinsulinaemia as neonates, but both Kir6.2 −/− and SUR1 −/− unexpectedly show glucose intolerance and loss of insulin secretion as adults [12][13][14]17].…”