ATP-sensitive K+ channels (KATP channels) play important roles in many cellular functions by coupling cell metabolism to electrical activity. The KATP channels in pancreatic beta-cells are thought to be critical in the regulation of glucose-induced and sulfonylurea-induced insulin secretion. Until recently, however, the molecular structure of the KATP channel was not known. Cloning members of the novel inwardly rectifying K+ channel subfamily Kir6.0 (Kir6.1 and Kir6.2) and the sulfonylurea receptors (SUR1 and SUR2) has clarified the molecular structure of KATP channels. The pancreatic beta-cell KATP channel comprises two subunits: a Kir6.2 subunit and an SUR1 subunit. Molecular biological and molecular genetic studies have provided insights into the physiological and pathophysiological roles of the pancreatic beta-cell KATP channel in insulin secretion.
T Miki, K Nagashima and S Seino
K Capito, S E Hansen and P Thams
The involvement of phosphatidylcholine (PC) hydrolysis in the regulation of insulin secretion was studied in mouse pancreatic islets prelabelled with [3H]choline. Phospholipase C (PLC) and phospholipase D (PLD) activities were demonstrated and also that of an enzyme that removes both fatty acids from PC and thus catalyses the production of [3H]glycerophosphorylcholine (GroPCho). After 2 min of incubation with 20 mm glucose a 35% increase in the content of [3H]GroPCho was observed in prelabelled islets, whereas the amount of [3H]lysoPC, [3H]phosphorylcholine (PCho) and [3H]choline was unaffected. After 30 min of incubation with 20 mm glucose, 0·2 mm tolbutamide, 40 mm KC1, 10 mm succinic acid monomethyl ester (SME) or 10 mm NaF, a 25-50% increase in [3H]GroPCho was observed. In the presence of 100 μm diazoxide or 35 μm RHC 80267 the glucose activation was attenuated. PLC was stimulated slightly by tolbutamide and 100 μm isoprenaline (isoproterenol), whereas SME decreased the amount of [3H]PCho by 10%. [3H]Choline content was increased by 25-40% in the presence of 0·16 μm 12-O-tetradecanoylphorbol 13-acetate (TPA), 10 mm NaF or 100 μm carbachol. This effect of fluoride was potentiated in the presence of 20 mm glucose. It is concluded that metabolism of PC to GroPCho may be involved in the regulation of glucose-stimulated insulin secretion, and that PLD may participate in insulin secretion evoked by TPA, carbachol and fluoride.
LC Bollheimer, S Troll, H Landauer, CE Wrede, J Scholmerich and R Buettner
Thiazolidinediones (TZDs) have been suggested to act beneficially on pancreatic islet function and on beta-cell viability but data concerning direct effects on isolated islets are controversial. Therefore, we have examined parameters of pancreatic insulin and glucagon secretion and biosynthesis in TZD-exposed rat pancreatic islets under physiological glucose level conditions and under conditions of glucolipotoxicity. Primary rat islets were incubated for 2.5 h with or without troglitazone (10 microM) in 5.6 mM glucose (standard glucose levels) and 16.7 mM glucose (high glucose levels); a subgroup was additionally treated with oleate (200 microM) to simulate acute glucolipotoxicity. Insulin and glucagon secretion, intracellular content and their respective mRNAs were quantified. Newly synthesized insulin was determined by pulse-labeling experiments. Troglitazone reduced insulin secretion at standard and high glucose levels by about one-third (P<or=0.05). Insulin content was decreased at 5.6 mM glucose but increased at 16.7 mM glucose by the presence of troglitazone (P<or=0.05). Newly synthesized insulin mRNA and preproinsulin mRNA decreased by about 20% at standard glucose levels (P<or=0.05). Glucagon secretion was augmented by troglitazone in islets under high glucose conditions by an additional 50% (P<or=0.05). No clear beneficial troglitazone effects were observed under glucolipotoxic conditions. The reduced insulin secretion and biosynthesis at standard glucose levels can be interpreted as an insulin-sparing effect. Troglitazone effects were less pronounced at high glucose alone or in combination with oleate. From a clinical point of view, these results indicate a greater benefit of troglitazone for beta-cell function in hyperinsulinemic, but normoglycemic patients with insulin resistance or early type 2 diabetes without major insulin secretion deficits and/or pronounced hyperglycemia.
J. U. Weaver, G. A. Hitman and P. G. Kopelman
Obesity is likely to be a multifactorial disease with an important genetic component. Animal models of genetic and experimentally induced obesity suggest that glucocorticoid receptor (GR) activity plays a role in the aetiology and maintenance of the obese state. Glucocorticoid activity appears to be essential for the development of hyperinsulinaemia and subsequent fat deposition. In humans, glucocorticoid excess is associated with central fat distribution. We have therefore investigated the restriction fragment length polymorphisms of the human GR gene locus (GRL) and have sought associations of specific alleles with anthropometric measurements and indices of insulin secretion and resistance in obesity.
Fifty-six extremely obese, unrelated, nondiabetic premenopausal British Caucasian females and 43 age-matched, normal weight controls were studied. The obese subjects were characterized by fat distribution (waist to hip ratio), insulin secretion and insulin resistance (fasting insulin (FI)), an index of insulin resistance (HOMA), stimulated insulin secretion during an oral glucose tolerance test and insulin-mediated glucose disposal, steady-state plasma glucose). A BclI polymorphism (fragments of 4·5 and 2·3 kb) demonstrated significant association with indices of glucose metabolism in obesity; those subjects homozygous for the 4·5 kb fragment had elevated FI (Pc=0·012) and HOMA (Pc=0·012) values. The genotypic and allelic frequencies of the GRL BclI polymorphism were otherwise similar in obese and normal weight subjects. We postulate that the GRL BclI polymorphism may directly affect GR gene expression, or be in linkage disequilibrium with a possible mutation within one of three exons of the GR gene, and thereby modulate GR transcriptional activity on target genes involved in glucose and insulin homeostasis.
KT Scougall, CA Maltin and JA Shaw
Long-term constitutive secretion of insulin by implantation of ex vivo transfected cells such as fibroblasts or myoblasts or in situ by intramuscular injection of naked plasmid DNA provides a potential approach to gene therapy for diabetes mellitus. A mechanism for regulating insulin secretion will be necessary to realize the therapeutic potential of this approach. A second obstacle is the inability of non-endocrine host cells to fully process proinsulin. Therefore, alteration of the wild-type cDNA will be necessary to achieve processing of proinsulin by endogenous endoproteases within these cells. The cDNAs for beta-galactosidase (beta), human wild-type proinsulin (hppI1) and a mutated construct (hppI4), in which the dibasic PC2 and PC3 cleavage sites had been altered to form furin cleavage sites, were sub-cloned into four vectors (pCR3, pVR1012, pIRES, pTRE), including a tetracycline responsive plasmid (pTRE) that requires co-transfection with another plasmid encoding a transactivator (pTet-off) for transgene expression. Transient transfection of the COS-7 fibroblast cell line with these constructs was performed using DEAE-dextran and liposomes. Analysis of vector efficiencies revealed that pTRE/pTet-off>pIRES>pCR3>pVR1012. Further analysis demonstrated total pro/insulin secretion of 2.33 ng/10(6) cells/24 h with > or =25% processed to insulin in hppI-1.pTRE/pTet-off-transfected cells compared with 0.39 ng/10(6) cells/24 h and >70% processing in hppI-4.pTRE/pTet-off-transfected cells. In co-transfection studies with pTRE-hppI1/pTet-off and pTRE-hppI4/pTet-off constructs, pro/insulin secretion was inhibited to 65-66% and 36-38% of control (100%) in the presence of 0.01 and 0.1 microg/ml tetracycline respectively over a 24-h incubation period. Furthermore, reversal of tetracycline inhibition was demonstrated for pTRE-hppI1/pTet-off- and pTRE-hppI4/pTet-off-transfected cells. After a 48-h incubation with 1.0 microg/ml tetracycline, total pro/insulin levels were 10 and 14% compared with untreated cells respectively. On tetracycline removal, total proinsulin levels increased and were equivalent to untreated groups 72 h later. In conclusion, regulation of fully processed human insulin secretion has been achieved in a transiently transfected non-endocrine cell line.
Mutations in the human genes encoding the tissue-specific transcription factors hepatocyte nuclear factor (HNF)1alpha, HNF1beta and HNF4alpha are responsible for maturity onset diabetes of the young (MODY), a monogenic dominant inherited form of diabetes mellitus characterized by defective insulin secretion of the pancreatic beta-cells. In addition, the mutated HNF1beta gene causes defective development of the kidney and genital malformation. This review summarizes the main features of these transcription factors and discusses potential events leading to the specific disease phenotypes.
L. Best, E. A. Bone, J. E. Meats and S. Tomlinson
Intracellular pH (pHi) was monitored in dispersed pancreatic islet cells from rats using the fluorescent dye 2′7′bis-carboxyethyl-5′(6′)-carboxyfluorescein. The addition of a weak acid (acetate, propionate or formate) provoked a rapid fall in pHi, corresponding to approximately 0·2 units, followed by a slower return to the basal value. Amiloride also caused a rapid fall in pHi, but no recovery occurred in this case. Addition of NH4Cl induced a rise in pHi. Of the nutrients tested, only glyceraldehyde produced a fall in pHi, both glucose and α-ketoisocaproate causing a gradual and sustained rise in pHi.
Insulin secretion and inositol lipid metabolism in response to nutrient stimuli were markedly inhibited by NH4Cl. The responses to non-nutrient stimuli were unaffected. Glucose-induced insulin secretion and inositol lipid metabolism were potentiated in the presence of amiloride. No such potentiation, however, was observed in the presence of weak acids.
Amiloride and weak acids shared the ability to reduce the fractional outflow rate of 45Ca2+.
It is concluded that pharmacological manipulations of pHi can influence certain aspects of islet cell function, such as calcium handling, though it seems unlikely that the stimulation of islets by nutrient secretagogues occurs as a result of changes in pHi.
M Tiedge and S Lenzen
RINm5F insulinoma cells show a defective physiological insulin secretory response to glucose stimulation. The short chain carbonic acid sodium butyrate induced a growth arrest during a 72-h tissue culture period. In contrast to control RINm5F cells, 2 mm glucose increased insulin secretion by more than 70% in these sodium butyrate-treated cells (1 mm) without any further increase of the secretory rate between 2 and 20 mm glucose. This effect of sodium butyrate on insulin secretion was assessed in comparison with its effect on gene expression of the GLUT1 and GLUT2 glucose transporter, hexokinase type I and type II, glucokinase and insulin. Sodium butyrate at a 1 mm concentration decreased GLUT1 gene expression by nearly 50%, but did not induce gene expression of the low-affinity GLUT2 glucose transporter above the detection limit. Furthermore, sodium butyrate increased glucokinase gene expression by more than 50% and hexokinase type II gene expression by more than 100%, while insulin gene expression was increased only by 24%. Hexokinase type II enzyme activity was increased by more than 100% without a concomitant significant change of the glucokinase enzyme activity. Sodium butyrate (2 mm) caused effects comparable with those of 1 mm sodium butyrate. Thus the improved insulin secretory responsiveness of RINm5F insulinoma cells after sodium butyrate treatment at low non-physiological millimolar glucose concentrations can be interpreted as a result of an increased hexokinase-mediated metabolic flux rate through the glycolytic chain.
NT Lam, AT Cheung, MJ Riedel, PE Light, CI Cheeseman and TJ Kieffer
Leptin suppresses insulin secretion by opening ATP-sensitive K(+) (K(ATP)) channels and hyperpolarizing beta-cells. We measured the intracellular concentration of ATP ([ATP](i)) in tumor-derived beta-cells, INS-1, and found that leptin reduced [ATP](i) by approximately 30%, suggesting that the opening of K(ATP) channels by leptin is mediated by decreased [ATP](i). A reduction in glucose availability for metabolism may explain the decreased [ATP](i), since leptin (30 min) reduced glucose transport into INS-1 cells by approximately 35%, compared to vehicle-treated cells. The twofold induction of GLUT2 phosphorylation by GLP-1, an insulin secretagogue, was abolished by leptin. Therefore, the acute effect of leptin could involve covalent modification of GLUT2. These findings suggest that leptin may inhibit insulin secretion by reducing [ATP](i) as a result of reduced glucose availability for the metabolic pathway. In addition, leptin reduced glucose transport by 35% in isolated rat hepatocytes that also express GLUT2, suggesting that glucose transport may also be altered by leptin in other glucose-responsive tissues such as the liver.
M. Welsh, D. L. Eizirik and E. Strandell
To elucidate the role of thermal stress on the function of pancreatic β cells, isolated mouse pancreatic islets were incubated for 30 min at 42°C. This resulted in decreased glucose-stimulated insulin secretion, inhibited total protein and pro-insulin synthesis and the induction of heat-shock proteins with molecular weights of 64 and 88 kDa. Six days later, the islets exposed to heat shock showed a lower DNA content, indicating islet cell death. However, the insulin secretory response and rates of oxygen consumption in the presence of glucose were normal. It is suggested that the induction of heat-shock proteins does not permanently impair β-cell function, but rather protects these cells from lasting damage.