Donna K Berner

Regulation of Glucose Metabolism in Pancreatic Islets

We evaluated the possible role of islet glucokinase in controlling the rate of islet glucose metabolism, and thereby the rate of glucose-induced insulin release. The activities of glucokinase, hexokinase, P-fructokinase, and glyceraldehyde-P dehydrogenase were quantitated in sonicated or isotonically homogenized islet preparations using pyridine nucleotide-dependent fluorometric assays. In sonicates, about ¼ of the islet glucose phosphorylating activity was due to an enzyme with kinetic properties similar to glucokinase; % of the activity was due to hexokinase. The procedure for determining islet glucokinase activity was improved by centrifuging isotonic islet homogenates at 12,000 × g. The supernatant fraction was enriched for glucokinase. About ½ of the glucose phosphorylating activity in this fraction was due to glucokinase and ½ was due to hexokinase. The glucokinase activity in islet homogenates was ⅓ of the activity of hexokinase, V40 of the activity of P-fructokinase, and 1/400 of the activity of glyceraldehyde-P dehydrogenase. Detailed concentration dependency curves of glucose and mannose utilization were also obtained with intact isolated pancreatic rat islets. Glucose and mannose usage in islets was governed by two superimposed hyperbolic systems differing in Km and Vmax. A high Km system (Km for glucose 11 mM and for mannose 21 mM) predominated. A low Km system (Km for glucose 215 and for mannose 530 μM) contributed about 15% to the total activity. The available data with intact islets could be rationalized by the existence of two distinct hexose phosphorylating enzymes with differing capacities and kinetic properties. These enzymes, tentatively identified as glucokinase and hexokinase, could coexist in the same cell or could be distributed among different cell types. The possible physiologic significance of these results is discussed,emphasizing the idea of dual control of glycolysis and insulin release by glucokinase and hexokinase. An earlier proposal that glucokinase serves as glucoreceptor of β-cells [J. Biol. Chem. 243:2730 (1968] is greatly strengthened by the present studies.

Chronic effect of fatty acids on insulin release is not through the alteration of glucose metabolism in a pancreatic beta-cell line (βHC9)

Summary Hyperinsulinaemia in the fasting state and a blunted insulin secretory response to acute glucose stimulation are commonly observed in obesity associated non-insulin-dependent diabetes mellitus. Hyperlipidaemia is a hallmark of obesity and may play a role in the pathogenesis of this beta-cell dysfunction because glucose metabolism in pancreatic beta cells may be altered by the increased lipid load. We tested this hypothesis by assessing the chronic effect of oleic acid on glucose metabolism and its relationship with glucose-induced insulin release in βHC9 cells in tissue culture. Our results show: (1) A 4-day treatment with oleic acid caused an enhancement of insulin release at 0–5 mmol/l glucose concentrations while a significant decrease in insulin release occurred when the glucose level was greater than 15 nmol/l; (2) Hexokinase activity was increased and a corresponding left shift of the dose-dependency curve of glucose usage was observed associated with inhibition of glucose oxidation in oleic acid treated βHC9 cells, yet the presumed glucose-related ATP generation did not parallel the change in insulin release due to glucose; (3) The rate of cellular respiration was markedly increased in oleic acid treated βHC9 cells both in the absence of glucose and at all glucose concentrations tested. This enhanced oxidative metabolism may explain the increased insulin release at a low glucose level but is clearly dissociated from the blunted insulin secretion at high glucose concentrations. We conclude that a reduction of oxidative metabolism in pancreatic beta cells is unlikely to be the cause of the dramatic effect that high levels of non-esterified fatty acids have on glucose-induced insulin release. [Diabetologia (1997) 40: 1018–1027]

Adaptation of Glycolytic Enzymes: Glucose Use and Insulin Release in Rat Pancreatic Islets During Fasting and Refeeding

Starvation refeeding experiments were conducted in rats to test the hypothesis that adaptation of glucokinase (the high Km component of glucose phosphorylation) could be the major determinant of glucose metabolism of pancreatic islet cells and of glucosestimulated insulin release. It was found that glucokinase of islet homogenates, glucose use by intact isolated islets, and glucose-induced insulin release as studied in a perifusion system were decreased after 24 h of fasting, whereas P-fructokinase and 3-P-glyceraldehyde DH were unaltered. After extended fasting (e.g., 120 h) all three enzymes were decreased but glucose use did not change any further. Refeeding normalized all parameters. These and previous results support the concept that glucokinase serves as the adaptive β-cell glucoreceptor relating blood glucose to insulin release.

Identification of Glucokinase as an Alloxan-Sensitive Glucose Sensor of the Pancreatic β-Cell

Alloxan inactivated glucokinase in intact, isolated pancreatic islets incubated in vitro. Inactivation of glucokinase was antagonized by 30 mM glucose present during incubation of islets with alloxan. Glucokinase partially purified from transplantable insulinomas or rat liver was inactivated by alloxan with a half-maximal effect at 2–4 μM alloxan. Inactivation of purified glucokinase was antagonized by glucose, mannose, and 2-deoxyglucose in order of decreasing potency but not by 3-O-methylglucose. Glucose anomers at 6 and 14 mM were discriminated as protecting agents, with the α-anomer more effective than the β-anomer. Glucokinase was not protected from alloxan inactivation by N-acetylglucosamine, indicating that the reactive site for alloxan is not the active site; therefore, glucose may protect glucokinase by inducing a conformational change. Glucokinase is thought to be the glucose sensor of the pancreatic β-cell. The finding that glucokinase is inactivated by alloxan and protected by glucose with discrimination of its anomers similar to inhibition of glucose-stimulated insulin secretion by alloxan supports this hypothesis and appears to explain the mechanism for inhibition of hexose-stimulated insulin secretion by this agent and the unique role of glucose and mannose as protecting agents.

Effects of Glucose on Insulin Secretion, Glucokinase Activity, and Transgene Expression in Transgenic Mouse Islets Containing an Upstream Glucokinase Promoter-Human Growth Hormone Fusion Gene

We have analyzed in organ culture the effects of glucose on glucose-induced insulin secretion, glucokinase (GK) activity, and human growth hormone (hGH) expression in pancreatic islets from transgenic mice containing an upstream GK promoter-hGH fusion gene. Freshly isolated islets from these mice had a normal insulin secretory response to glucose but showed subtle defects after culture in low or high glucose for 4 days that may have been due to the accumulation of hGH in the culture media. Islets cultured from both normal and transgenic mice had approximately a fourfold induction of GK activity in response to an increased concentration of glucose in the culture media, whereas no such change in total islet hGH production was observed. Immunocytochemical localization of hGH in islets cultured in 3 mM glucose showed a pattern similar to that in freshly isolated islets. However, after culture in 30 mM glucose, hGH immunostaining became strikingly more heterogeneous. We conclude 1) that GK-hGH transgene expression does not appear to adversely affect glucose-stimulated insulin secretion in vivo or in freshly isolated islets, 2) that glucose does not induce transgene expression, thus providing additional evidence against an effect of glucose on GK gene transcription in the islet, and 3) that glucose stimulates the co-release of hGH with insulin, thereby enhancing the heterogeneous staining pattern seen among pancreatic β-cells.

Regulatory Role of Fructose-2,6-Bisphosphate in Pancreatic Islet Glucose Metabolism Remains Unsettled

Fructose-2,6-P2 was measured in perifused, isolated rat pancreatic islets. Fructose-2,6-P2 was present in pancreatic islets at low levels approximately equal to fructose-2,6-P2 content of liver from fasted rats. In islets perifused with glucose at physiologic concentrations, fructose-2,6-P2 was increased from 0.8 μM in the presence of 5.5 mM glucose to 1.0 μM at 10 mM glucose and 1.3 μM at 16.7 mM glucose, but did not increase further at higher glucose concentration. Therefore, only modest increases in the phosphofructokinase-1 activator, fructose-2,6-P2, occur at glucose concentrations stimulating insulin secretion.

Fundamentals of Fuel Sensing and Intermediary Metabolism in Pancreatic A-and B-Cells

The endocrine cells of the pancreatic islet organ respond very effectively to the blood’s small nutrient molecules: Glucose and the mixture of 20 standard amino acids are potent stimulants at physiological concentrations, and fatty acids and ketone bodies also appear to be effective. The high efficacy of these molecules contrasts with the inertness of major metabolites, as for instance pyruvate, lactate, glycerol, uric acid, urea, and ammonia, which have no influence on these cells at physiological concentrations. The examination of this issue is naturally greatly influenced by the prevailing views about the biochemical nature of fuel sensing, in particular the essential role that intermediary metabolism plays in this process (1) as contrasting to concepts of fuel sensing mechanisms that might be based on direct fuel interactions with membrane receptors and ion channels (2), circumventing catabolism of the agonists. It is currently widely accepted that the effect of most nutrient agonists on pancreatic endocrine cells is a result of their catabolism (3). It is the purpose of this brief review to provide a perspective on this topic.