Karen Moens

Metabolic fate of glucose in purified islet cells. Glucose-regulated anaplerosis in beta cells.

Previous studies in rat islets have suggested that anaplerosis plays an important role in the regulation of pancreatic β cell function and growth. However, the relative contribution of islet β cells versus non-β cells to glucose-regulated anaplerosis is not known. Furthermore, the fate of glucose carbon entering the Krebs cycle of islet cells remains to be determined. The present study has examined the anaplerosis of glucose carbon in purified rat β cells using specific 14C-labeled glucose tracers. Between 5 and 20 mm glucose, the oxidative production of CO2 from [3,4-14C]glucose represented close to 100% of the total glucose utilization by the cells. Anaplerosis, quantified as the difference between14CO2 production from [3,4-14C]glucose and [6-14C]glucose, was strongly influenced by glucose, particularly between 5 and 10 mm. The dose dependence of glucose-induced insulin secretion correlated with the accumulation of citrate and malate in β(INS-1) cells. All glucose carbon that was not oxidized to CO2 was recovered from the cells after extraction in trichloroacetic acid. This indirectly indicates that lactate output is minimal in β cells. From the effect of cycloheximide upon the incorporation of 14C-glucose into the acid-precipitable fraction, it could be calculated that 25% of glucose carbon entering the Krebs cycle via anaplerosis is channeled into protein synthesis. In contrast, non-β cells (approximately 80% glucagon-producing α cells) exhibited rates of glucose oxidation that were 1 3 to 1 6 those of the total glucose utilization and no detectable anaplerosis from glucose carbon. This difference between the two cell types was associated with a 7-fold higher expression of the anaplerotic enzyme pyruvate carboxylase in β cells, as well as a 4-fold lower ratio of lactate dehydrogenase to FAD-linked glycerol phosphate dehydrogenase in β cells versus α cells. Finally, glucose caused a dose-dependent suppression of the activity of the pentose phosphate pathway in β cells. In conclusion, rat β cells metabolize glucose essentially via aerobic glycolysis, whereas glycolysis in α cells is largely anaerobic. The results support the view that anaplerosis is an essential pathway implicated in β cell activation by glucose.

Expression and Functional Activity of Glucagon, Glucagon-Like Peptide I, and Glucose-Dependent Insulinotropic Peptide Receptors in Rat Pancreatic Islet Cells

Rat pancreatic α- and β-cells are critically dependent on hormonal signals generating cyclic AMP (cAMP) as a synergistic messenger for nutrient-induced hormone release. Several peptides of the glucagon-secretin family have been proposed as physiological ligands for cAMP production in β-cells, but their relative importance for islet function is still unknown. The present study shows expression at the RNA level in β-cells of receptors for glucagon, glucose-dependent insulinotropic polypeptide (GIP), and glucagon-like peptide I(7-36) amide (GLP-I), while RNA from islet α-cells hybridized only with GIP receptor cDNA. Western blots confirmed that GLP-I receptors were expressed in β-cells and not in α-cells. Receptor activity, measured as cellular cAMP production after exposing islet β-cells for 15 min to a range of peptide concentrations, was already detected using 10 pmol/l GLP-I and 50 pmol/l GIP but required 1 nmol/l glucagon. EC50 values of GLP-I- and GIP-induced cAMP formation were comparable (0.2 nmol/l) and 45-fold lower than the EC50 of glucagon (9 nmol/l). Maximal stimulation of cAMP production was comparable for the three peptides. In purified α-cells, 1 nmol/l GLP-I failed to increase cAMP levels, while 10 pmol/l to 10 nmol/l GIP exerted similar stimulatory effects as in β-cells. In conclusion, these data show that stimulation of glucagon, GLP-I, and GIP receptors in rat β-cells causes cAMP production required for insulin release, while adenylate cyclase in α-cells is positively regulated by GIP.

Dual glucagon recognition by pancreatic beta-cells via glucagon and glucagon-like peptide 1 receptors

cAMP is required for normal glucose-induced insulin release by pancreatic beta-cells. In a previous study, we showed that cAMP production in beta-cells depends on the expression of receptors for glucagon, glucagon-like peptide 1(7-36) amide [GLP-1(7-36) amide], and glucose-dependent insulinotropic polypeptide. Although the latter two peptides are thought to amplify meal-induced insulin release (incretin effect), the role of glucagon in the regulation of insulin release remains elusive. In the present study, we analyzed the interaction of glucagon with its own receptor and with the glucagon-like peptide 1 (GLP-1) receptor using purified rat beta-cells. Glucagon binding was partially displaced by 1 micromol/l des-His1-[Glu9]glucagon-amide, a glucagon receptor antagonist, and by 1 micromol/l GLP-1. Conversely, GLP-1 binding was competitively inhibited by high glucagon concentrations (Ki = 0.3 micromol/l). Glucagon-induced cAMP production in beta-cells was inhibited both by 1 micromol/l des-His1-[Glu9]glucagon-amide and exendin-(9-39)-amide, a specific GLP-1 receptor antagonist, whereas GLP-1-induced cAMP formation was suppressed only by exendin-(9-39)-amide. Finally, addition of 1 micromol/l exendin-(9-39)-amide to 20 mmol/l glucose-stimulated beta-cells did not antagonize the potentiating effect of 1 nmol/l glucagon, although it prevented 45% of glucagon potentiation when the peptide was administered at 10 nmol/l. Our data suggest that glucagon recognition via two distinct receptors allows pancreatic beta-cells to detect this peptide both when diluted in the systemic circulation and when concentrated as local signal in the islet interstitium.

Cellular Origin of Hexokinase in Pancreatic Islets

Transgenic or tumoral pancreatic islet beta cells with enhanced expression of low K m hexokinases (HK) exhibit a leftward shift of the normal dose-response curve for glucose-induced insulin release. Furthermore, HK catalyzes roughly 50% of total glucose phosphorylation measured in extracts from freshly isolated rodent islets, suggesting that HK participates in the process of glucose sensing in beta cells. We previously observed that HK activity represents 20% of total glucose phosphorylation in purified rat beta cell preparations and that HK is not homogenously distributed over these cells. The present study provides several arguments for the idea that HK detected in freshly isolated rat islets or islet cell preparations originates mainly from contaminating exocrine cells. First, reverse transcriptase-polymerase chain reaction using isoform-specific primers allowed detection of hexokinase I and IV mRNA in rat beta cells, whereas the messenger levels encoding the hexokinase II and III isoforms were undetectably low. However, immunoblots indicated that hexokinase I protein was 10-fold more abundant in freshly isolated islets and flow-sorted exocrine cells than in purified rat beta cell preparations. Second, comparison of HK activity in the different pancreatic cell types resulted in 15–25-fold higher values in exocrine than in endocrine cells (acinar cells: 21 ± 3 pmol of glucose 6-phosphate formed/h/ng of DNA; duct cells: 30 ± 8 pmol/h/ng of DNA; islet beta cells: 1.2 ± 0.2 pmol/h/ng DNA; alpha cells: 0.9 ± 0.4 pmol/h/ng of DNA). Since freshly purified beta cell preparations contain 3 ± 1% exocrine cells, at least 50% of their HK activity can be accounted for by exocrine contamination. Third, after 5 days of culture of purified islet beta cells, both HK activity and the proportion of exocrine cells decreased by more than 1 order of magnitude, while the ratio of glucokinase over hexokinase activity increased more than 10-fold. Finally, preincubating the cells with 50 mmol/liter 2-deoxyglucose did not affect glucose stimulation of insulin biosynthesis and release. In conclusion, the observation that pancreatic exocrine cells are responsible for a major part of HK activity in islet cell preparations cautions against the use of HK measurements in islet extracts in the study of these enzymes in glucose sensing by pancreatic beta cells.

Glucose-Induced B-Cell Recruitment and the Expression of Hexokinase Isoenzymes

The balanced release of insulin and glucagon by the endocrine pancreas is an important feature of glucose homeostasis in mammals. Adaptation of hormonal output from the endocrine pancreas to physiological demands is thought to depend on a glucose sensor — also called glucostat — which not only measures the prevailing extracellular glucose concentration but also ensures the signal transduction mechanisms required for appropriate glucagon and insulin release by A-cells and B-cells respectively (1–4). The scope of this paper is restricted to review some recent research concerning glucose sensing in B-cells, neglecting the glucose sensor in glucagon-producing A-cells about which relatively little is known.