Previous studies in rat islets have suggested that anaplerosis plays an important role in the regulation of pancreatic ␤ cell function and growth. However, the relative contribution of islet ␤ cells versus non-␤ 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 ␤ cells using specific 14 C-labeled glucose tracers. Between 5 and 20 mM glucose, the oxidative production of CO 2 from [3,4-14 C]glucose represented close to 100% of the total glucose utilization by the cells. Anaplerosis, quantified as the difference between 14 CO 2 production from [3,4-14 C]glucose and [6-14 C]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 ␤(INS-1) cells. All glucose carbon that was not oxidized to CO 2 was recovered from the cells after extraction in trichloroacetic acid. This indirectly indicates that lactate output is minimal in ␤ cells. From the effect of cycloheximide upon the incorporation of 14 C-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-␤ cells (approximately 80% glucagon-producing ␣ 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 ␤ cells, as well as a 4-fold lower ratio of lactate dehydrogenase to FADlinked glycerol phosphate dehydrogenase in ␤ cells versus ␣ cells. Finally, glucose caused a dose-dependent suppression of the activity of the pentose phosphate pathway in ␤ cells. In conclusion, rat ␤ cells metabolize glucose essentially via aerobic glycolysis, whereas glycolysis in ␣ cells is largely anaerobic. The results support the view that anaplerosis is an essential pathway implicated in ␤ cell activation by glucose.Pancreatic ␤ cells are equipped with a sensing device that measures the levels of circulating nutrients by processes requiring cellular uptake and metabolism (Refs. 1 and 2; reviewed in Refs. 3-5). D-Glucose elicits insulin secretion only when extracellular levels exceed the basal threshold value of 3 mM (6). This feature has been largely attributed to the enzyme glucokinase, which is rate-limiting for overall glucose consumption in ␤ cells from rat (7) and human (8) islets of Langerhans. Although targeted gene disruption in mice (9) and mutations in human diabetes (10) have strengthened the concept that glucokinase is a glucose-sensing protein, the following evidence indicates that (post)mitochondrial events are important for glucose signaling (4, 5). First, up to 80% of glucose carbon is o...

Rat pancreatic a-and P-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 P-cells, but their relative importance for islet function is still unknown. The present study shows expression at the RNA level in p-cells of receptors for glucagon, glucose-dependent insulinotropic polypeptide (GIP), and glucagon-like peptide 1(7-36) amide (GLP-I), while RNA from islet a-cells hybridized only with GIP receptor cDNA. Western blots confirmed that GLP-I receptors were expressed in P-cells and not in a-cells. Receptor activity, measured as cellular cAMP production after exposing islet P-cells for 15 min to a range of peptide concentrations, was already detected using 10 pmol/1 GLP-I and 50 pmol/1 GIP but required 1 nmol/1 glucagon. EC 50 values of GLP-I-and GIP-induced cAMP formation were comparable (0.2 nmol/1) and 45-fold lower than the EC g0 of glucagon (9 nmol/1). Maximal stimulation of cAMP production was comparable for the three peptides. In purified a-cells, 1 nmol/1 GLP-I failed to increase cAMP levels, while 10 pmol/1 to 10 nmol/1 GIP exerted similar stimulatory effects as in P-cells. In conclusion , these data show that stimulation of glucagon,

Expression of glucokinase in hepatocytes and pancreatic 13-cells is of major physiologic importance to mammalian glucose homeostasis. Liver glucokinase catalyzes the first committed step in the disposal of glucose, and 18-cell glucokinase catalyzes a rate-limiting step required for glucose-regulated insulin release. The present study reports the expression of glucokinase in rat glucagon-producing a-cells, which are negatively regulated by glucose. Purified rat a-cells express glucokinase mRNA and protein with the same transcript length, nucleotide sequence, and immunoreactivity as the 13-cell isoform. Glucokinase activity accounts for more than 50% of glucose phosphorylation in extracts of a-cells and for more than 90% of glucose utilization in intact cells. The glucagon-producing tumor MSL-G-AN also contained glucokinase mRNA, protein, and enzymatic activity. These data indicate that glucokinase may serve as a metabolic glucose sensor in pancreatic a-cells and, hence, mediate a mechanism for direct regulation of glucagon release by extracellular glucose. Since these cells do not express Glut2, we suggest that glucose sensing does not necessarily require the coexpression of Glut2 and glucokinase.Control of blood glucose levels in mammals is dependent on hormonal and metabolic communication between pancreatic islets of Langerhans and the liver. Acute changes in blood glucose concentration are detected by the endocrine pancreas, which responds by secreting a hormonal mixture that is rich in either glucagon (fasting state) or insulin (during or just after meals). Glucagon stimulates the liver to mobilize glucose from intracellular glycogen stores, while insulin increases postprandial glucose extraction from the portal vein (1). Since glucose itself is the main physiological activator of insulin release (2) and a direct inhibitor of glucagon release (3-5), the system is controlled via short feedback loops that are typical for homeostasis in general.Glucokinase (hexokinase IV) is expressed in liver and islets of Langerhans where it has been proposed to regulate hepatic glucose disposal and pancreatic glucose sensing (6, 7). Unlike the other mammalian hexokinases, which all have high affinity for glucose, the Km of glucokinase is in the millimolar range. Consequently, glycolytic flux becomes proportional to the extracellular glucose concentration in glucokinase-expressing cells as long as glucose uptake is not rate-limiting for its further metabolism (6, 7). Furthermore, the enzyme is not sensitive to feedback inhibition by glucose 6-phosphate (G6P), allowing the liver to sustain high metabolic flux despite elevated intracellular concentration of G6P (7). Glucokinase gene expression level in 13-cells is correlated to cellular glucose sensitivity both in vivo (8, 9) and in vitro (10), suggesting that the enzyme is a constituent of the ,B-cell glucose sensor. Analogous to the situation in 13-cells, it is conceivable that glucokinase expression in pancreatic a-cells is required for glucose to suppress glucagon re...

1-Cells from rodents and humans express different receptors recognizing hormones of the secretin-glucagon family, which--when activated--synergize with glucose in the control of insulin release. We have recently reported that isolated islets from mice homozygous for a GLP-1 receptor null mutation (GLP-1R(-/-)) exhibit a well-preserved insulin-secretory response to glucose. This observation can be interpreted in two different ways: 1) the presence of GLP-1R is not essential for the secretory response of isolated islets to glucose alone; 2) beta-cells in GLP-1R(-/-) pancreases underwent compensatory changes in response to the null mutation. To explore these possibilities, we studied islets from control GLP-IR(+/+) mice in the absence or presence of 1 pmol/l exendin (9-39)amide, a specific and potent GLP-1R antagonist. Exendin (9-39)amide (15-min exposure) reduced glucose-induced insulin secretion from both perifused and statically incubated GLP-1R(+/+) islets by 50% (P < 0.05), and reduced islet cAMP production in parallel (P < 0.001). Furthermore, GLP-1R(-/-) islets exhibited: 1) reduced cAMP accumulation in the presence of 20 mmol/l glucose (knockout islets versus control islets, 12 +/- 1 vs. 27 +/- 3 fmol x islet(-1) x 15 min(-1); P < 0.001) and exaggerated acceleration of cAMP production by 10 nmol/l glucose-dependent insulinotropic peptide (GIP) (increase over 20 mmol/l glucose by GIP in knockout islets versus control islets: 66 +/- 5 vs. 14 +/- 3 fmol x islet(-1) x 15 min(-1); P < 0.001); 2) increased mean cytosolic [Ca2+] ([Ca2+]c) at 7, 10, and 15 mmol/l glucose in knockout islets versus control islets; and 3) signs of asynchrony of [Ca2+]c oscillations between different islet subregions. In conclusion, disruption of GLP-1R signaling is associated with reduced basal but enhanced GIP-stimulated cAMP production and abnormalities in basal and glucose-stimulated [Ca2+]c. These abnormalities suggest that GLP-1R signaling is an essential upstream component of multiple beta-cell signaling pathways.

Rat pancreatic a-and P-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 P-cells, but their relative importance for islet function is still unknown. The present study shows expression at the RNA level in p-cells of receptors for glucagon, glucose-dependent insulinotropic polypeptide (GIP), and glucagon-like peptide 1(7-36) amide (GLP-I), while RNA from islet a-cells hybridized only with GIP receptor cDNA. Western blots confirmed that GLP-I receptors were expressed in P-cells and not in a-cells. Receptor activity, measured as cellular cAMP production after exposing islet P-cells for 15 min to a range of peptide concentrations, was already detected using 10 pmol/1 GLP-I and 50 pmol/1 GIP but required 1 nmol/1 glucagon. EC 50 values of GLP-I-and GIP-induced cAMP formation were comparable (0.2 nmol/1) and 45-fold lower than the EC g0 of glucagon (9 nmol/1). Maximal stimulation of cAMP production was comparable for the three peptides. In purified a-cells, 1 nmol/1 GLP-I failed to increase cAMP levels, while 10 pmol/1 to 10 nmol/1 GIP exerted similar stimulatory effects as in P-cells. In conclusion, these data show that stimulation of glucagon,