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...

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Chronic exposure of pancreatic beta-cells to high glucose has pleiotropic action on beta-cell function. In particular, it induces key glycolytic genes, promotes glycogen deposition, and causes beta-cell proliferation and altered insulin secretion characterized by sensitization to low glucose. Postglycolytic events, in particular, anaplerosis and lipid signaling, are thought to be implicated in beta-cell activation by glucose. To understand the biochemical nature of the beta-cell adaptive process to hyperglycemia, we studied the regulation by glucose of lipogenic genes in the beta-cell line INS-1. A 3-day exposure of cells to elevated glucose (5-25 mmol/l) increased the enzymatic activities of fatty acid synthase 3-fold, acetyl-CoA carboxylase 30-fold, and malic enzyme 1.3-fold. Pyruvate carboxylase and citrate lyase expression remained constant. Similar observations were made at the protein and mRNA levels except for malic enzyme mRNA, which did not vary. Metabolic gene expression changes were associated with chronically elevated levels of citrate, malate, malonyl-CoA, and conversion of glucose carbon into lipids, even in cells that were subsequently exposed to low glucose. Similarly, fatty acid oxidation was suppressed and phospholipid and triglyceride synthesis was enhanced independently of the external glucose concentration in cells preexposed to high glucose. The results suggest that a coordinated induction of glycolytic and lipogenic genes in conjunction with glycogen and triglyceride deposition, as well as increased anaplerosis and altered lipid partitioning, contribute to the adaptive process to hyperglycemia and glucose sensitization of the beta-cell.

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The malonyl-CoAlcarnitine palmitoyl-transferase I (CPI I) interaction has emerged as a key component of a fuel "crosstalk" metabolic signaling system in a number of tissues including the liver, heart, and skeletal muscle [3, 6, 8]. With respect to the 13-cell, the hypothesis has been proposed that both accelerated acetyl-CoA production and anaplerosis are important events in glucose signaling [5]. Evidence has also been provided which suggests that malonyl-CoA, via its inhibitory action on CPT I and a resulting rise in cytosolic long chain acyl-CoA, is implicated in conjunction with the KATP channel pathway in the transduction mechanisms whereby nutrients induce the insulin secretory process [5]. The strength of this metabolic model of fuel sensing is not only in offering a plausible explanation for the mode of action of all nutrient stimuli but also in providing a framework for the understanding of the mechanism of 13-cell "glucotoxicity" and "lipotoxicity" [4, 6, 9].Chronic elevation in glucose has pleiotropic effects on the pancreatic 3-cell including a high rate of insulin secretion at low glucose, 13-cell hypertrophy and hyperplasia. These actions of glucose are expected to be associated with the modulation of the expression of a number of glucose-regulated genes which need to be identified. To investigate the molecular mechanisms implicated in these adaptation processes to hyperglycemia, we have studied the regulation of genes encoding key glycolytic enzymes in the glucose responsive f3-cell line INS-i. Glucose (5 to 25 mM) induced mRNAs of phosphofructokinase-I (PFK-1) isoform C, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (4 fold) and L-pyruvate-kinase (L-PK) (7 fold) [7]. In addition to the induction of these key glycolytic genes, long term exposure to high glucose caused a marked glycogen deposition which was readily mobilized upon lowering of ambient glucose [7]. The regulation by glucose of anaplerotic and lipogenic genes was also investigated. Elevated glucose induced mRNAs of pyruvate carboxylase (1.5 fold), malic enzyme (2 fold), acetyl-CoA carboxylase (ACC) (15 fold) and fatty acid synthase (4 fold). In contrast the expression of pyruvate dehydrogenase E-1 a, AlP-citrate lyase and CPT I transcripts remained constant. Following a 3-day exposure to elevated glucose similar inductions were observed at the protein and enzyme activity levels. Transcriptional induction by glucose of the GAPDH, L-PK and ACC genes was demonstrated using the run-on assay with isolated INS cells nuclei. These changes in enzyme expression were associated with 3 fold increases in insulin secretion and glucose oxidation at low (2-5 mM) glucose. Chronically elevations of citrate (2-fold), malate (8-fold) and malonyl-CoA (8-fold) were also observed in cells which were subsequently exposed to low glucose. Similarly, fatty acid oxidation was suppressed and phospholipid and triglyceride synthesis enhanced in cells preexposed to high glucose. The results suggest that a coordinated induction of glycolytic, anaplerotic and lipoge...