Metabolic Fate of Glucose in Purified Islet Cells

Abstract

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...

The intrauterine metabolic environment modulates the gene expression pattern in fetal rat islets: prevention by maternal taurine supplementation

“…The study design, however, does not allow any discrimination to be made between primary causative or secondary changes in gene expression leading to the phenotype modification. Normal beta cells have aerobic metabolism at least threefold higher than in most other cell types and may therefore be more susceptible to nutritional environment [32,33]. First, we identified that the lack of protein in the diet targeted cellular respiration mechanism, especially the TCA cycle, since ten of the 32 genes involved were upregulated in the LP fetal islets.…”

The role of rapid lipogenesis in insulin secretion: Insulin secretagogues acutely alter lipid composition of INS-1 832/13 cells

“…Glucose also increased the level of PS containing these fatty acid chains (Table 2), but PS comprised only 3.7% of the total PLs. Phospholipase C-mediated hydrolysis of PI catalyzes the formation of IP 3 and PIP 2 which have signaling roles in numerous types of cells including the pancreatic beta cell. Our observation that glucose decreased the amounts of individual and total PIs (Table 2) is consistent with results of studies of pancreatic islets with radioactive tracers, such as [ 3 H]myoinositol or 32 Pi, used to prelabel PLs in islets.…”

“…In addition to mitochondria providing ATP to power cellular processes and activate insulin exocytosis via ATP acting on the ATP-dependent potassium channel, it is clear that the net synthesis of citric acid cycle intermediates by mitochondria (anaplerosis) [1] is involved in insulin secretion. The evidence for anaplerosis is the high level of the anaplerotic enzyme pyruvate carboxylase in the pancreatic islet beta cell [2,3] that enables about 50% of pyruvate derived from glucose, the most potent insulin secretagogue, to be carboxylated to oxaloacetate [2,[4][5][6][7]. This permits the net synthesis of any citric acid cycle intermediate in beta cell mitochondria and indicates that secretagogue carbon is used for anaplerosis.…”

Insulin stimulates glucose metabolism via the pentose phosphate pathway in Drosophila Kc cells

“…Finally, the pieces of Whatman paper were removed and transferred to scintillation vials for radioactivity counting. [16,17]. The experimental procedures were similar to those described above for the determination of 14 CO 2 production from D-[U-14 C]glucose.…”