Study of hexose transport, glycerol phosphate shuttle and Krebs cycle in islets of adult rats injected with streptozotocin during the neonatal period (original) (raw)
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Diabetologia, 1990
Smmnary. Pancreatic islets removed from adult rats injected with streptozotocin during the neonatal period display an impaired secretory response to D-glucose and, to a lesser extent, to L-leucine. Despite normal to elevated hexokinase and glucokinase activities in the islets of these glucose-intolerant animals and despite normal mitochondrial binding of the hexokinase isoenzymes, the metabolic response to a high concentration of D-glucose is severely affected, especially in terms of D-[6-14C]glucose oxidation. Thus, the ratio in D-[6-14C]glucose oxidation/D-[5-3H]glucose utilization is much less markedly increased in response to a rise in hexose concentration and, at a high concentration of D-glucose (16.7 mmol/1), less markedly decreased by the absence of Ca 2t and presence of cycloheximide in diabetic than control rats. This metabolic defect contrasts with (1) a close-to-normal or even increased capacity of the islets of diabetic rats to oxidize D-[6-14C]glucose, [2-~4C]pyruvate, L-[U-~4C]glutamine and L-[U-~4C]leucine at low, non-insulinotropic, con-centrations of these substrates; (2) a lesser impairment of the oxidation of L-[U-14C]leucine tested in high concentration (20 mmol/1), the effect of Ca 2+ deprivation upon the latter variable being comparable in diabetic and control rats; (3) an unaltered transamination of either [2-~4C]pyruvate or L-[U-14C]leucine; and (4) a modest perturbation of glycotysis. The most obvious alteration in glycolysis consists in a lesser increase of the glycolytic flux in response to a rise of D-glucose concentration in diabetic than control rats, this coinciding with an apparent decrease in affinity of glucokinase for the hexose. It is speculated that the preferential impairment of the metabolic and secretory response to D-glucose may be mainly attributable to an altered coupling between calcium accumulation and the stimulation of oxidative events in Beta-cell mitochondria of diabetic rats.
Glucose Cycling in Islets From Healthy and Diabetic Rats
Diabetes, 1990
Pancreatic islets from healthy (control) and neonatally streptozocin-induced diabetic (STZ-D) rats, a model for non-insulin-dependent diabetes mellitus, were incubated with 3H2O and 5.5 or 16.7 m M glucose. At 5.5 mM glucose, no detectable [3H]glucose was formed. At 16.7 mM, 2.2 patom · islet−1 · h−1 of 3H was incorporated into glucose by the control islets and 5.4 · patom · islet−1 · h−1 by STZ-D islets. About 75% of the 3H was bound to carbon-2 of the glucose. Glucose utilization was 35.3 pmol · islet−1 · h−1 by the control and 19.0 pmol · islet−1 · h−1 by the STZ-D islets· Therefore, 4.5% of the glucose-6-phosphate formed by the control islets and 15.7% by the STZ-D islets was dephosphorylated. This presumably occurred in the β-cells of the islets catalyzed by glucose-6-phosphatase. An increased glucose cycling, i.e., glucose → glucose-6-phosphate → glucose, in islets of STZ-D rats may contribute to the decreased insulin secretion found in these animals.
Hexose metabolism in pancreatic islets. Feedback control of d-glucose oxidation by functional events
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1988
A rise in extraceUular v-glucose concentration in pancreatic Islet cells causes a greater relative increase in the oxidation of pyruvate and acet~l residues than in 81ycolysis. A possible explanation for such an unusual situation was sought in the present study. The preferential stimulation of mitochondrial oxidative events was found to dis#ay a sigmoidal dependency on hexose concentration, and an exlmnential time course during Igolunged exposure of the islets to a high concentration of v-81ueose. The preferential stimulation of mitochondrinl oxidative events was abolished in islets incubated in the presence of eyeloheximide and absance of Ca 2÷, in which case the oxidation of ~[6-14Clg]~ wits more severely inhibited than that of D-l&4-uClglucose. Likewise, the inhibitor of protein biosynthesis and the absence of Ca 2+ Mfeeted the oxidation of L-[U-14C]leucine preferentially, relative to that of L-[l-UC]leucine, in islets exposed to a high, but not a low, concentration of the amino acid, These results demonstrate that in pancreatic islets it is possible to dissociate bath glyecdysls from mitochondrial oxidative events and the oxidation of acetyi residues from their generation rate. Moreover, the experimental data suggest that nutrient-responsive and ATP-requiring functional precesses exert a feedback control on mitochonddul respiration in this fuel-sensor organ.
Molecular and Cellular Endocrinology, 1991
In rat pancreatic islets, a rise in D-@uCOSe concentrations increases the oxidation of hexose-derived acetyl residues relative to glycolytic flux, an effect possibly attributable, in part at least, to the activation of key mitochondrial dehydrogenases by Ca2+ accumulated in the mitochondria of glucose-stimulated islet cells. The effects of non-nutrient insuhnotropic agents upon D-[6-i4C]ghtcose oxidation and ~-[5-~H]glucase utilization were investigated. At an intermediate concentration of D-glucose (6 mM), the oxidation of D-[6-i4C]glucose was unaffected by hypoglycemic sulfonylureas, an organic Ca2+ agonist, a cholinergic agent, forskolin, theophylline and cytochalasin B. At a higher concentration of the hexose (17 mM), however, the i4C02/'H20 production rate was decreased by organic and inorganic Ca2+-antagonists and by ouabain, whilst being increased by NH: (10 mM) and aminooxyacetate. These findings suggest that the preferential stimulation of oxidative events in the Krebs cycle is largely independent of the rate of insulin release, and not merely consequential to the stimulation of Ca2+ inflow into the B-cell. It might be regulated, in a feedback process, by the rate of ATP utilization and, both directly and indirectly, by the mitochondrial redox state. The glucose-induced mitochondrial accumulation of Cat+ and subsequent activation of the Krebs cycle appear to require an increase in both cytosolic Ca2+ activity and ATP availability.
Glucose Metabolism in Goto-Kakizaki Rat Islets*
Endocrinology, 1998
Islets from Goto-Kakizaki (GK) rats from our colony, despite marked impairment of glucose-induced insulin release, used glucose and produced CO2 at a rate 3 times that of islets from control Wistar rats. Almost all glucose used was accounted for in CO2 and lactate production. The percentages of glucose carbon used collected in CO2 and lactate were similar for control and GK islets. GK islets also oxidized 40% more acetate and leucine to CO2 than did control islets. The fraction of carbon leaving the Krebs cycle relative to CO2 production was the same in GK and control islets. The capacities of mitochondria from GK islets to generate ATP from glutamate and malate were similar and that to generate ATP from succinate and rotenone was somewhat less from GK islets. The reason for the enhanced utilization of substrates by islets of the GK rat is not apparent. In conclusion, there is no decrease in islet glucose utilization, glucose oxidation, Krebs cycle function, or the electron transpor...
Diabetologia, 1993
In islets from both adult rats injected with streptozotocin during the neonatal period and spontaneously diabetic rats obtained by repeated selective breedings (GK rats), the ratio between D-[3, 4-14C]glucose oxidation and D-[5-3H]glucose conversion to 3HOH was 25 % lower than in islets from control rats, indicating an impaired contribution of oxidative to total glycolysis. No primary defect in the Krebs cycle was found in the islets of diabetic rats, as judged from the ratio between either D-[2-14C]glucose or D-[6-~4C]glucose and D-[3, 4-~4C]glucose oxidation. Therefore, we propose that a preferential alteration of oxidative glycolysis in the pancreatic beta cell may contribute to the impairment of glucose-induced insulin release not only in a cytotoxic but also in a spontaneous model of non-insulin-dependent diabetes mellitus.
Diabetes, 1991
We studied the possible relationships between the functional status of the p-cell and activities or mRNA contents of enzymes involved in the catabolism of glucose. Three different in vitro models with attenuated insulin response were used: rat islets cultured at a low glucose concentration, rat islets incubated in vitro with streptozocin, and fetal rat islets. The fetal and streptozocin-administered islets were compared with adult islets cultured in RPMI-1640 containing 11 mM glucose, and the effects of the in vitro glucose concentrations (3.3,11, and 28 mM) were assessed on adult islets only. Cellular mRNA levels for the mitochondrial DNA-encoded cytochrome b and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined by Northern-blot analysis. Enzymatic activities of high-K m (glucokinase) and Iow-K m (hexokinase) glucose-phosphorylating enzymes and succinate-cytochrome c reductase were also determined. Islets cultured at 3.3 mM glucose displayed a decreased activity of glucokinase compared with islets cultured at 28 mM glucose (23.3 ± 12%), whereas there was no difference in hexokinase activity or the level of GAPDH mRNA. The activity of succinate-cytochrome c reductase was similar in islets cultured at the different glucose concentrations. The level of cytochrome b mRNA increased at 28 mM glucose compared with islets cultured at 11 mM glucose (140 ± 14%). Islets incubated with streptozocin and subsequently cultured for 7 days at 11 mM glucose exhibited a decreased level of cytochrome b mRNA (65 ± 5%) and no differences in the activities of glucokinase, hexokinase, succinate-cytochrome c reductase, or the level of GAPDH mRNA. Fetal islets displayed increased activity of hexokinase (570 ± 130%
PLOS ONE, 2016
We have previously demonstrated that islet depolarization with 70 mM KCl opens Cx36 hemichannels and allows diffusion of small metabolites and cofactors through the β-cell plasma membrane. We have investigated in this islet "permeabilized" model whether glycolytic and citric acid cycle intermediates stimulate insulin secretion and how it correlates with ATP production (islet content plus extracellular nucleotide accumulation). Glycolytic intermediates (10 mM) stimulated insulin secretion and ATP production similarly. However, they showed differential sensitivities to respiratory chain or enzyme inhibitors. Pyruvate showed a lower secretory capacity and less ATP production than phosphoenolpyruvate, implicating an important role for glycolytic generation of ATP. ATP production by glucose-6-phosphate was not sensitive to a pyruvate kinase inhibitor that effectively suppressed the phosphoenolpyruvate-induced secretory response and islet ATP rise. Strong suppression of both insulin secretion and ATP production induced by glucose-6-phosphate was caused by 10 μM antimycin A, implicating an important role for the glycerophosphate shuttle in transferring reducing equivalents to the mitochondria. Five citric acid cycle intermediates were investigated for their secretory and ATP production capacity (succinate, fumarate, malate, isocitrate and αketoglutarate at 5 mM, together with ADP and/or NADP + to feed the NADPH re-oxidation cycles). The magnitude of the secretory response was very similar among the different mitochondrial metabolites but α-ketoglutarate showed a more sustained second phase of secretion. Gabaculine (1 mM, a GABA-transaminase inhibitor) suppressed the second phase of secretion and the ATP-production stimulated by α-ketoglutarate, supporting a role for the GABA shuttle in the control of glucose-induced insulin secretion. None of the other citric acid intermediates essayed showed any suppression of both insulin secretion or ATP-production by the presence of gabaculine. We propose that endogenous GABA metabolism in the "GABA-shunt" facilitates ATP production in the citric acid cycle for an optimal insulin secretion.
Metabolic Fate of Glucose in Purified Islet Cells
Journal of Biological Chemistry, 1997
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.