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A distinct difference in the metabolic stimulus-response coupling pathways for regulating proinsulin biosynthesis and insulin secretion that lies at the level of a requirement for fatty acyl moieties.

ABSTRACT: The regulation of proinsulin biosynthesis in pancreatic beta-cells is vital for maintaining optimal insulin stores for glucose-induced insulin release. The majority of nutrient fuels that induce insulin release also stimulate proinsulin biosynthesis, but since insulin exocytosis and proinsulin synthesis involve different cellular mechanisms, a point of divergence in the respective metabolic stimulus-response coupling pathways must exist. A parallel examination of the metabolic regulation of proinsulin biosynthesis and insulin secretion was undertaken in the same beta-cells. In MIN6 cells, glucose-induced proinsulin biosynthesis and insulin release shared a requirement for glycolysis to generate stimulus-coupling signals. Pyruvate stimulated both proinsulin synthesis (threshold 0.13-0.2 mM) and insulin release (threshold 0.2-0.3 mM) in MIN6 cells, which was eliminated by an inhibitor of pyruvate transport (1 mM alpha-cyano-4-hydroxycinnamate). A combination of alpha-oxoisohexanoate and glutamine also stimulated proinsulin biosynthesis and insulin release in MIN6 cells, which, together with the effect of pyruvate, indicated that anaplerosis was necessary for instigating secondary metabolic stimulus-coupling signals in the beta-cell. A consequence of increased anaplerosis in beta-cells is a marked increase in malonyl-CoA, which in turn inhibits beta-oxidation and elevates cytosolic fatty acyl-CoA levels. In the beta-cell, long-chain fatty acyl moieties have been strongly implicated as metabolic stimulus-coupling signals for regulating insulin exocytosis. Indeed, it was found in MIN6 cells and isolated rat pancreatic islets that exogenous oleate, palmitate and 2-bromopalmitate all markedly potentiated glucose-induced insulin release. However, in the very same beta-cells, these fatty acids in contrast inhibited glucose-induced proinsulin biosynthesis. This implies that neither fatty acyl moieties nor beta-oxidation are required for the metabolic stimulus-response coupling pathway specific for proinsulin biosynthesis, and represent an early point of divergence of the two signalling pathways for metabolic regulation of proinsulin biosynthesis and insulin release. Therefore alternative metabolic stimulus-coupling factors for the specific control of proinsulin biosynthesis at the translational level were considered. One possibility examined was an increase in glycerophosphate shuttle activity and change in cytosolic redox state of the beta-cell, as reflected by changes in the ratio of alpha-glycerophosphate to dihydroxyacetone phosphate. Although 16.7 mM glucose produced a significant rise in the alpha-glycerophosphate/dihydroxyacetone phosphate ratio, 1 mM pyruvate did not. It follows that the cytosolic redox state and fatty acyl moieties are not necessarily involved as secondary metabolic stimulus-coupling factors for regulation of proinsulin biosynthesis. However, the results indicate that glycolysis and the subsequent increase in anaplerosis are indeed necessary for this signalling pathway, and therefore an extramitochondrial product of beta-cell pyruvate metabolism (that is upstream of the increased cytosolic fatty acyl-CoA) acts as a key intracellular secondary signal for specific control of proinsulin biosynthesis by glucose at the level of translation.

SUBMITTER: Skelly RH

PROVIDER: S-EPMC1219388 | biostudies-other | 1998 Apr

REPOSITORIES: biostudies-other

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Project description:Aims/hypothesisStudies on beta cell metabolism are often conducted in rodent beta cell lines due to the lack of stable human beta cell lines. Recently, a human cell line, EndoC-βH1, was generated. Here we investigate stimulus-secretion coupling in this cell line, and compare it with that in the rat beta cell line, INS-1 832/13, and human islets.MethodsCells were exposed to glucose and pyruvate. Insulin secretion and content (radioimmunoassay), gene expression (Gene Chip array), metabolite levels (GC/MS), respiration (Seahorse XF24 Extracellular Flux Analyzer), glucose utilization (radiometric), lactate release (enzymatic colorimetric), ATP levels (enzymatic bioluminescence) and plasma membrane potential and cytoplasmic Ca2+ responses (microfluorometry) were measured. Metabolite levels, respiration and insulin secretion were examined in human islets.ResultsGlucose increased insulin release, glucose utilization, raised ATP production and respiratory rates in both lines, and pyruvate increased insulin secretion and respiration. EndoC-βH1 cells exhibited higher insulin secretion, while plasma membrane depolarization was attenuated, and neither glucose nor pyruvate induced oscillations in intracellular calcium concentration or plasma membrane potential. Metabolite profiling revealed that glycolytic and TCA-cycle intermediate levels increased in response to glucose in both cell lines, but responses were weaker in EndoC-βH1 cells, similar to those observed in human islets. Respiration in EndoC-βH1 cells was more similar to that in human islets than in INS-1 832/13 cells.Conclusions/interpretationFunctions associated with early stimulus-secretion coupling, with the exception of plasma membrane potential and Ca2+ oscillations, were similar in the two cell lines; insulin secretion, respiration and metabolite responses were similar in EndoC-βH1 cells and human islets. While both cell lines are suitable in vitro models, with the caveat of replicating key findings in isolated islets, EndoC-βH1 cells have the advantage of carrying the human genome, allowing studies of human genetic variants, epigenetics and regulatory RNA molecules.

Project description:1. The ability of a range of phenothiazines to inhibit activation of brain phosphodiesterase by purified calmodulin was studied. Trifluoperazine, prochlorperazine and 8-hydroxyprochlorperazine produced equipotent dose-dependent inhibition with half-maximum inhibition at 12mum. When tested at 10 or 50mum, 7-hydroxyprochlorperazine was a similarly potent inhibitor. However, trifluoperazine-5-oxide and N-methyl-2-(trifluoromethyl)phenothiazine were ineffective at concentrations up to 50mum, and produced only a modest inhibition at 100mum. 2. The same phenothiazines were tested for their ability to inhibit activation of brain phosphodiesterase by boiled extracts of rat islets of Langerhans. At a concentration of 20mum, 70-80% inhibition was observed with trifluoperazine, prochlorperazine, 7-hydroxyprochlorperazine or 8-hydroxyprochlorperazine, whereas trifluoperazine-5-oxide and N-methyl-2-(trifluoromethyl)phenothiazine were less effective. 3. The effect of these phenothiazines on insulin release from pancreatic islets was studied in batch-type incubations. Insulin release stimulated by glucose (20mm) was markedly inhibited by 10mum-trifluoperazine or -prochlorperazine and further inhibited at a concentration of 20mum. 8-Hydroxyprochlorperazine (20mum) was also a potent inhibitor but 7-hydroxyprochlorperazine (20mum) elicited only a modest inhibition of glucose-stimulated insulin release; no inhibition was observed with trifluoperazine-5-oxide or N-methyl-2-(trifluoromethyl)phenothiazine. 4. Trifluoperazine (20mum) markedly inhibited insulin release stimulated by leucine or 4-methyl-2-oxopentanoate in the absence of glucose, and both trifluoperazine and prochlorperazine (20mum) decreased insulin release stimulated by glibenclamide in the presence of 3.3mm-glucose. 5. None of the phenothiazines affected basal insulin release in the presence of 2mm-glucose. 6. Trifluoperazine (20mum) did not inhibit islet glucose utilization nor the incorporation of [(3)H]leucine into (pro)insulin or total islet protein. 7. Islet extracts catalysed the incorporation of (32)P from [gamma-(32)P]ATP into endogenous protein substrates. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis resolved several phosphorylated bands, but incorporation was slight. However, calmodulin in the presence of Ca(2+) greatly enhanced incorporation: the predominant phosphorylated band had an estimated mol.wt. of 55000. This enhanced incorporation was abolished by trifluoperazine, but not by cyclic AMP-dependent protein kinase inhibitor protein. 8. These results suggest that islet phosphodiesterase-stimulating activity is similar to, although not necessarily identical with, calmodulin from skeletal muscle; that islet calmodulin may play an important role in Ca(2+)-dependent stimulus-secretion coupling in the beta-cell; and that calmodulin may exert part at least of its effect on secretion via phosphorylation of endogenous islet proteins.

Project description:1. In isolated pancreatic islets, pyruvate causes a shift to the left of the sigmoidal curve relating the rate of insulin release to the ambient glucose concentration. The magnitude of this effect is related to the concentration of pyruvate (5--90 mM) and, at a 30 mM concentration, is equivalent to that evoked by 2 mM-glucose. Pyruvate also enhances insulin release in the presence of fructose, leucine and 4-methyl-2-oxopentanoate. 2. In the presence of glucose 8 mM), the secretory response to pyruvate is an immediate process, displaying a biphasic pattern. 3. The insulinotropic action of pyruvate coincides with an inhibition of 45Ca efflux and a stimulation of 45Ca net uptake. The relationship between 45Ca uptake and insulin release displays its usual pattern in the presence of pyruvate. 4. Exogenous pyruvate rapidly accumulates in the islets in amounts close to those derived from the metabolism of glucose. The oxidation of [2-14C]pyruvate represents 64% of the rate of [1-14C]pyruvate decarboxylation and, at a 30 mM concentration, is comparable with that of 8 mM-[U-14C]glucose. 5. When corrected for the conversion of pyruvate into lactate, the oxidation of 30 mM-pyruvate corresponds to a net generation of about 314 pmol of reducing equivalents/120 min per islet. 6. Pyruvate does not affect the rate of glycolysis, but inhibits the oxidation of glucose. Glucose does not affect pyruvate oxidation. 7. Pyruvate (30 mM) does not affect the concentration of ATP, ADP and AMP in the islet cells. 8. Pyruvate (30 mM) increases the concentration of reduced nicotinamide nucleotides in the presence but not in the absence of glucose. A close correlation is seen between the concentration of reduced nicotinamide nucleotides and the net uptake of 45Ca. Menadione inhibits the effect of pyruvate on insulin release, without altering its rate of oxidation. 9. Pyruvate, like glucose, modestly stimulates lipogenesis. 10. Pyruvate, in contrast with glucose, markedly inhibits the oxidation of endogenous nutrients. The latter effect accounts for the apparent discrepancy between the rate of pyruvate oxidation and the magnitude of its insulinotropic action. 11. Dichloroacetate fails to affect glucose oxidation and glucose-stimulated insulin release. 12. It is concluded that the effect of pyruvate to stimulate insulin release depends on its ability to increase the concentration of reduced nicotinamide nucleotides in the islet cells.

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A distinct difference in the metabolic stimulus-response coupling pathways for regulating proinsulin biosynthesis and insulin secretion that lies at the level of a requirement for fatty acyl moieties.

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