Synergistic potent insulin release by combinations of weak secretagogues in pancreatic islets and INS-1 cells

Insulin secretion by the beta cell depends on anaplerosis in which insulin secretagogues are metabolized by mitochondria into molecules that are most likely exported to the extramitochondrial space where they have signaling roles. However, very little is known about the products of anaplerosis. We discovered an experimental paradigm that has begun to provide new information about these products. When various intracellular metabolites were applied in combination to overnight-cultured rat or human pancreatic islets or to INS-1 832/13 cells, they interacted synergistically to strongly stimulate insulin release. When these same metabolites were applied individually to these cells, insulin stimulation was poor. Discerning the contributions of the individual compounds to metabolism has begun to allow us to dissect some of the pathways involved in insulin secretion, which was not possible from studying individual secretagogues. Monomethyl succinate (MMS) combined with a barely stimulatory concentration of alpha-ketoisocaproate (KIC) (2 mm) stimulated insulin release in cultured rat islets 18-fold (versus 21-fold for 16.7 mm glucose). MMS plus low glucose (2 mm) or pyruvate (5 mm) gave 11- and 9-fold stimulations. These agents also potentiated MMS-induced insulin release in fresh islets, and KIC plus MMS gave synergistic insulin release in cultured human islets. In INS-1 cells, neither MMS nor KIC (10 mm) was an insulin secretagogue, but when added together KIC (2 mm) and MMS stimulated insulin release 7-fold (versus 12-fold for glucose). In islets and INS-1 cells, conditions that stimulated insulin release caused large relative increases in acetoacetate, which is a precursor of pathways to short chain acyl-CoAs. Liquid chromatography-tandem mass spectrometry measurements of acetyl-CoA, acetoacetyl-CoA, succinyl-CoA, hydroxymethylglutaryl-CoA, and malonyl-CoA confirmed that they were increased by insulin secretagogues. The results suggest a new mechanism of insulin secretion in which anaplerosis increases short chain acyl-CoAs that have roles in insulin exocytosis.     

Feasibility of pathways for transfer of acyl groups from mitochondria to the cytosol to form short chain acyl-CoAs in the pancreatic beta cell

The mitochondria of pancreatic beta cells are believed to convert insulin secretagogues into products that are translocated to the cytosol where they participate in insulin secretion. We studied the hypothesis that short chain acyl-CoA (SC-CoAs) might be some of these products by discerning the pathways of SC-CoA formation in beta cells. Insulin secretagogues acutely stimulated 1.5-5-fold increases in acetoacetyl-CoA, succinyl-CoA, malonyl-CoA, hydroxymethylglutaryl-CoA (HMG-CoA), and acetyl-CoA in INS-1 832/13 cells as judged from liquid chromatography-tandem mass spectrometry measurements. Studies of 12 relevant enzymes in rat and human pancreatic islets and INS-1 832/13 cells showed the feasibility of at least two redundant pathways, one involving acetoacetate and the other citrate, for the synthesis SC-CoAs from secretagogue carbon in mitochondria and the transfer of their acyl groups to the cytosol where the acyl groups are converted to SC-CoAs. Knockdown of two key cytosolic enzymes in INS-1 832/13 cells with short hairpin RNA supported the proposed scheme. Lowering ATP citrate lyase 88% did not inhibit glucose-induced insulin release indicating citrate is not the only carrier of acyl groups to the cytosol. However, lowering acetoacetyl-CoA synthetase 80% partially inhibited glucose-induced insulin release indicating formation of SC-CoAs from acetoacetate in the cytosol is important for insulin secretion. The results indicate beta cells possess enzyme pathways that can incorporate carbon from glucose into acetyl-CoA, acetoacetyl-CoA, and succinyl-CoA and carbon from leucine into these three SC-CoAs plus HMG-CoA in their mitochondria and enzymes that can form acetyl-CoA, acetoacetyl-CoA, malonyl-CoA, and HMG-CoA in their cytosol.     

Studies with leucine, beta-hydroxybutyrate and ATP citrate lyase-deficient beta cells support the acetoacetate pathway of insulin secretion

We hypothesized that contrasting leucine with its non-metabolizable analog 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) might provide new information about metabolic pathways involved in insulin secretion. Both compounds stimulate insulin secretion by allosterically activating glutamate dehydrogenase, which enhances glutamate metabolism. However, we found that leucine was a stronger secretagogue in rat pancreatic islets and INS-1 cells. This suggested that leucine's metabolism contributed to its insulinotropism. Indeed, we found that leucine increased acetoacetate and was metabolized to CO(2) in pancreatic islets and increased short chain acyl-CoAs (SC-CoAs) in INS-1 cells. We then used the leucine-BCH difference to study the hypothesis that acyl groups derived from secretagogue carbon can be transferred as acetoacetate, in addition to citrate, from mitochondria to the cytosol where they can be converted to SC-CoAs. Since BCH cannot form sufficient acetoacetate from glutamate, transport of any glutamate-derived acyl groups to the cytosol in BCH-stimulated cells must proceed mainly via citrate. In ATP citrate lyase-deficient INS-1 cells, which are unable to convert citrate into cytosolic acetyl-CoA, insulin release by BCH was decreased and adding beta-hydroxybutyrate or alpha-ketoisocaproate, which increases mitochondrial acetoacetate, normalized BCH-induced insulin release. This strengthens the concept that acetoacetate-transferred acyl carbon can be converted to cytosolic SC-CoAs to stimulate insulin secretion.     

Anaplerosis from glucose, α-ketoisocaproate, and pyruvate in pancreatic islets, INS-1 cells and liver mitochondria

Methyl succinate (MS) and alpha-ketoisocaproate (KIC) when applied alone to cultured pancreatic islets or INS-1 832/13 cells do not stimulate insulin release. However, when the two metabolites are combined together they strongly stimulate insulin release. Studying the possible explanations for this complementarity has provided clues to the pathways involved in insulin secretion. MS increased carbon incorporation of KIC into acid-precipitable material and lipid in INS-1 cells. In isolated mitochondria, MS alone increased malate, but MS plus KIC increased citrate, alpha-ketoglutarate, and isocitrate. These data and the known pathways of their metabolism suggest that MS supplies the oxaloacetate component of citrate and KIC supplies the acetate component of citrate. Other citric acid cycle intermediates can be formed from citrate enabling anaplerosis to supply precursors for extramitochondrial pathways. In addition, KIC, glucose and pyruvate can be metabolized to acetoacetate. In an INS-1 cell line deficient in ATP citrate lyase, incorporation of carbon from pyruvate into acid-precipitable material and lipid was not lowered. This negative result is in agreement with our recent discovery that citrate is not the only carrier of acyl groups from the mitochondria to the cytosol in the beta cell and that acetoacetate can also transfer acyl carbon to the cytosol.     

Impaired anaplerosis and insulin secretion in insulinoma cells caused by small interfering RNA-mediated suppression of pyruvate carboxylase

Anaplerosis, the synthesis of citric acid cycle intermediates, by pancreatic beta cell mitochondria has been proposed to be as important for insulin secretion as mitochondrial energy production. However, studies designed to lower the rate of anaplerosis in the beta cell have been inconclusive. To test the hypothesis that anaplerosis is important for insulin secretion, we lowered the activity of pyruvate carboxylase (PC), the major enzyme of anaplerosis in the beta cell. Stable transfection of short hairpin RNA was used to generate a number of INS-1 832/13-derived cell lines with various levels of PC enzyme activity that retained normal levels of control enzymes, insulin content, and glucose oxidation. Glucose-induced insulin release was decreased in proportion to the decrease in PC activity. Insulin release in response to pyruvate alone, 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) plus glutamine, or methyl succinate plus beta-hydroxybutyrate was also decreased in the PC knockdown cells. Consistent with a block at PC, the most PC-deficient cells showed a metabolic crossover point at PC with increased basal and/or glucose-stimulated pyruvate plus lactate and decreased malate and citrate. In addition, in BCH plus glutamine-stimulated PC knockdown cells, pyruvate plus lactate was increased, whereas citrate was severely decreased, and malate and aspartate were slightly decreased. The incorporation of 14C into lipid from [U-14C]glucose was decreased in the PC knockdown cells. The results confirm the central importance of PC and anaplerosis to generate metabolites from glucose that support insulin secretion and even suggest PC is important for insulin secretion stimulated by noncarbohydrate insulin secretagogues.     

The export of metabolites from mitochondria and anaplerosis in insulin secretion

Combinations of insulin secretagogue-derived metabolites were added to microgram amounts of mitochondria obtained from rat and mouse pancreatic islets and the INS-1 cell line, and the export of citric acid cycle intermediates was surveyed to study anaplerosis in insulin secretion. Cellular levels of metabolites were also measured. In mitochondria from all three tissues, malate production was the most responsive to various substrates. The export of citrate and isocitrate in the presence of pyruvate and most other substrates was small and their levels in intact cells did not change with any secretagogue, except in INS-1 cells where citrate increased slightly. Changes in alpha-ketoglutarate and glutamate export from mitochondria and levels in intact cells indicate that glutamate can be consumed as a fuel secretagogue, but it is not likely produced as a messenger in insulin secretion. The citrate level may not need to increase in order to provide increased malonyl-CoA for signaling insulin secretion. Unlike some cells, insulin cells probably obtain cytosolic NADPH equivalents by exporting them from mitochondria to the cytosol via a pyruvate malate shuttle or an isocitrate shuttle. Only fuels that can enhance anaplerosis via pyruvate or alpha-ketoglutarate can be insulin secretagogues.     

Perspective: emerging evidence for signaling roles of mitochondrial anaplerotic products in insulin secretion

The importance of mitochondrial biosynthesis in stimulus secretion coupling in the insulin-producing beta-cell probably equals that of ATP production. In glucose-induced insulin secretion, the rate of pyruvate carboxylation is very high and correlates more strongly with the glucose concentration the beta-cell is exposed to (and thus with insulin release) than does pyruvate decarboxylation, which produces acetyl-CoA for metabolism in the citric acid cycle to produce ATP. The carboxylation pathway can increase the levels of citric acid cycle intermediates, and this indicates that anaplerosis, the net synthesis of cycle intermediates, is important for insulin secretion. Increased cycle intermediates will alter mitochondrial processes, and, therefore, the synthesized intermediates must be exported from mitochondria to the cytosol (cataplerosis). This further suggests that these intermediates have roles in signaling insulin secretion. Although evidence is quite good that all physiological fuel secretagogues stimulate insulin secretion via anaplerosis, evidence is just emerging about the possible extramitochondrial roles of exported citric acid cycle intermediates. This article speculates on their potential roles as signaling molecules themselves and as exporters of equivalents of NADPH, acetyl-CoA and malonyl-CoA, as well as alpha-ketoglutarate as a substrate for hydroxylases. We also discuss the "succinate mechanism," which hypothesizes that insulin secretagogues produce both NADPH and mevalonate. Finally, we discuss the role of mitochondria in causing oscillations in beta-cell citrate levels. These parallel oscillations in ATP and NAD(P)H. Oscillations in beta-cell plasma membrane electrical potential, ATP/ADP and NAD(P)/NAD(P)H ratios, and glycolytic flux are known to correlate with pulsatile insulin release. Citrate oscillations might synchronize oscillations of individual mitochondria with one another and mitochondrial oscillations with oscillations in glycolysis and, therefore, with flux of pyruvate into mitochondria. Thus citrate oscillations may synchronize mitochondrial ATP production and anaplerosis with other cellular oscillations.     

Differences between mouse and rat pancreatic islets: succinate responsiveness,malic enzyme,and anaplerosis

Succinic acid methyl esters are potent insulin secretagogues in rat pancreatic islets, but they do not stimulate insulin release in mouse islets. Unlike rat and human islets, mouse islets lack malic enzyme and, therefore, are unable to form pyruvate from succinate-derived malate for net synthesis of acetyl-CoA. Dimethyl-[2,3-(14)C]succinate is metabolized in the citric acid cycle in mouse islets to the same extent as in rat islets, indicating that endogenous acetyl-CoA condenses with oxaloacetate derived from succinate. However, without malic enzyme, the net synthesis from succinate of the citric acid cycle intermediates citrate, isocitrate, and alpha-ketoglutarate cannot occur. Glucose and other nutrients that augment alpha-ketoglutarate formation are secretagogues in mouse islets with potencies similar to those in rat islets. All cycle intermediates can be net-synthesized from alpha-ketoglutarate. Rotenone, an inhibitor of site I of the electron transport chain, inhibits methyl succinate-induced insulin release in rat islets even though succinate oxidation forms ATP at sites II and III of the respiratory chain. Thus generating ATP, NADH, and anaplerosis of succinyl-CoA plus the four-carbon dicarboxylic acids of the cycle and its metabolism in the citric acid cycle is insufficient for a fuel to be insulinotropic; it must additionally promote anaplerosis of alpha-ketoglutarate or two intermediates interconvertible with alpha-ketoglutarate, citrate, and isocitrate.     

Effect of beta-hydroxybutyrate and acetoacetate on insulin and glucagon secretion from perfused rat pancreas

To elucidate the physiological significance of ketone bodies on insulin and glucagon secretion, the direct effects of beta-hydroxybutyrate (BOHB) and acetoacetate (AcAc) infusion on insulin and glucagon release from perfused rat pancreas were investigated. The BOHB or AcAc was administered at concentrations of 10, 1, or 0.1 mM for 30 min at 4.0 ml/min. High-concentration infusions of BOHB and AcAc (10 mM) produced significant increases in insulin release in the presence of 4.4 mM glucose, but low-concentration infusions of BOHB and AcAc (1 and 0.1 mM) caused no significant changes in insulin secretion from perfused rat pancreas. BOHB (10, 1, and 0.1 mM) and AcAc (10 and 1 mM) infusion significantly inhibited glucagon secretion from perfused rat pancreas. These results suggest that physiological concentrations of ketone bodies have no direct effect on insulin release but have a direct inhibitory effect on glucagon secretion from perfused rat pancreas.     

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

Pancreatic beta cell mitochondria convert insulin secretagogues into products that support insulin exocytosis. We explored the idea that lipids are some of these products formed from acyl group transfer out of mitochondria to the cytosol, the site of lipid synthesis. There are two isoforms of acetyl-CoA carboxylase, the enzyme that forms malonyl-CoA from which C₂ units for lipid synthesis are formed. We found that ACC1, the isoform seen in lipogenic tissues, is the only isoform present in human and rat pancreatic islets and INS-1 832/13 cells. Inhibitors of ACC and fatty acid synthase inhibited insulin release in islets and INS-1 cells. Carbon from glucose and pyruvate were rapidly incorporated into many lipid classes in INS-1 cells. Glucose and other insulin secretagogues acutely increased many lipids with C₁₄-C₂₄ chains including individual cholesterol esters, phospholipids and fatty acids. Many phosphatidylcholines and phosphatidylserines were increased and many phosphatidylinositols and several phosphatidylethanolamines were decreased. The results suggest that lipid remodeling and rapid lipogenesis from secretagogue carbon support insulin secretion.     

13C NMR isotopomer analysis of anaplerotic pathways in INS-1 cells

Anaplerotic flux into the Kreb's cycle is crucial for glucose-stimulated insulin secretion from pancreatic beta-cells. However, the regulation of flux through various anaplerotic pathways in response to combinations of physiologically relevant substrates and its impact on glucose-stimulated insulin secretion is unclear. Because different pathways of anaplerosis generate distinct products, they may differentially modulate the insulin secretory response. To examine this question, we applied 13C-isotopomer analysis to quantify flux through three anaplerotic pathways: 1) pyruvate carboxylase of pyruvate derived from glycolytic sources; 2) pyruvate carboxylase of pyruvate derived from nonglycolytic sources; and 3) glutamate dehydrogenase (GDH). At substimulatory glucose, anaplerotic flux rate in the clonal INS-1 832/13 cells was approximately 40% of Kreb's cycle flux, with similar contributions from each pathway. Increasing glucose to 15 mm stimulated insulin secretion approximately 4-fold, and was associated with a approximately 4-fold increase in anaplerotic flux that could mostly be attributed to an increase in PC flux. In contrast, the addition of glutamine to the perfusion media stimulated GDH flux approximately 6-fold at both glucose concentrations without affecting insulin secretion rates. In conclusion, these data support the hypothesis that a signal generated by anaplerosis from increased pyruvate carboxylase flux is essential for glucose-stimulated insulin secretion in beta-cells and that anaplerosis through GDH does not play a major role in this process.     

Preferential utilization of ketone bodies in the brain and lung of newborn rats

Persistent mild hyperketonemia is a common finding in neonatal rats and human newborns, but the physiological significance of elevated plasma ketone concentrations remains poorly understood. Recent advances in ketone metabolism clearly indicate that these compounds serve as an indispensable source of energy for extrahepatic tissues, especially the brain and lung of developing rats. Another important function of ketone bodies is to provide acetoacetyl-CoA and acetyl-CoA for synthesis of cholesterol, fatty acids, and complex lipids. During the early postnatal period, acetoacetate (AcAc) and beta-hydroxybutyrate are preferred over glucose as substrates for synthesis of phospholipids and sphingolipids in accord with requirements for brain growth and myelination. Thus, during the first 2 wk of postnatal development, when the accumulation of cholesterol and phospholipids accelerates, the proportion of ketone bodies incorporated into these lipids increases. On the other hand, an increased proportion of ketone bodies is utilized for cerebroside synthesis during the period of active myelination. In the lung, AcAc serves better than glucose as a precursor for the synthesis of lung phospholipids. The synthesized lipids, particularly dipalmityl phosphatidylcholine, are incorporated into surfactant, and thus have a potential role in supplying adequate surfactant lipids to maintain lung function during the early days of life. Our studies further demonstrate that ketone bodies and glucose could play complementary roles in the synthesis of lung lipids by providing fatty acid and glycerol moieties of phospholipids, respectively. The preferential selection of AcAc for lipid synthesis in brain, as well as lung, stems in part from the active cytoplasmic pathway for generation of acetyl-CoA and acetoacetyl-CoA from the ketone via the actions of cytoplasmic acetoacetyl-CoA synthetase and thiolase.     

Effect of insulin on ketogenesis and fatty acid synthesis in rat hepatocytes incubated with dichloroacetate

In parenchymal liver cells isolated from fed rats, insulin increased the formation of 14CO2 from [1-14C]pyruvate (and presumably the flux through pyruvate dehydrogenase) by 14%. Dichloroacetate, an activator of the pyruvate dehydrogenase complex, stimulated this process by 133%. As judged from the conversion of [2-14C]pyruvate to 14CO2, the tricarboxylic acid cycle activity was not affected by insulin, but it was depressed by dichloroacetate. In hepatocytes from fed rats, incubated with glucose as the only carbon source, dichloroacetate caused a stimulation (31%) of fatty acid synthesis, measured as 3H incorporation from 3H2O into fatty acid, and an increased (134%) accumulation of ketone bodies (acetoacetate + D-3-hydroxybutyrate). Dichloroacetate did not affect ketone body formation from [14C]palmitate, suggesting that the increased accumulation of ketone bodies resulted from acetyl-CoA derived from pyruvate. Insulin stimulated fatty acid synthesis in hepatocytes from fed rats. In the combined presence of insulin plus dichloroacetate, fatty acid synthesis was more rapid than in the presence of either insulin or dichloroacetate, whereas the accumulation of ketone bodies was smaller than in the presence of dichloroacetate alone. Although pyruvate dehydrogenase activity, which is rate-limiting for fatty acid synthesis in hepatocytes from fed rats, is stimulated both by insulin and by dichloroacetate, the reciprocal changes in fatty acid synthesis and ketone body accumulation brought about by insulin in the presence of dichloroacetate suggest that insulin is also involved in the regulation of fatty acid synthesis at a mitochondrial site after pyruvate dehydrogenase, possibly at the partitioning of acetyl-CoA between citrate and ketone body formation.     

Citrate oscillates in liver and pancreatic beta cell mitochondria and in INS-1 insulinoma cells

Oscillations in citric acid cycle intermediates have never been previously reported in any type of cell. Here we show that adding pyruvate to isolated mitochondria from liver, pancreatic islets, and INS-1 insulinoma cells or adding glucose to intact INS-1 cells causes sustained oscillations in citrate levels. Other citric acid cycle intermediates measured either did not oscillate or possibly oscillated with a low amplitude. In INS-1 mitochondria citrate oscillations are in phase with NAD(P) oscillations, and in intact INS-1 cells citrate oscillations parallel oscillations in ATP, suggesting that these processes are co-regulated. Oscillations have been extensively studied in the pancreatic beta cell where oscillations in glycolysis, NAD(P)/NAD(P)H and ATP/ADP ratios, plasma membrane electrical activity, calcium levels, and insulin secretion have been well documented. Because the mitochondrion is the major site of ATP synthesis and NADH oxidation and the only site of citrate synthesis, mitochondria need to be synchronized for these factors to oscillate. In suspensions of mitochondria from various organs, most of the citrate is exported from the mitochondria. In addition, citrate inhibits its own synthesis. We propose that this enables citrate itself to act as one of the cellular messengers that synchronizes mitochondria. Furthermore, because citrate is a potent inhibitor of the glycolytic enzyme phosphofructokinase, the pacemaker of glycolytic oscillations, citrate may act as a metabolic link between mitochondria and glycolysis. Citrate oscillations may coordinate oscillations in mitochondrial energy production and anaplerosis with glycolytic oscillations, which in the beta cell are known to parallel oscillations in insulin secretion.     

beta-Cell adaptation to insulin resistance. Increased pyruvate carboxylase and malate-pyruvate shuttle activity in islets of nondiabetic Zucker fatty rats

The beta-cell biochemical mechanisms that account for the compensatory hyperfunction with insulin resistance (so-called beta-cell adaptation) are unknown. We investigated glucose metabolism in isolated islets from 10-12-week-old Zucker fatty (ZF) and Zucker lean (ZL) rats (results expressed per mg/islet of protein). ZF rats were obese, hyperlipidemic, and normoglycemic. They had a 3.8-fold increased beta-cell mass along with 3-10-fold increases in insulin secretion to various stimuli during pancreas perfusion despite insulin content per milligram of beta-cells being only one-third that of ZL rats. Islet glucose metabolism (utilization and oxidation) was 1.5-2-fold increased in the ZF islets despite pyruvate dehydrogenase activity being 30% lowered compared with the ZL islets. The reason was increased flux through pyruvate carboxylase (PC) and the malate-pyruvate and citrate-pyruvate shuttles based on the following observations (% ZL islets): increased V(max) of PC (160%), malate dehydrogenase (170%), and malic enzyme (275%); elevated concentrations of oxaloacetate (150%), malate (250%), citrate (140%), and pyruvate (250%); and 2-fold increased release of malate from isolated mitochondria. Inhibition of PC by 5 mm phenylacetic acid markedly lowered glucose-induced insulin secretion in ZF and ZL islets. Thus, our results suggest that PC and the pyruvate shuttles are increased in ZF islets, and this accounts for glucose mitochondrial metabolism being increased when pyruvate dehydrogenase activity is reduced. As the anaplerosis pathways are implicated in glucose-induced insulin secretion and the synthesis of glucose-derived lipid and amino acids, our results highlight the potential importance of PC and the anaplerosis pathways in the enhanced insulin secretion and beta-cell growth that characterize beta-cell adaptation to insulin resistance.     

Alpha-Ketoisocaproate-induced hypersecretion of insulin by islets from diabetes-susceptible mice

Most patients at risk for developing type 2 diabetes are hyperinsulinemic. Hyperinsulinemia may be a response to insulin resistance, but another possible abnormality is insulin hypersecretion. BTBR mice are insulin resistant and hyperinsulinemic. When the leptin(ob) mutation is introgressed into BTBR mice, they develop severe diabetes. We compared the responsiveness of lean B6 and BTBR mouse islets to various insulin secretagogues. The transamination product of leucine, alpha-ketoisocaproate (KIC), elicited a dramatic insulin secretory response in BTBR islets. The KIC response was blocked by methyl-leucine or aminooxyacetate, inhibitors of branched-chain amino transferase. When dimethylglutamate was combined with KIC, the fractional insulin secretion was identical in islets from both mouse strains, predicting that the amine donor is rate-limiting for KIC-induced insulin secretion. Consistent with this prediction, glutamate levels were higher in BTBR than in B6 islets. The transamination product of glutamate, alpha-ketoglutarate, elicited insulin secretion equally from B6 and BTBR islets. Thus formation of alpha-ketoglutarate is a requisite step in the response of mouse islets to KIC. alpha-Ketoglutarate can be oxidized to succinate. However, succinate does not stimulate insulin secretion in mouse islets. Our data suggest that alpha-ketoglutarate may directly stimulate insulin secretion and that increased formation of alpha-ketoglutarate leads to hyperinsulinemia.     

Both insulin and epidermal growth factor stimulate lipogenesis and acetyl-CoA carboxylase activity in isolated adipocytes. Importance of homogenization procedure in avoiding artefacts in acetyl-CoA carboxylase assay.

Epidermal growth factor (EGF) stimulates lipogenesis by 3-4-fold in isolated adipocytes, with a half-maximal effect at 10 nM-EGF. In the same batches of cells insulin stimulated lipogenesis by 15-fold. Freezing and prolonged homogenization of adipocytes results in release of large quantities of pyruvate carboxylase from broken mitochondria, and sufficient pyruvate can be carried through into assays for this enzyme to cause significant interference with assays of acetyl-CoA carboxylase in crude adipocyte extracts. This may account for the high amount of citrate-independent acetyl-CoA carboxylase activity reported to be present in adipocyte extracts in some previous publications. This problem may be eliminated by homogenizing very briefly without freezing. By using the modified homogenization procedure, EGF treatment of adipocytes was shown to produce an effect on acetyl-CoA carboxylase activity almost identical with that of insulin. Both messengers increase Vmax. without significant effect on the Ka for the allosteric activator, citrate.     

Effects of ketone bodies on insulin release and islet-cell metabolism in the rat.

Ketone bodies promote insulin secretion from isolated rat pancreatic islets in the presence of 5 mM-glucose, but are ineffective in its absence. At concentrations of 10 mM or less, the relative abilities of the ketone bodies to potentiate release are in the order D-3-hydroxybutyrate greater than DL-3-hydroxybutyrate greater than acetoacetate. The response curve relating insulin release to D-3-hydroxybutyrate concentration displays a threshold at 1 mM and a maximum at 10 mM. D-3-Hydroxybutyrate (5 mM, but not 10 mM) promotes insulin secretion in the presence of 5 mM concentrations of both L-arginine and DL-glyceraldehyde, but not with L-leucine, L-alanine, L-glutamate or 4-methyl-2-oxopentanoate. The oxidation rates of the exogenous ketone bodies do not correlate well with their capacities to promote insulin release. Moreover, the oxidation of 5 mM-D-3-hydroxybutyrate can be inhibited by 25% with methylmalonate (10 mM) without any diminution of release. The potentiation with D-3-hydroxybutyrate occurs without an observable increase in total islet cyclic AMP. However, a small net efflux matches the relative abilities of the ketone bodies to promote insulin release. With islets from 48 h-starved animals the insulin response is both diminished and less sensitive than in fed animals, since insulin secretion is not significantly raised until a threshold of 5 mM-D-3-hydroxybutyrate is reached. These results suggest that, in the rat at least, there should be a reappraisal of the physiological role of ketone bodies in the promotion of insulin release.     

Different secretory response of pancreatic islets and insulin secreting cell lines INS-1 and INS-1E to osmotic stimuli

Objective of this study was to characterize osmotically-induced insulin secretion in two tumor cell lines. We compared response of freshly isolated rat pancreatic islets and INS-1 and INS-1E tumor cell lines to high glucose, 30 % hypotonic medium and 20 % hypertonic medium. In Ca(2+)-containing medium glucose induced insulin release in all three cell types. Hypotonicity induced insulin secretion from islets and INS-1 cells but not from INS-1E cells, in which secretion was inhibited despite similar increase in cell volume in both cell types. GdCl(3) (100 micromol/l) did not affect insulin response from INS-1E cells to hypotonic challenge. Hypertonic medium inhibited glucose-induced insulin secretion from islets but not from tumor cells. Noradrenaline (1 micromol/l) inhibited glucose-induced but not swelling-induced insulin secretion from INS-1 cells. Surprisingly, perifusion with Ca(2+)-depleted medium showed distinct secretory response of INS-1E cells to hypotonicity while that of INS-1 cells was partially inhibited. Functioning glucose-induced insulin secretion is not sufficient prerequisite for hypotonicity-induced response in INS-1E cells suggesting that swelling-induced exocytosis is not essential step in the mechanism mediating glucose-induced insulin secretion. Both cell lines are resistant to inhibitory effect of hyperosmolarity on glucose-induced insulin secretion. Response of INS-1E cells to hypotonicity is inhibited by the presence of Ca(2+) in medium.     

Aspects of ketogenesis: Control and mechanism of ketone-body formation in isolated RAT-liver mitochondria

The synthesis of ketone bodies by intact isolated rat-liver mitochondria has been studied at varying rates of acetyl-CoA production and of acetyl-CoA utilization in the Krebs cycle. Factors which enhanced the rate of acetyl-CoA production caused an increase in the fraction of acetyl-CoA which was incorporated into ketone bodies. On the other hand, it was found that factors which stimulated the formation of citrate lowered the relative rate of ketogenesis. It is concluded that acetyl-CoA is preferentially used for citrate synthesis, if the level of oxaloacetate in the mitochondrial matrix space is adequate. The intramitochondrial level of oxaloacetate, which is determined by the malate concentration and the ratio of NADH over NAD+, is the main factor controlling the rate of citrate synthesis. The ATP/ADP ratio per se does not affect the activity of citrate synthase in this in vitro system. Ketogenesis can be described as an overflow of acetyl-groups: Ketone-body formation is stimulated only when the rate of acetyl-CoA production increases beyond the capacity for citrate synthesis. The interaction between fatty acid oxidation and pyruvate metabolism and the effects of long-chain acyl-CoA on mitochondrial metabolism are discussed. Ketone bodies which were generated during the oxidation of [1-14C] fatty acids were preferentially labelled in their carboxyl group. This carboxyl group had the same specific activity as the acetyl-CoA pool, whereas the specific activity of the acetone moiety of acetoacetate was much lower, especially at low rates of ketone-body formation. The activities of acetoacetyl-CoA deacylase and the hydroxymethylglutaryl-CoA (HMG-CoA) pathway were compared in soluble and mitochondrial fractions of rat- and cow-liver in different ketotic states. In rat-liver mitochondria, both pathways of acetoacetate synthesis were stimulated upon starvation or in alloxan diabetes. In cow liver, only the HMG-CoA pathway was increased during ketosis in the mitochondrial as well as in the soluble fraction.