Chronic exposure to beta-hydroxybutyrate inhibits glucose-induced insulin release from pancreatic islets by decreasing NADH contents

To investigate the effects of chronic exposure to ketone bodies on glucose-induced insulin secretion, we evaluated insulin release, intracellular Ca2+ and metabolism, and Ca2+ efficacy of the exocytotic system in rat pancreatic islets. Fifteen-hour exposure to 5 mM d-beta-hydroxybutyrate (HB) reduced high glucose-induced insulin secretion and augmented basal insulin secretion. Augmentation of basal release was derived from promoting the Ca2+-independent and ATP-independent component of insulin release, which was suppressed by the GDP analog. Chronic exposure to HB affected mostly the second phase of glucose-induced biphasic secretion. Dynamic experiments showed that insulin release and NAD(P)H fluorescence were lower, although the intracellular Ca2+ concentration ([Ca2+](i)) was not affected 10 min after exposure to high glucose. Additionally, [Ca2+](i) efficacy in exocytotic system at clamped concentrations of ATP was not affected. NADH content, ATP content, and ATP-to-ADP ratio in the HB-cultured islets in the presence of high glucose were lower, whereas glucose utilization and oxidation were not affected. Mitochondrial ATP production shows that the respiratory chain downstream of complex II is not affected by chronic exposure to HB, and that the decrease in ATP production is due to decreased NADH content in the mitochondrial matrix. Chronic exposure to HB suppresses glucose-induced insulin secretion by lowering the ATP level, at least partly by inhibiting ATP production by reducing the supply of NADH to the respiratory chain. Glucose-induced insulin release in the presence of aminooxyacetate was not reduced, which implies that chronic exposure to HB affects the malate/aspartate shuttle and thus reduces NADH supply to mitochondria.     

FKBP12.6 disruption impairs glucose-induced insulin secretion

Cyclic ADP-ribose (cADPR), accumulated in pancreatic β-cells in response to elevated ATP levels after glucose stimulation, mobilizes Ca²⁺ from the endoplasmic reticulum through the ryanodine receptor (RyR) and thereby induces insulin secretion. We have recently demonstrated in an in vitro study that cADPR activates RyR through binding to FK506-binding protein 12.6 (FKBP12.6), an accessory protein of RyR. Here we generated FKBP12.6-deficient (FKBP12.6^−/−) mice by homologous recombination. FKBP12.6^−/− mice showed glucose intolerance coupled to insufficient insulin secretion upon a glucose challenge. Insulin secretion in response to glucose was markedly impaired in FKBP12.6^−/− islets, while sulfonylurea- or KCl-induced insulin secretion was unaffected. No difference was found in the glucose oxidation rate between FKBP12.6^−/− and wild-type islets. These results indicate that FKBP12.6 plays a role in glucose-induced insulin secretion downstream of ATP production, independently of ATP-sensitive K⁺ channels, in pancreatic β-cells.     

Resistin induces insulin resistance in pancreatic islets to impair glucose-induced insulin release

An adipokine resistin, a small cysteine-rich protein, is one of the major risk factors of insulin resistance. In the present study, transiently resistin-expressing mice using adenovirus method showed an impaired glucose tolerance due to insulin resistance. We found that resistin-expressing mice exhibited impaired insulin secretory response to glucose. In addition, in vitro treatment with resistin for 1 day induced insulin resistance in pancreatic islets and impaired glucose-stimulated insulin secretion by elevating insulin release at basal glucose (2.8 mM) and suppressing insulin release at stimulatory glucose (8.3 mM). In addition, resistin inhibited insulin-induced phosphorylation of Akt in islets as well as other insulin target organs. Furthermore, resistin induced SOCS-3 expression in beta-cells. In conclusion, resistin induces insulin resistance in islet beta-cells at least partly via induction of SOCS-3 expression and reduction of Akt phosphorylation and impairs glucose-induced insulin secretion.     

The glucose-induced insulin release after adrenalectomy in the rat

Plasma glucose and insulin levels following glucose loading were investigated in adrenalectomized rats. Both oral and intravenous administration of glucose induced an elevation in plasma glucose and insulin level. The increases of plasma glucose and insulin concentrations were significantly higher in the adrenalectomized rats compared with the controls. We conclude, that corticoid hormones are capable of inhibiting glucose-induced insulin release in the rat.     

Tacrolimus suppresses glucose-induced insulin release from pancreatic islets by reducing glucokinase activity

Tacrolimus is widely used for immunosuppressant therapy, including various organ transplantations. One of its main side effects is hyperglycemia due to reduced insulin secretion, but the mechanism remains unknown. We have investigated the metabolic effects of tacrolimus on insulin secretion at a concentration that does not influence insulin content. Twenty-four-hour exposure to 3 nM tacrolimus reduced high glucose (16.7 mM)-induced insulin secretion (control 2.14 +/- 0.08 vs. tacrolimus 1.75 +/- 0.02 ng.islet(-1).30 min(-1), P < 0.01) without affecting insulin content. In dynamic experiments, insulin secretion and NAD(P)H fluorescence during a 20-min period after 10 min of high-glucose exposure were reduced in tacrolimus-treated islets. ATP content and glucose utilization of tacrolimus-treated islets in the presence of 16.7 mM glucose were less than in control (ATP content: control 9.69 +/- 0.99 vs. tacrolimus 6.52 +/- 0.40 pmol/islet, P < 0.01; glucose utilization: control 103.8 +/- 6.9 vs. tacrolimus 74.4 +/- 5.1 pmol.islet(-1).90 min(-1), P < 0.01). However, insulin release from tacrolimus-treated islets was similar to that from control islets in the presence of 16.7 mM alpha-ketoisocaproate, a mitochondrial fuel. Glucokinase activity, which determines glycolytic velocity, was reduced by tacrolimus treatment (control 65.3 +/- 3.4 vs. tacrolimus 49.9 +/- 2.8 pmol.islet(-1).60 min(-1), P < 0.01), whereas hexokinase activity was not affected. These results indicate that glucose-stimulated insulin release is decreased by chronic exposure to tacrolimus due to reduced ATP production and glycolysis derived from reduced glucokinase activity.     

The stimulus secretion coupling of glucose-induced insulin release.

In isolated rat pancreatic islets, exogenous l-lactate causes a dose-related enhancement of glucose-induced insulin release and shifts the sigmoidal curve relating insulin output to ambient glucose concentrations to the left. l-Lactate also enhances α-ketoisocaproate-induced insulin release and glucose-induced proinsulin biosynthesis. l-Lactate rapidly accumulates in the islet cells, is converted to pyruvate and CO₂, and raises the intracellular concentration of both ATP and NAD(P)H. On a molar basis, the insulinotropic capacity of nutrients ranges as follows d-glucose ? l-lactate > pyruvate = d/l-lactate > d-lactate and does not correlate with their respective oxidation rates. However, when allowance is made for the intracellular interconversion of these exogenous nutrients, for their reciprocal influence upon oxidation rates, and for their sparing action on the utilization of endogenous fuels, a close correlation is found between the aptitude of glucose, l-lactate, and pyruvate to generate reducing equivalents and to stimulate insulin release. It is proposed that the concentration of NAD(P)H in islet cells affects the ionophoretic fluxes of cations (K⁺, Ca²⁺) across membrane systems and, hence, regulates the net uptake of Ca²⁺ and subsequent release of insulin. The effect of l-lactate upon Ca²⁺ handling is sufficiently rapid to account for the immediate secretory response to this nutrient.     

Lipoxygenase-generated icosanoids inhibit glucose-induced insulin release from rat islets

Lipoxygenase-pathway metabolites of arachidonic acid are produced in pancreatic islets. They are are implicated in insulin release, since nonselective inhibitors of lipoxygenases inhibit glucose-induced insulin release. We studied the interplay in insulin release between glucose and selected icosanoids formed in 5-, 12- and 15-lipoxygenase pathways. Effects on immunoreactive insulin release of 10(7) to 10(6)-12-(R)-HETE, 12-(S)-HETE, hepoxilin A3, lipoxin B4, LTB4 or LTC4 were tested individually in 30-min incubations of freshly isolated young adult Wistar rat pancreatic islets, in the presence of 5.6 mM or 23 mM glucose. Basal insulin release (at 5.6 mM glucose) was stimulated by LTC4 and hepoxilin A3 (304% and 234% of controls at 5.6 mM glucose alone, respectively), inhibited by 12-(S)-HPETE (56%), and was not affected by 12-(R)-HETE, 12-(S)-HETE, lipoxin B4 or LTB4 (111%, 105%, 106% and 136%, respectively). Insulin release evoked by 23 mM glucose (190-320%) was inhibited (50-145%) by all icosanoids tested, except LTC4 (162%). We conclude that, among the lipoxygenase products tested, only leukotrienes and hepoxilin are candidates for a tonic-stimulatory influence on basal insulin release. Since glucose promotes icosanoid formation in islets, the observed inhibition of glucose-induced insulin release by lipoxygenase products suggests the existence of a negative-feedback system.     

Inhibition of high glucose-induced erythrocyte phosphatidylserine exposure by leupeptin and disaccharides

High glucose can lead to serious phosphatidylserine exposure of erythrocytes which may influence the protective effect of glucose on lyophilization of erythrocytes. In this study, caspase activation has not occurred during phosphatidylserine exposure of erythrocytes. However, leupeptin can efficiently inhibit phosphatidylserine exposure of erythrocytes induced by high glucose. With increase of the leupeptin concentrations, the percentages of cells with exposed phosphatidylserine were decreased steadily. In addition, trehalose and sucrose can significantly inhibit phosphatidylserine exposure and cell shrinkage of erythrocytes induced by high glucose through increasing tolerance to osmotic shock. When the disaccharide concentrations were more than 100 mM, the percentages of cells with exposed phosphatidylserine were similar to those of control cells. Moreover, addition of disaccharides in the glucose buffer can result in high osmotic pressure which may facilitate uptake of glucose and disaccharides into erythrocytes and higher cellular glucose and disaccharide concentrations can provide more protection for lyophilized erythrocytes. Although disaccharides can increase the osmotolerance and decrease the phosphatidylserine exposure of erythrocytes exposed to high glucose, whether disaccharides can prevent phosphatidylserine exposure of lyophilized erythrocytes still needs further researches.     

The stimulus-secretion coupling of glucose-induced insulin release. Insulin release due to glycogenolysis in glucose-deprived islets.

1. When pancreatic islets are preincubated for 20h in the presence of glucose (83.3mM) and thereafter transferred to a glucose-free medium, theophylline (1.4mM) provokes a dramatic stimulation of insulin release. This phenomenon does not occur when the islets are preincubated for either 20h at low glucose concentration (5.6mM) or only 30 min at the high glucose concentration (83.3mM). 2. The insulinotropic action of theophylline cannot be attributed to contamination of the islets with exogenous glucose and is not suppressed by mannoheptulose. 3. The secretory response to theophylline is an immediate phenomenon, but disappears after 60min of exposure to the drug. 4. The release of insulin evoked by theophylline is abolished in calcium-depleted media containing EGTA. Theophylline enhances the net uptake of 45Ca by the islets. 5. Glycogen accumulates in the islets during the preincubation period, as judged by both ultrastructural and biochemical criteria. Theophylline significantly increases the rate of glycogenolysis during the final incubation in the glucose-free medium. 6. The theophylline-induced increase in glycogenolysis coincides with a higher rate of both lactate output and oxidation of endogenous 14C-labelled substrates. 7. These data suggest that stimulation of glycolysis from endogenous stores of glycogen is sufficient to provoke insulin release even in glucose-deprived islets, as if the binding of extracellular glucose to hypothetical plasma-membrane glucoreceptors is not an essential feature of the stimulus-secretion coupling process.     

Chronic exposure to increasing background ozone impairs stomatal functioning in grassland species

Two species found in temperate calcareous and mesotrophic grasslands (Dactylis glomerata and Leontodon hispidus) were exposed to eight ozone treatments spanning preindustrial to post‐2100 regimes, and late‐season effects on stomatal functioning were investigated. The plants were grown as a mixed community in 14 L containers and were exposed to ozone in ventilated solardomes (dome‐shaped greenhouses) for 20 weeks from early May to late September 2007. Ozone exposures were based on O₃ concentrations from a nearby upland area, and provided the following seasonal 24 h means: 21.4, 39.9 (simulated ambient), 50.2, 59.4, 74.9, 83.3, 101.3 and 102.5 ppb. In both species, stomatal conductance of undamaged inner canopy leaves developing since a midseason cutback increased linearly with increasing background ozone concentration. Imposition of severe water stress by leaf excision indicated that increasing background ozone concentration decreased the ability of leaves to limit water loss, implying impaired stomatal control. The threshold ozone concentrations for these effects were 15–40 ppb above current ambient in upland UK, and were within the range of ozone concentrations anticipated for much of Europe by the latter part of this century. The potential mechanism behind the impaired stomatal functioning was investigated using a transpiration assay. Unlike for lower ozone treatments, apparently healthy green leaves of L. hispidus that had developed in the 101.3 ppb treatment did not close their stomata in response to 1.5 μm abscisic acid (ABA); indeed stomatal opening initially occurred in this treatment. Thus, ozone appears to be disrupting the ABA‐induced signal transduction pathway for stomatal control thereby reducing the ability of plants to respond to drought. These results have potentially wide‐reaching implications for the functioning of communities under global warming where periods of soil drying and episodes of high vapour pressure deficit are likely to be more severe.     

Lack of inhibitory effect of verapamil on glucose-induced insulin release in the rat

The effect of the calcium-antagonist Verapamil was investigated on the blood glucose and insulin release in rats. Verapamil induced a significant elevation of the blood glucose and increased the insulin release too, however, it was not able to inhibit the glucose-induced insulin release. The authors suggest, that the effect of glucose is not restricted for the calcium turnover of the islets of the pancreas, and may be there are other cellular mechanisms apart from the effect on the calcium movements by which glucose induces insulin release.     

Inhibition of glucose-induced insulin release by 3-O-methyl-D-glucose: Enzymatic,metabolic and cationic determinants

The analog of D-glucose, 3-O-methyl-D-glucose, is thought to delay the equilibration of D-glucose concentration across the plasma membrane of pancreatic islet B-cells, but not to exert any marked inhibitory action upon the late phase of glucose-stimulated insulin release. In this study, however, 3-O-methyl-D-glucose, when tested in high concentrations (30-80 mM) was found to cause a rapid, sustained and not rapidly reversible inhibition of glucose-induced insulin release in rat pancreatic islets. In relative terms, the inhibitory action of 3-O-methyl-D-glucose was more marked at low than high concentrations of D-glucose. It could not be attributed to hyperosmolarity and appeared specific for the insulinotropic action of D-glucose, as distinct from non-glucidic nutrient secretagogues. Although 3-O-methyl-D-glucose and D-glucose failed to exert any reciprocal effect upon the steady-state value for the net uptake of these monosaccharides by the islets, the glucose analog inhibited D-[5-3H]glucose utilization and D-[U-14C]glucose oxidation. This coincided with increased 86Rb outflow and decreased 45Ca outflow from prelabelled islets, as well as decreased 45Ca net uptake. A preferential effect of 3-O-methyl-D-glucose upon the first phase of glucose-stimulated insulin release was judged compatible with an altered initial rate of D-glucose entry into islet B-cells. The long-term inhibitory action of the glucose analog upon the metabolic and secretory response to D-glucose, however, may be due, in part at least, to an impaired rate of D-glucose phosphorylation. The phosphorylation of the hexose by beef heart hexokinase and human B-cell glucokinase, as well as by parotid and islet homogenates, was indeed inhibited by 3-O-methyl-D-glucose. The relationship between insulin release and D-glucose utilization or oxidation in the presence of 3-O-methyl-D-glucose was not different from that otherwise observed at increasing concentrations of either D-glucose or D-mannoheptulose. It is concluded, therefore, that 3-O-methyl-D-glucose adversely affects the metabolism and insulinotropic action of D-glucose by a mechanism largely unrelated to changes in the intracellular concentration of the latter hexose.     

Chronic hypoxia changes the ratio of endothelin to ATP release from rat aortic endothelial cells exposed to high flow.

Endothelial cells isolated from the thoracic aorta of normoxic and chronically hypoxic rats were perfused (0.5 ml min-1) and stimulated by increased flow rate (3.0 ml min-1). The release of ATP, endothelin and vasopressin was investigated. During periods of high flow rate, endothelial cells isolated from normoxic rats increased their release of ATP and endothelin. In comparison, in hypoxic rats, ATP release during the period of high flow rate was less, whereas endothelin release was greater. Vasopressin release was not increased during periods of stimulation in either group of animals. These results suggest that, under conditions of reduced arterial oxygen tension, a dynamic balance between ATP and endothelin release could regulate the response of vessels to shear stress.     

Chronic exposure to ketone bodies impairs glucose uptake in adult cardiomyocytes in response to insulin but not vanadate: the role of PI3-K

There is a strong positive correlation between insulin resistance and cardiac diseases. We have already shown that chronic exposure to the ketone body β-hydroxybutyrate (OHB) decreases insulin-mediated activation of protein kinase B (PKB) and glucose uptake in cardiomyocytes. To gain further insights into the mechanism underlying ketone body-induced insulin resistance, we examined whether OHB alters activation of the insulin-signaling cascade and whether the insulinomimetic agent vanadate could bypass insulin resistance and stimulate glucose uptake in these cells. Cardiomyocytes were incubated with 5 mM OHB, 50 μM vanadate or both for 16 h before the measurement of glucose uptake or the activation of insulin-signaling molecules. While chronic exposure to OHB did not alter insulin- or vanadate-mediated activation of the insulin receptor, it suppressed insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation in response to both agonists. Furthermore, this treatment decreased by 54 and 36% the phosphorylation of the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3-K) and PKB in response to insulin, whereas it did not alter vanadate-mediated activation of these enzymes. Although insulin did not significantly stimulate p38MAPK phosphorylation, vanadate increased it by 3.8-fold. Furthermore, chronic exposure to OHB potentiated vanadate’s action, resulting in a 250% increase in enzyme activation compared to control cells. Though OHB induced a 2.1-fold increase of basal ERK1/2 phosphorylation, inhibition of this enzyme with the MEK inhibitor PD98059 demonstrated that ERK1/2 did not participate in OHB-induced insulin resistance. In conclusion, ketone bodies promote insulin resistance probably through decreased activation of the PI3-K/PKB signaling cascade. Furthermore, vanadate can bypass insulin resistance and stimulate glucose uptake in OHB-treated cardiomyocytes.     

Potentiation of glucose-induced insulin release in islets by desHis1[Glu9]glucagon amide

Glucagon and secretin and some of their hybrid analogs potentiate glucose-induced release of insulin from isolated mouse pancreatic islets. It was recently shown that the synthetic glucagon analog, desHis1[Glu9]glucagon amide, does not stimulate the formation of cyclic adenosine monophosphate in the rat hepatocyte membrane, but binds well to the glucagon receptor and is a good competitive antagonist of glucagon. In the present study the effect of this analog on isolated islets was examined. desHis1-[Glu9]glucagon amide at 3 x 10(-7) M, in the presence of 0.01 M D-glucose, increased the release of insulin by 30% and maintained that level for the full 30-min test period. The rate of insulin release returned to the glucose-induced base line after removal of the peptide. The same insulin level was produced by 3 x 10(-9) M glucagon, and at 3 x 10(-7) M glucagon insulin release was enhanced 290% above the glucose base line.     

Attenuated anomeric difference of glucose-induced insulin release in the perfused pancreas of diazoxide-treated rats

Mild hyperglycemia was induced in normal rats by oral administration of both diazoxide and D-glucose. After 48 hours of such a treatment, the insulin and glucagon secretory responses of the perfused pancreas to alpha- and beta-D-glucose (3.3 mM) were examined in the presence of 10.0 mM L-leucine. The output of insulin, but not that of glucagon, and the perfusion pressure were higher in treated than control rats. The alpha-anomer of D-glucose was a more potent insulin secretagogue than beta-D-glucose in both control and treated rats. However, the alpha/beta ratio in insulin output was twice higher in control than treated rats. By analogy with other experimental models of diabetes, the attenuation in the anomeric difference of glucose-stimulated insulin output in the treated rats could reflect an altered secretory response to alpha- rather than beta-D-glucose. These findings suggest that hyperglycemia provokes, as a function of its severity and duration, first attenuation and then suppression, if not inversion, of the anomeric preference for alpha-D-glucose in insulin release. They are also compatible with the hypothesis that the anomeric malaise, associated with B-cell glucotoxicity, is caused by a progressive accumulation of glycogen in this cell.