Palmitate modulates the early steps of insulin signalling pathway in pancreatic islets

Insulin stimulates its own secretion and synthesis by pancreatic beta-cells. Although the exact molecular mechanism involved is unknown, changes in beta-cell insulin signalling have been recognized as a potential link between insulin resistance and its impaired release, as observed in non-insulin-dependent diabetes. However, insulin resistance is also associated with elevated plasma levels of free fatty acids (FFA) that are well known modulators of insulin secretion by pancreatic islets. This information led us to investigate the effect of FFA on insulin receptor signalling in pancreatic islets. Exposure of pancreatic islets to palmitate caused up-regulation of several insulin-induced activities including tyrosine phosphorylation of insulin receptor and pp185. This is the first evidence that short exposure of these cells to 100 microM palmitate activates the early steps of insulin receptor signalling. 2-Bromopalmitate, a carnitine palmitoyl-CoA transferase-1 inhibitor, did not affect the effect of the fatty acid. Cerulenin, an acylation inhibitor, abolished the palmitate effect on protein levels and phosphorylation of insulin receptor. This result supports the proposition that protein acylation may be an important mechanism by which palmitate exerts its modulating effect on the intracellular insulin signalling pathway in rat pancreatic islets.     

Anomeric dissociation between glucokinase activity and glycolysis in pancreatic islets

In pancreatic islet homogenates incubated in the presence of a high glucose concentration (40 mM), the beta-anomer of D-glucose is phosphorylated at a higher rate than the alpha-anomer, whether in the absence or presence of exogenous glucose 6-phosphate. However, in intact islets also exposed to 40 mM D-glucose, the production of 3H2O from D-[5-3H] glucose, the oxidation of D-[U-14C] glucose and the glucose-induced increment in either lactate production or 45Ca net uptake, as well as the release of insulin from isolated perfused pancreases, are not higher with beta- than alpha-D-glucose. It is concluded that the rate of glucose utilization by islet cells is not regulated solely by the activity of hexokinase and/or glucokinase.     

Calcium and glycolysis mediate multiple bursting modes in pancreatic islets

Pancreatic islets of Langerhans produce bursts of electrical activity when exposed to stimulatory glucose levels. These bursts often have a regular repeating pattern, with a period of 10-60 s. In some cases, however, the bursts are episodic, clustered into bursts of bursts, which we call compound bursting. Consistent with this are recordings of free Ca2+ concentration, oxygen consumption, mitochondrial membrane potential, and intraislet glucose levels that exhibit very slow oscillations, with faster oscillations superimposed. We describe a new mathematical model of the pancreatic beta-cell that can account for these multimodal patterns. The model includes the feedback of cytosolic Ca2+ onto ion channels that can account for bursting, and a metabolic subsystem that is capable of producing slow oscillations driven by oscillations in glycolysis. This slow rhythm is responsible for the slow mode of compound bursting in the model. We also show that it is possible for glycolytic oscillations alone to drive a very slow form of bursting, which we call "glycolytic bursting." Finally, the model predicts that there is bistability between stationary and oscillatory glycolysis for a range of parameter values. We provide experimental support for this model prediction. Overall, the model can account for a diversity of islet behaviors described in the literature over the past 20 years.     

Intrahepatic transplantation of pancreatic islets in the rat.

Islets of Langerhans from isogeneic donor rats were transplanted directly into the hepatic parenchyma of recipients which had been made severely diabetic by streptozotocin (glycaemia ranging between 400 and 1090 mg%). Complete control lasting up to 13 months was achieved in 65% of recipients by using 600-800 islets. Following intravenous glucose administration, each rat responded similarly to normal rats with a rapid but reduced release of insulin. Cytoimmunofluorescence and electron microscopic studies demonstrated the presence of both functional insulin and glucagon cells, within the transplanted islets. It is suggested that for various reasons direct intrahepatic transplantation might become the preferred method for islets.     

Impaired enzyme-to-enzyme channelling between hexokinase isoenzyme(s) and phosphoglucoisomerase in rat pancreatic islets incubated at a low concentration of D-glucose

It was recently proposed that in rat pancreatic islets exposed to 8.3 mM D-glucose, alpha-D-glucose-6-phosphate undergoes enzyme-to-enzyme channelling between hexokinase isoenzyme(s) and phosphoglucoisomerase. To explore the identity of the hexokinase isoenzyme(s) involved in such a tunnelling process, the generation of 3HOH from the alpha- and beta-anomers of either D-[2-3H]glucose or D-[5-3H]glucose was now measured over 60 min incubation at 4 degrees C in pancreatic islets exposed only to 2.8 mM D-glucose, in order to decrease the relative contribution of glucokinase to the phosphorylation of the hexose. Under these experimental conditions, the ratio for 3HOH production from D-[2-3H]glucose/D-[5-3H]glucose at anomeric equilibrium (39.7 +/- 11.6%) and the beta/alpha ratios for the generation of 3HOH from either the D-[2-3H]glucose anomers (70.9 +/- 12.6%) or the D-[5-3H]glucose anomers (59.6 +/- 12.4%) indicated that a much greater fraction of alpha-D-glucose-6-phosphate escapes from the process of enzyme-to-enzyme channelling in the islets exposed to 2.8 mM, rather than 8.3 mM D-glucose. These findings suggest, therefore, that the postulated process of enzyme-to-enzyme channelling involves mainly glucokinase.     

Hexose metabolism in pancreatic islets. Regulation of aerobic glycolysis and pyruvate decarboxylation.

1. D-Glucose (0.5-16.7 mM) preferentially stimulates aerobic glycolysis and D-[3,4-14C]glucose oxidation, relative to D-[5-3H]glucose utilization in rat pancreatic islets, the concentration dependency of such a preferential effect displaying a sigmoidal pattern. 2. Inorganic and organic calcium antagonists, as well as Ca2+ deprivation, only cause a minor decrease in the ratio between D-[3,4-14C]glucose oxidation and D-[5-3H]glucose utilization in islets exposed to a high concentration of the hexose (16.7 mM). 3. Non-glucidic nutrient secretagogues such as 2-aminobicyclo[2,2,1]heptane-2-carboxylate (BCH), 2-ketoisocaproate and 3-phenylpyruvate fail to stimulate aerobic glycolysis and D-[3,4-14C]glucose oxidation in islets exposed to 6.0 mM D-glucose. Nevertheless, BCH augments [1-14C]pyruvate and [2-14C]pyruvate oxidation. 4. The glucose-induced increment in the paired ratio between D-[3,4-14C]glucose oxidation and D-[5-3H]glucose utilization is impaired in the presence of either cycloheximide or ouabain. 5. These findings suggest that the preferential effect of D-glucose upon aerobic glycolysis and pyruvate decarboxylation is not attributable solely to a Ca(2+)-induced activation of FAD-linked glycerophosphate dehydrogenase and/or pyruvate dehydrogenase, but may also involve an ATP-modulated regulatory process.     

Remodelling sympathetic innervation in rat pancreatic islets ontogeny

Background  

Pancreatic islets are not fully developed at birth and it is not clear how they are vascularised and innervated. Nerve Growth Factor (NGF) is required to guide sympathetic neurons that innervate peripheral organs and also in cardiovascular system and ovary angiogenesis. Pancreatic beta cells of a transgenic mouse that over-expressed NGF in attracts sympathetic hyper-innervation towards them. Moreover, we have previously demonstrated that adult beta cells synthesize and secrete NGF; however, we do not know how is NGF secreted during development, nor if it might be trophic for sympathetic innervation and survival in the pancreas.     

pH is decreased in transplanted rat pancreatic islets

Recent studies of transplanted pancreatic islets have indicated incomplete revascularization. We investigated the pH, in relation to oxygen tension (Po(2)), in endogenous islets and islets syngeneically transplanted to the renal subcapsular site of nondiabetic and streptozotocin-diabetic recipients. Tissue pH and Po(2) were measured using microelectrodes. In the endogenous islets, tissue pH was similar to that in arterial blood. In the transplanted islets, tissue pH was 0.11-0.15 pH units lower. No differences in islet graft pH were seen between nondiabetic and diabetic animals, and none if the islet grafts were investigated 1 day or 1 mo posttransplantation. The Po(2) in the endogenous islets was approximately 35 mmHg. Transplanted islets had a markedly lower tissue Po(2) both 1 day and 1 mo after transplantation. A negative correlation between the tissue Po(2) and the hydrogen ion concentration was seen in the 1-mo-old islet transplants in diabetic animals. In conclusion, decreased Po(2) in transplanted islets is associated with a decreased tissue pH, suggesting a shift toward more anaerobic glucose metabolism after transplantation.     

Glucose raises cytosolic free calcium in the rat pancreatic islets

Cytosolic free calcium [( Ca2+]i) was measured using fura 2 in the whole pancreatic islets obtained from male Wistar rats by collagenase dispersion. The pattern of change of [Ca2+]i in response to high glucose, potassium (K+) depolarization or the removal of extracellular calcium was compared with the temporal profile of insulin secretion. Twenty-nine mM glucose produced a gradual increase in [Ca2+]i with approximately 1.5 min of latency period. It remained elevated until the end of observation period (25 min) during which period the first phase of insulin secretion ceased and the second phase of secretion gradually increased. Depolarizing concentration of KCl also produced an elevation of [Ca2+]i, without detectable latency period, which lasted at a sustained level for the entire observation period (30 min). KCl caused a rapid increase of insulin secretion followed by a gradually decreasing level of secretion. Elevated [Ca2+]i and insulin secretion in response to high glucose returned to the basal level when external calcium was removed by the addition of EGTA. We conclude that high glucose and K+ depolarization raise [Ca2+]i in the pancreatic islet. However, the elevation of [Ca2+]i and insulin secretion are not always correlated in the later period of stimulation.     

Active transport of myo-inositol in rat pancreatic islets.

myo-Inositol transport by isolated pancreatic islets was measured with a dual isotope technique. Uptake was saturable with a half-maximal response at approx. 75 microM. With 50 microM-inositol, uptake was linear for at least 2 h during which time the free intracellular concentration rose to double that of the incubation medium. Inositol transport is therefore active and probably energized by electrogenic co-transport of Na+ down its concentration gradient as uptake was inhibited by ouabain, Na+ removal or depolarizing K+ concentrations. Inositol transport was abolished by cytochalasin B which binds to hexose carriers, but not by carbamoylcholine or Li+ which respectively stimulate or inhibit phosphoinositide turnover. Uptake of inositol was not affected by 3-O-methylglucose or L-glucose (both 100 mM) nor by physiological concentrations of D-glucose. The results suggest that most intracellular inositol in pancreatic islets would be derived from the extracellular medium. Since the transport mechanism is distinct from that of glucose, inositol uptake would not be inhibited during periods of hyperglycaemia.     

Activity of prostaglandin biosynthetic pathways in rat pancreatic islets

Isolated pancreatic islets of the rat were either prelabeled with [3H]arachidonic acid, or were incubated over the short term with the concomitant addition of radiolabeled arachidonic acid and a stimulatory concentration of glucose (17mM) for prostaglandin (PG) analysis. In prelabeled islets, radiolabel in 6-keto-PGF1 alpha, PGE2, and 15-keto-13,14-dihydro-PGF2 alpha increased in response to a 5 min glucose (17mM) challenge. In islets not prelabeled with arachidonic acid, label incorporation in 6-keto-PGF1 alpha increased, whereas label in PGE2 decreased during a 5 min glucose stimulation; after 30-45 min of glucose stimulation labeled PGE levels increased compared to control (2.8mM glucose) levels. Enhanced labelling of PGF2 alpha was not detected in glucose-stimulated islets prelabeled or not. Isotope dilution with endogenous arachidonic acid probably occurs early in the stimulus response in islets not prelabeled. D-Galactose (17mM) or 2-deoxyglucose (17mM) did not alter PG production. Indomethacin inhibited islet PG turnover and potentiated glucose-stimulated insulin release. Islets also converted the endoperoxide [3H]PGH2 to 6-keto-PGF1 alpha, PGF2 alpha, PGE2 and PGD2, in a time-dependent manner and in proportions similar to arachidonic acid-derived PGs. In dispersed islet cells, the calcium ionophore ionomycin, but not glucose, enhanced the production of labeled PGs from arachidonic acid. Insulin release paralleled PG production in dispersed cells, however, indomethacin did not inhibit ionomycin-stimulated insulin release, suggesting that PG synthesis was not required for secretion. In confirmation of islet PGI2 turnover indicated by 6-keto-PGF1 alpha production, islet cell PGI2-like products inhibited platelet aggregation induced by ADP. These results suggest that biosynthesis of specific PGs early in the glucose secretion response may play a modulatory role in islet hormone secretion, and that different pools of cellular arachidonic acid may contribute to PG biosynthesis in the microenvironment of the islet.     

Prostaglandin synthesis and metabolism in isolated pancreatic islets of the rat

Isolated pancreatic islets of Langerhans of the rat which were sonicated and incubated with radiolabeled arachidonic acid for 1 hr synthesized several species of prostaglandins (PGs). Both thin-layer and high-performance liquid (HPLC) chromatographic techniques demonstrated the synthesis by islet sonicates of PGF_2α and PGE₂ equivalents, in addition to the 15-keto-13, 14-dihydro metabolites of these primary PGs. In addition, HPLC allowed the identification of 6-keto-PGF_1α (the metabolite of prostacyclin) as a major PG synthesized from arachidonate by this tissue. Islet vascular elements, as well as endocrine cells, may contribute to the synthesis of the latter compound. Lesser amounts of arachidonate were incorporated into PG-like compounds eluting as thromboxane. The synthesis of PGs was sensitive to the protein concentration of islet sonicate, and a five-fold dilution of protein resulted in a comparable reduction in arachidonate incorporation into PGs. Labeled arachidonate was also incorporated into compounds which elute as hydroxy or hydroperoxy-eicosatetrainoic acids on HPLC. Thus, isolated pancreatic islets synthesize a variety of PGs which may have a physiological role in hormone secretion form this endocrine organ.     

Plasma membrane calcium pump activity in rat pancreatic islets

The aim of this study was to quantify the glucose modulation of the plasma membrane calcium pump (PMCA) function in rat pancreatic islets. Ca²⁺-ATPase activity and levels of phosphorylated PMCA intermediates both transiently declined to a minimum in response to stimulation by glucose. Strictly dependent on Ca²⁺ concentration, this inhibitory effect was fully expressed at physiological concentrations of the cation (less than 0.5 μM), then progressively diminished at higher concentrations. These results, together with those previously reported on the effects of insulin secretagogues and blockers on the activity, expression and cellular distribution of the PMCA, support the concept that the PMCA plays a key role in the regulation of Ca²⁺ signaling and insulin secretion in pancreatic islets.     

Lactation-induced changes in calcium handling by rat pancreatic islets

Glucose-stimulated insulin release occurred at a lower rate in pancreatic islets removed from lactating than non-lactating rats. This defect was corrected in the presence of either gliclazide or a calcium-agonist. With both agents present, insulin release from islets of lactating rats was greater. When islets were prelabelled with 45calcium, gliclazide stimulated to the same extent 45Ca outflow in islets from lactating and non-lactating rats, respectively. However, when the islets were prelabelled with 45Ca in the presence of gliclazide, the administration of Ba2+ increased effluent radioactivity more markedly in islets from non-lactating than lactating rats. This suggests that lactation favours, in gliclazide-stimulated islets, the sequestration of 45Ca in non-labile subcellular pools. When D-glucose was used instead of Ba2+, the greater lability of 45Ca in islets from non-lactating animals was apparently masked by a lesser efficiency in the metabolism and cationic effects of D-glucose in the non-lactating rats. The calcium-ionophoretic effect of islet extracts was higher in lactating than non-lactating rats. These results support the view that a depletion of endogenous calcium stores accounts, in part at least, for the decreased insulin secretory responsiveness to D-glucose in lactation, since the latter apparently favours the function of those systems involved in either the entry of calcium into or its sequestration within the islet cells.     

Glucose-induced accumulation of glucose-1,6-bisphosphate in pancreatic islets : its possible role in the regulation of glycolysis

A rise in the extracellular concentration of glucose from zero to 5.6 and 16.7 mM caused a graded increase in the glucose-1,6-bisphosphate content of rat pancreatic islets. Glucose-1,6-bisphosphate activated phosphofructokinase in islet homogenates, when the reaction velocity was measured at low concentrations of fructose-6-phosphate. It is postulated that glucose-1, 6-bisphosphate participates, together with fructose-2,6-bisphosphate, in the regulation of glycolysis in intact islet cells.