Proinsulin biosynthesis in broken-cell preparations of islets of Langerhans.

1. Rabbit islets of Langerhans were disrupted by ultrasonic methods and the sonicated preparations were used to study proinsulin biosynthesis. 2. When [3h]leucine is incubated in such preparations, incorporation takes place into proinsulin, as evidenced by characterization on polyacrylamide gels, and by the conversion of this labelled material into insulin, by using trypsin. 3. The labelled proinsulin may also be purified by antiinsulin antibody bound to Sepharose. 4. With the broken-cell preparation it was shown that incorporation of leucine is accelerated by increasing the glucose content of the medium from 2mM to 16mM. However, 16mM-galactose or -sucrose did not stimulate incorporation significantly from basal values. This effect of glucose was abolished by cycloheximide. 5. The significance of these findings in relation to the mechanism of glucose stimulation of proinsulin biosynthesis is discussed.     

Ionophoretic activity in pancreatic islets.

Extracts of pancreatic islets stimulate the translocation of calcium from an aqueous into an organic immiscible phase. This ionophoretic activity, which is derived mainly from membrane-rich subcellular fractions, displays several features in common with that of A23187 in the same model. The phenomenon of calcium translocation caused by either the islet extract or the antibiotic ionophore represents a power function of the concentration of ionophoretic material; it is saturable at high calcium concentrations, affected by the concentration of Na⁺ and pH of the aqueous phase, increased at low temperature, and inhibited by suloctidil, the latter inhibitory effect being antagonized by calcium itself. These findings underline the potential significance of native ionophores in the regulation of calcium movements across membrane systems in the islet cells.     

Statistical geometry of pancreatic islets.

Quantitative histomorphometric studies of the dynamics of growth and development of pancreatic islets in normal and pathological states pose substantial methodological and conceptual problems. We address these problems with the geometry of random fractals, and apply our methods to the analysis of islet regeneration in the alloxan-treated guinea-pig. In both experimental islet-regenerated and control animals, islet centres are found to cluster in similar fractal subsets of dimension strictly less than 3, in agreement with the postulated origin of islets along a system of ductules, and suggesting that regeneration follows the same mathematical dynamics as original islet formation.     

Hexose metabolism in pancreatic islets. Insignificance of D-glucose futile cycling in rat islets.

When rat pancreatic islets were incubated in the presence of unlabelled D-glucose (16.7 mM) and 3HOH, the production of 3H-labelled material susceptible to be phosphorylated by yeast hexokinase and then detritiated by yeast phosphoglucoisomerase did not exceed 2.66 +/- 0.21 pmol/islet per 180 min, i.e. about 1% of the rate of exogenous D-[5-3H]glucose utilization. Such a material accounted for 43 +/- 4% of the total radioactivity, associated with tritiated hexose(s). It is proposed, therefore, that the futile cycling of D-glucose in the reactions catalyzed in the islet cells by the hexokinase isoenzymes and glucose-6-phosphatase represents a negligible fraction of the total rate of D-glucose phosphorylation.     

Glucose diffusion in pancreatic islets of Langerhans.

We investigate the time required for glucose to diffuse through an isolated pancreatic islet of Langerhans and reach an equilibrium. This question is relevant in the context of in vitro electrophysiological studies of the response of an islet to step changes in the bath glucose concentration. Islet cells are electrically coupled by gap junctions, so nonuniformities in islet glucose concentration may be reflected in the activity of cells on the islet periphery, where electrical recordings are made. Using a mathematical model of hindered glucose diffusion, we investigate the effects of the islet porosity and the permeability of a surrounding layer of acinar cells. A major factor in the determination of the equilibrium time is the transport of glucose into islet beta-cells, which removes glucose from the interstitial spaces where diffusion occurs. This transport is incorporated by using a model of the GLUT-2 glucose transporter. We find that several minutes are required for the islet to equilibrate to a 10 mM change in bath glucose, a typical protocol in islet experiments. It is therefore likely that in electrophysiological islet experiments the glucose distribution is nonuniform for several minutes after a step change in bath glucose. The delay in glucose penetration to the inner portions of the islet may be a major contributing factor to the 1-2-min delay in islet electrical activity typically observed after bath application of a stimulatory concentration of glucose.     

Hexose metabolism in pancreatic islets. Inhibition of hexokinase.

In islet homogenates, hexokinase-like activity (Km 0.05 mM; Vmax. 1.5 pmol/min per islet) accounts for the major fraction of glucose phosphorylation. Yet the rate of glycolysis in intact islets incubated at low glucose concentrations (e.g. 1.7 mM) sufficient to saturate hexokinase only represents a minor fraction of the glycolytic rate observed at higher glucose concentrations. This apparent discrepancy between enzymic and metabolic data may be attributable, in part at least, to inhibition of hexokinase in intact islets. Hexokinase, which is present in both islet and purified B-cell homogenates, is indeed inhibited by glucose 6-phosphate (Ki 0.13 mM) and glucose 1,6-bisphosphate (Ki approx. 0.2 mM), but not by fructose 2,6-bisphosphate. In intact islets, the steady-state content of glucose 6-phosphate (0.26-0.79 pmol/islet) and glucose 1,6-bisphosphate (5-48 fmol/islet) increases, in a biphasic manner, at increasing concentrations of extracellular glucose (up to 27.8 mM). From these measurements and the intracellular space of the islets, it was estimated that the rate of glucose phosphorylation as catalysed by hexokinase represents, in intact islets, no more than 12-24% of its value in islet homogenates.     

Phospholipid methylation in pancreatic islets

Glucose provokes a transient stimulation of phospholipid methylation in rat pancreatic islets, possibly by increasing phospholipid methyltransferase activity. The association of DL-homocysteine and 3-deazaadenosine inhibits phospholipid methylation. The methylation of phospholipids may play a role in the stimulus-secretion coupling for glucose-induced insulin release.     

Hexose metabolism in pancreatic islets

Summary In rat pancreatic islets, a rise in extracellular D-glucose concentration is known to cause a greater increase in the oxidation of D-[6-¹⁴C]glucose than utilization of D-[5-³H]glucose. In the present study, such a preferential stimulation of acetyl residue oxidation relative to glycolytic flux was mimicked by nutrient secretagogues such as 2-aminobicyclo[2,2,1]heptane-2-carboxylate, 3-phenylpyruvate, L-leucine, 2-ketoisocaproate, D-fructose and ketone bodies. The preferential stimulation of D-[6-¹⁴C]glucose oxidation by these nutrients was observed at all hexose concentrations (0.5, 6.0 and 16.7 mM), coincided with an unaltered rate of D-[3,4-¹⁴C]glucose oxidation, was impaired in the absence of extracellular Ca²⁺, and failed to be affected by NH₄ ⁺. Although the ratio between D-[6-¹⁴C]glucose oxidation and, D-[5-³H]glucose utilization in islets exposed to other nutrient secretagogues could be affected by factors such as isotopic dilution and mitochondrial redox state, the present data afford strong support to the view that the preferential stimulation of oxidative events in the Krebs cycle of nutrient-stimulated islets is linked to the activation of key mitochondrial dehydrogenases, e.g. 2-ketoglutarate dehydrogenase. The latter activation might result from the mitochondrial accumulation of Ca²⁺, as attributable not solely to stimulation of Ca²⁺ inflow into the islet cells but also to an increase in ATP availability.     

Transglutaminase activity in pancreatic islets

1. Pancreatic islet homogenates catalyze, in a Ca²⁺-dependent fashion, the incorporation of [2,5-³H]histamine, [1,4-¹⁴C]putrescine, [1,2-³H]agmatine, [¹⁴C]methylamine, L-[U-¹⁴C]lysine in N,N-dimethylcasein. 2. Using [2,5-³H]histamine as the amine donor, the K_m for Ca²⁺ and histamine amounts to 90μM and 0.7 mM, respectively. 3. The incorporation of [2,5-³H]histamine into N,N-dimethylcasein is inhibited by monodansylcadaverine, N-p-tosyl glycine, bacitracin and methylamine, the relative extent of inhibition depending on the respective concentrations of Ca²⁺, inhibitor and amine donor. 4. Bacitracin and methylamine, but not N-p-tosyl glycine, cause a dose-related inhibition of glucose-stimulated insulin release. 5. It is concluded that, in pancreatic islets, the Ca²⁺-responsive transglutaminase activity plays a critical role in the process of glucose-induced insulin release.     

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.     

Alloxan inhibits glucose oxidation in the pancreatic islets.

The effects of alloxan on glucose oxidation and the protection by anomers of D-glucose from alloxan inhibition of glucose oxidation in the pancreatic islets were investigated using in vitro incubation of rat isolated islets. The pretreatment by alloxan (5-30 mg/dl) for 6 minutes inhibits significantly 14CO2 formation from 14C-U-D-glucose (10 mM) and the addition of alpha-anomer of D-glucose (8.3 mM) to alloxan (20 mg/dl) completely reverses alloxan inhibition of glucose oxidation. These findings seem to be incompatible with the recent view that alloxan acts at the glucose receptor on the plasma membrane of pancreatic beta-cells without affecting glucose metabolism in the pancreatic islets.     

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.     

Lack of glyconeogenesis in pancreatic islets: expression of gluconeogenic enzyme genes in islets.

Studies were performed to obtain evidence for glyconeogenesis from pyruvate to the triose phosphates in pancreatic islets. Inability to show this evidence would be consistent with the fact that glyceraldehyde, but not pyruvate, is a potent insulin secretagogue. Synthesis of 14C-labelled glucose from 14C-labelled pyruvate could not be detected. Since this might have been due to lack of sensitivity required to measure 14C-glucose production in such a scarce tissue as islets, cDNA probes were used to estimate the relative expression of genes coding for gluconeogenic enzymes. Islets expressed pyruvate carboxylase mRNA, but even islets from rats which had been starved (a condition which induces phosphoenolpyruvate carboxykinase (PEPCK) in liver, kidney and adipose tissue) showed no PEPCK mRNA. This is consistent with our previous work showing the absence of PEPCK enzyme activity in islets. Therefore, islets can convert pyruvate to oxalacetate, but since they lack PEPCK, neither the beta nor alpha cell can convert oxalacetate to phosphoenolpyruvate and carry out glyconeogenesis. Pyruvate carboxylase mRNA was increased in islets that possessed the capacity for glucose-induced insulin release versus islets that lacked the capacity to respond to glucose, such as islets from fed rats (versus starved rats) and in islets cultured at a high concentration of glucose (versus at low glucose). Pyruvate carboxylase, therefore, must be involved in pyruvate metabolism and not glyconeogenesis in the pancreatic islet.     

Glucose metabolism in mouse pancreatic islets

1. Rates of glucose oxidation, lactate output and the intracellular concentration of glucose 6-phosphate were measured in mouse pancreatic islets incubated in vitro. 2. Glucose oxidation rate, measured as the formation of (14)CO(2) from [U-(14)C]glucose, was markedly dependent on extracellular glucose concentration. It was especially sensitive to glucose concentrations between 1 and 2mg/ml. Glucose oxidation was inhibited by mannoheptulose and glucosamine but not by phlorrhizin, 2-deoxyglucose or N-acetylglucosamine. Glucose oxidation was slightly stimulated by tolbutamide but was not significantly affected by adrenaline, diazoxide or absence of Ca(2+) (all of which may inhibit glucose-stimulated insulin release), by arginine or glucagon (which may stimulate insulin release) or by cycloheximide (which may inhibit insulin synthesis). 3. Rates of lactate formation were dependent on the extracellular glucose concentration and were decreased by glucosamine though not by mannoheptulose; tolbutamide increased the rate of lactate output. 4. Islet glucose 6-phosphate concentration was also markedly dependent on extracellular glucose concentration and was diminished by mannoheptulose or glucosamine; tolbutamide and glucagon were without significant effect. Mannose increased islet fructose 6-phosphate concentration but had little effect on islet glucose 6-phosphate concentration. Fructose increased islet glucose 6-phosphate concentration but to a much smaller extent than did glucose. 5. [1-(14)C]Mannose and [U-(14)C]fructose were also oxidized by islets but less rapidly than glucose. Conversion of [1-(14)C]mannose into [1-(14)C]glucose 6-phosphate or [1-(14)C]glucose could not be detected. It is concluded that metabolism of mannose is associated with poor equilibration between fructose 6-phosphate and glucose 6-phosphate. 6. These results are consistent with the idea that glucose utilization in mouse islets may be limited by the rate of glucose phosphorylation, that mannoheptulose and glucosamine may inhibit glucose phosphorylation and that effects of glucose on insulin release may be mediated through metabolism of the sugar.