IONIC EFFECTS ON THE UPTAKE OF CHLOROMERCURIBENZENE-p-SULPHONIC ACID BY PANCREATIC ISLETS

ABSTRACT Effects of inorganic ions on the uptake of chloromercuribenzene-p-sulphonic acid (CMBS) were studied in microdissected pancreatic islets of non-inbred ob/ob-mice. Na2SO4 stimulated the total islet cell uptake of CMBS but decreased the amount of CMBS remaining in islets after brief washing with L-cysteine. CaCl2 stimulated both the total and the cysteine-non-displaceable uptake; the stimulatory effect of CaCl2 on the cysteine-non-displaceable CMBS uptake was counteracted by Na2SO4. NaCl, KCl or choline chloride had no significant effect on the total islet cell uptake of CMBS, whereas LiCl was stimulatory. It is concluded that β-cells resemble erythrocytes in having a permeation path for CMBS that is inhibited by SO42−. By analogy with existing models of the erythrocyte membrane, it is suggested that the SO42−-sensitive path leads to sulphydryl groups controlling monovalent cationic permeability in β-cells.

GLUCOSE INHIBITION OF THE PERMEATION OF CHLOROMERCURIBENZENE-P-SULPHONIC ACID IN THE PLASMA MEMBRANE OF PANCREATIC β-CELLS

ABSTRACT The transport of chloromercuribenzene-p-sulphonic acid (CMBS) was studied in microdissected pancreatic islets of ob/ob mice incubated in Krebs-Ringer bicarbonate medium. Glucose inhibited the islet uptake of CMBS. The initial binding of CMBS to superficial thiol groups was inhibited to a lesser extent than was its further permeation into the interior of the plasma membrane. The non-metabolized glucose analogue 3-0-methyl glucose had no effect on the permeation of CMBS. Whereas mannoheptulose, epinephrine and diazoxide lacked any effect in themselves, they tended to counteract the glucose inhibition of CMBS permeation. The ability of glucose to inhibit CMBS permeation is related to its insulin-releasing action rather than to its membrane transport or further metabolism.

INFLUENCE OF THE PITUITARY GLAND ON INSULIN SECRETION IN THE GENETICALLY OBESE (ob/ob) MOUSE

SUMMARY The influence of the pituitary gland of lean and genetically obese (ob/ob) mice on insulin secretion from microdissected pancreatic islets of lean and ob/ob mice has been studied by perifusing the pituitaries of these animals in series with the isolated islets and measuring insulin secretion at 5-min intervals over a period of 60 min. It has been shown that the pituitary perifusate of both lean and obese mice stimulate insulin secretion from lean mouse islets but not from obese mouse islets. The maximum stimulation occurs in the first 10 min and with the lean mouse pituitaries returns to the basal level in about 20 min, whereas with the obese mouse pituitaries insulin secretion is about double that from the control islets even after 40 min. A concentration of pure porcine ACTH equivalent to about three times the amount released from the pituitary gland under the experimental conditions used, caused only a small stimulation of insulin release. Possible interpretations of these findings and further lines of investigation are discussed.

The pancreatic β-cell recognition of insulin secretagogues. Effects of calcium and sodium on glucose metabolism and insulin release

The transport and oxidation of glucose, the content of fructose 1,6-diphosphate, and the release of insulin were studied in microdissected pancreatic islets of ob/ob mice incubated in Krebs–Ringer bicarbonate medium. Under control conditions glucose oxidation and insulin release showed a similar dependence on glucose concentration with the steepest slope in the range 5–12mm. The omission of Ca2+, or the substitution of choline ions for Na+, or the addition of diazoxide had little if any effect on glucose transport. However, Ca2+ or Na+ deficiency as well as diazoxide (7-chloro-3-methyl-1,2,4-benzothiadiazine 1,1-dioxide) or ouabain partially inhibited glucose oxidation. These alterations of medium composition also increased the islet content of fructose 1,6-diphosphate, as did the addition of adrenaline. Phentolamine [2-N-(3-hydroxyphenyl)-p-toluidinomethyl-2-imidazoline] counteracted the effects of adrenaline and Ca2+ deficiency on islet fructose 1,6-diphosphate. After equilibration in Na+-deficient medium, the islets exhibited an increase in basal insulin release whereas the secretory response to glucose was inhibited. The inhibitory effects of Na+ deficiency on the secretory responses to different concentrations of glucose correlated with those on 14CO2 production. When islets were incubated with 17mm-glucose, the sudden replacement of Na+ by choline ions resulted in a marked but transient stimulation of insulin release that was not accompanied by a demonstrable increase of glucose oxidation. Galactose and 3-O-methylglucose had no effect on glucose oxidation or on insulin release. The results are consistent with a metabolic model of the β-cell recognition of glucose as insulin secretagogue and with the assumption that Ca2+ or Na+ deficiency, or the addition of adrenaline or diazoxide, inhibit insulin release at some step distal to stimulus recognition. In addition the results suggest that these conditions create a partial metabolic block of glycolysis in the β-cells. Hence the interrelationship between the processes of stimulus recognition and insulin discharge may involve a positive feedback of secretion on glucose metabolism.

Iodoacetamide-induced sensitization of the pancreatic β-cells to glucose stimulation

At a glucose concentration of 3mm or less, iodoacetamide had no effect on the release of insulin from microdissected pancreatic islets of ob/ob-mice. At higher glucose concentrations, iodoacetamide exerted both an initial stimulatory and a subsequent inhibitory action. When islets were perifused with 1mm-iodoacetamide and 17mm-glucose the inhibitory action predominated after about 15min of transient stimulation. With decreasing concentrations of iodoacetamide the stimulatory phase was gradually prolonged, and with 0.003–0.1mm-iodoacetamide stimulation only was observed for 75min. Prolonged stimulation was also noted after a short pulse of iodoacetamide. Similar responses to 0.1mm-iodoacetamide were observed with islets from normal mice. With islets from ob/ob-mice the effect of 0.1mm-iodoacetamide was reproduced with 0.1mm-iodoacetate, whereas 0.1mm-acetamide had no apparent effect. Iodoacetamide increased the Vmax. of glucose-stimulated insulin release without altering the apparent Km for glucose. Leucine, glibenclamide or theophylline could not replace glucose in this synergistic action with iodoacetamide. Iodoacetamide rather inhibited the insulin-releasing action of theophylline. Iodoacetamide-induced potentiation of the glucose-stimulated insulin release was rapidly and reversibly inhibited by mannoheptulose, adrenaline, or calcium deficiency. The potentiating effect on insulin release was not paralleled by effects on glucose oxidation or on islet fructose 1,6-diphosphate. However, the inhibitory action of iodoacetamide might be explained by inhibition of glycolysis as evidenced by an inhibition of glucose oxidation and a rise of fructose 1,6-diphosphate. The results support our previous hypothesis that thiol reagents can stimulate insulin release by acting on relatively superficial thiol groups in the β-cell plasma membrane. Glycolysis seems to be necessary in order for iodoacetamide to stimulate in this way.