A B S T R A C T Interactions between glucose and argiRline and a mixture of 20 amino acids found in normal rat serum were studied in the isolated perfused rat pancreas of normal rats, with release of immunoreactive glucagon and insulin as parameters. Secretion of both pancreatic hormones was low during the steady state, whether glucose (5 mMA) was included in the perfusion medium or not. This glucose concentration significantly stimulated insulin release twofold and resulted in an 80% inhibition of basal glucagon release. Arginine and the amino acid mixture were potent stimulants of both hormones. Secretion of both hormones followed identical biphasic response patterns after addition of arginine or the amino acid mixture. However, stimulation of insulin release occurred only when glucose was included, whereas both phases of glucagon release were elicited in the absence of glucose and markedly reduced in its presence. The dose-dependency curves of hormone release due to arginine on one hand and the amino acid mixture on the other differed substantially: with arginine, release of insulin and glucagon was linear between a concentration of 0.3 and 20 mM. In contrast, the amino acid mixture resulted in half-maximal release for both hormones between a concentration of 3 and 4.5 mAM, and maximal release between 6 and 8 mAM. The dose-dependencies of glucose modulation of a-and P-cell activity were also different: when the amino acid mixture was maintained at 15 mMI and glucose varied (0-6.25 mM), no insulin release occurred until glucose was above 2.5 mM, whereas incremental inhibition of glucagon occurred through the complete dose range. It was also observed that glucose inhibition of amino acid-stimulated glucagon reThis wdvork was presented in part at the meeting of the lease was dissociated from glucose-dependent increase of insulin release. These studies indicate that: (a) the a-cell, like the P-cell, secretes at a low basal rate; (b) hypoglycemia per se is a weak stimulus for glucagon secretion compared to the high efficacy of a physiologic amino acid mixture; (c) glucose plays opposite roles in the mechanisms leading to amino acid-induced hormone release from the a-and P-cells, functioning as an inhibitor in the first case and a permissive agent in the second, and (d) the data are compatible with the postulated existence of glucose and amino acid receptors in both the a-and P-cells.

A B S T R A C T The hyperglucagonemia that occurs in vivo in animals made diabetic with alloxan or streptozotocin is not suppressed by high glucose but is suppressed by exogenous insulin. These observations together with other studies suggested that insulin-dependent glucose transport and metabolism by the a-cells serves as the primary mechanism controlling glucagon secretion. This hypothesis was tested in the present investigation. The possible interactions between glucose, insulin, and a mixture of 20 amino acids at physiological proportions were examined in the isolated-perfused pancreas of normal, alloxan diabetic, and streptozotocin diabetic rats. Release of insulin and glucagon were used as indicators of A-cell and a-cell function. According to rigid criteria the diabetic animals entering the study were severely diabetic. It was found that in vitro: (a) basal glucagon release (measured in the absence of an a-cell stimulus or inhibitor) was extremely low, even lower (i.e. 10%) than the basal rates seen in controls; (b) the a-cells of alloxanized-and streptozotocin-treated rats responded with a biphasic glucagon release to stimulation by an amino acid mixture; (c) this a-cell response was reduced after both streptozotocin and alloxan; (d) glucose at 5 mM was a potent inhibitor of amino acid-induced glucagon secretion in both types of experimental diabetes; (e) in alloxan diabetes a-cell stimulation by amino acids can be curbed by exogenous This work was presented in part at the meetings

A B S T R A C T It has been suggested that the hyperglucagonemia observed in diabetic animals and man may be due to an impairment of glucose uptake and metabolism by the a-cells resulting in a decreased production of ATP. To test this hypothesis glucose, ATP, glucagon, and insulin were measured in pancreatic islets of normal and alloxan or streptozotocin diabetic rats. Two experimental approaches were used. In the first, the pancreas was perfused in vitro for assessing insulin and glucagon release due to 10 mM amino acids with and without 5 mM glucose. These perfusions were performed in the presence and absence of insulin. After perfusion, the pancreas was frozen and processed for analysis of islet glucose, ATP, insulin, and glucagon content. The second approach was to investigate the islet sucrose, urea, and glucose spaces together with ATP, insulin, and glucagon content in vivo in normal and in insulintreated and untreated streptozotocin diabetic rats.Perfusion of the pancreas in vitro with 5 mM glucose resulted in higher glucose content of normal islets than in alloxan and streptozotocin diabetic islets. Similarly in the in vivo studies, the intracellular glucose space of the streptozotocin diabetic islets was 30% the value found in normals. In the in vivo experiments, despite the relatively small intracellular glucose space of a-cell islets, the ATP content of these islets was only 15-20%/o lower than the ATP content of normal islets. In the in vitro experiments, perfusion with glucose resulted in ATP contents of a-cell islets and of normal mixed a-,1cell islets which were indistinguishable. However, the ATP INTRODUCTIONThe hyperglucagonemia that occurs in severe diabetes in man, and in animals made diabetic with alloxan or streptozotocin, is not suppressed by high glucose, but is suppressed after insulin treatment (1). On the basis of these observations, together with in vitro studies utilizing isolated islets, in which metabolic poisons seemed to interfere with glucose inhibition of glucagon secretion (2, 3), it has been suggested that the hyperglycemic suppression of glucagon release in the normal islet results from enhanced glucose transport with a subsequent increase in energy metabolism within the a-cell, and that the glucose metabolism of these cells is an insulin-requiring process. Similarly, the lack of glucose suppression of glucagon release in the diabetic state decrease in glucose uptake by the a-cell with a concomitant decrease in ATP availability (4). In a previous communication form this laboratory (5), it was demonstrated in both alloxan and streptozotocin diabetic rats that: (a) basal glucagon release from the isolated perfused pancreas (measured in the absence of an a-cell stimulus or inhibitor) was extremely low, about 1/10 the basal rates seen in controls; (b) the a-cells of both diabetic prototypes responded with a biphasic glucagon release to stimulation by an amino acid mixture; (c) the a-cell response to amino acids was reduced after both streptozotocin and alloxan as compared to c...

Iodoacetate and iodoacetamide were compared as to their capacity to block islet glycolysis and interfere with glucose inhibition of glucagon release and glucose stimulation of insulin release. Glycolysis was measured in isolated rat islet by the rate of lactate formation from 27 mM glucose. Hormone release was investigated by perfusing isolated rat pancreas with a 10 mM mixture of 19 amino acids, with and without 5 mM glucose. In perfusion experiments, lactate (2.5 mM) and pyruvate (0.5 mM) were present to provide alternate source of energy independent of glycolysis. Iodoacetate was about twice as potent as iodoacetamide in blocking glycolysis in islets, 0.2 and 0.5 mM, respectively being needed for complete inhibition of lactate production. Levels of either agent lower than 0.05 mM did not affect lactate accumulation. Iodoacetate, at the level which completely inhibited glycolysis did not interfere with the permissive action of glucose for insulin release. In contrast, iodoacetamide at a level (0.05 mM) which had no effect on lactate production, changed the response of the beta-cell dramatically: amino acids now released insulin even in the absence of glucose and insulin release by 5 mM glucose alone was greatly augmented. Both thiol reagents at 0.025 mM concentration completely prevented glucose from suppressing amino acid stimulated glucagon release, iodoacetamide being more potent than iodoacetate. These data indicate that the opposite physiological actions of glucose in alpha and beta-cells are in each case dissociable from the fuel function of the sugar molecule, and the results best support the concept that glucose and thiol reagents effect insulin and glucagon secretion by acting on sulfhydryl groups related to receptor sites in the alpha-and beta-cell membrane.

Metabolic interactions between glucose and amino acids were studied with isolated rat islets using glucose utilization and lactate formation as indicators. Certain amino acids (8-10 mM) are capable of greatly stimulating lactate formation from 5mM glucose. On a molar basis L-isoleucine is the most potent stimulator in a group of twenty-six amino acids. Aphysiological amino acid mixture (7.5-14 mM) or L-isoleucine (8 mM) profoundly altered the basic sigmoidal relation between glucose concentration in the medium and the rate of glucose utilization and lactate formation: with basal glucose (5 mM) both glucose utilization and lactate production were stimulated by the amino ACID MIXTURE and by L-isoleucine; at high glucose levels utilization was decreased by the amino acid mixture, but was unaffected by L-isoleucine, whereas lactate formation was decreased by both additions. The data indicate that amino acids may play a significant role in regulating the extent to which glucose serves as a fuel of pancreatic islet cells and in determining the pathways of glucose metabolism. In order to elucidate the mechanisms of the amino acid effect, studies with phloridzin, ouabain, iodoacetate, cytochalasin B, and Na+-deficiency were performed with the most effective amino acid, L-isoleucine. Each of these agents and Na+-deficiency substantially reduced or completely blocked the extra lactate formation induced by L-isoleucine (8-10 mM). The intracellular uptake of 14-CL-isoleucine by isolated islets was found to be Na+-independent, and uphill transport of this amino acid was not detectable, whether basal glucose was present in the medium or not. The action of iodoacetate in blocking glycolysis was reinvestigated. After forty-five minutes of exposure, 0.2mM iodoacetate completely blocks lactate formation as well as glucose utilization. Thisconfirms and extends earlier data for this laboratory and suggests that this SH-reagent indeed allows dissociation of the fuel and releasing functions of glucose.