Type 2 diabetes (T2D) is characterized by chronic hyperglycemia resulting from a deficiency in insulin signaling, because of insulin resistance and/or defects in insulin secretion; it is also associated with increases in glucagon and endogenous glucose production (EGP). Gliflozins, including dapagliflozin, are a new class of approved oral antidiabetic agents that specifically inhibit sodium-glucose co-transporter 2 (SGLT2) function in the kidney, thus preventing renal glucose reabsorption and increasing glycosuria in diabetic individuals while reducing hyperglycemia. However, gliflozin treatment in subjects with T2D increases both plasma glucagon and EGP by unknown mechanisms. In spite of the rise in EGP, T2D patients treated with gliflozin have lower blood glucose levels than those receiving placebo, possibly because of increased glycosuria; however, the resulting increase in plasma glucagon levels represents a possible concerning side effect, especially in a patient population already affected by hyperglucagonemia. Here we demonstrate that SGLT2 is expressed in glucagon-secreting alpha cells of the pancreatic islets. We further found that expression of SLC5A2 (which encodes SGLT2) was lower and glucagon (GCG) gene expression was higher in islets from T2D individuals and in normal islets exposed to chronic hyperglycemia than in islets from non-diabetics. Moreover, hepatocyte nuclear factor 4-α (HNF4A) is specifically expressed in human alpha cells, in which it controls SLC5A2 expression, and its expression is downregulated by hyperglycemia. In addition, inhibition of either SLC5A2 via siRNA-induced gene silencing or SGLT2 via dapagliflozin treatment in human islets triggered glucagon secretion through KATP channel activation. Finally, we found that dapagliflozin treatment further promotes glucagon secretion and hepatic gluconeogenesis in healthy mice, thereby limiting the decrease of plasma glucose induced by fasting. Collectively, these results identify a heretofore unknown role of SGLT2 and designate dapagliflozin an alpha cell secretagogue.

The release of insulin evoked by nutrients in the pancreatic beta-cell is attributed to either the activation of a stereospecific receptor by the nutrient molecule itself or the generation of one or more signal(s) through the intracellular metabolism of the nutrient secretagogue. The first of these hypotheses is apparently supported by the fact the nonmetabolized amino acids, especially the L-leucine analogue b(-)2-amino-bicyclo[2,2,1]heptane-2-carbocyclic acid (BCH), stimulate insulin release. However, we now report evidence in support of the second hypothesis. We present data consistent with the idea that BCH induces insulin release through the allosteric activation of glutamate dehydrogenase. This is compatible with the fuel hypothesis, which states that the secretory response to nutrient secretagogues depends always on an increase of catabolic fluxes in the islet cells.

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The diabetogenic agent alloxan exerts a preferential cytotoxic effect on the pancreatic B cell. The determinants of such a tissue specificity were investigated. Alloxan accumulated rapidly in liver and pancreatic islets but much more slowly in muscle. The activity of glutathione peroxidase and the resistance to exogenous peroxide were -20 times higher in liver and kidney than in islets, intermediate values being found in exocrine pancreas and muscle. These findings suggest that the selective cytotoxicity of alloxan to the pancreatic B cell is attributable to the conjunction oftwo features: a rapid cellular uptake ofthe drug and an exquisite sensitivity of the B cell to peroxide.

1 . The possible significance of the insular sorbitol pathway in glucose-induced insulin release was investigated.2. Glucose, but neither fructose nor galactose, stimulated sorbitol synthesis by isolated islets. Aldose reductase inhibitors such as 2,4-dimethylglutaric and 3,3-tetramethylene glutaric acid abolished the glucose-induced sorbitol formation, but failed to affect glucose-induced insulin release.About 75 % of the sorbitol formed by the islets was recovered in the incubation medium, the efflux of sorbitol occurring independently of insulin release. Exogenous fructose and sorbitol exerted a modest insulinotropic action characterized by a shift to the left of the sigmoidal curve relating insulin release to glucose concentration. The relative insulinotropic potency of glucose (1 00 %), fructose (16%) and sorbitol (7%) paralleled their rate of oxidation by the isolated islets.3. These data suggest that metabolism through the sorbitol pathway plays little role in the process of glucose-induced insulin release, although exogenous sorbitol exerts a modest insulinotropic action.In the present series of investigations, we have reached the tentative conclusion that the insulinotropic action of glucose is mediated through an inhibition of calcium outward transport across the plasma membrane of the /?-cell [l], leading to the cytosolic accumulation of calcium [2,3] and subsequent activation of a microtubular-microfilamentous system controlling the migration and extrusion of secretory granules [4-61. In this sequence of events, a critical step which remains to be elucidated concerns the modality of glucose recognition by the /?-cell and the link between such a process and the subsequent modification of cation transport across the cell membrane. As reviewed in detail elsewhere [7,8], the intimate nature and properties of the glucosensor device in the pancreatic /?-cell are indeed still a matter of debate, especially as far as the possible participation of different pathways of glucose metabolism in the mechanism of glucose identification is concerned. For instance, the recent description ofan insular sorbitol pathway [9] has led to speculations as to the possible role of sorbitol in the control of insulin secretion [lo]. The present report aims at characterizing the metabolism of sorbitol in isolated islets and its possible involvement in the process of glucose-induced insulin release. ~~This paper is the XVIIIth of a series. MATERIALS AND METHODS Sorbitol Formation by Isolated IsletsPaired groups of 95 to 180 islets each, obtained from fed female albino rats [ll], were incubated for 90 and occasionally 180 min at 37 "C in 1 .O ml bicarbonate-buffered medium [12], equilibrated against a mixture of 0, (95%) and C 0 2 (5%) and containing albumin (5 mg/ml; fraction V, Sigma Chemical Co., St. Louis, U.S.A.) and, as required, glucose, galactose, fructose, mannoheptulose, glutaric acid, 2,4-dimethylglutaric acid (Koch-Light Lab., Colnbrook, England) and 3,3-tetramethyleneglutaric acid (gift of Dr K. Gabbay, Boston, U.S.A.)...

The present report aims mainly at a reevaluation of salivary glucose concentration and excretion in unstimulated and mechanically stimulated saliva in both normal and diabetic subjects. In normal subjects, a decrease in saliva glucose concentration, an increase in salivary flow, but an unchanged glucose excretion rate were recorded when comparing stimulated saliva to unstimulated saliva. In diabetic patients, an increase in salivary flow with unchanged salivary glucose concentration and glucose excretion rate were observed under the same experimental conditions. Salivary glucose concentration and excretion were much higher in diabetic patients than in control subjects, whether in unstimulated or stimulated saliva. No significant correlation between glycemia and either glucose concentration or glucose excretion rate was found in the diabetic patients, whether in unstimulated or stimulated saliva. In the latter patients, as compared to control subjects, the relative magnitude of the increase in saliva glucose concentration was comparable, however, to that of blood glucose concentration. The relationship between these two variables was also documented in normal subjects and diabetic patients undergoing an oral glucose tolerance test.

1. In isolated pancreatic islets, pyruvate causes a shift to the left of the sigmoidal curve relating the rate of insulin release to the ambient glucose concentration. The magnitude of this effect is related to the concentration of pyruvate (5--90 mM) and, at a 30 mM concentration, is equivalent to that evoked by 2 mM-glucose. Pyruvate also enhances insulin release in the presence of fructose, leucine and 4-methyl-2-oxopentanoate. 2. In the presence of glucose 8 mM), the secretory response to pyruvate is an immediate process, displaying a biphasic pattern. 3. The insulinotropic action of pyruvate coincides with an inhibition of 45Ca efflux and a stimulation of 45Ca net uptake. The relationship between 45Ca uptake and insulin release displays its usual pattern in the presence of pyruvate. 4. Exogenous pyruvate rapidly accumulates in the islets in amounts close to those derived from the metabolism of glucose. The oxidation of [2-14C]pyruvate represents 64% of the rate of [1-14C]pyruvate decarboxylation and, at a 30 mM concentration, is comparable with that of 8 mM-[U-14C]glucose. 5. When corrected for the conversion of pyruvate into lactate, the oxidation of 30 mM-pyruvate corresponds to a net generation of about 314 pmol of reducing equivalents/120 min per islet. 6. Pyruvate does not affect the rate of glycolysis, but inhibits the oxidation of glucose. Glucose does not affect pyruvate oxidation. 7. Pyruvate (30 mM) does not affect the concentration of ATP, ADP and AMP in the islet cells. 8. Pyruvate (30 mM) increases the concentration of reduced nicotinamide nucleotides in the presence but not in the absence of glucose. A close correlation is seen between the concentration of reduced nicotinamide nucleotides and the net uptake of 45Ca. Menadione inhibits the effect of pyruvate on insulin release, without altering its rate of oxidation. 9. Pyruvate, like glucose, modestly stimulates lipogenesis. 10. Pyruvate, in contrast with glucose, markedly inhibits the oxidation of endogenous nutrients. The latter effect accounts for the apparent discrepancy between the rate of pyruvate oxidation and the magnitude of its insulinotropic action. 11. Dichloroacetate fails to affect glucose oxidation and glucose-stimulated insulin release. 12. It is concluded that the effect of pyruvate to stimulate insulin release depends on its ability to increase the concentration of reduced nicotinamide nucleotides in the islet cells.

The leucine analog -2-aminobicyclo[2.2. lheptane-2-carboxylic acid (BCH) activates glutamate dehydrogenase [L-glutamate:NAD+ oxidoreductase (deaminating), EC 1.4.1.2] in pancreatic islet homogenates. In intact islets, BCH increased the islet content or output ofNH4, 2-ketoglutarate, malate, pyruvate, and alanine. BCH caused a dose-related increase in 14CO2 output from islets prelabeled with L-[U-"4C]glutamine. BCH increased the islet content of ATP and stimulated both 'Ca net uptake and insulin release. The capacity of seven distinct amino'acids to activate glutamate dehydrogenase tightly correlated with their ability to augment 0CO2 outplt from islets prelabeled with [U-'4C]-glutamine and to stimulate insulin release in the presence of Lglutamine. The activation of glutamate dehydrogenase by BCH may thus account for the insulin-releasing capacity of the leucine analog.It is well establishedthat the nonmetabolizable L-leucine analog ,B-2-aminobicyclo[2.2. 1]heptane-2-carboxylic acid (BCH) stimulates insulin release, both in vitro and in vivo (1-4)..At first glance, the capacity ofBCH to stimulate insulin release appears to be incompatible with the current concept that thestimulant effect of nutrients upon,insulin secretion tightly depends on their capacity to be metabolized in the pancreatic islet cells (5, 6). However, it was recently proposed that the insulin secretory response to BCH is attributable to activation of glutamate dehydrogenase [L-glutamate:NAD' oxidoreductase (deaminating), EC 1.4.1.2].by the leucine analog (7). This hypothesis was based mainly on the existence ofa tight correlation between the capacity of distinct amino acids to activate glutamate dehydrogenase in islet homogenates and the capacityto stimulate insulin release in intact islets exposed to L-glutamine. In the present study, we have characterized the metabolic situation found in islets exposed to BCH, in order to further assess the validity of the above-mentioned hypothesis. MATERIALS AND METHODSAll experiments were performed with islets isolated from the pancreas of fed albino rats (8). For the measurement of NH4 production, groups of 20 islets each were incubated in 30 ,ul of a bicarbonate-buffered medium (9) containing bovine albumin (0.5 mg/ml). After incubation, the medium and islets were placed in liquid N2 and mixed with 10 A1 of HC1 (0.2 M). After mechanical disruption ofthe islets and heating for 5 min at 85°C, 30 ,ul ofthe homogenate was mixed with 60 u1l ofa solution containing Tris'HCl (75 mM, pH 7.5), 2-ketoglutarate (15 mM), ADP (1.5 mM), EDTA (7.5 mM), NADH (0.3 mM), and glutamate dehydrogenase (150 ,ug/ml). After 30-min incubation at -room temperature and termination of the reaction, the NAD+ formed was measured by fluorimetry (9). The methods used to measure the islet content and output of pyruvate (10), glutamate, 2-ketoglutarate, malate, alanine, oxaloacetate, and aspartate (I1) have been described. Unless otherwise mentioned, all these metabolites were measured in the islets and their incubation medium a...