Insulin release: reconciliation of the receptor and metabolic hypotheses Nutrient receptors in islet cells Willy J. Malaisse, Abdullah Sener and Francine Malaisse-Lagae

Laboratory of Experimental Medicine, Brussels University School of Medicine, Brussels, Belgium

Summary Nutrients which stimulate insulin secretion are currently thought to initiate the series of cellular events eventually leading to insulin release either by interacting with a stereospeciflc receptor system (the regulatory site hypothesis) or by acting as a fuel (the substrate site hypothesis) in the pancreatic B-cell. The latter hypothesis is supported by a number of observations indicating that the capacity of nutrients to stimulate insulin release is indeed highly dependent on their capacity to increase catabolic fluxes in isolated pancreatic islets. However, these observations do not rule out the existence of nutrient receptors in islet cells. For instance, a nonmetabolized analog of L-leucine stimulates insulin release by causing allosteric activation of glutamate dehydrogenase, which should be considered, therefore, as a receptor for certain amino acids. Likewise, the increase in glycolytic flux, which is associated with the process of glucose-stimulated insulin release, is attributable not solely to a mass action phenomenon but also to the activation of phosphofructokinase by fructose 2.6-bisphosphate. The biosynthesis of this activator may involve a glucose receptor system. The fact that certain nutrient secretagogues (e.g. D-glucose and L-leucine) act in the B-cell both as substrates and enzyme activators permits reconciliation of the substrate site and regulatory site hypotheses for insulin release.

1. Mechanism of insulin release 1.1. Insulin secretion: multifactorial regulation for a single process of release Insulin release by the pancreatic B-cell is regulated in both an immediate and direct manner by circulating nutrients, neurotransmitters and hormones and in a delayed or chronic fashion by ontogenic, endocrine and nutritional factors (1 3). The regulation of a single process of release by such a variety of factors implies that the B-cell is equipped with a number of distinct sensor systems able to identify each regulatory factor and, nevertheless, organized in such a manner that the messengers generated by distinct sensors eventually provide an univocal information to the effector system controlling the exocytosis of insulin

secretory granules (4). The common pathway through which all regulatory factors eventually affect the rate of insulin release involves the participation of Ca 2+ as a trigger mechanism for insulin release. It is indeed thought that the accumulation of ionized Ca 2+ in a critical site of the B-cell, presumably the cytosol and its ectoplasmic compartment, triggers the exocytosis of secretory granules by activating a microtubular-microfilamentous system, which itself controls the access of secretory granules to the exocytotic sites (5). At such a site, the fusion and fission of the granule-limiting membrane and plasma membrane may involve an anion-osmotic mechanism (6, 7), somehow comparable to the osmotic lysis ofliposomes fusing with a planar lipid bilayer (8).

Molecular and Cellular Biochemistry, 37, 157 165 (1981). 0300-8177/81/0373-0157/$ 1.80. 9 1981, Martinus Nijhoff/Dr W. Junk Publishers, The Hague. Printed in The Netherlands.

158 The cytosolic accumulation of Ca 2+ may be due to the facilitation of Ca 2+ entry into the B-cell, e.g. by gating of voltage-dependent Ca2+-channels (9, 10), a decreased rate of Ca 2+ outflow from the B-cell, e.g. by inhibition of a process of Na+-Ca 2+ countertransport (11, 12), a n d / o r an intracellular redistribution of Ca 2+ ions between the cytosol and other cellular pools, e.g. by the release of Ca 2+ from the vacuolar system (13, 14). Each of these three modalities have indeed been identified, under suitable experimental conditions, in stimulated B-cells. Distinct sensor systems may generate distinct messengers, which in turn may affect distinct Ca 2+ movements.

1.2. The coupling of sensor to effector systems: initiating, permissive and amplifying factors It is apparent that, even when insulin release is regulated by a single environmental agent (e.g. glucose), more than one messenger may be operative in coupling the activation of the sensor system to the activation of the effector system. For instance, when insulin release is stimulated by glucose, the generation rate of several potential coupling factors - namely reducing equivalents (NADH, N A D P H , glutathione), protons (H+), high-energy phosphate intermediates (ATP) and cyclic nucleotides (cyclic AMP) - is indeed increased (15, 16). At this point, however, a distinction ought to be made between initiating, permissive and aml~lifieation factors. Initiating factors are essential to the stimulus-secretion coupling process, because they are responsible for switching the B-cell from the resting to the stimulated functional state. Permissive factors are not indispensable for the latter switch to occur, but they may be required for the terminal event, i.e. insulin release from the stimulated B-cell, to take place. Amplifying factors are not required for stimulation of insulin release, but their participation ensures optimal magnitude of the secretory response. Although it could be argued that such a classification is more verbal than real, we wish to illustrate how it may help to localize the participation of a given factor at a specific locus in the secretory sequence. In the process of glucose-induced insulin release, the accumulation of Ca 2+ in the cytosol of the B-cell is currently attributed, mainly but not exclusively, to the facilitated entry of Ca 2+ into the

B-cell through gated voltage-dependent Ca 2+channels. The gating of these channels is itself attributed to electrical depolarization of the B-cell plasma membrane, as a result of a decrease in K + conductance (17). The mechanism responsible for the change in K + conductance is not fully elucidated, but the participation of reducing equivalents in such a process was proposed by several investigators (18, 19). If the latter hypothesis is correct, and it remains to be fully validated, the availability of reducing equivalents generated by the metabolism of glucose should be considered as an initiating factor. It is likely (but also not proved) that ATP is consumed at a distal step in the secretory sequence, e.g. to support the contraction of the microfilamentous cell web. In this respect ATP could be considered as a permissive factor. The latter view does not preclude that ATP simultaneously acts as an initiating factor at some other site in the secretory sequence. For instance, it was recently speculated that the glucose-induced decrease in K + conductance may be due to inactivation of a Ca 2+sensitive modality of K + extrusion from the islet cells (20). This situation could be secondary to a fall in the cytosolic concentration of Ca 2+ and the latter fall would itself depend on the generation of increased amounts of ATP, with concomitant facilitation of either Ca 2+ uptake by the vacuolar system or Ca 2+ outward transport across the plasma membrane, as mediated by suitable ATPases. The participation of cyclic A M P in the process of glucose-induced insulin release may serve to illustrate the role of an amplifying factor. It has been known for many years that increasing the islet cell content of cyclic A M P is usually not sufficient to provoke insulin release, but quite adequate to enhance secretion evoked by other secretagogues (21). It was recently reported that the islet cells contain the Ca2+-sensitive regulatory protein calmodulin (22, 23) and that calmodulin activates in a Ca2+-dependent fashion insular adenylate cyclase (23). These findings led us to propose that, when glucose (or some other secretagogues) is used to stimulate insulin release, the intracellular accumulation of Ca 2+ leads to self-amplication of the secretory response through activation by calmodulin of adenylate cyclase, subsequent stimulation of cyclic A M P synthesis and eventual

159 enhancement of the secretory rate (23). Incidentally, the enhancing action of cyclic A M P upon insulin release appears attributable, in part at least, to an intracellular redistribution of Ca 2+ between the cytosolic and vacuolar C sa 2+ pools (13). 1.3. N u t r i e n t sensor s y s t e m s

Keeping the background information reviewed briefly so far in mind, it is obvious that the identification and characterization of sensor systems represents a fundamental task in the field of islet research. In a few instances and by analogy with extensive kn6wledge gained in other tissues - it was proposed that the sensor system can be equated with receptor sites located at the plasma membrane. For instance, glucagon binds to islet cells (24) and the binding of glucagon to its receptor may initiate a cascade of events including activation of adenylate cyclase (25). In support of such a view, glucagon, like cyclic A M P , enhances stimulated insulin release but exerts little or no effect upon secretion in the absence of any other secretagogue (21). The sensor system(s) involved in the recognition by the B-cell of nutrient secretagogues, such as D-glucose or L-leucine, are less easy to define. For several years, two contrasting theories were considered (26). The first theory, often called the regulatory site hypothesis, postulated that nutrients such as glucose initiate the release of insulin by activation of a specific receptor in a manner analogous to that characterizing the interaction of peptide hormones with their tissue receptors. The second theory, or substrate site hypothesis, postulated that, in order to stimulate insulin release, glucose (or other nutrients) had to be metabolized in the islet cells, the secretory response being dependent on the availability of a metabolite or co-factor generated by the metabolism of glucose. This second or metabolic hypothesis has gained considerable support from a number of observations indicating that the secretory response to nutrients is indeed highly dependent on their metabolism in the islet cells (27, 28). For instance, the better aptitude of a-D-glucose compared to that of/%D-glucose to stimulate insulin release coincides with a higher rate of glycolysis in islets exposed to a-D-glucose, a situation itself attributable to the stereospecificity of the enzyme phosphoglucose isomerase, which utilizes preferentially a-D-glucose 6-phosphate as a substrate (29).

In two instances, it was even possible to dissociate the capacity of nutrients to be metabolized in islet cells and, hence, to stimulate insulin secretion from any effect they could conceivably exert through activation of a specific receptor site. In the first case, islets were pretreated to cause intracellular deposition of large amounts of glycogen and were incubated, thereafter, in the absence of extracellular glucose. When glycogenolysis was suitably stimulated, a rapid increase in insulin output was noticed (30). Thus, stimulation of glycogenolysis and glycolysis was associated with stimulation of insulin release even when no extracellular glucose was available to act upon hypothetical gluco-receptors. In the second case, islets were exposed to L-valine or other branched chain amino acids which are metabolized in the islet cells and, therefore, prevent the fall in A T P content otherwise found in islets deprived of exogenous nutrients (31, 32). In the presence of L-valine, the insulin secretory response to 2-ketoisocaproate was abolished, as a result of an increased conversion of the 2-keto acid to L-leucine and, hence, a decreased catabolism of 2-ketoisocaproate to CO2 and acetoacetate (33). Thus, two distinct molecules (i.e. 2ketoisocaproate and a branched chain amino acid), each of which could conceivably stimulate insulin release by activation of distinct receptors (34), were acting antagonistically when used in combination, this antagonism being attributable to a wellcharacterized metabolic interaction. F r o m such observations, it would be tempting to conclude that the insulinotropic action of nutrients indeed depends on their metabolism in islet cells and that no compelling reason exists to further consider the regulatory site or receptor hypothesis for insulin release (27). The aim of the present account, however, is to defend the opposite view, i.e. to demonstrate the existence of nutrient receptors in islet cells and to emphasize their significance in the regulation of insulin release. As such, the present report may help to reconciliate the receptor and metabolic theories for insulin release.

2. The L-leucine receptor The first demonstration of a nutrient receptor in islet cells was made in the course of investigations on the stimulus-secretion coupling of amino acidinduced insulin release.

160

2.1. The fuel hypothesis for L-leucine-induced insulin release Within the framework of the substrate site hypothesis, the knowledge that such nutrients as D-glucose and 2-ketoisocaproate (35) are potent insulin secretagogues - even though they are metabolized by distinct pathways at least up to the production of acetyl-coenzyme A - led us to propose that insulin release may depend on the capacity of nutrients to serve as fuel in islet cells and, hence, to increase the generation rate of reducing equivalents and high-energy phosphate intermediates, rather than being linked with the production of a specific metabolite in a given metabolic pathway (27). This view was then extrapolated to the process of L-leucine-induced insulin release (36). Indeed, L-leucine was found to be actively metabolized in islet cells and to affect several biochemical and biophysical variables of islet function in a manner comparable to that seen in islets stimulated with either D-glucose or 2ketoisocaproate. Modest differences in the insulinotropic capacity of 2-ketoisocaproate and L-leucine, relative to their respective oxidation rate, were attributed to a sparing action of L-leucine on the utilization of endogenous nutrients (36) and to some unfavourable effect of endogenously formed NH4 + upon the coupling of metabolic to ionic events (37).

islet cells. Freinkel et al. (40) first observed that BCH is able to evoke a 'phosphate flush' in isolated islets. This 'phosphate flush' consists of a dramatic but short-lived release of inorganic phosphate from perifused islets (41). It occurs within the few first minutes of exposure of the islets to nutrient secretagogues, such as D-glucose, L-leucine or 2-ketoisocaproate (41, 42). No phosphate flush is seen, however, when insulin release is stimulated by nonmetabolizable secretagogues such as hypoglycemic sulfonylureas or arginine (42). The fact that BCH evokes a phosphate flush suggests, therefore, that BCH, although nonmetabolized, affects islet metabolism in a manner analogous to that seen in the presence of metabolized nutrients. Hellerstr6m, et al. (43) then reported that BCH augments, at least transiently, O2 uptake by isolated islets, again suggesting that BCH somehow increases the oxidation of endogenous nutrients. The biochemical explanation for the latter increase appears to consist in an allosteric activation of the enzyme glutamate dehydrogenase by BCH, as supported by the following observations. BCH activates glutamate dehydrogenase whether the activity of the enzyme is tested with purified bovine liver glutamate dehydrogenase (44) or in an

100

2.2. Effect o f a nonmetabolizable analogue o f Lleucine upon islet function The concept that L-leucine-induced insulin release depends on the metabolism of this amino acid in the islet cells appears, at first glance, to be incompatible with the knowledge that a nonmetabolized analogue of L-leucine, namely b(-) 2amino-bicyclo-[2, 2, l]heptane-2-carboxylic acid (BCH) is able like L-leucine but to a lesser extent than the latter amino acid, to stimulate insulin release from islets deprived of other exogenous nutrient (38, 39). As a matter of fact, the mere observation that BCH stimulates insulin secretion appears incompatible with the fuel hypothesis for insulin release. Recent findings, however, suggest that even the process of BCH-induced insulin release is caused by an increase in the catabolic flux of nutrients in the

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161 islet homogenate (45). The b(-) stereo-isomer of BCH appears equally potent as L-leucine in causing activation of glutamate dehydrogenase. As shown in Fig. 1, when intact islets are incubated in the absence of exogenous nutrient, BCH augments the production of NH4 + by the islets. If the islets were preincubated with L-(U-14C)glutamine, BCH also augments the production of 14CO2 (46). It thus appears that BCH indeed stimulates the oxidative deamination of endogenous L-glutamate and, hence, augments CO2 production in further steps of a-ketoglutarate catabolism. 2.3. Glutamate dehydrogenase." a receptor f o r L-leucine

By analogy with the situation just described in islets exposed to BCH, it is conceivable that the release of insulin evoked by L-leucine is not attributable solely to the metabolism of this amino acid, but depends to a limited extent on an allosteric activation of glutamate dehydrogenase. In order to explore the latter mechanism, advantage was taken of the fact that L-glutamine, unlike L-glutamate, penetrates easily into islet cells and is converted to a large extent to L-glutamate, providing abundant

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substrate for the reaction catalyzed by glutamate dehydrogenas ~ (47). The conversion of L-glutamine to L-glutamate may occur by direct deamidation or via the ",/-glutamyl cycle, islets containing both glutaminase and 7-glutamyltranspeptidase activities (48). The interpretation of secretory data was facilitated by the fact that L-glutamine alone does not affect insulin release in the absence of other exogenous nutrient. This is not meant to deny that L-glutamine is metabolized in the islets. However, the catabolism of L,glutamine to a large extent merely compensates for a sparing action of the amino acid on the utilization of endogenous nutrients, so that the uptake of 02 is only slightly increased above basal value in the presence of L-glutamine (49). Incidentally, it is possible to unmask a modest insulinotropic action of L-glutamine by manipulating the ionic environment of the B-cell in a way which favours the cytosolic accumulation of Ca 2+ (50). Although, under normal environmental conditions, L-glutamine does not stimulate insulin release, this amino acid dramatically augmented insulin output evoked by L-leucine or BCH (45). When nine different amino acids were compared, a tight correlation was found between the ability of L-glutamine to augment insulin secretion in the presence of a given amino acid and the capacity of that given amino acid to act as an activator of glutamate dehydrogenase in islet homogenates (Fig. 2). The fact that, in the presence of Lglutamine, L-leucine and BCH were equally potent in stimulating insulin release represents one illustration of such a correlation, and fits with the observation that, in the presence of L-glutamine, the catabolism of L-leucine is severely impaired. In other words, when L-glutamine is present, the secretory response to L-leucine appears entirely attributable to the allosteric activation of glutamate dehydrogenase rather than to the catabolism of the branched chain amino acid. Nevertheless, the ionic response to the combination of L-leucine and Lglutamine still displays the characteristic features of a nutrient-induced stimulation of the B-cell, including a decrease in K + conductance, a gating of Ca2+-channels and the accumulation of Ca 2+ in the islet cells (51). These findings indicate that the release of insulin evoked by the combination of L-leucine and Lglutamine, although in good agreement with the

162 fuel hypothesis, is nevertheless essentially attributable to the interaction of L-leucine with its stereospecific receptor, namely the allosteric site of glutamate dehydrogenase.

3. The D-glucose receptor Whether the B-cell is equipped with a D-glucose receptor analogous to the L-leucine receptor just described remains an open question. However, recent findings argue in favour of the existence of such a D-glucose receptor.

3.1. Early steps of glucose metabolism in the islets The metabolism of glucose in islet cells displays several rather unusual features, which are well suited to provide rapid and ample changes in the rate of glycolysis as a function of the extracellular glucose concentration (52). Three of these attributes are listed below. First, the capacity of the carrier system that mediates the transport of glucose across the B-cell plasma membrane is one of the best so far studied (53). The intracellular concentration of free glucose in islet cells is thus immediately and permanently equal to that of extracellular glucose (32). This situation contrasts with that found in other tissues, e.g. muscle, in which the transport of glucose across the pl.asma membrane represents a rate-limiting step in the metabolism of the sugar. Second, islet cells, unlike most other tissues but like hepatocytes display, in addition to hexokinaselike activity with a Km for glucose close to 0.06 mM, glucokinase-like activity with a Km for glucose close to 10 mM or more (54, 55). This second phosphorylating enzymatic system may account for an increase in the rate of glucose phosphorylation when the extracellular, and hence intracellular, concentration of glucose is raised within the range of values (5-25 mM) in which glucose causes a dose-related stimulation of insulin release (56). Indeed, the concentration of glucose 6-phosphate in islet cells increases in a rapid and sustained manner when the concentration of extracellular glucose is increased from a low (e.g. 2.8 mM) to a high (e.g. 16.7 mM) value (57, 58). Last, the islets do not contain any significant fructose- 1.6-diphosphatase activity (52), so that the

net rate of conversion of fructose 6-phosphate to fructose-l.6-diphosphate depends exclusively on the velocity of the reaction catalyzed by phosphofructokinase. As in other tissues, the velocity of this reaction depends on the respective concentrations of fructose 6-phosphate, ATP and A M P (55). When the concentration of adenine nucleotides is optimal, the velocity of the reaction as a function of the concentration of fructose 6-phosphate displays a sigmoidal relationship (59). Within the range of fructose 6-phosphate concentrations to be found in islet cells (i.e. 0.03 0.07 mM at extracellular glucose concentrations ranging from 2.8 to 16.7 mM; see ref. 59), the velocity of the reaction catalyzed by phosphofructokinase in islet homogenates is surprisingly lower than that found in intact islets indeed exposed to high concentrations of glucose (59). Such a paradoxical situation strongly suggests that, in the islets exposed to high concentrations of glucose, phosphofructokinase must be somehow activated. The mechanism of such an activation will now be considered.

3.2. Fructose 2.6-bisphosphate, an activator of phosphofructokinase Van Schaftingen, Hue and Hers (60, 61, 62) have recently reported that in liver cells exposed to high concentrations of glucose, phosphofructokinase is rapidly activated. The activation phenomenon is characterized by the fact that the velocity of the reaction catalyzed by phosphofructokinase is considerably enhanced when measured at low concentrations of fructose 6-phosphate (e.g. 0.25 mM), whereas the maximal velocity measured at high concentrations of fructose 6-phosphate (e.g. 5.0 mM) appears unaffected. Thus, the ratio in reaction velocity at low and high concentration of fructose 6-phosphate, respectively, can be used to estimate the extent of enzymatic activation (60). The same investigators discovered that the glucoseinduced activation of the enzyme was attributable to a new hexose-phosphate that they identified as fructose 2.6-bisphosphate. Having achieved the chemical synthesis of this compound (63), Van Schaftingen and Hers were able to examine its effect upon purified phosphofructokinase (62). Fructose 2.6-bisphosphate indeed proved to be a potent activator of phosphofructokinase, with dramatic increases in reaction velocity at low

163 c o n c e n t r a t i o n s of fructose 6 - p h o s p h a t e but no o b v i o u s effect u p o n the m a x i m a l velocity. T h e a p p a r e n t K m for the a c t i v a t i o n p h e n o m e n o n corres p o n d s to a c o n c e n t r a t i o n of the a c t i v a t o r close to 0.1 # M . We have recently o b s e r v e d t h a t a c o m p a r able s i t u a t i o n is o p e r a t i v e in p a n c r e a t i c islets (59). In p a n c r e a t i c islet h o m o g e n a t e s , the velocity o f the r e a c t i o n c a t a l y z e d by p h o s p h o f r u c t o k i n a s e is m u c h h i g h e r in the presence t h a n in the a b s e n c e of fructose 2 . 6 - b i s p h o s p h a t e (Fig. 3). S u c h an increase in r e a c t i o n velocity is o b v i o u s at low c o n c e n t r a t i o n s o f f r u c t o s e 6 - p h o s p h a t e , b u t fades o u t at higher c o n c e n t r a t i o n s o f the latter substrate. Thus, the presence of a c t i v a t o r t r a n s f o r m s the r e l a t i o n s h i p between s u b s t r a t e c o n c e n t r a t i o n a n d r e a c t i o n velocity f r o m a s i g m o i d a l to a h y p e r b o l i c curve. The increase in r e a c t i o n velocity a t t r i b u t a b l e to fructose 2 . 6 - b i s p h o s p h a t e reaches half of its m a x i m a l value at a c o n c e n t r a t i o n o f the a c t i v a t o r close to 0.2 ~ M . These findings d o c u m e n t that f r u c t o s e 2 . 6 - b i s p h o s p h a t e is a p o t e n t a c t i v a t o r o f the islet p h o s p h o f r u c t o k i n a s e (59). ~.~x------~

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In o r d e r to investigate w h e t h e r glucose indeed causes a c t i v a t i o n of the e n z y m e in intact islet cells, p a n c r e a t i c islets were i n c u b a t e d for 60 min in the a b s e n c e o r presence o f D - g l u c o s e 16.7 m M . The islets were then h o m o g e n i z e d a n d the activity of p h o s p h o f r u c t o k i n a s e m e a s u r e d at two different c o n c e n t r a t i o n s of fructose 6 - p h o s p h a t e , 0.25 a n d 5.0 m M . F o r r e a s o n s a l r e a d y given, the r a t i o in r e a c t i o n velocity at these two c o n c e n t r a t i o n s of s u b s t r a t e was used as an i n d e x o f e n z y m e activation. E x p o s u r e o f the islets to glucose resulted in a c t i v a t i o n of p h o s p h o f r u c t o k i n a s e . Thus, the r a t i o in r e a c t i o n velocity a v e r a g e d 0.41 + 0.04 a n d 0.62 + 0.07 in islets p r e v i o u s l y d e p r i v e d of glucose a n d in islets first e x p o s e d to glucose, respectively (59).

3.3. Fructose 2.6-bisphosphate synthesis and the D-glucose receptor site N o i n f o r m a t i o n is as yet a v a i l a b l e c o n c e r n i n g the p a t h w a y for fructose 2 . 6 - b i s p h o s p h a t e b i o s y n thesis. H o w e v e r , the mere fact t h a t e x p o s u r e o f h e p a t o c y t e s , a n d a p p a r e n t l y also islet cells, to Dglucose increases the tissue c o n t e n t o f this newly d i s c o v e r e d h e x o s e - p h o s p h a t e suggests t h a t Dglucose acts s o m e h o w to f a v o u r the biosynthesis of f r u c t o s e 2 . 6 - b i s p h o s p h a t e . W h a t e v e r the precise m o d e of a c t i o n o f D - g l u c o s e in such a process, it is o b v i o u s f r o m the d a t a so far a v a i l a b l e that the effect of D - g l u c o s e to a u g m e n t glycolysis in islet cells and, hence, to s t i m u l a t e insulin release c a n n o t be solely a t t r i b u t e d to a mass a c t i o n p h e n o m e n o n in which D - g l u c o s e w o u l d act as the initial substrate. In a d d i t i o n , D - g l u c o s e is involved in the r e g u l a t i o n o f p h o s p h o f r u c t o k i n a s e activity, a r e g u l a t o r y effect m e d i a t e d by the a c t i v a t o r fructose 2.6-bisphosp h a t e . In o t h e r words, the changes in glycolytic rate e v o k e d by a rise in the e x t r a c e l l u l a r c o n c e n t r a t i o n o f D - g l u c o s e are a t t r i b u t a b l e to a d u a l effect o f the sugar, which acts b o t h as a s u b s t r a t e and as a r e g u l a t o r y factor. We wish to p o s t u l a t e , as a w o r k i n g hypothesis, that the latter effect involves a D - g l u c o s e r e c e p t o r site.

4. Conclusions The c a p a c i t y o f n u t r i e n t s to s t i m u l a t e insulin release f r o m the p a n c r e a t i c B-cell tightly correlates with their effect to a u g m e n t 02 u p t a k e by p e r i f u s e d

164 islets (49, 64). This finding taken together with other biochemical data, supports the view that the release of insulin evoked by nutrient secretagogues is invariably mediated by an increase in catabolic fluxes in the islet cells. Such a situation also applies to the release of insulin provoked by the nonmetabolized analogue of L-leucine BCH, reinforcing the validity of the fuel hypothesis for insulin release (27). The latter hypothesis, however, does not rule out that nutrient secretagogues, independently of or in concert with their fuel function, also act through specific receptor systems to facilitate their own catabolism or that of other nutrients. For instance, L-leucine or its nonmetabolized analogue b ( - ) B C H cause allosteric activation of glutamate dehydrogenase which should, therefore, be looked upon as a stereospecific receptor for these branched chain amino acids. Likewise, in islets exposed to D-glucose, the rate of glycolysis is increased not solely through a mass action phenomenon but also as a result of the activation of phosphofructokinase. The latter activation seems attributable to fructose 2.6-bisphosphate, the generation of which may involve a D-glucose receptor system. Finally, the present account indicates that the fuel hypothesis for nutrient-stimulated insulin release should be understood as including regulatory processes, e.g. the activation of enzymes acting as stereospecific receptors for certain nutrient molecules.

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Received January 20, 1981.