The amino acids that are derived from the protein meals, infusion of physiological amino acid mixtures, or certain individual amino acids have been shown to stimulate insulin release in humans (60,61). Amino acids are glucose-dependent secretory stimuli. Unlike glucose, they cannot by themselves cause insulin secretion from normal P-cells (3). These nutrients can only act as insulin secretagogues when the level of glucose is higher than 4-5 mM (61-64). This makes physiological sense, because it allows them to be mobilized from muscle and adipose tissue during starvation, when blood glucose levels are low. In this circumstance, insulin secretion ceases and glucose levels are then maintained by hepatic gluconeogenesis, which produces glucose from specific amino acids and lipid-derived glycerol. The changes of blood sugar levels markedly influence the responsiveness of the P-cells to individual amino acids. For example, hypoglycemia reduces insulin release to amino acid mixtures and most individual amino acids. However, chronic hypoglycemia in man greatly sensitizes the pancreatic P-cell to leucine when administered in high pharmacological dosages (61). A plausible explanation for these results is that amino acids are potent fuel stimulants of P-cells that activate metabolic signaling pathways, fundamentally similar to those discussed earlier for glucose but with characteristics that are special and unique for individual members of this nutrient class. Amino acids can be assumed to be transported into the P-cell by several transporters that are well characterized in other tissues and probably present in P-cells. Most of them are then likely to be transaminated by a family of pyridoxal-P-dependent enzymes that transfer the amino group to a-ketoglutarate to form glutamate (65). The carbon skeletons of the amino acids are converted to pyruvate, acetyl-CoA, a-ketoglutarate, succinyl-CoA, fumarate, and oxaloacetate and provide substrates for the citric acid cycle. Glutamate serves as the predominant final product of the transamination reactions and is oxidatively deaminated by glutamate dehydrogenase. This process is termed transdeamination. Studies with the isolated perfused rat pancreas and with isolated perifused rat islets have supported this view and have demonstrated, in addition, that physiological amino acid mixtures and even pharmacological concentrations of individual amino acids require the presence of permissive levels of glucose (i.e., 2.5-5.0 mM) to be effective P-cell stimulants (64). However, in view of the in vivo findings in man, it should not be surprising that leucine is an exception (66,67). When the isolated perfused pancreas or isolated islets are fuel deprived, most importantly when they are maintained in the absence of glucose, even if only for a period of 2 h or less, they are highly responsive to leucine at supraphysiological levels (10-20 mM). The findings with leucine as a secretagogue both in man and in a variety of experimental systems using rodent islet tissue were explained by a twofold action of leucine on P-cell metabolism and, consequently, insulin release (68-75). First, leucine may enter P-cell intermediary metabolism by the classical process of transamination and may thus act similar to other metabolic substrate stimuli. In this process, a-ketoglutarate serves as the predominant acceptor of the amino group to form glutamate, and the other product, a-ketoisocaproate (KIC), is thought to be oxidized by branched chain a-ketoacid dehydrogenase (BCKDH) to yield acetoacetate and acetyl-CoA. To complete the process of transdeamination, the product glutamate is reoxidized to a-ketoglutarate. Further catabolism of a-ketoglutarate results in the generation of GTP potentially important for signal transduction. Acetoacetate and actyl-CoA metabolism may result in the production of additional reducing equivalents and energy production. Second, leucine is also an effective allosteric activator of glutamate dehydrogenase (GDH), which generates a-ketoglutarate, NH3 and NAD(P)H. Leucine thus accelerates its own breakdown by removing glutamate and generating a-ketoglutarate.
In the normal pancreatic P-cell, the critical reaction of transdeamination, catalyzed by GDH, appears to be very strongly inhibited so that oxidation of glutamate is relatively slow. Thus, little ATP and malonyl-CoA would be generated from amino acids, explaining the lack of the response when amino acids are present in the absence of glucose. The activity of the GDH is tightly controlled by the energy potential of the cell (76). It is strongly inhibited by GTP, which is a downstream product and is probably in equilibrium with the phosphate potential ATP/(ADP x Pi). It is also activated by ADP, such that the GTP/ADP ratio of the mitochondria is a critical determinant of the enzyme's rate (77). The stimulation of insulin release by amino acids in the presence of glucose may be the result of enhanced production of pyruvate, a-ketoglutarate, and oxalacetate that would result from even a small increase in glucose metabolism. These metabolites can act as acceptors for the transamination reaction and should, therefore, enhance the degradation of amino acids, allowing them to potentiate the stimulatory effect of glucose.
It is difficult to quantify the relative contributions of these two actions of leucine for the stimulation of insulin secretion. However, activation of GDH by the amino acid is probably sufficient to stimulate the P-cells, given a permissively high glutamate supply. This conclusion is based on the stimulatory effect of the nonmetabolized leucine analog 2-amino-bicyclo-norbonane carboxylic acid (BCH) (69,70), which shares with leucine the GDH activating capacity and, therefore, its ability to stimulate insulin release. These results show that enhanced metabolism of the mitochondrial substrate glutamate is sufficient to cause insulin secretion.
We speculate here that the enzyme is very strongly inhibited at basal conditions but that it may be activated by at least two distinct pathways. First, GDH may be sensitized to its activator by P-cell fuel depletion, because of hypoglycemia, for example, as seen in insulinoma cases. As discussed earlier, patients with an insulinoma are leucine hypersensitive. It can be surmised that the GTP/ADP ratio of P-cells is low in this situation and that less leucine is required to overcome the block. The other pathway may be operative following a protein meal. Protein feeding results in elevated serum levels of the branched-chain amino acids and of glutamine and alanine, whereas neither the blood sugar nor the other amino acids change substantially (61,62). Glutamine, converted by P-cell glutaminase to glutamate, could increase the substrate level for GDH, whereas the branched-chain amino acids leucine, isoleucine, and valine could activate GDH and also serve as substrates for transamination, thus diverting and lowering a-ketoglutarate (a known inhibitor of GDH) (78).
The precision of this control mechanism is illustrated by a recently discovered syndrome of familial hyperinsulinemia associated with mild hyperammonemia (79,80). Patients with this syndrome present with fasting hypoglycemia characterized by inappropriately low P-hydroxybutyrate and free fatty acid levels and an unexpectedly large glycemic response to an injection of glucagon. The patients also have moderately elevated plasma ammonia levels unaffected by protein feeding or restriction. Treatment with diazoxide, which opens KATP channels and prevents insulin secretion, reliably improves the hypoglycemia. The underlying defect was speculated to involve a site that is common to the regulation of amino acid metabolism of both pancreatic P-cells and hepatocytes and was subsequently shown to be the result of mutations of GDH that interfere with the inhibition of the enzyme by GTP (80). The syndrome is thus best described as persistent GDH-linked hyperinsulinemic hypoglycemia in infancy (PHHI-GDH). The findings in these patients suggest that the P-cell GDH needs to be maintained in a low-activity state and that a high GTP/ADP ratio may accomplish that. It appears that the activation of P-cell GDH by lowering of blood glucose or by fuel depletion in general results physiologically in precisely adjusted flux in the glutaminol-ysis pathway, allowing the maintenance of critical levels of citric-acid-cycle intermediates and of a functionally appropriate resting phosphate potential, but avoiding a catabolic avalanche that would result in generation of inappropriately high levels of ATP or other coupling factors that might be generated by mitochondria. The actual rate of this basal flux in glutaminolysis remains to be determined. Hypersensitivity of the P-cell to pharmacological dosages of leucine as observed in hypoglycemia of otherwise normal individuals is interpreted as a manifestation of physiological deinhibition of the enzyme in fuel-depleted states, when glutamine may be an important substrate for subsistance metabolism of the cell.
Glutaminolysis in P-cells proceeds by several steps (81,82). Glutamine enters the cell via Na+-dependent and Na+-independent transport across the cell membrane, enters the mitochondria by a carrier system, and is then deaminated by glutaminase to glutamate. Glutamate serves as substrate for several enzymes: aminotransferases for various a-ketoacids, glutamate decarboxylase, which produces y-aminobutyrate, and GDH, which was discussed earlier. In view of the kinetic characteristics of the enzyme GDH (77), the results with its nonmetabolized activator BCH (69) and based on the findings in the PHHI-GDH syndrome (79,80), we suggest that GDH is the rate-controlling step for the glutaminolysis pathway in the pancreatic P-cell. It is further concluded that the activity of the enzyme is governed primarily by the energy status of the cell, dependent, most importantly, on the ambient glucose level and the activity of the glucose sensor glucokinase. It is concluded that it serves as an important anaplerotic step to maintain critical levels of citric-acid-cycle intermediates in the P-cell. This proposed role for GDH contrasts starkly with the situation in the liver where glutaminase is thought to be rate limiting (83).
Metabolism of leucine, isoleucine, and valine was also studied intensively. Impetus for such studies came, in part, from the finding that the corresponding a-ketoacids are potent stimulators of insulin release even in the absence of glucose (61,66,67,84,85). The interpretation of results obtained with these a-ketoacids is made difficult because transamination reactions may channel alanine (converted to pyruvate) or glutamate (converted to a-ketoglutarate) into the active metabolite pool. Furthermore, the branched-chain amino acids that are generated by transamination may stimulate GDH, as discussed earlier, and contribute indirectly to the generation of coupling factors from glutamate. The quantification of the relative contribution of these multiple auxiliary pathways has not been accomplished to date.
Even though leucine metabolism of P-cells is complex attempts have been made to identify critical enzymes that comprise and regulate relevant pathways (86-88). The design of such studies was influenced by observations showing that prior exposure of islet tissue to high glucose desensitized P-cells to stimulation by leucine in the absence of glucose. This contrasts markedly with the effect that pretreatment of islets with high glucose has on a subsequent glucose stimulation, which is greatly potentiated. In an attempt to find a molecular explanation for the phenomenon, MacDonald's laboratory discovered that high glucose markedly depressed the expression of the E1a-subunit of the BCKDH, contrasting with the induction of pyruvate dehydrogenase and pyruvate carboxylase (87). This led the investigators to the view that BCKDH determines leucine sensitivity of P-cells. The above studies are flawed by the omission of critical control experiments. For example, it was not determined whether insulin release as a result of KIC was impaired following high-glucose preincubation, as would be expected if reduced BCKDH is a critical event in the desensitization mechanism. Others found no evidence for reduced sensitivity to KIC in islets cultured for 6 d at the moderately high-glucose levels (88). MacDonald and collaborators (87) discounted the potential regulatory role of GDH in the desensitization process, because the activity of the enzyme measured in islet cell extracts was not affected by prior glucose treatment. Such a conclusion does not consider the potential for short-term allosteric regulation of GDH by potential activators and inhibitors (e.g., ADP and GTP, respectively).
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