Summary and Closing Remarks JAMES R. GAVIN III, M.D., PH.D. Ok/ahoma city, Ok/a/ma

T his symposium provided a comprehensive sum- mary of our current understanding of the mecha-

nisms of action of glyburide on the beta cell and in the extrapancreatic sites that play major roles in insulin- mediated glucose disposal in health and in disease, particularly non-insulin-dependent diabetes mellitus (NIDDM). What emerges from these studies is the demonstration of a continuum of biochemical actions involving this second-generation sulfonylurea at the cellular level. It is now reasonably clear that a drug such as glyburide acts initially by binding to specific high-affinity receptors, the cellular distribution of which may differ from tissue to tissue. Subsequent to receptor binding, glyburide initiates a cascade of events that may include many of the well-character- ized signaling mechanisms and pathways described for other agonist-receptor systems. Moreover, on the basis of studies reported in this supplement and else- where, it now appears entirely feasible that the chem- ical properties of glyburide may well enable this drug to access subcellular compartments in a way that facil- itates direct interactions between the drug and spe- cific enzymes and/or transport proteins. The finding that glyburide has direct or synergistic effects in mul- tiple tissues further supports the conjecture that it is capable of acting at heterogeneous loci in biologic tis- sues, possibly through the recognition of functionally conserved structures located in diverse metabolic pathways. It becomes important to identify, then, the structural features of glyburide that confer to this agent a greater binding potency than other sulfonyl- ureas, as well as more significant biologic activity in beta cells and in extrapancreatic sites. Conversely, it is just as important to ascertain the structural deter- minants on those proteins that recognize glyburide at the cellular level, such as the beta cell sulfonylurea receptor and other, perhaps similar, receptors in other tissues.

The studies summarized by Boyd et al have shed important light on the locus of the effect of glyburide in beta cells, the cascade of electrical and ion-trans- port events that follow glyburide binding, and the synergy that occurs between the sulfonylurea and glu- cose in mediating insulin secretion. The receptor has been identified as an adenosine triphosphate-sensitive potassium ion channel or a very closely associated plasma membrane protein. Glyburide inhibits or inac- tivates this channel, resulting in retention of potas- sium ion or a positive charge near the plasma mem- brane, a decrease in resting membrane potential with depolarization, and calcium ion influx through the voltage-dependent calcium channel. The findings de- scribed in this section show that glyburide has the greatest binding affinity for these receptor proteins, which provides a rationale for the well-known greater overall clinical potency of glyburide compared with other sulfonylureas. Isolation and characterization of the channel proteins required for this process will pro- vide important insights into the mechanism that in-

duces a change in the permeability of such proteins when they are bound to certain drugs. Moreover, we may gain better understanding of how these drugs help restore the ability of the beta cell to recognize glucose as a potent physiologic secretagogue. Such insight may lead to the discovery of those molecular defects in the beta cell that contribute to the failure of insulin secretion in persons with or susceptible to NIDDM. The recent finding of adenosine triphos- phate-sensitive potassium ion channels in heart mus- cle cells that can be inhibited in similar fashion by gly- buride (and activated by the antihypertensive vascu- lar smooth muscle relaxant cromakalim) suggests an excellent alternative system for further characteriza- tion and structural studies on these channels. Studies on cardiac muscle may provide an opportunity to an- swer some of the fundamental questions raised by Boyd et aZ, such as those regarding the location and orientation of sulfonylurea binding on the channel pro- tein in the plasma membrane. Likewise, the coupling of this protein to signaling mechanisms can be more fully explored in a nonpancreatic (although electrically excitable) tissue to obtain important clues for the study of these agents in other extrapancreatic tissues.

The in vitro beta cell studies have helped to shed some light on in viva observations, such as those re- ported by O’Meara et al in this supplement. These workers have shown that, in patients with NIDDM, glyburide produces a greater amplitude in insulin se- cretory pulses after lunch and dinner (although not after breakfast), but the drug fails to produce a larger number of pulses over 24 hours. Likewise, when such patients were subjected to hyperglycemic clamp con- ditions at 300 mg/dL, there was evidence of increased beta cell responsiveness to the prevailing glucose lev- els, insulin secretion having been found to be elevated in response to glyburide by 221 percent over the 3 hours of the study. There was minimal influence on insulin secretion in the basal state, and the intrinsic abnormality seen in the beta cells of persons with NIDDM was only partially reversed by the drug. Thus, the ability of glyburide to bind to sulfonylurea receptors, to alter the permeability of ion channels, and to enhance the secretagogue activity of glucose results in a clinically significant “correction” for those persons with NIDDM in whom the defects are blunted glucose recognition and inefficient stimulus-secretion coupling. These studies help provide a basis for the clinical observation wherein, upon short-term treat- ment with glyburide, there is a significant increase in ambient insulin levels; with continued glyburide ad- ministration there is a reduction of insulin levels with- out a deterioration of glycemic control. Shortly after the initiation of glyburide, there is an enhancement of beta cell sensitivity to the pre-existing hyperglyce- mia. However, the ensuing hyperinsulinemia does not precipitate hypoglycemia, because of the extent of peripheral insulin resistance in these persons. The process of enhanced beta cell sensitivity then dissi-

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pates, probably through depletion of some labile cellu- lar element(s) essential for effective stimulus-secre- tion coupling. Alternatively, there might be some “down-regulation” of glucose recognition elements (glucoreceptors) only when there is effective stimulus- secretion coupling in the beta cells, an outcome that would be facilitated by the use of glyburide. Although there are obviously many possibilities to account for these phenomena, little data exist to support any one particular mechanism. Thus, there remains a compel- ling need for further investigation to determine the precise biochemical components involved in the devel- opment of NIDDM. Given the strong contribution of obesity to disease expression, it is of interest to deter- mine which defects are genetic and how these defects may be translated into disease mechanisms by envi- ronmental stresses and insults. Once elucidated, such information may indicate which properties of glybu- ride are responsible for its partial reversal of the de- fects, facilitating the design of yet more potent and targeted drugs. This effort will certainly require con- tinued investigation in vitro, complemented by in vivo studies, so that clinical efficacy as well as the loci of drug effects may be ascertained.

Among those loci that must be a target of any agent designed to treat NIDDM, the liver is certainly to be considered a major tissue. Caro’s excellent summary of the effects of glyburide on carbohydrate metabo- lism and insulin action in the liver provides still an- other body of evidence that may lead to delineation of the actual mechanism or mechanisms responsible for the effects of glyburide action in peripheral tissue. The demonstrated effects of the drug on glycolysis and glycogen synthesis make it possible to identify specific enzymes in those metabolic pathways that are rate-limiting and that may serve as the most likely sites of direct action for this agent. Thus, we now see that further work will be required at the cellular and molecular level to assess the regulatory effects of gly- buride on phosphofructokinase 1 and 2 activity, on fructobisphosphatase activity, and on fructose 1,6- bisphosphatase activity. Are there direct effects of glyburide on these enzymes, and if so, what are the structural requirements for such interactions? Are there genetic defects in the expression of one or more of these enzymes in NIDDM whose deficits are partly reversible by glyburide? What are the similarities be- tween the actions of the drug in beta cells and in pe- ripheral tissues such as liver? The intriguing work under way in Caro’s laboratory on coupling of insulin- receptor activity to a guanine-nucleotide binding pro- tein may indeed prove to shed additional light on the actions of glyburide. Moreover, although the potent clinical benefits of glyburide may result from effects on diverse metabolic pathways, the actual biochemical mechanisms that bring about such effects may eventu- ally prove to be similar. Indeed, there are other en- zymes affected by glyburide and similar agents in vitro. One interesting alternative system that may provide useful information in this area is the enzyme acetolactate synthetase, an enzyme found in bacteria that is strongly inhibited by sulfonylurea-type drugs. This enzyme has been extensively studied in Salmo- nella typhimurium. Genetic mutants of this enzyme that lose their susceptibility for inhibition by these agents have been described, suggesting that informa- tion regarding the structural requirements for such

drug interactions at the molecular level may be read- ily discernible in a convenient and highly manipulable in vitro model.

Until such studies have been conducted in more de- tail, our explanations for the physiologic effects of gly- buride on glucose homeostasis will remain inadequate, as reported by MeGuinness and Cherrington. The ele- gant studies summarized by these investigators have clearly pointed to the central role played by the liver in the regulation of overall glucose homeostasis, espe- cially basal glucose metabolism. In the presence of glyburide, there is a potent augmentation of insulin- induced hepatic glucose suppression. The data indi- cate, however, that the effectiveness of glyburide in suppressing hepatic glucose production in the complex in vivo setting is dependent on the presence of serum insulin concentrations above a certain physiologic threshold. These data help explain why poorly in- sulinized type I diabetic patients have not benefited from sulfonylurea therapy. Likewise, they show that in the presence of markedly elevated insulin levels, there is no enhancement by glyburide of the inhibition by insulin of glucagon-stimulated hepatic glucose pro- duction. Overall, the effect of the compound in liver is to increase sensitivity without altering responsive- ness. This implies that glyburide and insulin are ex- erting their effects through similar or the same, rather than parallel, metabolic pathways. The in vivo studies provide an excellent means of assessing the integration of all the diverse ways in which glyburide affects insulin-mediated glucose metabolism.

The data reported in this supplement indicate that the effects. of glyburide in liver on glucose homeostasis largely influence the production of glucose rather than its utilization. The effects on muscle, conversely, af- fect glucose utilization. Smith’s report summarizes our current understanding of the effects of glyburide on skeletal muscle cells. Although the current body of data is limited, it is apparent that the sulfonylureas exert potent effects on isolated muscle preparations and in cultured muscle cells. Smith points out that the variability of the reported in vitro effects in cultured muscle preparations to date may be in part a reflection of differences in the types of preparations used. He also points out that cells with properties more closely related to smooth muscle may show more direct re- sponses to the sulfonylureas, perhaps due to the local production of insulin-like growth factors that could mediate some or all of the effects attributed to this class of drugs in these cells. However, the data on cultured skeletal muscle cell models are perhaps more directly relevant to the drug actions in vivo on skele- tal muscle tissue. Thus, it is of interest that glyburide augments the response of insulin in cultured skeletal muscle cells but does not have direct stimulatory ef- fects on glucose uptake. Glyburide appears to induce directly the formation of glucose transporters, al- though the presence of insulin is required for activa- tion of these transporters. Thus, there is evidence for a requisite synergism between glyburide and insulin in muscle for optimal effects on glucose uptake. Both agents stimulate the synthesis of transporters, but it remains unclear whether these transporters are of the same molecular species. Although much remains to be learned, the studies in this skeletal muscle model pro- vide important insights into the molecular basis of the actions of glyburide in the muscle tissue of patients

SYMPOSIUM ON GLYBURlDE/ GAVIN

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SYMPOSIUM ON GLYBURIDE I GAVIN

with NIDDM. The greater potency of this effect with glyburide, compared with other sulfonylureas that have similar actions, has yet to be explained and will be a focus of future studies.

The effectiveness of glyburide in enhancing muscle sensitivity to insulin in vivo was reviewed by Simon- son, who reported on his work in normal volunteers and in persons with insulin-dependent diabetes mel- litus or NIDDM. These studies have confirmed the ability of glyburide to enhance insulin action in both normal volunteers and in patients with NIDDM. No increase in insulin sensitivity was observed in patients with insulin-dependent diabetes mellitus who received glyburide. He concludes that the changes in insulin sensitivity in NIDDM may be due to the overall im- provement of metabolic control, since similar changes in peripheral tissue sensitivity have been observed in response to improved metabolic control attained with other means. He also suggests that there is a require- ment for endogenous insulin secretion for the benefi- cial effects of glyburide in humans. Although these studies are unable to provide direct evidence for spe- cific cellular mechanisms, they provide compelling corroboration for the tissues affected by glyburide in ways that result in clinically significant improvements of glucose metabolism in diabetes. They further define those clinical settings in which glyburide is expected to demonstrate clinical efficacy. Although it may be important to determine exactly what portion of the improved insulin sensitivity in glyburide-treated sub-

jects is due to overall improved metabolic status ver- sus direct effects of the drug, it may not be feasible to obtain this information with our current technology.

In closing, the reports presented in this supplement provide strong evidence that extrapancreatic actions of glyburide and other sulfonylureas, particularly in the liver, play an important role in the glucose-lower- ing effects of these agents. Although in vitro findings indicate that these extrapancreatic effects may be di- rect? the variability of such findings may be the result of differences in experimental protocols and heteroge- neity of cell types used. Much of the in vivo data indi- cate that the presence of insulin is required for the optimal beneficial effects of glyburide. The question of the extent to which improved insulin sensitivity ob- served in humans is due to improved overall metabolic control or improved pancreatic insulin secretion must for the moment remain unresolved. Nevertheless, much new insight has been gained concerning many of the molecular mechanisms through which glyburide exerts its actions in diverse tissues. The heterogene- ity of the NIDDM population makes it essential to characterize the full spectrum of variations that may exist in response to glyburide. The appropriate combi- nation of in vitro and in vivo studies, such as those reported here, will be essential to guide us to even more exciting and unique directions for the use of gly- buride and other, perhaps future, sulfonylureas in the treatment of NIDDM, one of the most serious chronic diseases of our time.

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