Pathways in ␤-Cell Stimulus-Secretion Coupling as Targets for Therapeutic Secretagogues Jean-Claude Henquin

Physiologically, insulin secretion is subject to a dual, useful tools to unravel the mechanisms of stimulus-secre- hierarchal control by triggering and amplifying path- tion coupling. Until recently, however, hypoglycemic sul- ؉ ways. By closing ATP-sensitive K channels (KATP chan- fonylureas were the only drugs used to stimulate insulin nels) in the plasma membrane, glucose and other secretion in patients with type 2 diabetes. metabolized nutrients depolarize ␤-cells, stimulate The story of started in 1942, in Montpellier ؉ Ca2 influx, and increase the cytosolic concentration of (rev. in 3). Marcel Janbon and his colleagues recognized 2؉ 2؉ free Ca ([Ca ]i), which constitutes the indispensable that some patients receiving a sulfonamide (2254RP) for triggering signal to induce exocytosis of insulin gran- the treatment of typhoid fever were experiencing severe ules. The increase in ␤-cell metabolism also generates -؉ hypoglycemia. Auguste Loubatie`res rapidly confirmed ex amplifying signals that augment the efficacy of Ca2 on the exocytotic machinery. Stimulatory hormones and perimentally that the drug was causing hypoglycemia and, neurotransmitters modestly increase the triggering sig- in 1946, at the end of a remarkable series of experiments nal and strongly activate amplifying pathways biochem- for the time, concluded that the underlying mechanism ically distinct from that set into operation by nutrients. was a direct stimulation of insulin secretion by 2254RP. In Many drugs can increase insulin secretion in vitro, but 1955, in Berlin, Franke and Fuchs reported that another only few have a therapeutic potential. This review iden- antibacterial sulfonamide, carbutamide, also caused hypo- tifies six major pathways or sites of stimulus-secretion glycemia. The drug was rapidly used to treat diabetic coupling that could be aimed by potential insulin-secret- patients who did not require insulin, and was followed 1 ing drugs and describes several strategies to reach these year later by (3). The discovery of hypoglyce- targets. It also discusses whether these perspectives are mic sulfonylureas was thus serendipitous. Although many realistic or theoretical only. These six possible ␤-cell targets are 1) stimulation of metabolism, 2) increase of drugs have since been reported to exert hypoglycemic side 2؉ ؉ effects, none has had such a prolific progeny. The reason [Ca ]i by closure of K ATP channels, 3) increase of ,2؉ of the success of sulfonylureas is obvious. They all [Ca ]i by other means, 4) stimulation of amplifying pathways, 5) action on membrane receptors, and 6) including the mother compound 2254RP (3), act on ATP- ϩ action on nuclear receptors. The theoretical risk of sensitive K channels (KATP channels), which play a inappropriate insulin secretion and, hence, of hypogly- central role in the regulation of insulin secretion by cemia linked to these different approaches is also envis- glucose itself. aged. Diabetes 53 (Suppl. 3):S48–S58, 2004 Nowadays, search for novel insulin secretagogues is guided by our knowledge of stimulus-secretion coupling in ␤-cells. In this review, I shall first outline the major mechanisms regulating insulin secretion before discussing ptimal treatment of type 2 diabetes is difficult how distinct pathways or sites of action could serve as because of the complex pathogenesis of the therapeutic targets. disease (1,2). Pharmacological agents improv- Oing the action of insulin on its target tissues and agents correcting the deficient secretion of insulin by THE PHYSIOLOGICAL CONTROL OF INSULIN ␤-cells both have a place in our armamentarium. Many SECRETION chemicals can increase insulin secretion in vitro and are Insulin secretion is subject to tight control by glucose, other nutrients, neurotransmitters, and hormones. Al- From the Unite´ d’Endocrinologie et Me´tabolisme, University of Louvain though numerous and complex, the mechanisms underly- Faculty of Medicine, Brussels, Belgium. ing this multifactorial regulation can be schematized by a Address correspondence and reprint requests to J.C. Henquin, Unite´ hierarchical interaction between triggering and amplifying d’Endocrinologie et Me´tabolisme, UCL 55.30, avenue Hippocrate 55, B-1200 Brussels, Begium. E-mail: [email protected]. pathways (Fig. 1) (4,5). Received for publication 8 April 2004 and accepted in revised form 20 May When the concentration of glucose increases, ␤-cell 2004. metabolism accelerates, leading to closure of K chan- This article is based on a presentation at a symposium. The symposium and ATP the publication of this article were made possible by an unrestricted educa- nels in the plasma membrane. These channels are com- ϩ tional grant from Servier. posed of the pore-forming K IR6.2 and the regulatory [Ca2ϩ] , free cytosolic Ca2ϩ concentration; Epac, exchange protein activated i receptor 1 (SUR1). Binding of intracellular by cAMP; GEF, guanine nucleotide exchange factor; GIP, glucose-dependent ϩ insulinotropic polypeptide; GLP-1, glucagon-like peptide 1; GTP, guanosine ATP to K IR6.2 closes the channel, whereas binding of ϩ triphosphate; KATP channel, ATP-sensitive K channel; PKA, protein kinase A; MgADP to SUR1 opens the channel. The increase in the PKC, protein kinase C; PPAR, peroxisome proliferator–activated receptor; SUR1, sulfonylurea receptor 1. ATP/ADP ratio resulting from the metabolism of glucose © 2004 by the American Diabetes Association. thus closes the channel (Fig. 1). The consequence is a

S48 DIABETES, VOL. 53, SUPPLEMENT 3, DECEMBER 2004 J.-C. HENQUIN

FIG. 1. Schematic representation of the triggering and amplifying pathways involved in the control of insulin secretion by glucose and other agents, and identification of potential sites of action (1–6) for pharmacological insulin secretagogues. ؉, stimulation; ؊, inhibition. See also Table 1. depolarization of the plasma membrane, with opening of nisms. They also produce major amplifying signals, mainly voltage-dependent Ca2ϩ channels, acceleration of Ca2ϩ through activation of protein kinases, in particular protein influx, and increase in the concentration of cytosolic free kinase A (PKA) and protein kinase C (PKC) (14–16,18–19). 2ϩ 2ϩ Ca ([Ca ]i) that is necessary and sufficient to trigger In addition to PKA, cAMP-regulated guanine nucleotide insulin secretion (6–10). However, this triggering signal exchange factors (GEFs, or Epac) might mediate part of alone is poorly effective. Its efficacy is augmented by an the effects of cAMP on insulin secretion (16,20). Activation amplifying pathway also using signals issued from glucose of PKA or PKC, or of GEF/Epac, augments the efficacy of metabolism. The nature of these signals and their intracel- Ca2ϩ on exocytosis. The biochemical mechanisms of this lular targets are still unclear, but a role of ATP and ADP is type of amplification are, however, distinct from those plausible (11–13). The same dual regulation applies to all implicated in the amplifying pathway of glucose and other nutrients that are actively metabolized and increase the nutrients (5). ATP/ADP ratio in ␤-cells (5). Although no direct evidence Finally, inhibitory hormones and neurotransmitters also is as yet available, both clinical investigation of diabetic act via two pathways. They depress insulin secretion patients and experimental studies of animal models sug- partly by decreasing the triggering signal (via membrane gest that the two pathways, triggering and amplifying, may repolarization) and mainly by reducing the efficacy of Ca2ϩ be impaired in ␤-cells affected by type 2 diabetes (4). on exocytosis (attenuating pathway operating via kinases 2ϩ Importantly, the triggering signal (rise in [Ca ]i) can or small guanosine triphosphate [GTP]-binding proteins) also be produced or augmented by mechanisms that are (21). independent of KATP channels (Fig. 1). Some hormones and neurotransmitters mobilize Ca2ϩ from intracellular stores (14–16). Agents acting on various ionic channels HOW CAN DRUGS FOOL ␤-CELLS TO SECRETE (e.g., inhibitors of voltage-dependent Kϩ channels) can EXCESSIVE AMOUNTS OF INSULIN? augment glucose-induced depolarization, thereby potenti- Hypoglycemia induced by excessive insulin secretion is a 2ϩ ating the [Ca ]i rise (17). Cationic amino acids, like major complication of current pharmacological treatments arginine, are poorly metabolized but depolarize ␤-cells of type 2 diabetes. Under physiological conditions, it is the because of their entry in a positively charged form, thus triggering pathway that determines whether insulin is without direct interaction with an ionic channel (5). secreted. The amplifying pathway serves to optimize the Stimulatory hormones and neurotransmitters, such as secretory response induced by the triggering signal but 2ϩ glucagon-like peptide 1 (GLP-1) and acetylcholine, usually does not induce secretion if [Ca ]i is not increased (5). potentiate insulin secretion by a dual action. They moder- This strict hierarchy between the two pathways can be 2ϩ ately increase the triggering signal (rise in [Ca ]i) through perturbed by pathological defects or by drugs. Theoreti- complex, variable, but largely glucose-dependent mecha- cally, actions on either the triggering or amplifying path-

DIABETES, VOL. 53, SUPPLEMENT 3, DECEMBER 2004 S49 PHARMACOLOGICAL INSULIN SECRETAGOGUES

TABLE 1 Site 1: Improvement of ␤-cell metabolism. Alterations Potential sites of action of insulin-secreting drugs of glucose metabolism often characterize ␤-cells from ␤ Site 1: Stimulation of ␤-cell metabolism animal models of type 2 diabetes and most likely -cells Activation of glucokinase from type 2 diabetic patients (23). Correction of specific Inhibition of glucose-6-phosphatase defects would obviously be optimal, but a global improve- Alternative fuels ment of glucose/fuel metabolism in ␤-cells could also have Inhibition of mitochondrial Naϩ/Ca2ϩ exchanger several advantages: restoration of normal triggering and 2؉ ␤ Site 2: Increase of -cell [Ca ]i by blockade of KATP amplifying signals, stimulation of proinsulin biosynthesis channels with lesser danger of store exhaustion, and little risk of Interaction with SUR1 ϩ hypoglycemia if the control steps are not offset. Interaction with K IR6.2 ␤ ؉ Site 3: Increase of [Ca2 ] by action at sites other than Theoretically, -cell metabolism might be boosted by a i selective action on an identified perturbed site (e.g., hypo- KATP channels Blockade of other Kϩ channels or hyperactive enzyme due to a mutation or a change in Activation of Ca2ϩ channels expression), by supply with alternative fuels that bypass a Action on other ionic channels defective enzyme or pathway, and by less specific, indirect ͓ 2ϩ͔ Inhibition of Ca i-lowering processes measures to accelerate global metabolism. Some of these ␤ Site 4: Stimulation of amplifying pathways in -cells approaches have already been tested experimentally. Activation of the nutrient-mediated amplification Allosteric activation of glucokinase. At the entry of Inhibition of AMP kinase Inhibition of 11␤-hydroxysteroid dehydrogenase type 1 glycolysis, glucokinase plays a primary regulatory role in Sensitization to Ca2ϩ the control of glucose metabolism in ␤-cells (10). Loss of Inhibition of cAMP degradation function and gain of function of glucokinase respectively Activation of the PKC pathway cause deficient and excessive insulin secretion in type 2 Site 5: Action on ␤-cell membrane receptors maturity-onset diabetes of the young (MODY2) patients Antagonists of inhibitory receptors and in certain infants with persistent hyperinsulinemic Agonists of stimulatory receptors ␤ hypoglycemia (24,25). An allosteric activator of glucoki- Site 6: Action on -cell nuclear receptors nase (compound RO-28-1675) has been developed recently (26). It activates glucose metabolism and lowers the threshold concentration for glucose-induced insulin secre- ways could result in hypoglycemia. Excessive insulin tion in rat islets. It also increases plasma insulin and secretion will occur if a drug increases the triggering decreases plasma glucose levels in normal mice and signal even when the glucose concentration is low, and promotes glucose usage in the liver (26). This novel family ␤ 2ϩ does so to such an extent that -cell [Ca ]i remains of drugs opens interesting perspectives for the develop- effective on exocytosis in face of the decrease in amplifi- ment of original antidiabetic agents. One should, however, cation that automatically accompanies a fall in blood be aware that patients taking excessive doses of such a glucose. This is how potent and long-acting sulfonylureas compound will mimic the activating mutations of glucoki- sometimes provoke hypoglycemia. Excessive insulin se- nase and thus incur a risk of hypoglycemia. cretion can also occur if a drug produces an amplifying 2ϩ Inhibition of glucose-6-phosphatase. Glucose-6-phos- signal that makes a rise in [Ca ]i unnecessary to trigger phatase is essential for glucose production by the liver, but insulin secretion. This is how strong and combined acti- its operation in ␤-cells creates a futile cycle of glucose vation of PKA and PKC increases insulin secretion at very ϩ phosphorylation and dephosphorylation, with reduction of low ␤-cell [Ca2 ] in vitro (22), but it is unlikely that i glucose usage and insulin secretion as consequences (27). amplifying pathways can ever be stimulated to such an The activity of the enzyme is insignificant in normal extent in vivo. The risk of a moderate stimulation of ␤-cells, and the possibility that an increase in activity amplifying pathways is minimal because excessive de- contributes to abnormal insulin secretion in animal mod- crease in blood glucose automatically stops generation of els of diabetes remains controversial (28–30). Available the triggering signal and thus the insulin-secreting effect of drugs inhibiting hepatic glucose-6-phosphatase are inac- the amplifying drug. This safeguard would however be lost tive on the high enzyme activity in ob/ob mouse islets (31). if glucose no longer could modulate the magnitude of the For this approach to be successful, it should first be triggering signal because another drug has taken over established that glucose-6-phosphatase is so overactive in control of the K channels. The combination of drugs ATP ␤-cells from type 2 diabetic patients that it eventually acting potently on both triggering and amplifying path- impairs oxidative glucose metabolism and that the enzyme ways might thus be dangerous. can be inhibited independently from its hepatic congener. Alternative fuels. Esters of carboxylic metabolites are THEORETICAL AND REALISTIC ␤-CELL TARGETS FOR effective insulin secretagogues in vivo and in vitro (32). In THERAPEUTIC DRUGS contrast to succinic, glutamic, and pyruvic acid, their Figure 1 schematizes the control of insulin secretion in a methyl or ethylesters enter ␤-cells, where the carboxylic normal ␤-cell and indicates six possible pathways or sites moiety is metabolized in the Krebs cycle and thus by- that could be targeted by drugs designed to increase passes the early and possibly defective steps of glucose secretion. Several strategies can theoretically be followed metabolism. Whereas problems linked to the mode of to reach these targets (Table 1), but we will see that they administration in vivo (route, amount) and to the produc- are not equivalent and that a number of shortcomings tion of methanol from the methylesters can be circum- often preclude transposition of theory into practice. vented (32), it remains to be established that undesirable

S50 DIABETES, VOL. 53, SUPPLEMENT 3, DECEMBER 2004 J.-C. HENQUIN effects on glucose usage/production by the liver and sulfonylureas do not promote proinsulin biosynthesis, it peripheral tissues do not outweigh favorable effects on has been suggested that, like glucose, they also stimulate diabetic ␤-cells. insulin secretion through an amplifying pathway (41,42). Monosaccharide esters, such as the pentaacetate ester This is based on electrophysiological studies in which of ␣-D-glucose, enter ␤-cells independently of the glucose single ␤-cells are usually patch-clamped in the whole cell transporters and stimulate insulin secretion (33). Because mode (permitting unrestricted exchange between cyto- it is unlikely that glucose transport ever becomes rate- plasm and pipette milieu), and exocytosis of insulin gran- limiting in human ␤-cells, the potential interest of these ules is monitored as changes in membrane capacitance. In 2ϩ compounds is elsewhere. Only minute amounts (much less the presence of fixed [Ca ]i, intracellularly applied sulfo- than of glucose itself) are indeed sufficient to increase nylureas increase exocytosis (41,42). It is therefore pro- plasma insulin levels in normal rats. Unexpectedly, in vitro posed that sulfonylureas penetrate ␤-cells and interact stimulation of insulin secretion is also observed with low with SUR1 or a related protein in the membrane of the concentrations of pentaacetate esters of nonmetabolized insulin granules to confer them release competence by hexoses such as L-glucose or 2-deoxyglucose. The effects facilitating their acidification. Surprisingly, this effect per- of these ester compounds are not attributed to an increase sists in Sur1 KO ␤-cells, implying that the intracellular in ␤-cell metabolism, which is rather inhibited, but to an binding protein is not SUR1 (43). It would be expected interaction with a still unidentified receptor (33). Develop- therefore that sulfonylureas retain an effect on insulin ments in this area await further studies on the mode of secretion—via the intracellular binding sites—in intact ␤ action and potential negative effects of these compounds, -cells without KATP channels. as well as demonstration that their insulin-releasing activ- Figure 2 shows that unstimulated (1 mmol/l glucose) ity is retained in models of type 2 diabetes. and stimulated (15 mmol/l glucose) insulin secretion was Inhibition of the mitochondrial Na؉/Ca2؉ ex- increased by 10 and 50 nmol/l in control ␤ 2ϩ changer. The rise in -cell [Ca ]i produced by glucose mouse islets. In Sur1 KO islets, insulin secretion was and other secretagogues is followed by an influx of Ca2ϩ already high in 1 mmol/l glucose and was further increased into mitochondria. It has been proposed that, by activating by 15 mmol/l glucose, which reflects the operation of the matrix dehydrogenases, Ca2ϩ augments the production of triggering pathway and the effectiveness of the amplifying ATP and other putative messenger molecules (34,35). The pathway of glucose in the absence of SUR1 (44). Gliben- exit of Ca2ϩ from mitochondria is mediated by a Naϩ/Ca2ϩ clamide, however, was completely ineffective, even at 1 exchanger, whose blockade might thus be expected to ␮mol/l, a concentration that largely exceeds those reached promote metabolism. Compound CGP 37157 was devel- in vivo (Fig. 2). Extracellular tolbutamide was also without oped for this purpose and was indeed found to increase effect on insulin secretion in islets without KATP channels 2ϩ 2ϩ ϩ mitochondrial [Ca ], ATP production, cytosolic [Ca ]i, due to a knockout of Sur1 (44,45) or K IR6.2 (46) at both and insulin secretion in INS-1 cells incubated in the low and high glucose levels. presence of glucose (36). It also increased glucose-in- In conclusion, intracellularly applied sulfonylureas in- duced insulin secretion by rat islets and augmented teract with a binding site probably on the insulin granule plasma insulin levels at certain time points of a hypergly- membrane (42). It is plausible that this site is somehow cemic clamp. Although encouraging, these preliminary implicated in the response to physiological secretagogues, results should be confirmed with related compounds, and but it does not seem relevant to the therapeutic action of the consequences of their application for longer periods sulfonylureas. should be evaluated in primary ␤-cells, before deciding Glinides. The nonsulfonylurea (benzamido) part of glib- whether this new approach is worth pursuing. The issue of enclamide, known as or HB-699, possesses tissue selectivity may also be difficult to solve. blood glucose–lowering properties that have been attrib- uted to stimulation of insulin secretion (47). Twenty years Site 2: Increasing the triggering signal by acting on ago it was shown that meglitinide mimics the sequence of KATP channels events by which tolbutamide and glibenclamide trigger Sulfonylureas. The binding of sulfonylureas to SUR1 insulin secretion (48). A number of other nonsulfonylurea leads to closure of KATP channels with subsequent depo- compounds have been developed more recently, some of larization of the plasma membrane, activation of Ca2ϩ which are already in clinical use (49). The best known are 2ϩ influx, and rise of [Ca ]i. These drugs mimic the effect of (AGEE-623), (A-4166), and glucose in the generation of the triggering signal, but do so (KAD-1229 or S-21403). They are functionally independently of changes in ␤-cell metabolism. The differ- related but structurally different. The common family ent sites of sulfonylurea binding to SUR1 and the molec- name of “glinides” derives from their trade name but does ular mechanisms of its transduction into closure of not refer to any specific chemical structure. Except for ϩ K IR6.2 have been reviewed recently (37–40). In brief, repaglinide, it is incorrect to call them “meglitinide ana- SUR1 possesses an “A” site to which binds tolbutamide logs” or “benzamido compounds.” Meglitinide and repa- and the half of the glibenclamide molecule containing the glinide bind to the “B” site in SUR1, whereas nateglinide sulfonylurea group, and a “B” site to which binds the and mitiglinide bind to the “A” site (39,40). Whatever the nonsulfonylurea half of glibenclamide. Binding to one of binding site, their eventual effect is similar to that of these two sites is sufficient to produce the same final sulfonylureas: depolarization of ␤-cells, with subsequent 2ϩ effects: closure of KATP channels, membrane depolariza- rise in [Ca ]i and triggering of insulin secretion. 2ϩ ␤ tion, and rise in [Ca ]i. Measurements of -cell capacitance have also been used Whereas it is widely accepted that, unlike glucose, to assess whether glinides amplify insulin secretion at an

DIABETES, VOL. 53, SUPPLEMENT 3, DECEMBER 2004 S51 PHARMACOLOGICAL INSULIN SECRETAGOGUES

FIG. 2. Lack of effect of glibenclamide on insulin

secretion by mouse islets lacking KATP channels (Sur1KO mice). Islets from 10-month-old control and Sur1KO mice (45) were cultured overnight in RPMI medium containing 10 mmol/l glucose before being incubated (batches of three islets) for1hinKrebs medium containing 1 or 15 mmol/l glucose and the indicated concentration of glibenclamide. Insulin was measured in the incubation medium. Values are means ؎ SE for 17 batches of islets from three separate preparations. The islet insulin content was 138 ؎ 4 and -ng/islet for controls and Sur1KO mice, respec 4 ؎ 145 tively.

ϩ intracellular site. Nateglinide, but not repaglinide, pro- with K IR6.2 (64). This effect largely explains their stimu- duces such an effect (50), but, again, this conclusion is not lation of insulin secretion. However, efaroxan and novel supported by experiments using intact islets. Thus, nateg- imidazoline compounds also or exclusively act on an linide, repaglinide, and mitiglinide do not increase insulin amplifying pathway, as will be discussed below. secretion from islets depolarized with KCl (51). As for From a mechanistic point of view, it is unimportant sulfonylureas, I conclude that the intracellular effect of whether drugs close KATP channels directly by an interac- ␤ ϩ some glinides, observed in dialysed single -cells, is not tion with the pore formed by K IR6.2 or indirectly by an therapeutically important. interaction with the regulatory subunit SUR1. The net In conclusion, the value of the glinides as insulin secre- result is the same: membrane depolarization, influx of tagogues does not reside in an original mode of action but Ca2ϩ, and generation of the triggering signal. The major in pharmacokinetic properties associated with a rapid difference, however, is the distribution of the two targets. onset of action and a rapid reversibility of action, at least The much more restricted distribution of SUR1 than ϩ for mitiglinide and nateglinide. Both also display an advan- K IR6.2 considerably increases the tissue specificity of the ϩ tageous greater selectivity for SUR1 than SUR2A or drugs acting through it. Moreover, drugs closing K IR6.2 ␤ SUR2B (38) and, hence, for KATP channels of the -cell. In directly usually also affect other channels (61). this context, it has been speculated that drugs closing KATP ␤ ϩ channels in -cells (SUR1/K IR6.2) and opening KATP Site 3: Increasing the triggering signal without acting ϩ channels in vascular muscles (SUR2B/K IR6.2) would be on KATP channels. In vitro, all depolarizing agents in- interesting to treat type 2 diabetic patients with hyperten- crease insulin secretion regardless of the mechanism sion. Agents like sulfate (52) and MCC-134 (53) underlying the depolarization. However, this approach has have such a profile, but their opposite effects on the two limited clinical applications notably because of the diffi- isoforms of the channel do not occur within the same culty to achieve tissue selectivity. A further underesti- ␤ concentration range. Blockade of the -cell KATP channel mated problem is that the depolarizing action of a requires concentrations that largely exceed those needed substance may be markedly dependent on the electrical to produce vasorelaxation. resistance of the ␤-cell membrane, which is essentially ؉ Drugs interacting with K IR 6.2. Many structurally determined by the extent of KATP channel closure. One different drugs used for the treatment of diseases other instructive example is that of arginine and other cationic than diabetes occasionally produce hypoglycemia. In vitro amino acids that depolarize ␤-cells because of their entry studies have shown that they increase insulin secretion by in a positively charged form (Fig. 1). The effects of arginine ␤ 2ϩ closing -cell KATP channels through a direct interaction on membrane potential and [Ca ]i increase with the ϩ with K IR6.2. Among these drugs are antimalarial quino- glucose concentration (65), which partially accounts for lines such as quinine and mefloquine (54,55), antibacterial the glucose dependency of arginine-induced insulin secre- ␤ fluoroquinolones such as norfloxacin and lomefloxacin tion. Defective closure of KATP channels in diabetic -cells (56), antiarrhythmic agents such as and may be expected to impair the production of a triggering cibenzoline (57–59), and others (60). signal by agents that depolarize by activating an inward More interest has been paid to drugs with an imidazoline current. The second mechanism explaining the glucose structure (phentolamine, antazoline, midaglizole), which dependency of insulin secretion induced by arginine and ␤ also inhibit KATP channels in -cells (61–63) by interacting related agents is the amplifying action of glucose (5).

S52 DIABETES, VOL. 53, SUPPLEMENT 3, DECEMBER 2004 J.-C. HENQUIN

؉ Blockers of K channels other than KATP channels. suggestions for the development of insulin-releasing drugs The major voltage-dependent Kϩ channel in ␤-cells, Kv 2.1, specifically acting through that pathway are premature. participates in the repolarization of the membrane during One candidate effector is the SUR1-like protein of the spike generation (17). Its blockade augments the influx of insulin granule membrane (42). Whereas this protein is Ca2ϩ and has long been shown to increase insulin secre- unlikely to contribute to the insulin-releasing action of tion (66). The wide tissue distribution of these channels is sulfonylureas (see above), it might participate in the a major limitation in their use as a target for insulin- amplifying action of glucose and could thus become the secreting drugs in vivo. However, because the activity of target of novel drugs. SUR1 itself is also present in the these channels may be subject to modulation by different granule membrane (74), but the amplification of insulin subunits and to regulation by hormonal or metabolic secretion by nutrients is not altered in its absence (44). signals, it has been speculated that the development of Another possible target is AMP-activated protein kinase. selective blockers of ␤-cell voltage-dependent Kϩ channels Inhibitors of AMP activated protein kinase. Islet AMP is not unrealistic (17). kinase activity is inhibited by glucose, which raises the Agonists of Ca2؉ channels. The main voltage-depen- possibility that the enzyme participates in a long- or 2ϩ dent Ca channels in ␤-cells are of the L-type and are short-term control of ␤-cell function, including insulin inhibited by dihydropyridines, which decrease insulin se- secretion (75). Activation of AMP kinase by AICAR or 2ϩ cretion by lowering the triggering signal [Ca ]i (67). Some overexpression of a constitutively active form of AMP dihydropyridines ( or CGP2832) instead act as kinase-␣1 inhibited glucose-stimulated insulin secretion agonists. Almost 20 years ago they were shown to increase but also virtually abolished glucose metabolism and the 2ϩ ␤ 2ϩ ␤ Ca influx in -cells and insulin secretion in vitro (60). [Ca ]i rise in -cells (75), making it difficult to identify the Since then, no progress has been made in the field and, site(s) of action. Conversely, a dominant negative form of because of insufficient tissue selectivity, the development AMP kinase increased basal insulin secretion, but this of this class of compounds for clinical use has been increase was Ca2ϩ-independent (75), which is in sharp discontinued. contrast with all effects of glucose (4). Thus, it is prema- Other ionic channels. ␤-cells are equipped with many ture to suggest that ␤-cell selective inhibitors of AMP other ionic channels that may subtly modulate the changes kinase might become insulin secretagogues. in membrane potential induced by glucose and other On the other hand, because the widely used secretagogues. None appears to have a sufficiently impor- can activate AMP kinase (76), it is important to discuss tant role in stimulus-secretion coupling or a sufficiently whether the drug exerts deleterious effects in ␤-cells. restricted tissue distribution to qualify as a target for Culture of human islets for 16 h in the presence of 1 insulin-secreting drugs. mmol/l metformin (ϳ100-fold the plasma concentration in 2؉ 2ϩ Inhibition of [Ca ]i-lowering processes. The [Ca ]i patients) (77) virtually abolished glucose-induced insulin rise induced by glucose and other secretagogues must be secretion (78), whereas no effect was observed after counterbalanced by Ca2ϩ sequestration in intracellular culture with 20 ␮mol/l metformin (79). Permeation of organelles and by Ca2ϩ extrusion to the extracellular metformin into cells is admittedly slow, but the key space. These tasks are achieved by several Ca2ϩ-ATPases unanswered question is whether in vitro exposure to a and Naϩ/Ca2ϩ exchangers (68–70). Inhibition of one or very high or to a therapeutic concentration of metformin several of these processes is followed by a variable rise of for a few hours only is equivalent to chronic exposure to 2ϩ [Ca ]i and increase in insulin secretion (70,71). However, the drug in vivo. When patients are treated with met- should effective blockers of the plasma membrane Ca2ϩ- formin, their plasma insulin level often decreases. This is ATPase and Naϩ/Ca2ϩ exhanger become available, tissue attributed to improvement of the metabolic state rather selectivity will still remain a serious issue. Moreover, the than to inhibition of insulin secretion by the drug (77). This endoplasmic reticulum stress provoked by blockade of interpretation is supported by a study showing that 2 intracellular Ca2ϩ-ATPases is likely to have deleterious weeks of metformin administration to subjects without consequences (72) that preclude this approach. glucose intolerance did not change their insulin response to a hyperglycemic clamp (80). Site 4: Activation of amplifying pathways Inhibition of 11␤-hydroxysteroid dehydrogenase Nutrient-mediated amplification. All measures result- type 1. Glucocorticoids impair glucose homeostasis ing in acceleration of ␤-cell metabolism (see above) in- mainly by opposing the effects of insulin on hepatic crease insulin secretion by a dual action on the triggering glucose production and peripheral glucose uptake. Al- and amplifying pathways. It is accepted that the ATP/ADP though plasma insulin is elevated in glucocorticoid- ratio serves as a second messenger in the generation of the induced insulin resistance, the direct effect of glucocorti- triggering signal not only because its changes occur over a coids on insulin secretion is inhibitory (81). Overexpres- wide physiological range of glucose concentrations (73), sion of the glucocorticoid receptor in ␤-cells leads to but also because the transducing molecules (SUR1 and hyperglycemia and hypoinsulinemia after several months ϩ K IR6.2) are known. In contrast, the molecular mecha- (82). The mechanisms of this inhibition of insulin secre- nisms of the amplifying pathway remain unclear, and the tion may be multifactorial (82), but interference with the possibility that several factors are involved cannot be amplifying action of glucose is probable (81). Local trans- ruled out (Fig. 1). Our proposal (4,73) that variations in the formation of inactive cortisone into active cortisol by ATP/ADP ratio (or associated changes in AMP or the 11␤-hydroxysteroid dehydrogenase type 1 could play a GTP/GDP ratio) are implicated suffers from one shortcom- pathogenic role in the metabolic syndrome, and inhibition ing: the effector molecules have not been identified, so that of the enzyme is envisaged as a potential therapy in type 2

DIABETES, VOL. 53, SUPPLEMENT 3, DECEMBER 2004 S53 PHARMACOLOGICAL INSULIN SECRETAGOGUES diabetes and obesity (83,84). 11␤-Hydroxysteroid dehydro- ␤-cells and from islets depolarized with KCl, two prepara- 2ϩ genase type 1 is present in mouse and human islets (85), tions in which [Ca ]i is clamped (96). A novel imidazoline and its activity is increased in proportion to hyperglycemia (BL11282) has no effects on KATP channels but increases in the islets from diabetic ZDF fa/fa rats (86). Inhibition of insulin secretion in vitro (97). Three compounds, the enzyme in mouse islets prevents precursors of active RX871024 (98), efaroxan (N.G. Morgan, personal commu- corticosteroids from decreasing insulin secretion in vitro nication), and BL11282 (S. Efendic, personal communica- (85). This approach deserves further investigation. tion) increase insulin secretion from ␤-cells deficient in Calcium sensitizers. Drugs are being developed with KATP channels. All these observations point to an intracel- the aim of increasing the action of Ca2ϩ on contractile lular site of action of imidazolines. This site is still proteins, in particular to improve myocardial performance unidentified and has tentatively been designated “I3-imida- (87). One of these, pimobendan, increases insulin secre- zoline binding site” (62). A number of experimental argu- tion by isolated rat islets. Because it remains effective ments suggest that it is involved in the amplification of ␤ 2ϩ when -cell [Ca ]i is clamped by high KCl, the drug insulin secretion by PKA and mainly PKC (62,97) and, indeed behaves like a Ca2ϩ sensitizer (88). However, this thus, not in the amplifying pathway of glucose, which is study did not investigate whether the effect could be independent of these kinases (4). Although structurally mediated by the phosphodiesterase inhibition that pimo- unrelated to imidazolines, ␤-carbolines might be endoge- bendan can also cause (87). Effects of levosimendan, nous ligands of the “imidazoline binding sites.” Interest- another available Ca2ϩ sensitizer, on the endocrine pan- ingly, two ␤-carbolines, harmane and pinoline, possess creas have not been reported. insulin-releasing properties in vitro (99). Agents acting on the cAMP pathway. Cyclic AMP is a In summary, the family of insulin-releasing imidazolines potent amplifier of insulin secretion (14,18,89). Inhibition is functionally heterogeneous. Older members (phentol- of its degradation by methylxanthines has long been amine, antazoline) owe their property to blockade of KATP known to increase plasma insulin concentrations (90). In channels (triggering pathway), whereas newer ones ␤-cells, this degradation is mainly achieved by phosphodi- (BL11282) increase the action of Ca2ϩ on exocytosis esterase 3B, the inhibition of which strongly augments (amplifying action). Because these novel compounds do insulin secretion in vitro (89). Conversely, overexpression not produce a triggering signal, their effect on insulin of phosphodiesterase 3B in ␤-cells reduces in vivo and in secretion is more strongly glucose dependent (97). How- vitro insulin secretion in response to glucose and gluca- ever, the advantage of this gain in safety could be can- gon-like peptide-1 (GLP-1) and impairs glucose tolerance celled by loss of tissue specificity. Identification of their (91). Unfortunately, phosphodiesterase 3B is present in intracellular site of action (I3-site) and investigation of many other tissues, including hepatocytes and adipocytes their possible effects on kinases in other tissues are where its blockade exerts anti-insulin effects. There is necessary before deciding whether imidazolines have a presently no evidence that cAMP degradation can be future as insulin secretagogues. selectively prevented in ␤-cells by pharmacological agents (89). Hormones acting on ␤-cell membrane receptors Site 5: Inhibitory and stimulatory membrane recep- linked to adenylyl cyclase are more promising insulin tors secretagogues (see below). Antagonists of inhibitory receptors. ␤-Cells are As already mentioned, the increase in insulin secretion equipped with a number of inhibitory receptors whose produced by cAMP is not exclusively mediated by PKA but activation decreases insulin secretion. The major ones are ␣ also involves GEF/Epac (20). Novel cAMP analogs activate the 2-adrenergic, galanin, and somatostatin receptors GEF/Epac selectively and increase insulin secretion in (100). Their inhibitory effects are mediated by complex vitro (16,43). However GEF/Epac is operative in other mechanisms that include partial repolarization with a 2ϩ tissues, and it is uncertain whether some of these analogs small decrease in [Ca ]i, inhibition of adenylyl cyclase will show enough tissue selectivity to be of therapeutic with suppression of the amplifying effects of cAMP, as well interest. as a poorly explained but major interference with the Imidazolines and the PKC pathway. The interest in action of Ca2ϩ on exocytosis (21). imidazoline compounds (such as phentolamine) as poten- The use of antagonists of inhibitory receptors could be tial insulin secretagogues started with the idea that their justified if tonic activation of these receptors contributed ␣ ability to block 2-adrenoceptors could relieve diabetic to the impairment of insulin secretion in pathological ␤-cells from a tonic sympathetic inhibition (92,93). Subse- states. Studies in the 1970s suggested that excessive quently, it was shown that the increase in insulin secretion adrenergic activity could impede insulin secretion in type that phentolamine, efaroxan, and other imidazolines pro- 2 diabetic patients (92,93), but this was not confirmed duce in vitro and in vivo is largely mediated by a blockade (101). Moreover, the plasma insulin increase that some ␤ ␣ of KATP channels in -cells (see above) rather than of 2-adrenoceptor blockers (e.g., phentolamine) produce is ␣ 2-adrenoceptors (61–63,94). now ascribed to a direct inhibition of KATP channels (see However, different experimental approaches and the above). Finally, because adrenergic inhibition of insulin development of novel compounds have shown that the secretion is a safeguard against hypoglycemia during mode of action of imidazolines is complex. Compound exercise, blockade of this action may not be without risk. KU14R, the imidazol analog of efaroxan, does not interfere Effects of antagonists of somatostatin or galanin recep- with the action of the latter on KATP channels, yet it tors on insulin secretion have not been reported. Mice inhibits its action on insulin secretion (95). Compound with a general knock-out of the somatostatin receptor type RX871024 increases insulin release from permeabilized 5, the major type in ␤-cells, do not show significant

S54 DIABETES, VOL. 53, SUPPLEMENT 3, DECEMBER 2004 J.-C. HENQUIN

alterations of in vivo insulin secretion and glucose ho- glucose. Second, despite some inhibitory action on KATP meostasis. However, when their pancreas is perfused in channels, GLP-1 hardly produces a triggering signal at low vitro, insulin secretion is increased compared with con- glucose for two reasons. It mainly acts on ionic channels trols, particularly in the basal state (102). Inactivation of (voltage-dependent Ca2ϩ and Kϩ channels) that become the galanin gene unexpectedly reduces insulin secretion in operative only when the ␤-cell membrane has been depo- vivo and impairs glucose tolerance. The inhibition of larized by glucose (19). Moreover, the mobilization of insulin secretion persists in vitro (103). Overall, there are intracellular Ca2ϩ that GLP-1 promotes results from a no convincing arguments suggesting that blockade of Ca2ϩ-induced Ca2ϩ release, which itself depends on glu- somatostatin or galanin receptors in ␤-cells could be cose-induced influx of Ca2ϩ from the extracellular medium useful for increasing insulin secretion in type 2 diabetic (14,16). This glucose dependency of GLP-1 stimulation of patients. insulin secretion might however be lost if the hormone Agonists of stimulatory receptors. ␤-Cells possess were combined with a potent and long-acting sulfonylurea. numerous membrane receptors for stimulatory hormones or neurotransmitters, and several have already been con- Site 6: Nuclear receptors. Several nuclear receptors, sidered as potential therapeutic targets, but only few may including the liver X receptor (LXR) and the peroxisome qualify. proliferator–activated receptors (PPARs), play a central Acetylcholine is a physiologically important and potent role in lipid and carbohydrate metabolism in liver, mus- amplifyer of insulin secretion, but its effects in ␤-cells are cles, and adipose tissue (112,113). Their importance for mediated by muscarinic receptors of the M3 type, whose ␤-cell function is less clear. However, preliminary evi- characteristics and tissue distribution are such that selec- dence suggests that activation of LXR in ␤-cells may tivity of an agonist for insulin secretion is most unlikely influence insulin secretion (114). (15). PPAR-␣ and -␥ are moderately expressed in rodent and Extracellular glutamate increases insulin secretion from human islets (30,115–117) and may exert a long-term the perfused pancreas by acting on AMPA receptors, and influence on insulin secretion by controlling the expres- improves insulin secretion and glucose tolerance in vivo in sion of enzymes involved in fuel metabolism (118). Their rats (104). However, this approach is unlikely to be useful role in ␤-cell adaptation to metabolic perturbations (e.g., because of rapid desensitization of the receptor and insulin resistance) is under investigation, but it is known undesirable extrapancreatic effects of glutamate or ana- that PPAR-␣ is downregulated and PPAR-␥ upregulated in logs. islets from Zucker diabetic fatty rats (116). Similar Activation of purinergic receptors of the P2Y type changes are induced by exposure to hyperglycemia in vivo increases insulin secretion from rodent and human islets. or in vitro (30,117). Two families of clinically useful It is unclear, however, whether agonists with sufficient compounds, fibrates and , are synthetic ␤-cell selectivity can be designed (105). ligands of PPAR-␣ and -␥, respectively. Although they are Pituitary adenylate cyclase–activating polypeptide not prescribed with the specific aim of changing insulin (PACAP) exerts its effects through a PACAP-preferring secretion, such changes do occur. It is not always easy to receptor (PAC1) and two receptors shared by vasoactive determine if these effects are indirect or direct. intestinal peptide (VPAC1 and VPAC2). VPAC1 mediates PPAR-␣ and fibrates. An acute (Ͻ1 h) increase of stimulation of glucose production by the liver, whereas insulin secretion by fibrates has been observed in vitro, VPAC2 in ␤-cells mediates stimulation of insulin secretion but neither the mechanisms nor the specificity of the effect (106). A synthetic agonist selective for VPAC2 increased has been assessed (119). Culture of normal rat islets with plasma insulin and improved glucose disposal in normal fibrates increased PPAR-␣ expression and exerted variable rats (107). Confirmation of these effects in models of type effects on enzymes of pyruvate metabolism and fatty acid 2 diabetes and demonstration that no side effects occur oxidation, as well as on insulin secretion (116,119). The remain to be seen. interpretation of these experiments is problematic be- Glucose-dependent insulinotropic polypetide (GIP) and cause supratherapeutic drug concentrations were used. GLP-1 are the two major intestinal hormones released In vivo treatment of normal rats with a PPAR-␣ agonist during meals that strongly potentiate nutrient-induced for 24 h increased PPAR-␣ and pyruvate dehydrogenase insulin secretion (incretin effect) (108). In type 2 diabetic kinase 4 expression in islets, but did not affect glucose- patients, secretion of GIP is normal or slightly increased, induced insulin secretion ex vivo (120). A similar treat- but the efficacy of the hormone on ␤-cells is inexplicably ment exerted distinct effects in two models of insulin impaired, which renders GIP administration inefficient resistance. Hypersecretion of insulin by islets from high- treatment (109,110). fat-fed rats was reversed (120), whereas that from preg- The situation is very different with GLP-1. Its secretion nant rats was unaffected (121), suggesting an extra-islet is impaired but its action is preserved in type 2 diabetic site of action of the drug. patients. Therapeutic substitution may thus be justified PPAR-␥ and thiazolidinediones. Treatment with thia- and has indeed already proved successful, in particular zolidinediones is often accompanied by improved glucose- when the short half-life of the peptide is extended (109– stimulated insulin secretion in type 2 diabetic patients and 111). Marked glucose dependency is an important feature various animal models of the disease (118). In vitro studies of GLP-1 effects on insulin secretion. The increase in are contradictory. and have cAMP produced by GLP-1 activates a PKA- and GEF/Epac- been reported to induce a glucose-dependent increase in dependent amplifying pathway (14,16,19) that augments insulin secretion by HIT cells, rat islets, or the perfused rat 2ϩ the efficacy of [Ca ]i, the rise of which depends on pancreas (122–124). These acute effects were too rapid

DIABETES, VOL. 53, SUPPLEMENT 3, DECEMBER 2004 S55 PHARMACOLOGICAL INSULIN SECRETAGOGUES

(Ͻ1 h) for nuclear receptor involvement. They were cholinergic control of pancreatic beta-cell function. Endocr Rev 22:565– variably attributed to binding to SUR1 without closure of 604, 2001 2ϩ 16. Holz GG: Epac: A new cAMP-binding protein in support of glucagon-like the KATP channel, direct activation of Ca channels, and peptide-1 receptor-mediated signal transduction in the pancreatic ␤-cell. stimulation of phosphatidylinositol (PI) 3-kinase (122– Diabetes 53:5–13, 2004 124). A more recent study, however, did not find any acute 17. MacDonald PE, Wheeler MB: Voltage-dependent Kϩ channels in pancre- effect of rosiglitazone on insulin secretion by perifused rat atic beta cells: role, regulation and potential as therapeutic targets. islets (125). There also seems to be agreement that culture Diabetologia 46:1046–1062, 2003 (24–48 h) of normal human or rat islets with thiazo- 18. Jones PM, Persaud SJ: Protein kinases, protein phosphorylation, and the regulation of insulin secretion from pancreatic beta-cells. Endocr Rev lidinediones did not influence the subsequent secretory 19:429–461, 1998 response (115,126). Finally, insulin secretion was not 19. MacDonald PE, El-Kholy W, Riedel MJ, Salapatek AM, Light PE, Wheeler altered in mice with a selective knockout of PPAR-␥ in MB: The multiple actions of GLP-1 on the process of glucose-stimulated ␤-cells (127). When these mice were placed on a high-fat insulin secretion. Diabetes 51 (Suppl. 3):S434–S442, 2002 diet, the beneficial effects of rosiglitazone on insulin 20. Ozaki N, Shibasaki T, Kashima Y, Miki T, Takahashi K, Ueno H, Sunaga Y, Yano H, Matsuura Y, Iwanaga T, Takai Y, Seino S: cAMP-GEFII is a direct resistance and glucose tolerance was preserved despite target of cAMP in regulated exocytosis. Nat Cell Biol 2:805–811, 2000 the absence of PPAR-␥ in ␤-cells (127). It is thus unlikely 21. Sharp GW: Mechanisms of inhibition of insulin release. Am J Physiol that PPAR-␣ or -␥ qualify as primary targets for drugs 271:C1781–C1799, 1996 destined to stimulate insulin secretion. 22. Sato Y, Nenquin M, Henquin JC: Relative contribution of Ca2ϩ-dependent and Ca2ϩ-independent mechanisms to the regulation of insulin secretion by glucose. FEBS Lett 421:115–119, 1998 ACKNOWLEDGMENTS 23. Fernandez-Alvarez J, Conget I, Rasschaert J, Sener A, Gomis R, Malaisse The experimental work performed in my laboratory was WJ: Enzymatic, metabolic and secretory patterns in human islets of type supported by the Universite´ Catholique de Louvain, Fonds 2 (non-insulin-dependent) diabetic patients. Diabetologia 37:177–181, 1994 National de la Recherche Scientifique a` Bruxelles, Direc- 24. Velho G, Petersen KF, Perseghin G, Hwang JH, Rothman DL, Pueyo ME, tion Ge´ne´rale de la Recherche Scientifique de la Commu- Cline GW, Froguel P, Shulman GI: Impaired hepatic glycogen synthesis in naute´ Franc¸aise de Belgique et Programme des Poˆ les glucokinase-deficient (MODY-2) subjects. J Clin Invest 98:1755–1761, d’attraction interuniversitaire–Politique scientifique Fe´de´- 1996 rale. 25. Glaser B, Kesavan P, Heyman M, Davis E, Cuesta A, Buchs A, Stanley CA, I am grateful to J. Bryan for providing Sur1KO mice and Thornton PS, Permutt MA, Matschinsky FM, Herold KC: Familial hyper- insulinism caused by an activating glucokinase mutation. N Engl J Med to J. Bryan, S. Efendic, N.G. Morgan, U. Quast, and M.C. 338:226–230, 1998 Sugden for discussions and communication of unpub- 26. Grimsby J, Sarabu R, Corbett WL, Haynes NE, Bizzarro FT, Coffey JW, lished data. I thank V. Lebec for editorial assistance. Guertin KR, Hilliard DW, Kester RF, Mahaney PE, Marcus L, Qi L, Spence CL, Tengi J, Magnuson MA, Chu CA, Dvorozniak MT, Matschinsky FM, Grippo JF: Allosteric activators of glucokinase: potential role in diabetes REFERENCES therapy. Science 301:370–373, 2003 1. Ferrannini E: Insulin resistance versus insulin deficiency in non-insulin- 27. Trinh K, Minassian C, Lange AJ, O’Doherty RM, Newgard CB: Adenovirus- dependent diabetes mellitus: problems and prospects. Endocr Rev 19: mediated expression of the catalytic subunit of glucose-6-phosphatase in 477–490, 1998 INS-1 cells: effects on glucose cycling, glucose usage, and insulin secre- 2. Kahn SE: The relative contributions of insulin resistance and beta-cell tion. J Biol Chem 272:24837–24842, 1997 dysfunction to the pathophysiology of type 2 diabetes. Diabetologia 28. Ostenson CG, Khan A, Abdel-Halim SM, Guenifi A, Suzuki K, Goto Y, 46:3–19, 2003 Efendic S: Abnormal insulin secretion and glucose metabolism in pan- 3. Henquin JC: The fiftieth anniversary of hypoglycaemic sulphonamides: creatic islets from the spontaneously diabetic GK rat. Diabetologia how did the mother compound work? Diabetologia 35:907–912, 1992 36:3–8, 1993 4. Henquin JC: Triggering and amplifying pathways of regulation of insulin 29. Portha B, Giroix MH, Serradas P, Gangnerau MN, Movassat J, Rajas F, secretion by glucose. Diabetes 49:1751–1760, 2000 Bailbe D, Plachot C, Mithieux G, Marie JC: ␤-Cell function and viability in 5. Henquin JC, Ravier MA, Nenquin M, Jonas JC, Gilon P: Hierarchy of the the spontaneously diabetic GK rat: information from the GK/Par colony. beta-cell signals controlling insulin secretion. Eur J Clin Invest 33:742– Diabetes 50 (Suppl. 1):S89–S93, 2001 50, 2003 30. Laybutt DR, Sharma A, Sgroi DC, Gaudet J, Bonner-Weir S, Weir GC: 6. Aguilar-Bryan L, Bryan J: Molecular biology of adenosine triphosphate- Genetic regulation of metabolic pathways in ␤-cells disrupted by hyper- sensitive potassium channels. Endocr Rev 20:101–135, 1999 glycemia. J Biol Chem 277:10912–10921, 2002 7. Ashcroft FM, Gribble FM: ATP-sensitive Kϩ channels and insulin secre- 31. Khan A, Ling ZC, Pukk K, Herling AW, Landau BR, Efendic S: Effects of tion: their role in health and disease. Diabetologia 42:903–919, 1999 3-mercaptopicolinic acid and a derivative of chlorogenic acid (S-3483) on 8. Seino S, Iwanaga T, Nagashima K, Miki T: Diverse roles of K(ATP) hepatic and islet glucose-6-phosphatase activity. Eur J Pharmacol 349: channels learned from Kir6.2 genetically engineered mice. Diabetes 325–331, 1998 49:311–318, 2000 32. Malaisse WJ: The esters of carboxylic nutrients as insulinotropic tools in 9. Gilon P, Ravier MA, Jonas JC, Henquin JC: Control mechanisms of the non-insulin-dependent diabetes mellitus. Gen Pharmac 26:1133–1141, oscillations of insulin secretion in vitro and in vivo. Diabetes 51 (Suppl. 1995 1):S144–S151, 2002 33. Malaisse WJ: Insulinotropic action of monosaccharide esters: therapeutic 10. Matschinsky FM: Regulation of pancreatic beta-cell glucokinase: from perspectives. Diabetologia 42:286–291, 1999 basics to therapeutics. Diabetes 51 (Suppl. 3):S394–S404, 2002 34. Kennedy HJ, Pouli AE, Ainscow EK, Jouaville LS, Rizzuto R, Rutter GA: ϩ 11. Sato Y, Henquin JC: The K ATP channel-independent pathway of regula- Glucose generates sub-plasma membrane ATP microdomains in single tion of insulin secretion by glucose: in search of the underlying mecha- islet ␤-cells. J Biol Chem 274:13281–13291, 1999 nism. Diabetes 47:1713–1721, 1998 35. Wollheim CB: Beta-cell mitochondria in the regulation of insulin secre- 12. Aizawa T, Sato Y, Komatsu M: Importance of nonionic signals for tion: a new culprit in type II diabetes. Diabetologia 43:265–277, 2000 glucose-induced biphasic insulin secretion. Diabetes 51 (Suppl. 1):S96– 36. Lee B, Miles PD, Vargas L, Luan P, Glasco S, Kushnareva Y, Kornbrust ES, S98, 2002 Grako KA, Wollheim CB, Maechler P, Olefsky JM, Anderson CM: Inhibi- 13. Straub SG, Sharp GW: Glucose-stimulated signaling pathways in biphasic tion of mitochondrial Naϩ-Ca2ϩ exchanger increases mitochondrial me- insulin secretion. Diabete Metab Res Rev 18:451–463, 2002 tabolism and potentiates glucose-stimulated insulin secretion in rat 14. Gromada J, Holst JJ, Rorsman P: Cellular regulation of islet hormone pancreatic islets. Diabetes 52:965–973, 2003 secretion by the incretin hormone glucagon-like peptide 1. Pflugers Arch 37. Meyer M, Chudziak F, Schwanstecher C, Schwanstecher M, Panten U: 435:583–594, 1998 Structural requirements of sulphonylureas and analogues for interaction 15. Gilon P, Henquin JC: Mechanisms and physiological significance of the with sulphonylurea receptor subtypes. Br J Pharmacol 128:27–34, 1999

S56 DIABETES, VOL. 53, SUPPLEMENT 3, DECEMBER 2004 J.-C. HENQUIN

38. Proks P, Reimann F, Green N, Gribble F, Ashcroft F: Sulfonylurea Kϩ channels in mouse pancreatic B-cells. Br J Pharmacol 101:115–120, stimulation of insulin secretion. Diabetes 51 (Suppl. 3):S368—S376, 2002 1990 39. Gribble FM, Reimann F: Differential selectivity of insulin secretagogues: 62. Morgan NG, Chan SLF: Imidazoline binding sites in the endocrine mechanisms, clinical implications, and drug interactions. J Diabetes pancreas: can they fulfill their potential as targets for the development of Complications 17:11–15, 2003 new insulin secretagogues? Curr Pharm Design 7:1413–1431, 2001 ␣ 40. Bryan J, Crane A, Vila-Carriles W, Babenko AP, Aguilar-Bryan L: Insulin 63. Jonas JC, Plant TD, Henquin JC: Imidazoline antagonists of 2-adreno- ϩ secretagogues, sulfonylurea receptors and KATP channels. Curr Pharma ceptors increase insulin release in vitro by inhibiting ATP-sensitive K Design. In press channels in pancreatic ␤-cells. Br J Pharmacol 107:8–14, 1992

41. Barg S, Renstrom E, Berggren PO, Bertorello A, Bokvist K, Braun M, 64. Proks P, Ashcroft FM: Phentolamine block of KATP channels is mediated Eliasson L, Holmes WE, Kohler M, Rorsman P, Thevenod F: The stimu- by Kir6.2. Proc Natl Acad SciUSA94:11716–11720, 1997 latory action of tolbutamide on Ca2ϩ-dependent exocytosis in pancreatic 65. Hermans MP, Schmeer W, Henquin JC: The permissive effect of glucose, ␤ cells is mediated by a 65-kDa mdr-like P-glycoprotein. Proc Natl Acad tolbutamide and high Kϩ on arginine stimulation of insulin release in SciUSA96:5539–5544, 1999 isolated mouse islets. Diabetologia 30:659–665, 1987 42. Renstrom E, Barg S, Thevenod F, Rorsman P: Sulfonylurea-mediated 66. Henquin JC: Role of voltage- and Ca2ϩ-dependent Kϩ channels in the stimulation of insulin exocytosis via an ATP-sensitive Kϩ channel- control of glucose-induced electrical activity in pancreatic ␤-cells. independent action. Diabetes 51 (Suppl. 1):S33–S36, 2002 Pflu¨gers Arch 416:568–572, 1990 43. Eliasson L, Ma X, Renstrom E, Barg S, Berggren PO, Galvanovskis J, 67. Satin LS: Localized calcium influx in pancreatic beta-cells: its significance Gromada J, Jing X, Lundquist I, Salehi A, Sewing S, Rorsman P: SUR1 for Ca2ϩ-dependent insulin secretion from the islets of Langerhans. regulates PKA-independent cAMP-induced granule priming in mouse Endocrine 13:251–262, 2000 pancreatic B-cells. J Gen Physiol 121:181–197, 2003 68. Varadi A, Molnar E, Ostenson CG, Ashcroft SJ: Isoforms of endoplasmic 44. Nenquin M, Szollosi A, Aguilar-Bryan L, Bryan J, Henquin JC: Both reticulum Ca2ϩ-ATPase are differentially expressed in normal and dia- triggering and amplifying pathways contribute to fuel-induced insulin betic islets of Langerhans. Biochem J 319:521–527, 1996 secretion in the absence of sulfonylurea receptor-1 in pancreatic ␤-cells. 69. Ximenes HM, Kamagate A, Van Eylen F, Carpinelli A, Herchuelz A: J Biol Chem 279:32316–32324, 2004 Opposite effects of glucose on plasma membrane Ca2ϩ-ATPase and Na/Ca 45. Seghers V, Nakazaki M, DeMayo F, Aguilar-Bryan L, Bryan J: Sur1 exchanger transcription, expression, and activity in rat pancreatic ␤-cells.

knockout mice: a model for KATP channel-independent regulation of J Biol Chem 278:22956–22963, 2003 insulin secretion. J Biol Chem 275:9270–9277, 2000 70. Mitchell KJ, Tsuboi T, Rutter GA: Role for plasma membrane-related 46. Miki T, Nagashima K, Tashiro F, Kotake K, Yoshitomi H, Tamamoto A, Ca2ϩ-ATPase-1 (ATP2C1) in pancreatic ␤-cell Ca2ϩ homeostasis revealed Gonoi T, Iwanaga T, Miyazaki J, Seino S: Defective insulin secretion and by RNA silencing. Diabetes 53:393–400, 2004

enhanced insulin action in KATP channel-deficient mice. Proc Natl Acad 71. Henquin JC, Ishiyama N, Nenquin M, Ravier MA, Jonas JC: Signals and SciUSA95:10402–10406, 1998 pools underlying biphasic insulin secretion. Diabetes 51 (Suppl. 1):S60– 47. Geisen K, Hu¨ bner M, Hitzel V, Hrstka VE, Pfaff W, Bosies E, Regitz G, S67, 2002 Ku¨ hnle HF, Schmidt FH, Weyer R: Acylaminoalkyl-substituierte benzoe- 72. Harding HP, Ron D: Endoplasmic reticulum stress and the development und phenylalkansa¨uren mit blutglukose-senkender wirkung. Arzneim of diabetes. Diabetes 51 (Suppl. 3):S455–S461, 2002 Forsch 28:1081–1083, 1978 73. Detimary P, Van den Berghe G, Henquin JC: Concentration dependence 48. Garrino MG, Schmeer W, Nenquin M, Meissner HP, Henquin JC: Mecha- and time course of the effects of glucose on adenine and guanine nism of the stimulation of insulin release in vitro by HB 699, a benzoic nucleotides in mouse pancreatic islets. J Biol Chem 271:20559–20565, acid derivative similar to the non-sulphonylurea moiety of glibenclamide. 1996 Diabetologia 28:697–703, 1985 74. Geng X, Li L, Watkins S, Robbins PD, Drain P: The insulin secretory 49. Dornhorst A: Insulinotropic meglitinide analogues. Lancet 358:1709– granule is the major site of KATP channels of the endocrine pancreas. 1716, 2001 Diabetes 52:767–776, 2003 50. Bokvist K, Hoy M, Buschard K, Holst JJ, Thomsen MK, Gromada J: 75. Da Silva Xavier G, Leclerc I, Varadi A, Tsuboi T, Moule SK, Rutter GA: Selectivity of prandial glucose regulators: nateglinide, but not repaglinide, Role for AMP-activated protein kinase in glucose-stimulated insulin accelerates exocytosis in rat pancreatic A-cells. Eur J Pharmacol 386: secretion and preproinsulin gene expression. Biochem J 371:761–774, 105–111, 1999 2003 51. Malaisse WJ: Insulinotropic action of meglitinide analogues: modulation 76. Fryer LGD, Parbu-Patel A, Carling D: The anti-diabetic drugs rosiglitazone by an activator of ATP-sensitive Kϩ channels and high extracellular Kϩ and metformin stimulate AMP-activated protein kinase through distinct signaling pathways. J Biol Chem 277:25226–25232, 2002 concentrations. Pharmacol Res 32:111–114, 1995 ϩ 77. Bailey CJ, Turner RC: Metformin. N Engl J Med 334:574–579, 1996 52. Garrino MG, Plant TD, Henquin JC: Effects of putative activators of K 78. Leclerc I, Woltersdorf WW, Da Silva Xavier G, Rowe RL, Cross SE, channels in mouse pancreatic ␤-cells. Br J Pharmacol 98:957–965, 1989 Korbutt GS, Rajotte RV, Smith R, Rutter GA: Metformin, but not leptin, 53. Shindo T, Katayama Y, Horio Y, Kurachi Y: MCC-134, a novel vascular regulates AMP-activated protein kinase in pancreatic islets: impact on relaxing agent, is an inverse agonist for the pancreatic-type ATP-sensitive glucose-stimulated insulin secretion. Am J Physiol Endocrinol Metab Kϩchannel. J Pharmacol Exp Ther 292:131–135, 2000 286:E1023–E1031, 2004 54. Henquin JC: Quinine and the stimulus-secretion coupling in pancreatic 79. Lupi R, Del Guerra S, Tellini C, Giannarelli R, Coppelli A, Lorenzetti M, ␤ -cells: glucose-like effects on potassium permeability and insulin release. Carmellini M, Mosca F, Navalesi R, Marchetti P: The compound Endocrinology 110:1325–1332, 1982 metformin prevents desensitization of human pancreatic islets induced 55. Gribble FM, Davis TM, Higham CE, Clark A, Ashcroft FM: The antima- by high glucose. Eur J Pharmacol 364:205–209, 1999 larial agent mefloquine inhibits ATP-sensitive K-channels. Br J Pharma- 80. Binnert C, Seematter G, Tappy L, Giusti V: Effect of metformin on insulin col 131:756–760, 2000 sensitivity and insulin secretion in female obese patients with normal 56. Zunkler BJ, Wos M: Effects of lomefloxacin and norfloxacin on pancreatic glucose tolerance. Diabete Metab 29:125–132, 2003 ϩ ␤-cell ATP-sensitive K channels. Life Sci 73:429–435, 2003 81. Lambillotte C, Gilon P, Henquin JC: Direct glucocorticoid inhibition of 57. Hayashi S, Horie M, Tsuura Y, Ishida H, Okada Y, Seino Y, Sasayama S: insulin secretion: an in vitro study of dexamethasone effects in mouse ϩ Disopyramide blocks pancreatic ATP-sensitive K channels and en- islets. J Clin Invest 99:414–423, 1997 hances insulin release. Am J Physiol 265:C337–C342, 1993 82. Davani B, Portwood N, Bryzgalova G, Reimer MK, Heiden T, Ostenson 58. Bertrand G, Gross R, Petit P, Loubatieres-Mariani MM, Ribes G: Evidence CG, Okret S, Ahren B, Efendic S, Khan A: Aged transgenic mice with for a direct stimulatory effect of cibenzoline on insulin secretion in rats. increased glucocorticoid sensitivity in pancreatic ␤-cells develop diabe- Eur J Pharmacol 214:159–163, 1992 tes. Diabetes 53 (Suppl. 1):S51–S59, 2004 59. Kakei M, Nakazaki M, Kamisaki T, Nagayama I, Fukamachi Y, Tanaka H: 83. Alberts P, Engblom L, Edling N, Forsgren M, Klingstrom G, Larsson C, Inhibition of the ATP-sensitive by class I antiarrhyth- Ronquist-Nii Y, Ohman B, Abrahmsen L: Selective inhibition of 11␤- mic agent, cibenzoline, in rat pancreatic ␤-cells. Br J Pharmacol 109: hydroxysteroid dehydrogenase type 1 decreases blood glucose concen- 1226–1231, 1993 trations in hyperglycaemic mice. Diabetologia 45:1528–1532, 2002 60. Henquin JC: Established, unsuspected and novel pharmacological insulin 84. Andrews RC, Rooyackers O, Walker BR: Effects of the 11 ␤-hydroxy- secretagogues. In New Antidiabetic Drugs. Bailey CJ, Flatt PR, Eds. steroid dehydrogenase inhibitor carbenoxolone on insulin sensitivity in London, Smith-Gordon and Company, 1990, p. 93–106 men with type 2 diabetes. J Clin Endocrinol Metab 88:285–291, 2003 61. Plant TD, Henquin JC: Phentolamine and yohimbine inhibit ATP-sensitive 85. Davani B, Khan A, Hult M, Martensson E, Okret S, Efendic S, Jornvall H,

DIABETES, VOL. 53, SUPPLEMENT 3, DECEMBER 2004 S57 PHARMACOLOGICAL INSULIN SECRETAGOGUES

Oppermann UC: Type 1 11␤-hydroxysteroid dehydrogenase mediates ate cyclase-activating polypeptide and islet function. Diabetes 50:1959– glucocorticoid activation and insulin release in pancreatic islets. J Biol 1969, 2001 Chem 275:34841–34844, 2000 107. Tsutsumi M, Claus TH, Liang Y, Li Y, Yang L, Zhu J, Dela Cruz F, Peng X, 86. Duplomb L, Lee Y, Wang MY, Park BH, Takaishi K, Agarwal AK, Unger Chen H, Yung SL, Hamren S, Livingston JN, Pan CQ: A potent and highly RH: Increased expression and activity of 11␤-HSD-1 in diabetic islets and selective VPAC2 agonist enhances glucose-induced insulin release and prevention with troglitazone. Biochem Biophys Res Commun 313:594– glucose disposal: a potential therapy for type 2 diabetes. Diabetes 599, 2004 51:1453–1460, 2002 2ϩ 87. Lehmann A, Boldt J, Kirchner J: The role of Ca sensitizers for the 108. Fehmann HC, Goke R, Goke B: Cell and molecular biology of the incretin treatment of heart failure. Curr Opin Crit Care 9:337–344, 2003 hormones glucagon-like peptide-1 and glucose-dependent insulin releas- 88. Fujimoto S, Ishida H, Kato S, Okamoto Y, Tsuji K, Mizuno N, Ueda S, ing polypeptide. Endocr Rev 16:390–410, 1995 Mukai E, Seino Y: The novel insulinotropic mechanism of pimobendan: 109. Nauck MA, Meier JJ, Creutzfeldt W: Incretins and their analogues as new direct enhancement of the exocytotic process of insulin secretory gran- antidiabetic drugs. Drug News Perspect 16:413–422, 2003 2ϩ ules by increased Ca sensitivity in beta-cells. Endocrinology 139:1133– 110. Visboll T, Holst JJ: Incretins, insulin secretion and type 2 diabetes 1140, 1998 mellitus. Diabetologia 47:357–366, 2004 89. Pyne NJ, Furman BL: Cyclic nucleotide phosphodiesterases in pancreatic 111. Drucker DJ: Development of glucagon-like peptide-1-based pharmaceuti- Diabetologia islets. 46:1179–1189, 2003 cals as therapeutic agents for the treatment of diabetes. Curr Pharm Des 90. Cerasi E, Luft R: The effect of an adenosine-3Ј,5Ј-monophosphate dies- 7:1399–1412, 2001 terase inhibitor (aminophylline) on the insulin response to glucose 112. Steffensen KR, Gustafsson JA: Putative metabolic effects of the liver X infusion in prediabetic and diabetic subjects. Horm Metab Res 1:162–168, receptor (LXR). Diabetes 53 (Suppl. 1):S36–S42, 2004 1969 113. Ferre P: The biology of peroxisome proliferator-activated receptors: 91. Ha¨rndahl L, Wierup N, Enerback S, Mulder H, Manganiello VC, Sundler F, relationship with lipid metabolism and insulin sensitivity. Diabetes 53 Degerman E, Ahren B, Holst LS: ␤-cell-targeted overexpression of phos- (Suppl. 1):S43–S50, 2004 phodiesterase 3B in mice causes impaired insulin secretion, glucose intolerance and deranged islet morphology. J Biol Chem 279:15214– 114. Efanov AM, Sewing S, Bokvist K, Gromada J: Liver X receptor activation 15222, 2004 stimulates insulin secretion via modulation of glucose and lipid metabo- ␤ 92. Efendic S, Cerasi E, Luft R: Effect of blockade of the alpha-adrenergic lism in pancreatic -cells. Diabetes 53 (Suppl. 3):S75–S78, 2004 receptors on insulin response to glucose infusion in prediabetic subjects. 115. Dubois M, Pattou F, Kerr-Conte J, Gmyr V, Vandewalle B, Desreumaux P, Acta Endocrinol 74:542–547, 1973 Auwerx J, Schoonjans K, Lefebvre J: Expression of peroxisome prolif- 93. Robertson RP, Halter JB, Porte D: A role for alpha-adrenergic receptors erator-activated receptor ␥ (PPAR␥) in normal human pancreatic islet in abnormal insulin secretion in diabetes mellitus. J Clin Invest 57:791– cells. Diabetologia 43:1165–1169, 2000 795, 1976 116. Zhou YT, Shimabukuro M, Wang MY, Lee Y, Higa M, Milburn JL, Newgard 94. Schulz A, Hasselblatt A: An insulin-releasing property of imidazoline CB, Unger RH: Role of peroxisome proliferator-activated receptor ␣ in derivatives is not limited to compounds that block alpha-adrenoceptors. disease of pancreatic ␤-cells. Proc Natl Acad SciUSA95:8898–8903, Naunyn Schmiedebergs Arch Pharmacol 340:321–327, 1989 1998

95. Chan SLF, Mourtada M, Morgan NG: Characterization of a KATP channel- 117. Roduit R, Morin J, Masse´ F, Segall L, Roche E, Newgard CB, Assimaco- independent pathway involved in potentiation of insulin secretion by poulos-Jeannet F, Prentki M: Glucose down-regulates the expression of efaroxan. Diabetes 50:340–347, 2001 the peroxisome proliferator-activated receptor-␣ gene in the pancreatic 96. Efanov AM, Zaitsev SV, Mest HJ, Raap A, Appelskog IB, Larsson O, ␤-cells. J Biol Chem 275:35799–35806, 2000 Berggren PO, Efendic S: The novel imidazoline compound BL11282 118. Kim HI, Ahn YH: Role of peroxisome proliferator-activated receptor-␥ in potentiates glucose-induced insulin secretion in pancreatic ␤-cells in the the glucose-sensing apparatus of liver and ␤-cells. Diabetes 53 (Suppl.

absence of modulation of KATP channel activity. Diabetes 50:797–802, 1):S60–S65, 2004 2001 119. Yoshikawa H, Tajiri Y, Sako Y, Hashimoto T, Umeda F, Nawata H: Effects 97. Efendic S, Efanov AM, Berggren PO, Zaitsev SV: Two generations of of bezafibrate on ␤-cell function of rat pancreatic islets. Eur J Pharmacol insulinotropic imidazoline compounds. Diabetes 51 (Suppl. 3):S448–S454, 426:201–206, 2001 2002 120. Holness MJ, Smith ND, Greenwood GK, Sugden MC: Acute (24 h) 98. Efanov AM, Hoy M, Branstrom R, Zaitsev SV, Magnuson MA, Efendic S, activation of peroxisome proliferator-activated receptor-␣ (PPAR␣) re- Gromada J, Berggren PO: The imidazoline RX871024 stimulates insulin verses high-fat feeding-induced insulin hypersecretion in vivo and in secretion in pancreatic beta-cells from mice deficient in K(ATP) channel perifused pancreatic islets. J Endocrinol 177:197–205, 2003 function. Biochem Biophys Res Commun 284:918–22, 2001 121. Sugden MC, Greenwood GK, Smith ND, Holness MJ: Peroxisome prolif- 99. Cooper EJ, Hudson AL, Parker CA, Morgan NG: Effects of the ␤-carbo- erator-activated receptor-␣ activation during pregnancy attenuates glu- lines, harmane and pinoline, on insulin secretion from isolated human cose-stimulated insulin hypersecretion in vivo increasing insulin islets of Langerhans. Eur J Pharmacol 482:189–196, 2003 sensitivity, without impairing pregnancy-induced increases in ␤-cell glu- 100. Ahren B: Autonomic regulation of islet hormone secretion: implications cose sensing and responsiveness. Endocrinology 144:146–153, 2003 for health and disease. Diabetologia 43:393–410, 2000 122. Masuda K, Okamoto Y, Tsuura Y, Kato S, Miura T, Tsuda K, Horikoshi H, ␣ 101. Ostenson CG, Pigon J, Doxey JC, Efendic S: 2-Adrenoceptor blockade Ishida H, Seino Y: Effects of troglitazone (CS-045) on insulin secretion in does not enhance glucose-induced insulin release in normal subjects or isolated rat pancreatic islets and HIT cells: an insulinotropic mechanism patients with noninsulin-dependent diabetes. J Clin Endocrinol Metab distinct from glibenclamide. Diabetologia 38:24–30, 1995 67:1054–1059, 1988 123. Ohtani KI, Shimizu H, Sato N, Mori M: Troglitazone (CS-045) inhibits 102. Norman M, Moldovan S, Seghers V, Wang XP, DeMayo FJ, Brunicardi FC: ␤-cell proliferation rate following stimulation of insulin secretion in HIT-T Sulfonylurea receptor knockout causes glucose intolerance in mice that 15 cells. Endocrinology 139:172–178, 1998 is not alleviated by concomitant somatostatin subtype receptor 5 knock- 124. Yang C, Chang TJ, Chang JC, Liu MW, Tai TY, Hsu WH, Chuang LM: out. Ann Surg 235:767–774, 2002 Rosiglitazone (BRL 49653) enhances insulin secretory response via 103. Ahren B, Pacini G, Wynick D, Wierup N, Sundler F: Loss-of-function phosphatidylinositol 3-kinase pathway. Diabetes 50:2598–2602, 2001 mutation of the galanin gene is associated with perturbed islet function in 125. Zawalich WS, Tesz G, Zawalich KC: Contrasting effects of nateglinide and mice. Endocrinology 145:3190–3196, 2004 rosiglitazone on insulin secretion and phospholipase C activation. Metab- 104. Bertrand G, Puech R, Loubatieres-Mariani MM, Bockaert J: Glutamate olism 52:1393–1399, 2003 stimulates insulin secretion and improves glucose tolerance in rats. Am J 126. Shimabukuro M, Zhou YT, Lee Y, Unger RH: Troglitazone lowers islet fat Physiol 269:E551–E556, 1995 and restores beta cell function of zucker diabetic fatty rats. J Biol Chem 105. Petit P, Hillaire-Buys D, Loubatie`res-Mariani MM, Chapal J: Purinergic 273:3547–3550, 1998 receptors and the pharmacology of type 2 diabetes. In Handbook of 127. Rosen ED, Kulkarni RN, Sarraf P, Ozcan U, Okada T, Hsu CH, Eisenman Experimental Pharmacology: Purinergic and Pyrimidinergic Signal- D, Magnuson MA, Gonzalez FJ, Kahn CR, Spiegelman BM: Targeted ing. Abbrachio MP, Williams M, Eds. New York, Springer-Verlag, 2001, p. elimination of peroxisome proliferator-activated receptor ␥ in ␤-cells 377–391 leads to abnormalities in islet mass without compromising glucose 106. Filipsson K, Kvist-Reimer M, Ahren B: The neuropeptide pituitary adenyl- homeostasis. Mol Cell Biol 23:7222–7229, 2003

S58 DIABETES, VOL. 53, SUPPLEMENT 3, DECEMBER 2004