I. Introduction

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Diabetes mellitus (DM¹) is a chronic condition that is diagnosed by a blood test and requires life-long management (American Diabetes Association, 2002a; The Expert Committee on the Diagnosis and Classification of Diabetes Mellitus, 2002). It stands apart because of the importance of patient education (Nicollerat, 2000). For a detailed description of the etiological classification of the disease we refer the reader to Table 1 of the report of the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus (The Expert Committee on the Diagnosis and Classification of Diabetes Mellitus, 2002). The more patients understand about the disease the better they are enabled to make good decisions on its management. Dietary therapy and exercise are critical both in preventing and managing DM, and the results of the Diabetes Prevention Program Research Group indicate that changes in lifestyle (7% weight loss and 150 min of physical activity per week) reduced the incidence of diabetes by 58% (Knowler et al., 2002). Smoking should be avoided as it not only increases the prevalence of diabetes, regardless of exercise, strict diet, and low body mass index, but it greatly increases the probability of patients developing large vessel disease in the presence of DM (Wannamethee et al., 2001; American Diabetes Association, 2002b). In type 1 DM, where there is an absolute deficiency of insulin, insulin replacement forms a major component of treatment. In type 2 DM, insulin release from the pancreas is altered and may also be absolutely deficient in amount, and therefore its replacement also plays a part in management, especially when DM has been present for a long time.

Many new pharmacological agents have been added to our armamentarium of treatments for DM in the last decade. The goal of all treatments is the same irrespective of the cause of the DM: namely, to normalize blood glucose. Treating DM is not at all as simple as treating hypothyroidism, where replacing the missing hormone by the correct amount so that thyroid hormone and thyroid stimulating hormone levels are in the physiological range is all that is required. For normal metabolism insulin must be released from the pancreas in an exquisitely exact amount, at the correct time and in a correct pattern. The normal pancreas also senses the fasting and fed state as well as the energy content of the meals eaten. So far, no pharmacological agent can take the place of or restore this exquisite sensing capacity when it is diseased, and no agent can restore the exact pattern of insulin kinetics. The presence of so many types of insulin available to treat type 1 DM, and the availability of so many compounds to treat type 2 DM, is a testament to the complexity involved in normalizing blood glucose.

At any one time glucose homeostasis is maintained by a balance between insulin secretion and insulin action. Because of robustness within the system in nondiabetic subjects, alteration in one of these will lead to compensation by the other. As a general concept, type 2 DM occurs because insulin secretion no longer compensates for insulin resistance due to increasing obesity, aging, illness, etc. (Elahi et al., 2002). Before the onset of biochemical type 2 DM, prediabetes exists in that the pancreas is secreting an increasing amount of insulin, in the face of increasing insulin resistance, to maintain nondiabetic levels of blood glucose; at that point in the maintenance of glucose homeostasis, the balance is fragile. Therefore, any pharmacological agent that has a negative impact on this fragile balance can cause type 2 DM to occur. An example is the use of the immunosuppressant tacrolimus, which inhibits calcineurin, decreases insulin secretion, and causes -cell damage. Similarly, in the presence of known type 2 DM, the introduction of any agent that has a negative impact on glucose homeostasis could increase blood glucose and require adjustments to the diabetes treatment regimen. Therefore, a knowledge of possible or even theoretical interactions of pharmacologic agents that have an impact on glucose homeostasis would be beneficial in managing patients.

Pharmacological agents that are known to directly influence insulin secretion can be divided into two groups: 1) those prescribed because of their insulinotropic (i.e., insulin-releasing) properties are used in treating type 2 DM; and 2) those used for nondiabetes-related indications but have as their side effects direct negative or positive modulating effects on insulin release from the -cells of the pancreas. A number of agents, including those used in the treatment of type 2 DM, have an impact on insulin action at the insulin receptor level, thus indirectly influencing the amount of insulin secreted. We will not address such indirect mechanisms, but will focus instead on agents proven to have direct actions on the -cell. Similarly, insulin itself, when used as a pharmacological agent in the treatment of type 2 DM, has an impact on the release of endogenous insulin from -cells, but we refer the reader to a past review in this journal for further information on exogenous insulins (Vajo and Duckworth, 2000).

Pharmacological agents may alter insulin secretion by influencing the myriad of regulated physiologic molecular processes in the -cell or modifying insulin secretion by cytolytic or cytotoxic means. At the molecular level a drug may influence insulin secretion by 1) acting primarily on the ion channels of the -cell, and/or 2) by influencing the variety of second messenger pathways and the secretory machinery in the -cell. For example, in the case of the sulfonylureas the perturbation occurs only on the -cell K_ATP channel of the plasma membrane and mitochondrial membrane, but some receptor ligands, such as acetylcholine, have pleiotropic effects ranging from stimulation of the G-protein-coupled pathway, to activation of serine/threonine kinases, to changes in intracellular calcium levels, to growth effects, to effects on many systems besides -cells. We begin with an outline of the known mechanisms and molecular processes involved in insulin secretion. We then categorize drugs in terms of their primary mode of therapeutic use or class, discussing how their pharmacological action can modify insulin secretion from the -cell.

	II. Insulin Synthesis and Secretion

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A. Stimulus Secretion Coupling/the Metabolism of Glucose

1. The Basic Mechanism of Glucose-Induced Insulin Secretion. Blood glucose levels are very tightly controlled by rapid pulsatile release of insulin from -cells (Fig. 1). Glucose equilibrates through the GLUT2 transporter across the plasma membrane of the -cell. It is rapidly phosphorylated to glucose 6-phosphate by glucokinase, which thereafter determines the rate of glycolysis, i.e., acts as the glucose sensor and pyruvate generation for entry into the tricarboxylic acid (TCA) cycle in mitochondria. Subsequent oxidative metabolism provides the link between the products of glucose metabolism and insulin secretion. The resultant increase in the ATP/ADP ratio in the cytosol causes depolarization of the plasma membrane by closure of the ATP-sensitive K⁺ channels. This permits opening of voltage-dependent Ca²⁺ channels and an increase in cytosolic Ca²⁺, which then triggers fusion of insulin-containing secretory vesicles to the plasma membrane, and exocytosis of insulin follows rapidly. Besides activating K⁺ channels, ATP appears to be a major permissive factor for movement of insulin vesicles toward the plasma membrane and for priming of exocytosis (Eliasson et al., 1997) and, as will be discussed under Section II.B.2.a.i., it provides the phosphate for protein kinase A (PKA)-mediated phosphorylation of proteins important in exocytosis.

View larger version (48K): [in this window] [in a new window]   Fig. 1.   A schematic showing some of the main molecular events occurring during glucose-induced insulin secretion in the -cell. Glucose equilibrates across the plasma membrane via the glucose transporters. It is phosphorylated to glucose 6-phosphate by glucokinase, and this determines the rate of glycolysis and the rate of pyruvate generation for entry into the mitochondria. Therefore, when blood glucose is high, the rate of glycolysis will increase. In the mitochondria, pyruvate is the substrate for both pyruvate carboxylase and dehydrogenase. Ultimately, ATP is generated from substrate oxidation in the TCA cycle and activation of the electron transport chain, and it is then exported to the cytoplasm. The increase in the cytoplasmic ATP/ADP ratio closes the KATP channels and depolarizes the plasma membrane, allowing opening of voltage-dependent calcium channels and a rapid influx of Ca2+. This is a key step by which glucose regulates insulin exocytosis from primed secretory vesicles, as the increase in cytosolic Ca2+ is the main trigger in glucose-dependent insulin secretion. Intracellular calcium ([Ca2+]i) levels can also increase because of release from organelles. Activation of G (by 1 or muscarinic receptor ligands, for example) leads to hydrolysis of phosphatidyl inositol bisphosphate to produce diacylglycerol and IP3. IP3, in turn, can also increase [Ca2+]i by mobilizing endoplasmic reticulum calcium stores. DAG activates protein kinase C, at least partly by sensitizing it to Ca2+. Activation of the Ca2+/calmodulin kinases also occurs with the rise of [Ca2+]i. Many drugs may interact with the ion channels or with the G-protein-coupled family of receptors on the -cell. The Gs protein is coupled to the subfamily B G-protein-coupled receptors and 2 adrenergic receptors and leads to activation of adenylyl cyclase and subsequently of PKA, whereas the somatostatin, purinergic (P1), and 2 adrenergic receptors are coupled to Gi, leading to inhibition of adenylyl cyclase. Activated Ca2+/calmodulin kinases, PKC, and PKA can lead to phosphorylation of a myriad of proteins throughout the -cell associated with the insulin secretory vesicles, the ion channels, and the cytoskeletal structure, and not all of the reactions have been fully characterized. Phosphorylation and dephosphorylation reactions initiated through these G-coupled pathways also ultimately regulate transcription of genes involved in the regulation of insulin secretion..

2. MitochondriaCalcium Effects and Metabolism. The mitochondria, specifically the TCA cycle and electron transport that occur within them, are extremely important in the regulation of insulin secretion. Electron microscopy of the rat -cell reveals that the mitochondria are in close proximity to the insulin secretory vesicles. They essentially act as fuel sensors coupling the nutrient metabolism to the process of exocytosis (see Section II.C.2.). They are the primary source of energy within the -cell-regulating recovery from membrane depolarization, protein synthesis, and vesicle transport. In the 1970s it was demonstrated that mitochondrial poisons inhibit glucose-induced insulin secretion (Hellman, 1970). In 1992 a form of type 2 DM, maternally inherited diabetes and deafness, was linked to mutations in the mitochondrial genome (Ballinger et al., 1992). Both of these facts point to the importance of the mitochondria in the process of insulin secretion. The TCA cycle metabolizes the products of glycolysis and provides the reducing equivalents that activate the electron transport chain, resulting in hyperpolarization of the mitochondrial membrane and generation of ATP, which is exported to the cytosol, thus increasing the ATP/ADP ratio.

B. Components of the Insulin Secretory Pathway

1. Ion Channels. a. The Potassium Channels. The K_ATP channel on the -cell consists of a hetero-octomeric complex of four pore subunits, which are known as K_ir6.2, and four regulatory, or SUR1, subunits (Aguilar-Bryan and Bryan, 1999). The K_ir6.2 does not form functional channels in the absence of the SUR protein. Thus, the SUR protein may serve as a chaperon protein for K_ir6.2 (Zerangue et al., 1999) and there is evidence that the reverse may also be true (Clement et al., 1997). Both subunits contain binding sites for ligands that initiate conformational changes in the channel and can effect the channel's sensitivity to other ligands and/or open or close the channel.

b. The Voltage-Dependent Ca²⁺ Channels. With the exception of mouse -cells, two types of voltage-dependent calcium channels have been identified on the -cell. These can be distinguished from each other by their kinetics and pharmacology (Ashcroft et al., 1990

2. Second Messengers. a. G-Protein-Coupled Receptor Systems. In terms of insulin secretion the most important transducer of ligand activation is the guanyl-nucleotide-binding (GTP) protein system or G-protein-coupled system. This consists of a seven-transmembrane receptor that is coupled to a heterotrimeric G-protein; i.e., it consists of three subunits: the -subunit, which contains the guanine nucleotide binding site, and the tightly associated - and -subunits. The activated receptor induces a conformational change in the G-protein -subunit-releasing guanosine diphosphate (GDP) followed by binding of GTP. The GTP-bound form of the -subunit dissociates from the receptor and from the stable -dimer. Both the GTP-bound -subunit and the released -dimer can either stimulate or inhibit an effector enzyme on the inner surface of the membrane that converts precursor molecules into second messengers. In the case of the -cell there are two membrane-bound enzyme systems: adenylyl cyclase (AC), which converts adenosine triphosphate (ATP) into cyclic AMP (cAMP); and phospholipase C (PLC), which cleaves phosphoinositides into two second messengers, inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG), which are involved in Ca²⁺ release and the activation of PKC (serine/threonine kinases), respectively.

Three G-protein subfamilies are found in -cells: G_s, which stimulates AC and so increases cAMP production; G_i/G_o, which inhibits adenylyl cyclase; and the G_q subfamily, which is associated with the phosphatidylinositol system alone. The difference between the different G-protein-coupled receptors is determined by the structure of the -subunit that is unique to each subfamily. Table

View this table: [in this window] [in a new window]   TABLE 1 Outline of the G-protein-coupled receptors present on the -cell

i. Adenylyl Cyclase System. The amount of intracellular cAMP is regulated by the balance between the activity of two enzyme systems, AC, activation of which generates cAMP and PPi from ATP, and the cyclic nucleotide phosphodiesterases (PDEs), which metabolize cAMP. Activation of AC and the consequent rise in cAMP results in a significant up-regulation of the activity of the cAMP-dependent protein kinase PKA family, comprising ubiquitous serine/threonine phosphorylating enzymes. This leads to a cascade in which phosphorylation of vesicular and plasma membrane proteins, voltage-dependent calcium channels, and potentially other ion channels (possibly even the GLUT2 transporter) results in augmentation of glucose-induced exocytosis of insulin, as well as phosphorylation of transcription factors, which increases insulin gene promoter activity and therefore will influence insulin secretion long-term (reviewed in Jones and Persaud, 1998). Within the -cell, stimulation of AC through G under normal physiological circumstances occurs mainly via the gut hormone receptors for glucose-dependent insulinotropic factor (GIP) and glucagon-like peptide 1 (GLP-1), whose levels increase dramatically after eating (see Section II.B.1.b.i.). Somatostatin (SST), released from the entero-endocrine cells and from -cells of the islets, inhibits AC by activating G_i (see Section II.B.1.b.i.). cAMP synthesis plays a major role in augmenting glucose-induced insulin release. The importance of this system is clearly demonstrated in type 2 DM, where -cells of the pancreas have become resistant to the insulinotropic effects of GIP (Elahi et al., 1994), probably because of a maladaption within the AC/PKA system because of the high glucose (Livak and Egan, 2002).

ii. Calcium/Phosphatidylinositol System. Of the four main phosphoinositide-specific PLCs (PLC-, -, -, and -) only the PLC- isozymes are known to be activated by G-protein-coupled receptors (Williams, 1999). The collective evidence weighs in favor of the presence of all four PLC- isozymes in the pancreas (PLC- 1, 2, 3, and 4), based on data from rat pancreas (Kim et al., 2001; Wang et al., 2000). However, it is not yet known which of these isozymes are coupled to the muscarinic receptor in the -cell. As with the AC second messenger system, the presence of the various isozymes raises the possibility that neurohumoral agonists such as acetylcholine activate different PLC isozymes from nutrients such as glucose or fatty acids, thus allowing for differential regulation. There is evidence that PKC, which is activated by DAG, is instrumental in second-phase insulin secretion, as the general PKC inhibitors staurosporin and Gö 6976 are known to inhibit this phase (Zawalich and Zawalich, 2001).

b. G-Protein-Coupled Receptors on the -Cell

i. Gut Hormone Receptors. The G-protein-coupled receptors present on the plasma membrane of the -cell in this category belong to the class B family of G-protein-coupled receptors. Included in this family are GLP-1, glucagon, GIP, secretin, pituitary adenylyl cyclase activating peptide, calcitonin, latrophilin, and parathyroid hormone and calcitonin gene-related peptide receptors. These peptide hormones are ligands for hormone-specific seven-transmembrane receptors that are coupled to the G_s protein and stimulate cAMP production in the -cell. In general, there is very little cross-reactivity among these receptors, e.g., glucagon binds with 100-1000-fold less affinity to the GLP-1 receptor than does GLP-1 itself (Fehmann et al., 1994). Of these peptide hormones only GLP-1 has so far been used clinically to stimulate insulin secretion (see Section III.D.1.) and glucagon is used to treat hypoglycemia secondary to exogenous insulin and as a test of -cell reserve.

ii. Muscarinic Receptors. Acetylcholine (ACh), the major parasympathetic neurotransmitter, is released from intrapancreatic nerve endings. The arrival of an action potential at the nerve terminal triggers ACh release due to the influx of calcium. The effects of ACh at the effector cell are mediated by muscarinic cholinergic receptors, of which five subtypes are known to exist (M₁-M₅). The M₂ and M₄ subtypes are known to be coupled to G_i and are pertussis toxin-sensitive, while the remaining subtypes (M₁, M₃, and M₅) are coupled to G_q. Activation of the muscarinic receptors on the -cell of the pancreas are not pertussis toxin-sensitive, and thus their activation is mediated by the G_q-coupled M₁/M₃/M₅ category and not the M₂/M₄ adenylyl cyclase-linked pertussis toxin-sensitive pathway (reviewed in Gilon and Henquin, 2001). Reverse transcriptase-polymerase chain analysis of extracts of rat islets indicates the presence of all three G_q-coupled subtypes with a predominance of M₁ and M₃, which are expressed approximately to the same extent (Iismaa et al., 2000). Presently we do not know of any muscarinic receptor ligands that are used specifically to modulate insulin secretion; neither have we encountered any reports of the use of agonists the muscarinic receptors as potential agents to enhance insulin secretion and hence treat diabetes. It is known that the drug tacrine, an acetylcholinesterase inhibitor, affects insulin secretion (see Section IV.H.).

iii. Adrenergic Receptors. There is physiologic and pharmacologic evidence for the presence of inhibitory and stimulatory adrenoreceptors on the -cell. The inhibitory -adrenoreceptor has been characterized as being of the ₂-subtype (Cherksey et al., 1983) and the stimulatory -adrenoreceptor as the ₂-subtype (Fyles et al., 1986). The ₂-subtype is coupled to G_i/G_o, and the ₂ is coupled to the G_s protein. Stimulation of the ₂-adrenoreceptors in the -cell is believed to directly activate a G-protein-gated K⁺ channel, thereby inhibiting exocytosis (Rorsman et al., 1991). Epinephrine, which activates both types of adrenoreceptors, inhibits glucose-induced insulin secretion (Cawthorn and Chan, 1991) indicating that ₂ is the predominant adrenergic receptor in -cells. Such an inhibitory action is suppressed when islets are exposed to pertussis toxin (which causes irreversible ADP-ribosylation of the G_a-subunit and prevents it interaction with the receptor). Interestingly, however, selective ₂ blockade with deriglidole, while preventing epinephrine-induced inhibition of insulin secretion, did not potentiate basal or intravenous or oral glucose tolerance-induced insulin release in nondiabetic humans (Natali et al., 1998). This leads to the assumption that in healthy people neither basal nor postabsorptive insulin secretion is under tonic adrenergic tone. This may not be the case in type 2 diabetes, as it was shown that acute -adrenergic blockade improved glucose-potentiated insulin secretion in that specific group of people (Broadstone et al., 1987). While plasma norepinephrine levels were increased by the blockade in both control and diabetic subjects, it was increased much more in the diabetic condition, so it is conceivable that synaptic cleft levels of norepinephrine are higher in diabetic than nondiabetic subjects. There are a number of ₂-adrenoreceptor agonists and antagonists that are used in the treatment of diseases of the circulatory system (see Section IV.D.).

iv. Purinergic Receptors. There are two main classes of purinergic receptors: those stimulated by adenosine are classified as P₁ receptors and those that respond to ATP are known as P₂ receptors. There are four subtypes within the P₁ class: A₁, A_2a, A_2b, and A₃. The A₂ subtypes are coupled to G_s and stimulate adenylyl cyclase, whereas A₁ and A₃ are coupled to G_o/G_i and inhibit adenylyl cyclase. Stimulation of the A₁ receptor on the -cell inhibits insulin secretion (Bertrand et al., 1989). Using two stable P₂ receptor agonists, ,-methylene ATP and ADPS, which are more specific for the P_2X and the P_2Y receptor agonists, respectively, Petit and colleagues have shown that both of these receptors exist on the -cell (Petit et al., 1998). Their action is to potentiate glucose-stimulated insulin secretion. Of the purinergic receptors only the A₁ has been shown to be important in pharmacological action in the -cell, as it is antagonized by the group of compounds known as the methylxanthines (see Section IV.E.). There are as yet no known pharmacological agents that stimulate ATP production, nor are there any pharmacological agents in use that are known to be effective at the P₂ receptors on the -cell. However, the P2 receptors are the targets of research for future treatments for diabetes (see Section III.D.3.).

C. Insulin Synthesis

1. Transcriptional and translational regulation. The -cell is the only cell in the adult body that can make proinsulin mRNA. Other cells have been programmed to transcribe proinsulin (Laub and Rutter, 1983) and other neuroendocrine cells are able to process proinsulin to mature insulin secretory vesicles and secrete insulin in a regulated manner (Moore et al., 1983), but the initiation of transcription of the preproinsulin gene is unique to the -cell of the pancreas. PDX-1 is perhaps the most extensively studied insulin transcription factor because it is essential for the maintenance of the -cell phenotype and pancreatic invagination and development (Jonsson et al., 1996). It binds to the A-box motifs of the insulin promoter and is involved in glucose- and GLP-1-mediated up-regulation of the insulin gene (Macfarlane et al., 1999). Glucose induces translocation of PDX-1 to the nucleus within 15 to 30 min (Wang et al., 1999). This mechanism is PKA-independent (Wang et al., 2000) but is known to be sensitive to wortmannin-LY 294002 (PI 3-kinase inhibitors) and has been shown to require PI 3-kinase activation (Rafiq et al., 2000). Previously it was believed that the restriction of insulin transcription to this single cell type was dependent on the expression of a unique compliment of transcription factors (including PDX-1) within the -cell. However, PDX-1 / mice do not have a pancreas, but insulin-positive cells are observed in the rudimentary bud of the organ (Offield et al., 1996). Consequently, PDX-1 is not necessary for transcription because there are other transcription factors that can bind to the A-box element of the insulin promoter and cooperate with the coactivator 2/NeuroD to activate transcription of insulin and thus act in place of PDX-1 when it is not expressed. In this case the Lim-homeodomain proteins Lmx1.1 and Lmx1.2 are capable of forming a more effective transcription factor complex with 2/NeuroD than is PDX-1 (Ohneda et al., 2000). Thus, factors that may be influential in reserving insulin transcription to the -cell alone could include the presence, in other cell types, of inhibitory proteins that bind to and inactivate key factors and/or the absence of certain transcription factors upstream of those directly involved in insulin transcription. For a comprehensive review of the transcription factors that bind to the insulin promoter we refer the reader to the following reviews: Melloul et al. (2002); Ohneda et al. (2000); and Sander and German (1997).

2. Endoplasmic Reticulum, Insulin Secretory Vesicles and Transportation, and Exocytosis. Preproinsulin, once synthesized in the endoplasmic reticulum, exists for about 30 to 60 s before the pre-portion is removed enzymatically, and proinsulin is then transported along the microtubule network in transport vesicles to the cis part of the Golgi apparatus. This latter transportation step is GTP- and calcium-dependent (Beckers and Balch, 1989). It is in the trans network of the Golgi apparatus that proinsulin is converted by the prohormone-converting endopeptidases PC3 (also known as PC1) and PC2, and the exoprotease, carboxypeptidase H into insulin and the inactive byproduct C-peptide. Once in the cisternae of the Golgi apparatus insulin is packaged into secretory vesicles ready for export to the plasma membrane. The grains of insulin accumulate in the cisternae of the Golgi apparatus, where they initially form immature clathrin-coated vesicles. Most of the insulin molecules are channeled into vesicles and hence are secreted in the regulated pathway with only about 1% being secreted through the constitutive pathway (Rhodes and Halban, 1987). From the trans-Golgi network the secretory vesicles are carried via the microtubules that form part of the cytoskeleton of the -cell. The cytoskeleton of the -cell is an important component of insulin secretion, and disruption of this network hinders the post-translation processing and mobilization of insulin to the plasma membrane. This network consists of polymerized structures of actin filaments and microtubules and they form an important bridge between the endoplasmic reticulum and the Golgi apparatus and the plasma membrane. The microtubules consist of polymerized tubulin, and the application of glucose to the cells is known to increase the amount of polymerized tubulin in the -cell (Montague et al., 1976). The polymerization of tubulin and mobilization of vesicles through the cytoskeletal network is regulated by proteins that bind to tubulin, known as microtubule-associated proteins. The microtubule-associated protein or proteins that promote polymerization of tubulin are believed to be phosphorylated by cAMP-responsive protein kinases (reviewed in Howell and Tyhurst, 1986 and more recently in Easom, 2000). Likewise, the amount of polymerized actin in islet cells increases from roughly 40% to about 70% upon glucose-stimulated insulin secretion (Howell and Tyhurst, 1986; Swanston-Flatt et al., 1980). The force generating microtubule-associated adenosine triphosphatase (ATPase), kinesin, has been identified as important in the mobilization of insulin secretory vesicles. It has been found on both the microtubulin network (Balczon et al., 1992; Meng et al., 1997) and on the vesicles themselves (Donelan et al., 2002), and consists of an ATPase portion, a tubulin binding site and a vesicle binding site (Hirokawa, 1998). In the resting -cell kinesin is phosphorylated by casein kinase 2, but with increasing calcium levels (as with glucose-induced insulin secretion) it is rapidly dephosphorylated by calcineurin (Donelan et al., 2002).

	III. Pharmaceutical Agents Active in the Treatment of Disorders of Glucose Homeostasis

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Of all compounds known to directly positively modulate insulin release, the sulfonylureas are the most studied. Their insulinotropic properties have been much exploited by the drug industry in the treatment of type 2 DM. As sulfonylureas require intact -cells, they have no value in treating type 1 DM. They are named for their common core configuration, which consists of a sulfonylurea group attached via the sulfur to a benzene ring (Fig. 2). They are derivatized by varying the substituents on the nitrogen of the urea group (R₂) and on the para position of the benzene ring (R₁). In the case of first-generation sulfonylureas (chlorpropamide, tolbutamide, tolazamide, and acetohexamide) the R₁ substituents are small and polar, and therefore render the aryl-sulfonylurea more water-soluble. In the second-generation sulfonylureas (glyburide, glipizide, gliclazide, and glimepiride) the substituents are large, nonpolar, lipophilic groups that more readily penetrate cell membranes and are thus more potent.

View larger version (7K): [in this window] [in a new window]   Fig. 2.   Core chemical structure of the sulfonylurea drugs. R1 are substituents in the para position of the benzene ring and R2 represents substituents on the nitrogen of the urea group.

Sulfonylureas release insulin by binding to the SUR subunit of the K_ATP channel and reducing its probability of opening (see Section II.B.1.i.). They are exogenous ligands of the SUR subunit, closing the channel and eliciting insulin release regardless of plasma glucose concentrations. In the presence of sulfonylureas K_ATP channel activity is disconnected from glucose sensing, so hypoglycemia resulting from hyperinsulinemia may occur in the fasting state (Ferner and Neil, 1988; Seltzer, 1989). The longer the half-life of a particular analog the greater will be its probability of inducing hypoglycemia. There is evidence that some sulfonylureas actually enhance the usual inhibitory action that MgADP has on the nucleotide binding site of the K_ir6.2 subunit and so this may increase insulin release even more under hypoglycemic conditions. MgADP effects on the K_ATP channel are 2-fold: it inhibits the K_ir6.2 subunit and stimulates the SUR1 activity. There is therefore usually a balance between these effects. In the presence of some sulfonylureas (notably tolbutamide and glyburide), however, the interaction of MgADP with SUR1 is diminished, resulting in its unopposed inhibitory effect on K_ir6.2 (Gribble et al., 1997).

In 1996 it was first reported that the action of sulfonylureas may not be entirely limited to closure of the K_ATP channels, but that they may have a direct effect on the exocytotic machinery of the -cell (Eliasson et al., 1996). In individual mouse -cells that were voltage-clamped with the membrane potential held at 70 mV, tolbutamide, glibenclamide, and glipizide all caused a 2- to 3-fold increase in exocytosis (Eliasson et al., 1996). This observation has been made in at least one other laboratory (Tian et al., 1998), but the details of the mechanism remain controversial. The effect is PKC-dependent (Eliasson et al., 1996) but may not involve activation of PKC (Tian et al., 1998). Renstrom and colleagues have proposed that it is associated with the maintenance in the insulin vesicles of a pH and ion balance conducive to exocytosis (Renstrom et al., 2002). Acidification of the internal milieu of the insulin vesicle occurs via uptake of H⁺ and is a necessary priming step for release of insulin. This increase in positive charge within the vesicle must be balanced by an influx of Cl to prevent an excessive build-up of positive charge and thus permit vesicle acidification. Sulfonylureas are known to bind to a 65-kDa protein found on the vesicle fraction of the -cell (Kramer et al., 1994) and are thought to modulate the activity of the granular ClC-3Cl channel, which is instrumental in the acidification of the insulin vesicle (Renstrom et al., 2002).

Much has been written on the pharmacology and treatment regimens of the various generations of sulfonylureas used in the maintenance of euglycemia in the diabetic state and the combination of these drugs with other oral agents and insulin in the treatment of diabetes and its complications (DeFronzo, 1999; Inzucchi, 2002; Holmboe, 2002). In particular, many of the publications continuing to emanate from the United Kingdom Prospective Study on Diabetes (UKPDS) provide recent information on the efficacy of intensive combination therapy; these publications are listed on the homepage of the UKPDS website (http://www.dtu.ox. ac. uk/index. html).

A new class of drugs that also close the K_ATP channels has been discovered, and arose from the observation that the second-generation sulfonylurea, glibenclamide, binds to a low-affinity binding site on the SUR1 subunit. These benzoic acid derivatives (also referred to as the meglitinides) are structurally distinct from the sulfonylureas but show some chemical resemblance to the nonsulfonylurea part of glibenclamide. Two benzoic acid derivatives are currently in use to treat type 2 DM. They are repaglinide and nateglinide with a third, mitiglinide, still in clinical trials (at the time of writing). Many studies have been performed comparing these drugs with each other and with glibenclamide both in vitro and in vivo. Both repaglinide and nateglinide are potent K_ATP channel blockers, with repaglinide being 10-fold more potent than glibenclamide (Fuhlendorff et al., 1998) and the rank order of potency being repaglinide>glibenclimide>nateglinide (Hu et al., 2000). Binding studies on repaglinide and glibenclamide in an insulinoma cell line (TC-3 cells) indicate that there are probably three distinct binding sites for these two compounds: a high-affinity repaglinide site and two lower-affinity sites for glibenclamide (Fuhlendorff et al., 1998). Repaglinide, in contrast to glibenclamide, did not stimulate insulin secretion in islets in the absence of glucose and is more effective than glibenclamide at higher glucose concentrations (16.7 mM; Fuhlendorff et al., 1998). However, nateglinide (30 µM) shows a glucose-induced insulin secretion profile similar to that of glibenclamide (0.3 µM) (Ikenoue et al., 1997). Nateglinide has the advantage in that it rapidly dissociates from the SUR1 and displays a more rapid onset of channel inhibition and faster reversal of same than rapaglinide (Hu et al., 2000).

Because the effects of both drugs are rapid and short-lived they are used to curtail postprandial excursions in glucose (de Souza et al., 2001; Kalbag et al., 2001). The plasma half-life of both compounds in healthy human volunteers is between 1 and 1.5 h, with nateglinide producing a much more rapid rise in insulin postprandially than does rapaglinide, when the agents are administered 30 min before eating (Kalbag et al., 2001). Thus the risk of hypoglycemia when treating with these drugs is lower than with traditional sulfonylureas. When nateglinide (120 mg) was administered to fasted type 2 DM patients 15 min before receiving an i.v. bolus of glucose (300 mg/kg body weight), it produced an incremental response in the first 10 min of 788 pM/min versus 303 pM/min for glyburide (10 mg; Kahn et al., 2001). For nateglinide the incremental response was greatest at 60-120 min, whereas for glyburide it was greatest at 120-300 min after the intravenous glucose tolerance test (IVGTT). At the end of the 300-min observation period plasma insulin levels in the patients who received nateglinide had returned to prevailing basal levels, whereas those for glyburide were still 2-fold basal values. Thus, due its rapid action on the -cell, administration of nateglinide to diabetic individuals preprandially produces a more physiologically normal insulin response than is seen with the sulfonylureas. There is a rapid 5-fold increase of insulin above basal values within 30 min of eating, which represents the prompt release of the insulin in the vesicles of the RRP followed by a decline within 120 min to values 2-fold above basal. It must be noted, however, that this does not necessarily reflect a restoration of normal insulin secretory kinetics, as the early rise in insulin secretion was seen with nateglinide in the presence or absence of glucose, and it slowly decreased, most likely representing an exhaustion of the primed insulin secretory vesicles in the -cell.

Drugs in the class of insulin sensitizers known as thiazolidinediones (Berger and Moller, 2002) act primarily on the proxisome proliferator activated receptor-. They are used to treat insulin resistance, therefore we will not address them here but refer only to two reports where there is an indication of improvement of -cell function and consequently insulin secretion following treatment with this class of drugs. Two groups have measured the ratio of proinsulin/insulin, sometimes used as an indication of -cell insulin secretory dysfunction (Roder et al., 2000), with an elevation in the ratio associated with type 2 DM. In one study it was found that after 52 weeks of rosiglitazone therapy the proinsulin/insulin ratio was significantly decreased (Porter et al., 2000). The second study showed that a decrease in the ratio was associated with troglitazone therapy (Prigeon et al., 1998).

Glucagon is a polypeptide hormone that consists of 29 amino acids. In its endogenous form it is naturally secreted by the -cells of the islets and stimulates glycogenolysis in the liver resulting in increased blood glucose levels. At physiological concentrations it augments glucose-induced insulin secretion by stimulating the glucagon G-protein-coupled receptor on the -cell (Huypens et al., 2000) resulting in increased intracellular cAMP levels (Henquin, 1985). When used as an emergency treatment of hypoglycemia due to hyperinsulinemia it is administered as a 1-mg dose usually intramuscularly or subcutaneously, but may also be given intravenously (Hall-Boyer et al., 1984). At these concentrations it counteracts the anabolic effect of insulin on the hepatocytes and stimulates glycogenolysis. This latter effect counteracts the increased endogeno