Human Molecular Genetics, 2004, Vol. 13, Review Issue 1 DOI: 10.

1093/hmg/ddh066 Advance Access published on January 20, 2004

R21R31

Type 2 diabetes mellitus: not quite exciting enough?

Frances Ashcroft1,* and Patrik Rorsman2
1

University Laboratory of Physiology, Parks Road, Oxford OX1 3PT, UK and 2Oxford Centre for Diabetes, Endocrinology and Metabolism, Churchill Hospital, Oxford OX3 7LJ, UK

Received December 12, 2003; Revised and Accepted January 12, 2004

Type 2 diabetes mellitus is a serious metabolic disease that aficts around 5% of the population in Western societies and over 150 million people worldwide. It is characterized by elevation of the blood glucose concentration, usually presents in middle age, and is exacerbated by obesity. Both genetic and environmental factors contribute to the disease but in the vast majority of cases the aetiology is still not understood. Here we present a novel hypothesis for the aetiology of type 2 diabetes. We postulate that the electrical activity of the insulin-secreting b-cells of the pancreas acts to integrate the genetic and environmental factors that predispose to disease risk. Our hypothesis is supported by a substantial amount of data gathered from a range of different disciplines and makes predictions that can be tested experimentally both in vitro and in man.

Downloaded from http://hmg.oxfordjournals.org/ by guest on November 4, 2013

INTRODUCTION
Natural history of type 2 diabetes Diabetes mellitus refers to a range of conditions that are all characterized by elevation of the blood glucose level. It may be roughly divided into two principal varieties, type 1 and type 2 diabetes, which have very different aetiologies (1). Type 1 diabetes presents in childhood as an autoimmune attack on the pancreatic b-cells that results in their complete destruction: consequently, the patient must take insulin for the rest of their life (2). It accounts for <10% of all diabetes and will not be considered further here. Type 2 diabetes is a very different disease that is reaching epidemic proportions in Western societies and is predicted to affect 300 million people worldwide by 2025 (24). Impaired glucose tolerance, which precedes diabetes and is a risk factor for the disease, currently affects a further 200 million worldwide. Until recent years, type 2 diabetes was rarely observed in individuals under the age of 50, but increasing numbers of children are now being diagnosed with the disease (5). This probably reects the growing prevalence of childhood obesity, as type 2 diabetes is exacerbated by obesity and a sedentary lifestyle. Diabetes leads to a reduced life expectancy and quality of life, and a greater risk of heart disease, stroke, peripheral neuropathy, renal disease, blindness and amputation (6). The direct health care costs of the disease are also considerable, and have been estimated at around 5% of the total annual expenditure on health in Western societies.

Both insulin secretion and insulin action are impaired in type-2 diabetes (reviewed in 7,8). Their relative importance has been hotly debated, but it is now recognized that b-cell dysfunction is a key element in the development of the disease (810). Abnormalities in insulin secretion precede the onset of type-2 diabetes and may be present even when subjects show normal glucose tolerance (1114). By the time of diagnosis, insulin secretion is signicantly reduced and it continues to diminish inexorably throughout the course of the disease (11). Type 2 diabetes can also occur in the absence of insulin resistance (15) and, conversely, some severe forms of insulin resistance (such as those caused by mutations in the insulin receptor) may not be accompanied by diabetes (16,17). It now appears that insulin resistance only leads to diabetes if combined with a genetically determined propensity to b-cell dysfunction (15,18). In these individuals, however, insulin resistance plays an important role in the development of diabetes by placing an increased demand upon the b-cell that it is unable to match. Theoretically, the insulin secretory defect could result from either a failure of b-cell function or a reduction in b-cell mass (due to increased apoptosis or reduced b-cell replication). Most quantitative estimates of b-cell density in post-mortem tissue indicate that type-2 diabetics have either no change or <30% reduction in b-cell mass (19,20), that is independent of the duration of the disease (21,22). A substantial reduction in b-cell mass is only found in association with amyloidosis, which occurs at later stages of the disease (23,24). In baboons,

*To whom correspondence should be addressed. Tel: 44 01865285810; Fax: 44 01865285812; Email: frances.ashcroft@physiol.ox.ac.uk

Human Molecular Genetics, Vol. 13, Review Issue 1 # Oxford University Press 2004; all rights reserved

R22

Human Molecular Genetics, 2004, Vol. 13, Review Issue 1

a decrease in b-cell mass of >50% is require to cause diabetes (25) and hyperglycaemia does not occur in type-1 diabetics as long as b-cell mass remains above 3050% (26). It therefore seems that the insulin secretory defect in type 2 diabetes does not result primarily from insufcient b-cell mass but rather from impaired insulin secretion. Electrical activity and insulin release Electrical activity plays a critical role in the regulation of insulin secretion (27). No insulin is secreted in the absence of b-cell electrical activity, and there is a direct correlation between the extent of b-cell electrical activity, once initiated, and the amount of insulin secretion (28). Thus changes in electrical activity are immediately mirrored by changes in insulin secretion. The stimulatory actions of almost all insulin secretagogues, including glucose, neurotransmitters and hormones, converge at the level of electrical activity, which serves to integrate the effects of all these agents. Although most secretagogues have additional downstream effects on insulin secretion (29,30), none are effective in the absence of electrical activity. Thus, b-cell electrical activity is an essential step in insulin release. In the b-cell, electrical activity is determined by a complex interplay between several different types of ion channels (27). Tiny changes in the activity of these ion channels can have dramatic effects on electrical activity and thus on insulin secretion (Fig. 1). This is especially the case around the threshold for electrical activity, where almost undetectable changes in current amplitude can make the difference between ring and non-ring, and consequently between insulin secretion and no secretion at all. Thus, changes in ionic currents switch electrical activity (and insulin release) on and off, as the blood glucose concentration uctuates around the fasting blood glucose level. Because electrical activity is so responsive to tiny changes in current amplitude, it also serves as an exquisitely sensitive detector of small changes in b-cell metabolism and blood hormone levels (which regulate ion channel activity). Here, we propose that the insulin secretory defect that occurs in type 2 diabetes is the result of inadequate b-cell electrical activity in response to secretagogues. This reduction in electrical activity may result from polymorphisms in genes encoding ion channels, or in genes that regulate ion channel function (e.g. metabolic genes), membrane targeting or expression (e.g. transcription factors), as well as environmental factors such as age and obesity. Evidence in support of this hypothesis is accumulating, and includes much recent data, which are summarized here. Mechanism of insulin release Insulin is stored in secretory vesicles, which are released in response to elevation of intracellular Ca2. This results principally from Ca2 inux across the plasma membrane through voltage-gated Ca2 channels (27). Opening of these channels is triggered by the electrical activity of the cell. Glucose modulates electrical activity principally via metabolically induced changes in the activity of ATP-sensitive K (KATP) channels (31). In the absence of glucose (Fig. 2A), these channels are open and the ensuing K efux keeps the membrane hyperpolarised so that voltage-gated Ca2 channels

Figure 1. The input resistance of the b-cell membrane determines the ease with which electrical activity can be initiated. It is largely determined by the activity of the KATP channel and is low when KATP channels are open and high when they are shut. From Ohms Law (V IR), it is evident that the same magnitude of current (I) will produce a much greater change in membrane potential (DV), and thus in insulin secretion, when KATP channels are closed (R is high) than when they are open (R is low). At low glucose (blue trace) the input resistance of the membrane (Ri) is low, so that injection of a small current (I, black trace above) only depolarizes the membrane by a small amount (DV1). At high glucose (red trace), the input resistance is higher (5Ri), so that the same current depolarizes the cell by a much larger amount (DV2), and may be sufcient to trigger an action potential (dotted line). Potentiators of insulin secretion such as acetylcholine and arginine produce small inward currents (126,127). They are therefore ineffective in the absence of glucose, when the activity of the KATP-channels is high (i.e. R is low), but are able to stimulate electrical activity and insulin secretion at glucose concentrations that shut most KATP channels (i.e. R is high) (125128). In vivo, blood glucose levels rarely fall below 34 mM in man and thus the b-cell input resistance is always relatively high. This explains why acetylcholine, which is released in response to the sight and smell of food, is able stimulate insulin secretion even before blood glucose levels rise. Likewise, the presence of food in the gut releases incretins, such as GLP-1 and GIP, which also initiate insulin secretion prior to elevation of blood glucose. Because arginine and GLP-1 can stimulate insulin secretion even in type 2 diabetes, the input resistance must already be relatively high at fasting blood glucose concentrations. However, the fact that these agents are less effective in diabetes also suggests that, at the same glucose concentration, the input resistance is not as high as that in non-diabetic b-cells.

remain shut. Glucose metabolism closes KATP channels and depolarizes the b-cell (31). In turn, this leads to opening of voltage-gated Ca2 channels and initiation of electrical activity (Fig. 2B). The mechanism by which KATP channel closure is coupled to glucose metabolism remains unclear, although it is believed to involve changes in the intracellular concentrations of the adenine nucleotides ATP and ADP (32). The KATP channel is also the molecular target for common antidiabetic drugs, such as the sulfonylureas (33), which have been widely used to treat type 2 diabetes for more than half a century (34). Sulfonylureas close these channels independently of glucose metabolism, and thereby elicit electrical activity and insulin secretion (32,35).

Human Molecular Genetics, 2004, Vol. 13, Review Issue 1

R23

Figure 2. Role of ion channels in regulating b-cell electrical activity and insulin secretion. Insulin secretion is triggered by an increase in intracellular calcium ([Ca2]i), which is, in turn, regulated by the electrical activity of the b-cell. When metabolism is low (A), KATP channels are open keeping the membrane hyperpolarized and Ca2 channels closed, so that [Ca2]i remains low. When metabolism increases (B), KATP channels shut depolarizing the b-cell membrane potential, thereby eliciting electrical activity and opening voltage-gated Ca2channels. This leads to Ca2 inux and an increase in [Ca2]i that triggers exocytosis of insulin granules. (C) Membrane potential recorded from a b-cell within a mouse islet at 5 mM and 10 mM glucose, concentrations above (10 mM) and below (5 mM) the threshold for electrical activity and insulin release. Electrical activity consists of bursts (slow waves) of Ca2action potentials during which Ca2enters the cell and triggers insulin secretion (see text for further details).

Glucose not only initiates b-cell electrical activity: it also modulates action potential frequency, and thereby Ca2-inux and insulin secretion. Electrical activity exhibits a characteristic bursting pattern, which consists of slow oscillations in membrane potential between a depolarized plateau, on which Ca2dependent action potentials are superimposed, and a hyperpolarized electrically silent interval (Fig. 2C) (27,28). As glucose is increased, the duration of the plateau phase increases and that of the silent interval decreases so that electrical activity becomes continuous at 20 mM glucose (36). This produces a graded increase in action potential ring and Ca2 inux that accounts for the glucose-dependence of insulin secretion (EC50, 10 15 mM glucose). A complex interaction between KATP channels, voltage-gated Ca2 channels, Ca2-activated Kchannels, Ca2 pumps and metabolism is responsible for this bursting pattern of b-cell electrical activity and its modulation by glucose (27,37). The b-cells are electrically coupled to one another, so that the islet functions as an electrical syncytium (3840). This serves to coordinate electrical activity and insulin secretion across the islet and accounts for the fact that glucose-stimulated insulin secretion from a single islet is pulsatile and mirrors the slow waves of electrical activity (41).

In addition to glucose, insulin secretion is stimulated by amino acids (e.g. arginine), incretins [e.g. glucagon-like peptide 1 (GLP-1)], neurotransmitters (e.g. acetylcholine) and drugs (e.g. sulfonylureas) (27). Some of these agents, like sulfonylureas, can stimulate release in the absence of glucose because they close KATP channels, depolarize the b-cell membrane and elicit electrical activity. In contrast, incretins and neurotransmitters are ineffective in the absence of glucose, but are able to amplify insulin secretion produced by an initiator and can even initiate insulin secretion at glucose concentrations just below threshold. Their action is dependent on the ambient level of KATP channel activity, which confers their dependence on the glucose concentration (Fig. 1).

WHY IS ELECTRICAL ACTIVITY USED TO CONTROL INSULIN RELEASE? Why is electrical activity used to control insulin release? After all, it would be possible to achieve a graded response to glucose simply by direct metabolic regulation of exocytosis. Indeed, glucose is still able to elicit a graded increase in insulin

R24

Human Molecular Genetics, 2004, Vol. 13, Review Issue 1

secretion when changes in electrical activity are bypassed: for example, by continuous depolarization of the b-cell (42,43). The answer seems to be that electrical activity enables insulin secretion to be switched on and off quickly and precisely. This is particularly important in the case of insulin, as inappropriate release of the hormone can lead to life-threatening hypoglycaemia. Hypoglycaemia is a common complication in diabetic patients taking sulfonylureas, or patients with congenital hyperinsulinaemia, where insulin secretion cannot be switched off rapidly when blood glucose levels fall. The rapid onoff switch of insulin secretion provided by electrical activity also enables insulin secretion to be pulsatile (44), and so avoids down-regulation of insulin receptors in target tissues. This may explain why metabolic regulation of insulin secretion alone is not sufcient to control blood glucose and underscores the importance of electrical activity in insulin release.

tions (compared with the non-diabetic b-cell), which leads to reduced Ca2-dependent electrical activity and insulin release.

DO CHANGES IN ELECTRICAL ACTIVITY UNDERLIE THE IMPAIRED INSULIN SECRETION IN TYPE 2 DIABETES? b-Cell dysfunction in type 2 diabetes manifests as a reduction in the insulin secretory response not only to glucose, but also to potentiators of insulin release such arginine, hormones, incretins and sulfonylureas (4548). Any mechanistic explanation of the disease must also account for all these secretory abnormalities. The permissive role of electrical activity in modulating insulin secretion to both initiators and potentiators of release suggests that the abnormalities in hormone release found in type 2 diabetes might be secondary to changes in electrical activity. The most direct way to test our hypothesis would be to compare electrical activity in non-diabetic and diabetic human b-cells. Unfortunately, such in vitro data are extremely limited, because of the difculty in obtaining viable islets from type 2 diabetics. However, we recently found that glucose fails to elicit electrical activity, Ca2 inux and insulin secretion in b-cells and islets isolated from a type 2 diabetic (unpublished data). This is consistent with earlier reports that glucose did not elevate intracellular Ca2 (49), and that glucosestimulated insulin secretion was impaired (50), in type 2 diabetic islets. Clinical studies are also consistent with the idea that b-cell electrical activity may be impaired in type 2 diabetes. The fact that sulfonylureas and glinides, which close KATP channels (32), are effective in the treatment of type 2 diabetes suggests that KATP-channel closure is not complete in diabetes. On the other hand, the nding that arginine and GLP-1 stimulate insulin secretion in type 2 diabetic patients (4547) argues that even in diabetic b-cells most KATP channels must be closed at stimulatory glucose concentrations. This is because these agents are only effective when the membrane resistance is high (Fig. 1). As neither potentiator is as potent in type 2 diabetics as in healthy individuals (4547), however, membrane resistance and electrical activity are likely to be reduced. Thus, we hypothesize that glucose intolerance and type 2 diabetes are associated with a lower electrical resistance of the b-cell membrane in the presence of stimulatory glucose concentra-

DIABETES CORRELATES WITH MUTATIONS/ POLYMORPHISMS IN GENES REGULATING ISLET CELL ELECTRICAL ACTIVITY It is well established that type 2 diabetes is a polygenic disease (7). It has been linked to polymorphisms in many genes, but how these polymorphisms relate to one another, and to the disease phenotype, has not been established. We postulate that the collective action of several common gene variants, and lifestyle factors, combine to produce a small decrease in b-cell electrical activity, and thus a reduction in insulin secretion. We anticipate that the functional consequences of each individual gene variant will be small, so that a single polymorphism, by itself, is unlikely to result in diabetes. However, the cumulative effect of several such polymorphisms will increase disease risk, and in combination with age and/or obesity lead to overt disease. In effect, electrical activity serves as a bottleneck at which the effects of many different genes and lifestyle factors converge.
Impaired ion channels Polymorphisms in genes encoding ion channels are obvious causative candidates for the reduction in electrical activity that we propose underlies type 2 diabetes. Importantly, a number of such polymorphisms have been identied. One that has received much recent attention is a polymorphism (E23K) in the Kir6.2 subunit of the KATP channel, with a prevalence of 34% in Caucasians (51). Although early studies failed to show a signicant association with type 2 diabetes (51,52), metaanalysis and more recent studies using larger sample sizes have revealed that the K allele accounts for between 11 and 15% of the population risk of type 2 diabetes in Caucasians, with an odds ratio of around 1.5 (5359). When heterologously expressed in mammalian cells, the E23K polymorphism leads to a 2-fold reduction in the ATP sensitivity of the KATP channel (54) and enhanced activation by MgGDP (55,60). It is therefore expected to reduce b-cell electrical activity and insulin secretion. That such small changes in KATP-channel regulation can indeed exert a marked effect on b-cell function is exemplied by the fact that a 4-fold reduction in KATP channel ATP sensitivity causes severe neonatal diabetes in transgenic mice (61). The functional effects of the E23K polymorphism in man are controversial. Some studies report that insulin secretion is impaired (56), whereas others have failed to nd a difference in insulin secretion (62,63), but report that the ability of glucose to inhibit glucagon secretion is impaired (63). It is possible that these differences relate to the genetic background of the patients examined, but more studies are clearly needed. Polymorphisms in SUR1 (e.g. S1369A; 59) have also been linked to type 2 diabetes, but as yet no functional consequences of these polymorphisms have been identied. Overactivity of KATP-channels is not the only mechanism that may be expected to impair electrical activity. Polymorphisms in genes encoding the voltage-gated Ca2- and K-channels involved in generating b-cell action potentials that lead to

Human Molecular Genetics, 2004, Vol. 13, Review Issue 1

R25

decreased or increased channel activity, respectively, could also reduce b-cell electrical activity. Indeed, overexpression of the voltage-gated K-channel Kv1.5 decreases the secretory response of islets (64), and deletion of the L-type Ca2-channel in mice results in reduced insulin secretion and glucose intolerance (65). Furthermore, a 7 mV shift in the threshold for L-type Ca2-channel activation towards more negative membrane potentials enhanced b-cell electrical activity (66), and led to increased serum insulin levels and hypoglycaemia in mice (67). This suggests that a small positive shift in the activation threshold for these channels might increase the threshold for glucose-dependent electrical activity and thus impair insulin secretion. There is also some evidence that voltage-gated Ca2 channels are inuenced by b-cell metabolism (68), so that it is possible that polymorphisms in metabolic genes may modulate electrical activity and insulin release via channels other than the KATP channel. It is of interest that the b-subunit of the voltage-gated calcium channel (CACNB2) lies with the region of chromosome 10p that was linked to permanent neonatal diabetes in a genome-wide linkage analysis of a large family (69). Finally, cytoplasmic Ca2 inuences the activity of small conductance Ca2-activated K channels, providing a feedback mechanism that links changes in Ca2 handling by the b-cell with electrical activity and insulin secretion (7072). However, with the exception of the KATP channel subunits Kir6.2 and SUR1, the association of polymorphisms in genes encoding b-cell ion channels with type 2 diabetes has not yet been widely studied. Polymorphisms in genes encoding proteins that regulate ion channel transcription, membrane targeting or activity may also be expected to inuence b-cell electrical activity and thereby insulin secretion. In this context it is interesting that another candidate gene within the chromosome 10p locus is PIP5K2A (59), a phosphatidyl 4-phosphate 5-kinase type 2, which may be expected to inuence the level of PIP2 in the membrane and thereby KATP channel activity (73). Impaired metabolic regulation of ion channels Because metabolism regulates b-cell electrical activity, polymorphisms in metabolic genes may also be expected to inuence the ability of glucose to stimulate electrical activity and insulin secretion. Support for the idea that impaired metabolic regulation of electrical activity occurs in type 2 diabetes comes from a number of rare (15%) monogenic forms of diabetes (7). These are collectively referred to as maturity-onset diabetes of the young (MODY) because they present early in life. The rst of these to be identied (MODY2) results from inactivating mutations in glucokinase, the high Km enzyme that phosphorylates glucose in b-cells and is rate-limiting for glucose metabolism (10) (Fig. 3). All MODY2 patients are heterozygotes: permanent neonatal diabetes results from homozygous mutations (74). Other forms of MODY are due to mutations in genes (e.g. HNF1a, HNF4a, IpF1) encoding a transcriptional network that regulates the expression of several genes critical for glucose sensing (75 80). Studies on knock-out animals have shown the reduced bcell glycolytic metabolism caused by MODY mutations leads to a decrease in KATP channel closure and electrical activity in response to glucose, which in turn causes less insulin release

(81,82). Mutations in mitochondrial DNA (most frequently A3243G in the leucine tRNA gene) cause maternally inherited diabetes, probably by impairing b-cell metabolism and so reducing electrical activity (8385). These account for a further 12% of diabetic cases. There is a demonstrable overlap between MODY and multifactorial type 2 diabetes, and mutations in IpF-1 (MODY4) have been associated with type 2 diabetes (86). As yet, there is no evidence that polymorphisms in other MODY genes contribute to type 2 diabetes. It is pertinent, however, that a mutation in the HNF-1a (MODY3) gene accelerates the onset of type 2 diabetes in the OjiCree population by 7 years, and is found in 40% of diabetic OjiCree patients (87). Likewise, a 30G/A variant in the b-cell promoter of the glucokinase (MODY2) gene has been implicated in impaired glucose tolerance (88). Furthermore, a polymorphism in HNF4a has recently been found to associate with reduced risk of diabetes (59). Mitochondrial metabolism generates substantially more ATP than glycolysis and the production of mitochondrial ATP is critical for both glucose-dependent insulin secretion and KATP channel closure (85,89). It is therefore pertinent that polymorphisms in genes that regulate mitochondrial ATP production are associated with type 2 diabetes. UCP2 is an uncoupling protein that resides in the inner mitochondrial membrane and uncouples electron transport from ATP synthesis (90) (Fig. 3). Thus it tends to lower cytosolic ATP levels. Deletion of the UCP2 gene in mice enhances islet ATP production and insulin secretion in response to glucose (91). Conversely, overexpression of UCP2 in b-cells attenuates ATP generation and insulin secretion during glucose stimulation (92). This suggests that the level of UCP2 expression may inuence insulin secretion in man. Consistent with this idea, a common polymorphism in the UCP2 promoter (866G/A) causes a 2-fold increase in the risk of type 2 diabetes in obese white Europeans (93). The b-cell transcription factor PAX6 preferentially binds to, and trans activates, the 866A variant in insulin-secreting (INS-1) cells and is expected to increase UCP2 mRNA expression in islet b-cells, thereby reducing ATP levels, electrical activity and insulin release. The frequency of the 866A variant in the European population is 37% (94), suggesting it may make a signicant contribution to type 2 diabetes. Recent studies further suggest that the 866A variant is associated with differences in glucose-stimulated insulin secretion in glucose-tolerant human subjects both in vivo and in isolated islets (95). A common variant in mitochondrial DNA itself (16189) is also associated with type 2 diabetes (96). This variant causes a T to C transition in a region of mtDNA that lies close to control sequences governing replication and transcription. It is associated with increased fasting plasma insulin in several populations, maternal restraint of fetal growth and thinness at birth (97). Consequently, it has been suggested that it may serve as (one of) the gene(s) underlying the thrifty phenotype that is thought to predispose to type 2 diabetes (98,99). It is estimated that about 4% of diabetes in the UK diabetic population is attributable to this gene variant. Because its prevalence is higher in Polynesians (93%) and Pima Indians (50%) the 16189 variant may have a substantially larger effect in these populations (100).

R26

Human Molecular Genetics, 2004, Vol. 13, Review Issue 1

Figure 3. b-Cell metabolism and the role of UCP2. Glucose enters the b-cell via the high capacity glucose transporter GLUT2 (Km 50 mM) which ensures that the intracellular glucose concentration tracks the plasma glucose level. Glucose is then phosphorylated in a reaction catalysed by glucokinase (Gk), which serves as the rate-limiting step in glycolysis. The importance of Gk in glucose metabolism is illustrated by the fact that mutations in Gk cause MODY2. Glycolysis converts glucose-6-phosphate to pyruvate, which enters the mitochondria where it is metabolized within the TCA cycle and the respiratory chain, leading to the establishment of a proton gradient across the mitochondrial membrane. ATP is generated by the F1F0-ATPase, using energy derived from this proton gradient, and is transported to the cytoplasm. The importance of mitochondrial ATP generation in insulin secretion is illustrated by the fact that mutations in mitochondrial DNA cause maternally inherited diabetes with deafness. UCP2 is a proton channel that dissipates the mitochondrial proton gradient and so reduces ATP synthesis. Changes in the expression levels of UCP2 inuence ATP levels and thereby electrical activity and insulin secretion (the higher the expression, the less ATP, electrical activity and secretion). UCP2, uncoupling protein 2. ANT, adenine nucleotide transferase.

Loss-of-function mutations in the transcription factor peroxisome proliferator-activated receptor (PPARg) cause severe insulin resistance and type 2 diabetes, but are very rare (101). A polymorphism in this gene (P12A) is found in many ethnic groups. The more common P allele (frequency 85%) is associated with a 1.25-fold increase in diabetes risk and a population risk of 25% (102). Although the less common A12 allele is associated with a reduced risk of diabetes in the general population (103), in diabetics it is associated with decreased b-cell function and increased disease severity (104). In b-cells, PPARg enhances the expression (and activity) of a number of genes involved in glucose sensing, including glucokinase (105) and GLUT2 (106). However, it also upregulates UCP2 (107). These data suggest that the A12 allele, which has reduced transcriptional activity (102), may have opposing effects on b-cell metabolism, acting to reduce ATP levels by decreasing glycolytic activity and at the same time tending to enhance ATP levels by decreasing expression of UCP2. Whether or not the overall result is impaired b-cell metabolism (and thus diabetes) will depend on the relative strengths of these effects, and may also be inuenced by the presence of other gene variants or environmental factors.

Whatever the underlying mechanism, however, the data support the view that defective metabolic regulation of b-cell electrical activity is involved in type 2 diabetes.

THE INSIDIOUS INFLUENCE OF AGE AND OBESITY Any explanation of type 2 diabetes must account for the fact that disease develops with age and that it is enhanced by obesity. There is evidence that the interaction of such life style factors with genetic ones may also occur at the level of b-cell electrical activity. In mice, for example, ageing causes a reduction in the sensitivity of the KATP channel, and thereby electrical activity, to glucose, an effect that appears to be mediated by decreased b-cell metabolism rather than changes in the KATP channel itself (81). This may reect the well-documented decline in mitochondrial function with age, that is believed to result from accumulating mutations in mitochondrial DNA (84,108). A recent in vivo study showed a 40% decline in mitochondrial oxidative and phosphorylation function in muscle between 27 and 70 years (109). In addition, ageing

Human Molecular Genetics, 2004, Vol. 13, Review Issue 1

R27

reduces insulin sensitivity (8), thereby placing a greater secretory demand on the b-cell. In part, this may also be related to the decline in mitochondrial function (109). Obesity promotes insulin resistance, which can lead to insulin insufciency if the secretory capacity of the b-cell is already lower than normal. However, obesity may also reduce insulin secretion via changes in b-cell electrical activity. Obese individuals (110) and type-2 diabetics (111) have higher circulating levels of free fatty acids (FFAs), which are taken up and metabolized by b-cells. Chronic exposure to FFAs leads to the accumulation of long chain acyl CoAs (LC-CoAs) within the b-cell. LC-CoAs both enhance KATP channel activity and reduce its ATP sensitivity (112,113), thereby reducing glucosedependent closure of KATP channels, electrical activity and insulin secretion. KATP channel activity may also be enhanced by up-regulation of UCP2 in obesity and a consequent decrease in ATP synthesis (91,114,115). This is mediated by FFA stimulation of UCP2 expression (116), probably via PPARg (107). Mice that lack UCP2 show an enhanced insulin secretory capacity compared with wild-type mice when maintained on a high-fat diet (114). Changes in b-cell metabolism as a consequence of age and/or obesity will translate into reduced b-cell electrical activity and insulin secretion, not only to glucose but also to incretins and sulfonylureas.

THE YIN AND YANG OF b-CELL ELECTRICAL ACTIVITY Finally, we point out that diabetes may not only result from mutations that reduce electrical activity. It is also possible for mutations that lead to excessive b-cell electrical activity to cause diabetes, albeit by a different mechanism. Congenital hyperinsulinism (CHI) is associated with constant b-cell depolarization due to permanent KATP channel closure (117). This results from loss-of-function mutations in genes encoding the b-cell KATP channel subunits Kir6.2 and SUR1, or activating mutations in the metabolic enzymes glucokinase and pyruvate dehydrogenase (118,119). The unregulated insulin secretion characteristic of CHI necessitates subtotal pancreatectomy early in life in most patients. However, a dominant mutation in SUR1 produces a milder disease that can be managed without surgery (120). Interestingly, many of these patients develop a progressive insulin insufciency and diabetes in later life. A similar pattern is seen in mice in which the Kir6.2 gene has been disrupted in b-cells (121). These cells are continuously electrically active and show enhanced apoptosis, probably as a consequence of increased Ca2 inux. It therefore seems possible that b-cell loss underlies the diabetes of CHI patients, an idea that is supported by studies showing increased b-cell apoptosis in pancreatic tissue removed from patients with focal CHI (122,123). Thus, although it is clear that common type 2 diabetes is not associated with a reduced b-cell mass (see earlier), in some unusual forms of the disease it appears possible that excessive electrical activity leads to b-cell destruction and thereby reduced insulin secretion. However, not all dominant CHI mutations result in diabetes in later life (124).

A MODEL FOR TYPE 2 DIABETES We propose that individuals at risk of type 2 diabetes carry one or more polymorphisms in ion channel genes, or in genes regulating their activity, membrane targeting or transcription. The functional effect of these gene variants is a small reduction in b-cell electrical activity, which results in decreased Ca2 inux and insulin secretion (Fig. 4). This does not cause diabetes in early life, because glucose homeostasis is tightly controlled and feedback loops ensure that the b-cell secretory output is adjusted. With age, b-cell metabolic function declines, leading to a reduction in glucose sensitivity, electrical activity and insulin secretion. In non-diabetics this does not pose a problem, as the b-cell adjusts its secretory output. However, in individuals who already have reduced b-cell function, the further reduction in electrical activity means that b-cell is no longer able to compensate, so that insulin secretion declines and glucose intolerance, and subsequently overt diabetes, develop. Obesity exacerbates the situation both by causing insulin resistance (increasing the demand on the b-cell) and by further decreasing insulin secretion from the b-cell. This is mediated, at least in part, by a reduction in b-cell electrical activity. The cellular defects culminating in reduced electrical activity and insulin secretion have been analysed by mathematical modelling (125). It seems possible that, once sufcient experimental data have been accumulated, such models may help to determine in silico how combinations of several gene variants, that individually only have minor effects on b-cell metabolism, electrical activity or secretion, can collectively lead to type 2 diabetes.
Predictions To be of value, a hypothesis should make a number of testable predictions. Our hypothesis predicts that:  The risk of type 2 diabetes should be increased in individuals who carry disease-promoting polymorphisms in one or more genes. The more polymorphisms an individual carries, the greater the risk of type 2 diabetes and the earlier the onset of the disease.  These polymorphisms will be concentrated in genes that, directly or indirectly, regulate b-cell electrical activity. This is not limited to genes encoding ion channels but includes genes that regulate ion channel activity (e.g. metabolic genes, kinases), membrane trafcking and expression (e.g. transcription factors).  Detailed analysis of genes associated with permanent neonatal diabetes, or MODY, will be of value, as is its possible that mutations producing severe functional effects cause neonatal diabetes, whereas polymorphisms having only mild effects enhance susceptibility to type 2 diabetes.  Glucose will stimulate electrical activity less effectively, if at all, in type 2 diabetic b-cells than in non-diabetic b-cells. Likewise, glucose will cause less elevation of [Ca2]i.  Although sulfonylureas, arginine and incretins will still stimulate electrical activity in diabetic b-cells, they will be less effective than in non-diabetic b-cells (at the same glucose concentration).  The ability of glucose to close KATP channels and stimulate electrical activity will decline with age in both diabetic and non-diabetic b-cells, as a consequence of increasing

R28

Human Molecular Genetics, 2004, Vol. 13, Review Issue 1

Figure 4. Model showing how decreased b-cell electrical activity leads to glucose intolerance and diabetes. See text for details.

impairment of mitochondrial function. At the same age, diabetes-prone individuals may have a lower mitochondrial function than those not at risk of the disease.  Obesity will result in a reduced ability of glucose to stimulate electrical activity in b-cells.  Cells that possess similar glucose-sensing properties to bcells, such as the GLP-1 producing L-cells and (probably) glucose-sensing neurones in the brain, will also show impaired electrical activity and disturbed hormonal secretion in type 2 diabetes. Similar candidate genes will be involved. When assessing the effects of gene polymorphisms on diabetes risk, it is important to be aware that large sample sizes will be essential, as gene variants are likely to have small effects on b-cell function. This holds for functional as well as genetic studies. Furthermore, some gene variants will enhance disease risk, while others may reduce it. Indeed, a single gene variant may have both benecial and deleterious effects on risk, resulting from its expression in different tissues. Such multiplicity of actions may help explain why the aetiology of diabetes has proved so difcult to sort out.

supported by the Wellcome Trust, the Royal Society, Diabetes UK, the European Union, Juvenile Diabetes Research Foundation, the Swedish Research Council, the Swedish Diabetic Association and the Go ran Gustafsson Foundation in Natural Sciences and Medicine. F.M.A. is a Royal Society Research Professor.

REFERENCES
1. Ashcroft, F.M. and Ashcroft, S.J.H. (1992) InsulinMolecular Biology to Pathology. Oxford University Press, Oxford. 2. Zimmet, P., Alberti, K.G. and Shaw, J. (2001) Global and societal implications of the diabetes epidemic. Nature, 414, 782787. 3. World Health Organization (1985) Diabetes Mellitus: a Report of a World Study Group. WHO, Geneva. 4. King, H., Aubert, R.E. and Herman, W.H. (1998) Global burden of diabetes, 19952025: prevalence, numerical estimates, and projections. Diabetes Care, 21, 14141431. 5. Rosenbloom, A.L., Joe, J.R., Young, R.S. and Winter, W.E. (1999) Emerging epidemic of type 2 diabetes in youth. Diabetes Care, 22, 345354. 6. Brownlee, M. (2001) Biochemistry and molecular cell biology of diabetic complications. Nature, 414, 813820. 7. Bell, G.I. and Polonsky, K.S. (2001) Diabetes mellitus and genetically programmed defects in b-cell function. Nature, 414, 788791. 8. Kahn, S.E. (2003) The relative contributions of insulin resistance and bcell dysfunction to the pathophysiology of Type 2 diabetes. Diabetologia, 46, 319. 9. Jensen, C.C., Cnop, M., Hull, R.L., Fujimoto, W.Y. and Kahn, S.E. (2002) b-cell function is a major contributor to oral glucose tolerance in high-risk relatives of four ethnic groups in the US. Diabetes, 51, 21702178. 10. Bell, G.I., Pilkis, S.J., Weber, I.T. and Polonsky, K.S. (1996) Glucokinase mutations, insulin secretion, and diabetes mellitus. A. Rev. Physiol., 58, 171186. 11. UKPDS Group (1995) UK Prospective Diabetes Study 16. Overview of 6 years therapy of type II diabetes: a progressive disease. Diabetes, 44, 12491258. 12. UKPDS Group (1988) UK Prospective Diabetes Study. V. Characteristics of newly presenting type 2 diabetic patients: estimated insulin sensitivity and islet 4-cell function. Multi-centre study. Diabet. Med., 5, 444448. 13. Pimenta, W., Korytkowski, M., Mitrakou, A., Jenssen, T., Yki-Jarvinen, H., Evron, W., Dailey, G. and Gerich, J. (1995) Pancreatic b-cell dysfunction as the primary genetic lesion in NIDDM. Evidence from studies in normal glucose-tolerant individuals with a rst-degree NIDDM relative. JAMA, 273, 18551861. 14. van Haeften, T.W., Dubbeldam, S., Zonderland, M.L. and Erkelens, D.W. (1998) Insulin secretion in normal glucose-tolerant relatives of type 2 diabetic subjects. Assessments using hyperglycemic glucose clamps and oral glucose tolerance tests. Diabetes Care, 21, 278282.

CONCLUDING REMARKS The data discussed here are consistent with the idea that impaired regulation of b-cell electrical activity explains the defective insulin secretion found in type 2 diabetes. Electrical activity serves as a common conduit through which effects of polymorphisms in genes involved in metabolism, and lifestyle factors such as age and obesity are integrated and translated into changes in insulin release. Changes in electrical activity can account not only for impaired insulin secretion in response to glucose, but also for the reduced response to incretins, neurotransmitters and sulfonylureas that occurs in type 2 diabetes. We hope that our hypothesis will provoke debate and experiment, and provide increased impetus for the study of type 2 diabetic islets. It will stand or fall on the fruits of this research. ACKNOWLEDGEMENTS We thank our colleagues in Oxford and Lund for much stimulating debate and criticism. The work of our groups is

Human Molecular Genetics, 2004, Vol. 13, Review Issue 1

R29

15. Gerich, J.E. (1998) The genetic basis of type 2 diabetes mellitus: impaired insulin secretion versus impaired insulin sensitivity. Endocr. Rev., 19, 491503. 16. Taylor, S.I. (1992) Lilly Lecture: molecular mechanisms of insulin resistance. Lessons from patients with mutations in the insulin-receptor gene. Diabetes, 41, 14731490. 17. Chalkley, S.M., Hettiarachchi, M., Chisholm, D.J. and Kraegen, E.W. (2002) Long-term high-fat feeding leads to severe insulin resistance but not diabetes in Wistar rats. Am. J. Physiol. Endocrinol. Metab., 282, E12311238. 18. Gerich, J.E. (2002) Is reduced rst-phase insulin release the earliest detectable abnormality in individuals destined to develop type 2 diabetes? Diabetes, 51, S117S121. 19. Rahier, J., Goebbels, R.M. and Henquin, J.C. (1983) Cellular composition of the human diabetic pancreas. Diabetologia, 24, 366371. 20. Stefan, Y., Orci, L., Malaisse-Lagae, F., Perrelet, A., Patel, Y. and Unger, R.H. (1982) Quantitation of endocrine cell content in the pancreas of nondiabetic and diabetic humans. Diabetes, 31, 694700. 21. Clark, A., Jones, L.C., de Koning, E., Hansen, B.C. and Matthews, D.R. (2001) Decreased insulin secretion in type 2 diabetes: a problem of cellular mass or function? Diabetes, 50, S169S171. 22. Porte, D. Jr and Kahn, S.E. (2001) b-cell dysfunction and failure in type 2 diabetes: potential mechanisms. Diabetes, 50, S160S163. 23. Clark, A., Wells, C.A., Buley, I.D., Cruickshank, J.K., Vanhegan, R.I., Matthews, D.R., Cooper, G.J., Holman, R.R. and Turner, R.C. (1988) Islet amyloid, increased A-cells, reduced B-cells and exocrine brosis: quantitative changes in the pancreas in type 2 diabetes. Diabetes Res., 9, 151159. 24. Butler, A.E., Janson, J., Bonner-Weir, S., Ritzel, R., Rizza, R.A. and Butler, P.C. (2003) b-cell decit and increased b-cell apoptosis in humans with type 2 diabetes. Diabetes, 52, 102110. 25. McCulloch, D.K., Koerker, D.J., Kahn, S.E., Bonner-Weir, S. and Palmer, J.P. (1991) Correlations of in vivo b-cell function tests with b-cell mass and pancreatic insulin content in streptozocin-administered baboons. Diabetes, 40, 673679. 26. Foulis, A.K. and Stewart, J.A. (1984) The pancreas in recent-onset type 1 (insulin-dependent) diabetes mellitus: insulin content of islets, insulitis and associated changes in the exocrine acinar tissue. Diabetologia, 26, 456461. 27. Ashcroft, F.M. and Rorsman, P. (1989) Electrophysiology of the pancreatic b-cell. Prog. Biophys. Mol. Biol., 54, 87143. 28. Henquin, J.C. and Meissner, H.P. (1984) Signicance of ionic uxes and changes in membrane potential for stimulus-secretion coupling in pancreatic B-cells. Experientia, 40, 10431052. 29. Henquin, J.C. (2000) Triggering and amplifying pathways of regulation of insulin secretion by glucose. Diabetes, 49, 17511760. 30. Rorsman, P. and Renstrom, E. (2003) Insulin granule dynamics in pancreatic b cells. Diabetologia, 46, 10291045. 31. Ashcroft, F.M., Harrison, D.E. and Ashcroft, S.J. (1984) Glucose induces closure of single potassium channels in isolated rat pancreatic b-cells. Nature, 312, 446448. 32. Ashcroft, F.M. and Gribble, F.M. (1999) ATP-sensitive K channels and insulin secretion: their role in health and disease. Diabetologia, 42, 903919. 33. Aguilar-Bryan, L., Nichols, C.G., Wechsler, S.W., Clement, J.P.t., Boyd, A.E. III, Gonzalez, G., Herrera-Sosa, H., Nguy, K., Bryan, J. and Nelson, D.A. (1995) Cloning of the b cell high-afnity sulfonylurea receptor: a regulator of insulin secretion. Science, 268, 423426. 34. Henquin, J.C. (1992) The ftieth anniversary of hypoglycaemic sulphonamides. How did the mother compound work? Diabetologia, 35, 907912. 35. Trube, G., Rorsman, P. and Ohno-Shosaku, T. (1986) Opposite effects of tolbutamide and diazoxide on the ATP-dependent K channel in mouse pancreatic b-cells. Pugers Arch., 407, 493499. 36. Sanchez-Andres, J.V., Gomis, A. and Valdeolmillos, M. (1995) The electrical activity of mouse pancreatic b-cells recorded in vivo shows glucose-dependent oscillations. J. Physiol., 486, 223228. 37. Kanno, T., Rorsman, P. and Gopel, S.O. (2002) Glucose-dependent regulation of rhythmic action potential ring in pancreatic b-cells by KATP-channel modulation. J. Physiol., 545, 501507. 38. Meissner, H.P. (1976) Electrophysiological evidence for coupling between b cells of pancreatic islets. Nature, 262, 502504. 39. Eddlestone, G.T., Goncalves, A., Bangham, J.A. and Rojas, E. (1984) Electrical coupling between cells in islets of Langerhans from mouse. J. Membr. Biol., 77, 114.

40. Santos, R.M., Rosario, L.M., Nadal, A., Garcia-Sancho, J., Soria, B. and Valdeolmillos, M. (1991) Widespread synchronous [Ca2]i oscillations due to bursting electrical activity in single pancreatic islets. Pugers Arch., 418, 417422. 41. Bergsten, P. (1995) Slow and fast oscillations of cytoplasmic Ca2 in pancreatic islets correspond to pulsatile insulin release. Am. J. Physiol., 268, E282E287. 42. Seghers, V., Nakazaki, M., DeMayo, F., Aguilar-Bryan, L. and Bryan, J. (2000) Sur1 knockout mice. A model for KATP channel-independent regulation of insulin secretion. J. Biol. Chem., 275, 92709277. 43. Gembal, M., Gilon, P. and Henquin, J.C. (1992) Evidence that glucose can control insulin release independently from its action on ATP-sensitive K channels in mouse B cells. J. Clin. Invest., 89, 12881295. 44. Porksen, N., Hollingdal, M., Juhl, C., Butler, P., Veldhuis, J.D. and Schmitz, O. (2002) Pulsatile insulin secretion: detection, regulation, and role in diabetes. Diabetes, 51, S245S254. 45. Ward, W.K., Bolgiano, D.C., McKnight, B., Halter, J.B. and Porte, D. Jr (1984) Diminished B cell secretory capacity in patients with noninsulin-dependent diabetes mellitus. J. Clin. Invest., 74, 13181328. 46. Fritsche, A., Stefan, N., Hardt, E., Haring, H. and Stumvoll, M. (2000) Characterisation of b-cell dysfunction of impaired glucose tolerance: evidence for impairment of incretin-induced insulin secretion. Diabetologia, 43, 852858. 47. Kjems, L.L., Holst, J.J., Volund, A. and Madsbad, S. (2003) The inuence of GLP-1 on glucose-stimulated insulin secretion: effects on b-cell sensitivity in type 2 and nondiabetic subjects. Diabetes, 52, 380386. 48. Stumvoll, M., Fritsche, A. and Haring, H.U. (2002) Clinical characterization of insulin secretion as the basis for genetic analyses. Diabetes, 51, S122S129. 49. Kindmark, H., Kohler, M., Arkhammar, P., Efendic, S., Larsson, O., Linder, S., Nilsson, T. and Berggren, P.O. (1994) Oscillations in cytoplasmic free calcium concentration in human pancreatic islets from subjects with normal and impaired glucose tolerance. Diabetologia, 37, 11211131. 50. Fernandez-Alvarez, J., Conget, I., Rasschaert, J., Sener, A., Gomis, R. and Malaisse, W.J. (1994) Enzymatic, metabolic and secretory patterns in human islets of type 2 (non-insulin-dependent) diabetic patients. Diabetologia, 37, 177181. 51. Sakura, H., Wat, N., Horton, V., Millns, H., Turner, R.C. and Ashcroft, F.M. (1996) Sequence variations in the human Kir6.2 gene, a subunit of the b-cell ATP-sensitive K-channel: no association with NIDDM in while Caucasian subjects or evidence of abnormal function when expressed in vitro. Diabetologia, 39, 12331236. 52. Inoue, H., Ferrer, J., Warren-Perry, M., Zhang, Y., Millns, H., Turner, R.C., Elbein, S.C., Hampe, C.L., Suarez, B.K., Inagaki, N. et al. (1997) Sequence variants in the pancreatic islet b-cell inwardly rectifying K channel Kir6.2 (Bir) gene: identication and lack of role in Caucasian patients with NIDDM. Diabetes, 46, 502507. 53. Hani, E.H., Boutin, P., Durand, E., Inoue, H., Permutt, M.A., Velho, G. and Froguel, P. (1998) Missense mutations in the pancreatic islet b cell inwardly rectifying K channel gene (KIR6.2/BIR): a meta-analysis suggests a role in the polygenic basis of Type II diabetes mellitus in Caucasians. Diabetologia, 41, 15111515. 54. Schwanstecher, C., Meyer, U. and Schwanstecher, M. (2002) K(IR)6.2 polymorphism predisposes to type 2 diabetes by inducing overactivity of pancreatic b-cell ATP-sensitive K channels. Diabetes, 51, 875879. 55. Schwanstecher, C., Neugebauer, B., Schulz, M. and Schwanstecher, M. (2002) The common single nucleotide polymorphism E23K in K(IR)6.2 sensitizes pancreatic b-cell ATP-sensitive potassium channels toward activation through nucleoside diphosphates. Diabetes, 51, S363S367. 56. Nielsen, E.M., Hansen, L., Carstensen, B., Echwald, S.M., Drivsholm, T., Glumer, C., Thorsteinsson, B., Borch-Johnsen, K., Hansen, T. and Pedersen, O. (2003) The E23K variant of Kir6.2 associates with impaired post-OGTT serum insulin response and increased risk of type 2 diabetes. Diabetes, 52, 573577. 57. Love-Gregory, L., Wasson, J., Lin, J., Skolnick, G., Suarez, B. and Permutt, M.A. (2003) E23K single nucleotide polymorphism in the islet ATP-sensitive potassium channel gene (Kir6.2) contributes as much to the risk of Type II diabetes in Caucasians as the PPARg Pro12Ala variant. Diabetologia, 46, 136137.

R30

Human Molecular Genetics, 2004, Vol. 13, Review Issue 1

58. Gloyn, A.L., Weedon, M.N., Owen, K.R., Turner, M.J., Knight, B.A., Hitman, G., Walker, M., Levy, J.C., Sampson, M., Halford, S. et al. (2003) Large-scale association studies of variants in genes encoding the pancreatic b-cell KATP channel subunits Kir6.2 (KCNJ11) and SUR1 (ABCC8) conrm that the KCNJ11 E23K variant is associated with type 2 diabetes. Diabetes, 52, 568572. 59. Barroso, I., Luan, J., Middelberg, R.P., Harding, A.H., Franks, P.W., Jakes, R.W., Clayton, D., Schafer, A.J., ORahilly, S. and Wareham, N.J. (2003) Candidate gene association study in type 2 diabetes indicates a role for genes involved in b-cell function as well as insulin action. PLoS Biol., 1, 4155. 60. Schwanstecher, C. and Schwanstecher, M. (2002) Nucleotide sensitivity of pancreatic ATP-sensitive potassium channels and type 2 diabetes. Diabetes, 51, S358S362. 61. Koster, J.C., Marshall, B.A., Ensor, N., Corbett, J.A. and Nichols, C.G. (2000) Targeted overactivity of b cell KATP channels induces profound neonatal diabetes. Cell, 100, 645654. 62. tHart, L.M., van Haeften, T.W., Dekker, J.M., Bot, M., Heine, R.J. and Maassen, J.A. (2002) Variations in insulin secretion in carriers of the E23K variant in the KIR6.2 subunit of the ATP-sensitive K channel in the b-cell. Diabetes, 51, 31353138. 63. Tschritter, O., Stumvoll, M., Machicao, F., Holzwarth, M., Weisser, M., Maerker, E., Teigeler, A., Haring, H. and Fritsche, A. (2002) The prevalent Glu23Lys polymorphism in the potassium inward rectier 6.2 (KIR6.2) gene is associated with impaired glucagon suppression in response to hyperglycemia. Diabetes, 51, 28542860. 64. Philipson, L.H., Rosenberg, M.P., Kuznetsov, A., Lancaster, M.E., Worley, J.F. III, Roe, M.W. and Dukes, I.D. (1994) Delayed rectier K channel overexpression in transgenic islets and b-cells associated with impaired glucose responsiveness. J. Biol. Chem., 269, 2778727790. 65. Schulla, V., Renstrom, E., Feil, R., Feil, S., Franklin, I., Gjinovci, A., Jing, X.J., Laux, D., Lundquist, I., Magnuson, M.A. et al. (2003) Impaired insulin secretion and glucose tolerance in b cell-selective Ca(v)1.2 Ca2 channel null mice. EMBO J., 22, 38443854. 66. Larsson-Nyren, G., Sehlin, J., Rorsman, P. and Renstrom, E. (2001) Perchlorate stimulates insulin secretion by shifting the gating of L-type Ca2 currents in mouse pancreatic B-cells towards negative potentials. Pugers Arch., 441, 587595. 67. Larsson-Nyren, G. (1996) Perchlorate is hypoglycaemic by amplifying glucose-stimulated insulin secretion in mice. Acta Physiol. Scand., 158, 7176. 68. Smith, P.A., Rorsman, P. and Ashcroft, F.M. (1989) Modulation of dihydropyridine-sensitive Ca2 channels by glucose metabolism in mouse pancreatic b-cells. Nature, 342, 550553. 69. Sellick, G.S., Garrett, C. and Houlston, R.S. (2003) A novel gene for neonatal diabetes maps to chromosome 10p12.1p13. Diabetes, 52, 26362638. 70. Kanno, T., Gopel, S.O., Rorsman, P. and Wakui, M. (2002) Cellular function in multicellular system for hormone-secretion: electrophysiological aspect of studies on a- b- and d-cells of the pancreatic islet. Neurosci. Res., 42, 7990. 71. Gopel, S.O., Kanno, T., Barg, S., Eliasson, L., Galvanovskis, J., Renstrom, E. and Rorsman, P. (1999) Activation of Ca2-dependent K channels contributes to rhythmic ring of action potentials in mouse pancreatic b cells. J. Gen. Physiol., 114, 759770. 72. Goforth, P.B., Bertram, R., Khan, F.A., Zhang, M., Sherman, A. and Satin, L.S. (2002) Calcium-activated K channels of mouse b-cells are controlled by both store and cytoplasmic Ca2: experimental and theoretical studies. J. Gen. Physiol., 120, 307322. 73. Shyng, S.L. and Nichols, C.G. (1998) Membrane phospholipid control of nucleotide sensitivity of KATP channels. Science, 282, 11381141. 74. Gloyn, A.L. (2003) Glucokinase (GCK) mutations in hyper- and hypoglycemia: maturity-onset diabetes of the young, permanent neonatal diabetes, and hyperinsulinemia of infancy. Hum. Mutat., 22, 353362. 75. Yamagata, K., Oda, N., Kaisaki, P.J., Menzel, S., Furuta, H., Vaxillaire, M., Southam, L., Cox, R.D., Lathrop, G.M., Boriraj, V.V. et al. (1996) Mutations in the hepatocyte nuclear factor-1a gene in maturity-onset diabetes of the young (MODY3). Nature, 384, 455458. 76. Yamagata, K., Furuta, H., Oda, N., Kaisaki, P.J., Menzel, S., Cox, N.J., Fajans, S.S., Signorini, S., Stoffel, M. and Bell, G.I. (1996) Mutations in the hepatocyte nuclear factor-4a gene in maturity-onset diabetes of the young (MODY1). Nature, 384, 458460.

77. Fajans, S.S., Bell, G.I. and Polonsky, K.S. (2001) Molecular mechanisms and clinical pathophysiology of maturity-onset diabetes of the young. New. Engl. J. Med., 345, 971980. 78. Frayling, T.M., Evans, J.C., Bulman, M.P., Pearson, E., Allen, L., Owen, K., Bingham, C., Hannemann, M., Shepherd, M., Ellard, S. et al. (2001) b-cell genes and diabetes: molecular and clinical characterization of mutations in transcription factors. Diabetes, 50, S94S100. 79. Wang, H., Hagenfeldt-Johansson, K., Otten, L.A., Gauthier, B.R., Herrera, P.L. and Wollheim, C.B. (2002) Experimental models of transcription factor-associated maturity-onset diabetes of the young. Diabetes, 51, S333S342. 80. Shih, D.Q., Screenan, S., Munoz, K.N., Philipson, L., Pontoglio, M., Yaniv, M., Polonsky, K.S. and Stoffel, M. (2001) Loss of HNF-1a function in mice leads to abnormal expression of genes involved in pancreatic islet development and metabolism. Diabetes, 50, 24722480. 81. Sakura, H., Ashcroft, S.J., Terauchi, Y., Kadowaki, T. and Ashcroft, F.M. (1998) Glucose modulation of ATP-sensitive K-currents in wild-type, homozygous and heterozygous glucokinase knock-out mice. Diabetologia, 41, 654659. 82. Dukes, I.D., Sreenan, S., Roe, M.W., Levisetti, M., Zhou, Y.P., Ostrega, D., Bell, G.I., Pontoglio, M., Yaniv, M., Philipson, L. et al. (1998) Defective pancreatic b-cell glycolytic signaling in hepatocyte nuclear factor-1a-decient mice. J. Biol. Chem., 273, 2445724464. 83. van den Ouweland, J.M., Lemkes, H.H., Ruitenbeek, W., Sandkuijl, L.A., de Vijlder, M.F., Struyvenberg, P.A., van de Kamp, J.J. and Maassen, J.A. (1992) Mutation in mitochondrial tRNA(Leu)(UUR) gene in a large pedigree with maternally transmitted type II diabetes mellitus and deafness. Nat. Genet., 1, 368371. 84. Maechler, P. and Wollheim, C.B. (2001) Mitochondrial function in normal and diabetic b-cells. Nature, 414, 807812. 85. Wollheim, C.B. (2000) b-Cell mitochondria in the regulation of insulin secretion: a new culprit in type II diabetes. Diabetologia, 43, 265277. 86. Weng, J., Macfarlane, W.M., Lehto, M., Gu, H.F., Shepherd, L.M., Ivarsson, S.A., Wibell, L., Smith, T. and Groop, L.C. (2001) Functional consequences of mutations in the MODY4 gene (IPF1) and coexistence with MODY3 mutations. Diabetologia, 44, 249258. 87. Triggs-Raine, B.L., Kirkpatrick, R.D., Kelly, S.L., Norquay, L.D., Cattini, P.A., Yamagata, K., Hanley, A.J., Zinman, B., Harris, S.B., Barrett, P.H. et al. (2002) HNF-1a G319S, a transactivation-decient mutant, is associated with altered dynamics of diabetes onset in an Oji-Cree community. Proc. Natl Acad. Sci. USA, 99, 46144619. 88. Stone, L.M., Kahn, S.E., Fujimoto, W.Y., Deeb, S.S. and Porte, D. Jr (1996) A variation at position -30 of the b-cell glucokinase gene promoter is associated with reduced b-cell function in middle-aged JapaneseAmerican men. Diabetes, 45, 422428. 89. Kennedy, E.D., Maechler, P. and Wollheim, C.B. (1998) Effects of depletion of mitochondrial DNA in metabolism secretion coupling in INS-1 cells. Diabetes, 47, 374380. 90. Saleh, M.C., Wheeler, M.B. and Chan, C.B. (2002) Uncoupling protein-2: evidence for its function as a metabolic regulator. Diabetologia, 45, 174187. 91. Zhang, C.Y., Baffy, G., Perret, P., Krauss, S., Peroni, O., Grujic, D., Hagen, T., Vidal-Puig, A.J., Boss, O., Kim, Y.B. et al. (2001) Uncoupling protein-2 negatively regulates insulin secretion and is a major link between obesity, b-cell dysfunction, and type 2 diabetes. Cell, 105, 745755. 92. Chan, C.B., De Leo, D., Joseph, J.W., McQuaid, T.S., Ha, X.F., Xu, F., Tsushima, R.G., Pennefather, P.S., Salapatek, A.M. and Wheeler, M.B. (2001) Increased uncoupling protein-2 levels in b-cells are associated with impaired glucose-stimulated insulin secretion: mechanism of action. Diabetes, 50, 13021310. 93. Krempler, F., Esterbauer, H., Weitgasser, R., Ebenbichler, C., Patsch, J.R., Miller, K., Xie, M., Linnemayr, V., Oberkoer, H. and Patsch, W. (2002) A functional polymorphism in the promoter of UCP2 enhances obesity risk but reduces type 2 diabetes risk in obese middle-aged humans. Diabetes, 51, 33313335. 94. Esterbauer, H., Schneitler, C., Oberkoer, H., Ebenbichler, C., Paulweber, B., Sandhofer, F., Ladurner, G., Hell, E., Strosberg, A.D., Patsch, J.R. et al. (2001) A common polymorphism in the promoter of UCP2 is associated with decreased risk of obesity in middle-aged humans. Nat. Genet., 28, 178183.

Human Molecular Genetics, 2004, Vol. 13, Review Issue 1

R31

95. Sesti, G., Cardellini, M., Marini, M.A., Frontoni, S., DAdamo, M., Del Guerra, S., Lauro, D., De Nicolais, P., Sbraccia, P., Del Prato, S. et al. (2003) A common polymorphism in the promoter of UCP2 contributes to the variation in insulin secretion in glucose-tolerant subjects. Diabetes, 52, 12801283. 96. Poulton, J., Luan, J., Macaulay, V., Hennings, S., Mitchell, J. and Wareham, N.J. (2002) Type 2 diabetes is associated with a common mitochondrial variant: evidence from a population-based casecontrol study. Hum. Mol. Genet., 11, 15811583. 97. Casteels, K., Ong, K., Phillips, D., Bendall, H. and Pembrey, M. (1999) Mitochondrial 16189 variant, thinness at birth, and type-2 diabetes. ALSPAC study team. Avon Longitudinal Study of Pregnancy and Childhood. Lancet, 353, 14991500. 98. Hales, C.N., Barker, D.J., Clark, P.M., Cox, L.J., Fall, C., Osmond, C. and Winter, P.D. (1991) Fetal and infant growth and impaired glucose tolerance at age 64. Br. Med. J., 303, 10191022. 99. Poulton, J. (1998) Does a common mitochondrial DNA polymorphism underlie susceptibility to diabetes and the thrifty genotype? Trends Genet., 14, 387389. 100. Miller, K.W., Dawson, J.L. and Hagelberg, E. (1996) A concordance of nucleotide substitutions in the rst and second hypervariable segments of the human mtDNA control region. Int. J. Legal Med., 109, 107113. 101. Barroso, I., Gurnell, M., Crowley, V.E., Agostini, M., Schwabe, J.W., Soos, M.A., Maslen, G.L., Williams, T.D., Lewis, H., Schafer, A.J. et al. (1999) Dominant negative mutations in human PPARg associated with severe insulin resistance, diabetes mellitus and hypertension. Nature, 402, 880883. 102. Deeb, S.S., Fajas, L., Nemoto, M., Pihlajamaki, J., Mykkanen, L., Kuusisto, J., Laakso, M., Fujimoto, W. and Auwerx, J. (1998) A Pro12Ala substitution in PPARg2 associated with decreased receptor activity, lower body mass index and improved insulin sensitivity. Nat. Genet., 20, 284287. 103. Altshuler, D., Hirschhorn, J.N., Klannemark, M., Lindgren, C.M., Vohl, M.C., Nemesh, J., Lane, C.R., Schaffner, S.F., Bolk, S., Brewer, C. et al. (2000) The common PPARg Pro12Ala polymorphism is associated with decreased risk of type 2 diabetes. Nat. Genet., 26, 7680. 104. Mori, H., Ikegami, H., Kawaguchi, Y., Seino, S., Yokoi, N., Takeda, J., Inoue, I., Seino, Y., Yasuda, K., Hanafusa, T. et al. (2001) The Pro12!Ala substitution in PPAR-_ is associated with resistance to development of diabetes in the general population: possible involvement in impairment of insulin secretion in individuals with type 2 diabetes. Diabetes, 50, 891894. 105. Kim, H.I., Cha, J.Y., Kim, S.Y., Kim, J.W., Roh, K.J., Seong, J.K., Lee, N.T., Choi, K.Y., Kim, K.S. and Ahn, Y.H. (2002) Peroxisomal proliferator-activated receptor-g upregulates glucokinase gene expression in b-cells. Diabetes, 51, 676685. 106. Kim, H.I., Kim, J.W., Kim, S.H., Cha, J.Y., Kim, K.S. and Ahn, Y.H. (2000) Identication and functional characterization of the peroxisomal proliferator response element in rat GLUT2 promoter. Diabetes, 49, 15171524. 107. Patane, G., Anello, M., Piro, S., Vigneri, R., Purrello, F. and Rabuazzo, A.M. (2002) Role of ATP production and uncoupling protein-2 in the insulin secretory defect induced by chronic exposure to high glucose or free fatty acids and effects of peroxisome proliferator-activated receptor-g inhibition. Diabetes, 51, 27492756. 108. Michikawa, Y., Mazzucchelli, F., Bresolin, N., Scarlato, G. and Attardi, G. (1999) Aging-dependent large accumulation of point mutations in the human mtDNA control region for replication. Science, 286, 774779. 109. Petersen, K.F., Befroy, D., Dufour, S., Dziura, J., Ariyan, C., Rothman, D.L., DiPietro, L., Cline, G.W. and Shulman, G.I. (2003) Mitochondrial dysfunction in the elderly: possible role in insulin resistance. Science, 300, 11401142. 110. Golay, A., Chen, Y.D. and Reaven, G.M. (1986) Effect of differences in glucose tolerance on insulins ability to regulate carbohydrate and free fatty acid metabolism in obese individuals. J. Clin. Endocrinol. Metab., 62, 10811088.

111. Reaven, G.M., Hollenbeck, C., Jeng, C.Y., Wu, M.S. and Chen, Y.D. (1988) Measurement of plasma glucose, free fatty acid, lactate, and insulin for 24 h in patients with NIDDM. Diabetes, 37, 1020 1024. 112. Gribble, F.M., Proks, P., Corkey, B.E. and Ashcroft, F.M. (1998) Mechanism of cloned ATP-sensitive potassium channel activation by oleoyl-CoA. J. Biol. Chem., 273, 2638326387. 113. Larsson, O., Deeney, J.T., Branstrom, R., Berggren, P.O. and Corkey, B.E. (1996) Activation of the ATP-sensitive K channel by long chain acyl-CoA. A role in modulation of pancreatic b-cell glucose sensitivity. J. Biol. Chem., 271, 1062310626. 114. Joseph, J.W., Koshkin, V., Zhang, C.Y., Wang, J., Lowell, B.B., Chan, C.B. and Wheeler, M.B. (2002) Uncoupling protein 2 knockout mice have enhanced insulin secretory capacity after a high-fat diet. Diabetes, 51, 32113219. 115. Koshkin, V., Wang, X., Scherer, P.E., Chan, C.B. and Wheeler, M.B. (2003) Mitochondrial functional state in clonal pancreatic b-cells exposed to free fatty acids. J. Biol. Chem., 278, 1970919715. 116. Lameloise, N., Muzzin, P., Prentki, M. and Assimacopoulos-Jeannet, F. (2001) Uncoupling protein 2: a possible link between fatty acid excess and impaired glucose-induced insulin secretion? Diabetes, 50, 803809. 117. Kane, C., Shepherd, R.M., Squires, P.E., Johnson, P.R., James, R.F., Milla, P.J., Aynsley-Green, A., Lindley, K.J. and Dunne, M.J. (1996) Loss of functional KATP channels in pancreatic b-cells causes persistent hyperinsulinemic hypoglycemia of infancy. Nat. Med., 2, 13441347. 118. Thomas, P.M., Cote, G.J., Wohllk, N., Haddad, B., Mathew, P.M., Rabl, W., Aguilar-Bryan, L., Gagel, R.F. and Bryan, J. (1995) Mutations in the sulfonylurea receptor gene in familial persistent hyperinsulinemic hypoglycemia of infancy. Science, 268, 426429. 119. Glaser, B., Thornton, P., Otonkoski, T. and Junien, C. (2000) Genetics of neonatal hyperinsulinism. Arch. Dis. Child Fetal Neonatal Edn, 82, F79F86. 120. Huopio, H., Otonkoski, T., Vauhkonen, I., Reimann, F., Ashcroft, F.M. and Laakso, M. (2003) A new subtype of autosomal dominant diabetes attributable to a mutation in the gene for sulfonylurea receptor 1. Lancet, 361, 301307. 121. Miki, T., Iwanaga, T., Nagashima, K., Ihara, Y. and Seino, S. (2001) Roles of ATP-sensitive K channels in cell survival and differentiation in the endocrine pancreas. Diabetes, 50, S48S51. 122. Glaser, B., Ryan, F., Donath, M., Landau, H., Stanley, C.A., Baker, L., Barton, D.E. and Thornton, P.S. (1999) Hyperinsulinism caused by paternal-specic inheritance of a recessive mutation in the sulfonylurea-receptor gene. Diabetes, 48, 16521657. 123. Kassem, S.A., Ariel, I., Thornton, P.S., Scheimberg, I. and Glaser, B. (2000) b-Cell proliferation and apoptosis in the developing normal human pancreas and in hyperinsulinism of infancy. Diabetes, 49, 1325 1333. 124. Thornton, P.S., MacMullen, C., Ganguly, A., Ruchelli, E., Steinkrauss, L., Crane, A., Aguilar-Bryan, L. and Stanley, C.A. (2003) Clinical and molecular characterization of a dominant form of congenital hyperinsulinism caused by a mutation in the high-afnity sulfonylurea receptor. Diabetes, 52, 24032410. 125. Giugliano, M., Bove, M. and Grattarola, M. (2000) Insulin release at the molecular level: metabolic-electrophysiological modeling of the pancreatic b-cells. IEEE Trans. Biomed. Eng., 47, 611623. 126. Smith, P.A., Sakura, H., Coles, B., Gummerson, N., Proks, P. and Ashcroft, F.M. (1997) Electrogenic arginine transport mediates stimulus-secretion coupling in mouse pancreatic b-cells. J. Physiol., 499, 625635. 127. Rolland, J.F., Henquin, J.C. and Gilon, P. (2002) G protein-independent activation of an inward Na current by muscarinic receptors in mouse pancreatic b-cells. J. Biol. Chem., 277, 3837338380. 128. Fernandez, J. and Valdeolmillos, M. (1999) Glucose-dependent stimulatory effect of glucagon-like peptide 1(736) amide on the electrical activity of pancreatic b-cells recorded in vivo. Diabetes, 48, 754757.