Diabetes Mellitus Type 2

INSULIN RESISTANCE IN TYPE 2 DIABETES

Insulin resistance is literally a lowered sensitivity/responsiveness of a tissue or multiple tissues to insulin.

However, in the context of type 2 diabetes, it is defined as impaired insulin-mediated glucose clearance into skeletal muscle, usually (but not always) with dysregulation of hepatic glucose production by insulin. This is because early studies showed dual effects of the insulin response to a meal for control of postmeal glycemia: activation of glucose transport into skeletal muscle, which is the major site of insulin-mediated clearance of a glucose load, and deactivation of hepatic glucose production. Both of these effects are impaired in type 2 diabetes, which explains how the term was first applied. This does not imply that insulin signaling in other tissues is intact.

In the last decade, the intracellular signaling cascade that is downstream from the insulin receptor has, to a large degree, been mapped. Unexpectedly, this cascade is present in tissues other than the classic insulin-regulated end-organs, such as islet ß-cells, endothelial cells, neurons, etc. In addition, tissue specific knockout mouse studies have confirmed the presence of important physiologic effects of insulin in these tissues, with speculation that hyperglycemia causes dysregulation. However, the term “insulin resistance” still typically focuses only on muscle and liver, with endothelial dysfunction increasingly being added because of the presumed link between insulin resistance and cardiovascular disease in type 2 diabetes.

Simplistically, in the fasting state, the degree of hyperglycemia is directly determined by the rate of glucose overproduction by the liver. With eating, failure of adequate insulin-mediated nutrient-clearance into skeletal muscle combined with an attenuated deactivation of hepatic glucose production causes postprandial hyperglycemia (122).

Recent investigation has focused on defining the cellular defects, aided by powerful new technologies, including glucose clamping with muscle biopsies, NMR analysis of cellular metabolic pathways, genetic mapping of target and novel mutations, and knockout mouse models (often tissue specific) for most of the key enzymes and transcription factors in the intracellular insulin action cascade. The major defect in muscle is impaired glucose transport into the cell combined with defective storage as glycogen (123). Initially, it was assumed that genetic defects would be discovered in the glucose transport machinery, the insulin receptor, or its downstream signaling cascade. This has not been the case. Instead, current hypotheses mainly focus on disruption of the cellular insulin signaling cascade by external factors. Several mechanisms are under investigation:

1. Serine phosphorylation of IRS-1. IRS-1 (Insulin Receptor Substrate-1, a component of the insulin signaling cascade that is immediately downstream from the insulin receptor) plays a key role in insulin signaling in skeletal muscle. The insulin signal propagates from the insulin receptor through IRS-1 to the distal signaling peptides, mainly through phosphorylation of tyrosines. Normoglycemic relatives of persons with type 2 diabetes have decreased insulin-stimulated IRS-1 tyrosine phosphorylation (124). A potential explanation relates to the recent finding that serine phosphorylation of IRS-1 attenuates insulin signaling, perhaps normally to turn off the insulin response, and that states of insulin resistance are characterized by enhanced serine phosphorylation of IRS-1 (125–128). Another idea is that degradation of IRS-1 is accelerated (129).

2. Excess glucosamine: Glucose is mainly metabolized through glycolysis, but a small percentage forms UDPacetylglucosamine.

Increased flux through this pathway has been shown to impair insulin-mediated glucose transport in adipocytes (130). Subsequent studies in mice whose livers overexpress the hexosamine biosynthesis enzyme, fructose-6-phosphate amidotransferase, showed enhanced glycogen storage and the metabolic syndrome (obesity, hyperlipidemia, and, glucose intolerance) (131), and rats fed glucosamine developed skeletal muscle insulin resistance (132). It is currently thought that this system normally acts as a cellular nutrient sensor, but goes awry when flux is excessive (133). At present, it remains unclear what role this pathway plays in human type 2 diabetes (134).

3. Defective mitochondria: There is great interest in mitochondrial dysfunction as a cause of skeletal muscle insulin resistance. This was highlighted in an important study that used state of the art NMR technology to examine normal weight, normoglycemic, insulin resistant offspring of parents with type 2 diabetes, finding defective skeletal muscle mitochondrial function (135). Increased storage of triglyceride in muscle and liver has recently been proposed to be a marker of insulin resistance (136,137). Petersen et al have speculated that mitochondrial dysfunction explains both the excess triglyceride accumulation and the defective glucose uptake that characterize muscle-related insulin resistance in type 2 diabetes because of decreased fatty acid oxidation and ATP production (135). Additional findings that support mitochondrial dysfunction are more type IIb muscle fibers (nonoxidative type) in persons with type 2 diabetes (138), and a reduced function and number of skeletal muscle mitochondria (139) that improved in tandem with an increased insulin sensitivity after weight loss and dietary therapy (140).

4. Fatty acid-induced insulin resistance and a role for inflammation. As discussed earlier, another aspect of the diabetes phenotype is hyperlipidemia. There is now strong experimental support for insulin resistance-inducing effects of excess fatty acids from both lipid infusion studies in healthy man and in vitro studies (141–144). It was initially assumed that the mechanism was a competition between fatty acids and glucose oxidation (the Randle cycle), but a much more complex effect of fatty acids on insulin signaling has evolved. Considerable evidence now supports fatty acids interfering with insulin signaling through a cascade of effects that includes protein kinase C (PKC)-induced serine phosphorylation of IRS-1 (PKC-theta knockout mice are resistant to fat-induced insulin resistance (145)), the proinflammatory mediators c-Jun N-terminal kinase (JNK) and IkappaB/NfkappaB (146), and suppressor of cytokine signaling 3(SOCS-3), which impairs insulin signaling at several sites (147). High dose aspirin ameliorates insulin resistance in animals by interfering with IkappaB kinase beta (IKKbeta) (148), and multicenter human trials to test that effect are underway.

5. Alternate fatty acid effects. Other aspects of lipotoxicity-induced insulin resistance are being investigated, including induction of oxidative stress (95) and malonyl-CoA-induced alterations in AMP kinase (149). The latter is proposed to be a site of action of the oral agent metformin (150).

6. Altered adipokine regulation. The last decade has seen the discovery that adipose tissue is far more complex than simply acting as a storage site for triglyceride. Adipocytes are now known to produce many proteins (cytokines and adipokines) that have effects on a number of tissues, including skeletal muscle and liver, and concurrently on insulin sensitivity (151). Of particular interest regarding the effect on skeletal muscle are TNF (152) and adiponectin (153–155), as well as the recently described retinol binding protein 4 (156). Another adipocyte-related factor of current interest is resistin, which was initially linked to the insulin resistance of obesity and diabetes (157). Subsequently its pathological role has been questioned (158). However, interest in resistin has returned, as resistin null mice have been shown to become hypoglycemic during fasting, and are protected against glucose intolerance and insulin resistance during fat feeding, confirming a physiologic effect (159). This study localized the action of resistin to the liver, showing that it de-activates AMP-kinase, impairing transcriptional regulation of gluconeogenic enzymes. Subsequent studies using knockout mice, transgenic resistin overexpressing  ice, adenoviral overexpression systems, interfering RNA, etc. confirmed and expanded this hypothesis (160–162). The results unequivocally show that resistin has an important regulatory role over hepatic glucose production in health and disease, at least in rodents. However, the role of resistin has not yet been elucidated in humans.

There has been intense study of potential cellular mechanisms of the ß-cell dysfunction in type 2 diabetes.

As already discussed, the inability to get islet tissue from free-living humans is a major impediment. Thus, with rare exceptions, these studies have been carried out in vitro using isolated islets from animals, clonal ß-cell lines exposed to high glucose and/or fatty acid levels, or by studying isolated islets from diabetic animals. There are a few studies of isolated islets from brain dead donors with type 2 diabetes (17,90,96–99). Also, an emerging technique that holds considerable promise is laser capture microdissection to carve out islet-cells from pancreas slides of autopsy material, followed by mRNA amplification and expression profiling. Still, work with islet tissue from humans with type 2 diabetes is just beginning, and there remains a concern that islets obtained at the time of death (for any number of medical reasons) may be misleading in terms of the observed ß-cell physiology compared with the average otherwise healthy subject with type 2 diabetes. Thus current concepts of potential mechanisms are generally based on nonhuman systems. Several have been proposed (66,100):

1. Glucose toxicity. This concept implies a direct effect of a high glucose level to impair one or more necessary aspects of ß-cell signaling, gene expression, cell architecture, etc, for normal insulin secretion. The list of reported ß-cell effects from experimental high glucose is lengthy; essentially every major ß-cell metabolic pathway, key enzyme, and important gene has been reported to be altered (66). A variation on this concept is a series of papers performed in normal rats made hyperglycemic by glucose-infusion or partial pancreatectomy, showing a profoundly altered pattern of transcriptional expression of important ß-cell genes, termed “ß-cell dedifferentiation” (101,102).

2. ß-cell exhaustion: This term implies an indirect effect of hyperglycemia to impair ß-cell function by way of the initial compensatory increase in insulin secretion depleting a key substance, metabolite, etc., below a crucial level that is required for continued insulin secretion. It is differentiated from glucose toxicity with an inhibitor of insulin secretion, such as diazoxide. Conceptually, with glucose toxicity, hyperglycemia, and consequently the ß-cell dysfunction, would worsen, but, with exhaustion, the “beta-cell rest” improves ß-cell function regardless of the blood glucose level. There is strong experimental support in both animal models and humans with type 2 diabetes for the exhaustion concept (100,103–106).

3. Impaired proinsulin biosynthesis: Extensive in vitro data support a hyperglycemia-induced defect in proinsulin transcription (107), although this requires high levels of glycemia (101) (i.e., this is one of the late-onset acquired ß-cell defects). Also, an added effect of excess fatty acids to impair proinsulin transcription, is potentially important (108).

4. Lipotoxicity: There has been great interest in the concept that excess fatty acids are harmful to ß-cell function and viability, so-called lipotoxicity (42). The working concept is that metabolic products of excess fatty acids, such as ceramides or other mediators of oxidative stress, cause ß-cell dysfunction and death (94,109). However, this idea remains controversial, in part because the cellular systems and animal models used to study the subject are often so extreme that the relevance to human type 2 diabetes is unclear. Using in vitro culture of rat islets with high levels of fatty acids, Liu et al found no insulin secretory dysfunction or ß-cell death. Instead, they identified a system in normal ß-cells that protects against fatty acid-induced reductions in glucose metabolism that occur in other tissues (so-called “Randle effect”). In those tissues, excess fatty acids impair the activation of pyruvate  ehydrogenase and retard glucose oxidation. In contrast, a relatively specialized feature of ß-cells is the high expression of a second pyruvate metabolism enzyme (pyruvate carboxylase), that allows the block in pyruvate metabolism to be bypassed (110). Furthermore, pyruvate carboxylase is the entry step to mitochondrial metabolic pathways in ß-cells that are believed to be important signals for glucose-induced insulin secretion (111). Therefore, Leahy and his collaborators have proposed that the heightened flow through pyruvate carboxylase not only protects against the detrimental effect of the excess fatty acids on glucose metabolism, but also provides a mechanism for the compensatory increase in insulin secretion that normally accompanies insulin resistance. Subsequent studies in a  ormoglycemic, insulin resistant rat model, Zucker fatty rats, support this theory (112,113). Thus, excess fatty acids, in concert with normoglycemia, appear to augment ß-cell function, whereas excess fatty acids in the setting of hyperglycemia impair ß-cell function, so-called glucolipotoxicity (43,44). This theory shares with the lipotoxicity hypothesis the concept that excess production of fatty acid metabolites such as ceramides causes ß-cell dysfunction and death.

However, in this concept a high glucose level must be present, as an increased level of the mitochondrial metabolic product of glucose, malonyl-CoA, is needed to inhibit fatty acid oxidation. Otherwise, the excess fatty acids would be oxidized, and thus detoxified. This combined hypothesis is particularly attractive, as it explains the compensatory [1]-cell hyperfunction of insulin resistance without diabetes (as occurs in obese people who are normoglycemic) and the toxic effect of the same level of hyperlipidemia in type 2 diabetes.

5. Impaired incretin effect: Incretin hormones are gut peptides that are released with eating and have a multitude of effects, including stimulating both the secretion and biosynthesis of insulin (114–116). The best known are glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP). A characteristic feature of type 2 diabetes is a reduced incretin effect through 2 known mechanisms. The first is a lowered insulinotropic effectiveness of GIP (117). The time of onset of this change is unclear, as it is present in some nondiabetic firstdegree relatives of persons with type 2 diabetes (118), but not women with a history of gestational diabetes (119).

The mechanism is not known, although one proposal is defective expression of GIP receptors on ß-cells (120).

In contrast, sensitivity to GLP-1 is mainly intact in type 2 diabetes (117). However, a second mechanism for defective incretin regulation in type 2 diabetes is a decrease in the secretion of GLP-1 (121). As yet, the molecular mechanisms for both of these observations, when they first occur, and their importance to the ß-cell dysfunction of type 2 diabetes, have not been determined.

In addition to [1]-cell dysfunction, reduced ß-cell mass may also contribute to the development of type 2 diabetes. Measurement of ß-cell mass in humans is technically difficult and must be done on autopsy specimens; until recently, there were few studies, with a limited number of subjects. Furthermore, in many of these studies, controls were poorly matched. An increased ß-cell mass is part of the normal ß-cell compensation to insulin resistance.

Weight-matching of control and diabetic subjects is now mandatory to minimize differences in insulin sensitivity, but was often not done in older studies. An important recent study by Butler et al of nearly 160 weight-matched control and type 2 diabetes subjects reported a 40–60% lowered ß-cell mass in this disease (Fig. 3) along with a 3-fold increase in ß-cell apoptosis (77). Also, a recent study from Korea reported a large reduction in ß-cell mass in type 2 diabetic subjects, and also reported the novel finding that the mass of the glucagon-producing cells was increased (78). Notwithstanding the limitations of the older studies, these studies confirmed the conclusion of most, but not all, prior work that ß-cell mass is lowered in type 2 diabetes.

The study by Butler et al (77) was the first study to provide data on subjects with IGT, finding a 40% reduced ß-cell mass. This novel observation is important for our understanding of the pathogenesis of type 2 diabetes.

The sequence of events suggests that reduced [1]-cell mass may cause the earliest hyperglycemia, when the blood glucose begins to rise but is still within the normal glucose tolerance range, initiating in some way defective first phase insulin secretion. Postmeal glycemia then rises to a level defined as IGT, with the potential for more acquired defects, worsening of glycemia, and progression to diabetes. Although no additional human data exist, studies of partially pancreatectomized rats support that pattern. After a 60% pancreatectomy, rats normally compensate for the ß-cell loss through a combination of partial ß-cell regeneration and hyperfunction of the remaining ß-cells, and thus remain normoglycemic under normal circumstances. The partial ß-cell regeneration results in the ß-cell mass rising from 40% immediately after the surgery to 60% of normal, and remains at this level indefinitely. The ß-cell compensatory capacity of these rats to a minor dietary change was studied by adding some sugar (10%) to the water supply from which they drank freely (79). Nonpancreatectomized rats given the sugar water drank it identically to the pancreatectomized rats. It is important to appreciate that the diet change was extremely modest: nonpancreatectomized rats given the sugar water over the 6 weeks of the study showed no obesity, hyperinsulinemia, or other metabolic difference compared to rats given tap water. In contrast, 60% pancreatectomy rats given sugar water developed mild hyperglycemia after a few weeks, with morning blood glucose values rising by 15 mg/dL. This small increase in glycemia was associated with a profound (75%) reduction in glucose-induced insulin secretion, analogous, it is proposed, to the process in humans that initiates progression to IGT and subsequent diabetes. Thus, one scenario for “susceptible ß-cells” is a reduced ß-cell mass that is incapable of maintaining normoglycemia when faced with environmental factors that have no detrimental metabolic effect when the ß-cell mass is normal.

Considerable current research is exploring the pathogenic basis for the lowered ß-cell mass in type 2 diabetes and prediabetes, with several proposed mechanisms:

1. Amyloid plaques occur in the islets of persons with type 2 diabetes, along with distorted and shrunken ß-cells (80). The amyloid protein, islet associated polypeptide (IAPP), is a 37 amino-acid ß-cell-specific protein that is normally packaged in insulin granules and co-secreted with insulin (81,82). The 25- to 28-amino acid sequence is the amyloidogenic portion. It is conserved in many species, all of which develop islet amyloid and diabetes.

However, rodents lack the sequence, allowing the creation of transgenic mice that overexpress human IAPP to test the plausibility of IAPP-induced ß-cell destruction. Some, but not all, transgenic mice develop islet amyloid plaques with accelerated ß-cell apoptosis and diabetes (83,84), engendering substantial interest in a pathogenic role for islet amyloid in type 2 diabetes (85). Lorenzo et al (86) cocultured islets with exogenous IAPP, causing ß-cell death, which suggested that external amyloid plaques are cytotoxic. Also, this finding implied that amyloid deposition must be an end-stage part of the disease, not involved in the ß-cell reduction in IGT, as islet amyloid is not yet present in autopsy specimens from these subjects. The current view, however, has evolved to small intracellular microfibrils of IAPP being cytotoxic through mitochondrial damage or endoplasmic reticulum stress (87,88), which is more in line with how other amyloid diseases, such as Alzheimer’s, are thought to occur. In animal  tudies, microfibrils occur long before the extracellular amyloid plaques. Unfortunately, showing their presence in human autopsy tissue is an inexact science, and it remains unknown if they are present in IGT. Also, IAPP is normally produced and secreted. The cause of the microfibrils and large amyloid plaques in type 2 diabetes remains unknown.

It is not related to the rate of IAPP secretion, as normally glucose tolerant obese subjects with long-term ß-cell compensatory hyperfunction for both insulin and IAPP lack islet amyloid at autopsy. Neither is it hyperglycemia per se, as amyloid plaques are often found in insulinomas (89). Genetic mutations in IAPP have been sought, but rarely found. Instead, the expectation is that mutations of other important proteins that normally keep IAPP soluble will be found, for example folding proteins, or others that prevent amyloid formation.

2. Pathological studies of ß-cells in type 2 diabetes have reported increased apoptosis as the cause of the lowered ß-cell mass (77,90). Many mechanisms of cellular apoptosis are known: ER stress from misfolded proteins, oxidative stress, inflammatory mediators, glucolipotoxicity, etc. All are being studied for relevance to type 2 diabetes (91–95).

3. There is no evidence to suggest that the cause of the lowered ß-cell mass is immune-mediated ß-cell destruction analogous to type 1 diabetes, as careful studies have shown the 2 types of diabetes are pathologically distinct.

Beta cell dysfunction in type 2 diabetes

Studies over many years have described the ß-cell dysfunction in type 2 diabetes (66). The major defects are:

1. Insulin is normally secreted in a pulsatile fashion, with oscillations every 11–14 minutes that provide for normal regulation of hepatic glucose production (67,68). Also large bursts (termed ultradian oscillations) occur several times daily, especially after meals, and maximize nutrient clearance (69). The pulsatile patterns are disrupted early in type 2 diabetes, with near-total elimination of the oscillations even in normoglycemic first degree relatives (70,71).

2. An acute rise in glucose normally causes a burst of insulin secretion lasting 5–10 minutes (“first phase”), followed by another rise in insulin output lasting the duration of the hyperglycemic stimulus (“second phase”). The characteristic ß-cell defect in type 2 diabetes is loss of the first phase (48,66), which occurs early in the course of the disease, with the first phase being reduced in half with fasting blood glucose levels above 100 mg/dL, and absent at values greater than 115 mg/dL (46). The first phase serves an important role during food ingestion, to control the postmeal glycemic excursion. Selectively disrupting the first phase in healthy subjects causes glucose intolerance (72,73), whereas restoring it in persons with type 2 diabetes markedly improves postprandial glycemia (74). Importantly, Vague and Moulin (75) found a substantial recovery of the first phase following a period of intensive glucose control. As such, loss of first phase insulin secretion is the earliest identified aspect of the previously discussed acquired ß-cell defects. Furthermore, this defect provides a pathophysiological explanation for the transition from normal glucose tolerance to IGT.

3. As the disease progresses and hyperglycemia worsens over time, additional ß-cell defects occur. Indeed, a defining feature of type 2 diabetes is a relentless slow deterioration of ß-cell function that is blamed for the typical clinical course of eventual waning of responses to oral antidiabetic agents (76). Also, this worsening explains why so many patients ultimately require insulin therapy for glucose control. These defects have been investigated almost exclusively in diabetic animals and cell systems. (A major obstacle to a better understanding of the ß-cell dysfunction is an inability to perform human pancreas biopsies because of risk of pancreatitis and/or leakage of digestive juices.) Animal studies have shown that there is a hierarchy of ß-cell defects at different glucose levels: modest hyperglycemia coexists with impaired glucose-induced insulin secretion that mimics human type 2 diabetes, and higher levels are associated with additional defects in proinsulin biosynthesis and ß-cell viability (66).

 Insulin resistance vs beta-cell dysfunction

One of the most controversial issues during the 1980s and 1990s was whether insulin resistance or ß-cell dysfunction was the main cause of type 2 diabetes. The fact that persons with type 2 diabetes, and also those with IGT, invariably have both defects fueled the debate. Several highly discussed studies of people at presumed high-risk for type 2 diabetes, but still normoglycemic (high risk ethnic groups such as Pima Indians, those with both parents having type 2 diabetes, and women with prior gestational diabetes), attempted to identify the operative pathogenic elements before glucose values become abnormal. These studies generally reported that insulin resistance was present, but not ß-cell dysfunction (45), resulting in a common belief at the time that insulin resistance was the earlier (and thus dominant) defect in this disease.

These conclusions were based on an experimental measure of ß-cell function that was later shown to be misinterpreted: the 2-hour insulin value postmeal or during an oral glucose tolerance test (OGTT). Insulin resistance was relatively easily measured, either by using the euglycemic glucose clamp, which is labor intensive and usually done with a limited number of subjects, or computer models that can be applied to large experimental groups. In contrast, the measurement of ß-cell function is highly complex. The insulin response to a meal normally is biphasic, with the amount released depending on many factors, such as the size and composition of the meal, prevailing glycemia, the subject’s insulin sensitivity, etc. As glucose tolerance moves from normal to impaired, insulin release during the first 30 minutes of eating (“first phase”) falls, and is absent by the time fasting glucose exceeds 115 mg/dL (46). The later insulin secretion (“second phase”) paradoxically becomes greater than normal in response to the hyperglycemia. The early studies concluded that the supernormal 2-hour insulin value, attributed to insulin resistance, proved that there was no ß-cell dysfunction at that stage. This misinterpretation was corrected by later studies that found reduced 30-minute and elevated 2-hour postmeal insulin values in IGT and early type 2 diabetes (47), and others demonstrating that defective first phase insulin responses to intravenous glucose is a characteristic feature of type 2 diabetes (48).

Investigators next turned to cross-sectional and natural history studies of ß-cell function versus insulin resistance.

They confirmed that insulin resistance is already present when glucose values are within the normal glucose tolerance range (49,50). There are a number of potential reasons: in some people this is presumably owing to a genetic abnormality that affects insulin sensitivity, and in others lifestyle factors, such as obesity, lack of exercise, high fat diets, aging, etc., may play a major role. Thereafter, insulin resistance is relatively unchanging.

Therefore, a change in the degree of insulin resistance could not explain blood glucose values progressing from normal to IGT to diabetes. Instead, worsening ß-cell function is causative. These natural history studies observed a biphasic pattern: initial hyperinsulinemia, with blood glucose values maintained in the normal range or only mildly impaired, and, subsequently a falling insulin level (“[1]-cell failure”), resulting in rising glycemia (49,50).

Thus, the concept of type 2 diabetes began to change, with insulin resistance being an important risk factor for type 2 diabetes, but ß-cell function determining glycemia in persons genetically at risk for the disease.

The most recent studies have returned to the question of the priority of these abnormalities, in part reflecting better techniques to assess ß-cell function. One of the most used is the disposition index, based on the understanding that ß-cell function is dependent on the degree of insulin sensitivity. In other words, the insulin response to a meal or other stimulus is normally less in an insulin sensitive person such as a marathon runner than for a normoglycemic insulin resistant subject (51). Thus, in normoglycemic subjects, insulin levels are more reflective of insulin sensitivity than ß-cell function. The relationship between experimentally measured insulin sensitivity and first phase insulin secretion as a measure of ß-cell function has been mapped out in a large number of normoglycemic subjects to derive the normal curve that is called the “disposition index” (52,53) (in Fig. 2, the hyperbolic curved lines are the experimentally derived normal curve). It is important to realize this is the normal system – everyone experiences insulin resistance at some time (puberty, pregnancy, aging), with most maintaining normoglycemia because of this ß-cell compensation. Indeed, many consider diabetes a failure of ß-cell compensation (54). It is a commonly used research technique to plot where subjects with varying degrees of glucose tolerance fall on the disposition index to identify the relative roles of insulin resistance versus ß-cell dysfunction (55).

A well-known study that used this method was performed in 48 normally glucose tolerant Pima Indians (a population with the highest worldwide incidence of type 2 diabetes), who were studied over an average of 5 years

(56). Seventeen developed type 2 diabetes (progressors) whereas 31 maintained normal glucose tolerance (nonprogressors).

The groups were comparably obese and insulin resistant at the start of the study. Nonprogressors gained a little weight, and became a little more insulin resistant during the study, but stayed on the disposition curve, i.e., had perfect ß-cell compensation (Fig. 2). Progressors instead started below the insulin secretion part of the curve, and fell even further as their glycemia worsened, clearly showing that their deterioration in glucose tolerance resulted from worsening ß-cell function. It is particularly notable that the progressors started the study already off the normal curve, showing that despite their entering the study with glucose tolerance in the normal range, there was already subtle ß-cell dysfunction that had not yet resulted in a measured degree of glucose intolerance.

This latter concept has been observed in many other populations. One notable series of studies cross-sectionally examined Mexican-Americans and Caucasians across a wide range of glycemia from normal glucose tolerance to diabetes (57,58). ß-cell function was determined as the insulin response to an OGTT that was adjusted for each subject’s insulin sensitivity (based on glucose clamp testing) and 2-hour postmeal glucose values. ß-cell function was observed to fall as glycemia rose ever so slightly within the normal glucose tolerance range; subjects with 2-hour glucose values of 101–120 mg/dL had 60% lower adjusted mealtime insulin responses than those with 2-hour glucose values <100 mg/dL (normal glucose tolerance is defined by a 2-hour value of <140 mg/dL). Studies in other populations (59,60), and a cross-sectional analysis of fasting glucose values (61), had similar results.

Thus, it is now clear that insulin resistance and ß-cell dysfunction both precede measured defects in glucose tolerance. Defective ß-cell mass or function must be present for blood glucose values to rise even minimally above normal, given the precision of a healthy glucose homeostasis system. Therefore, the current definition of normal glucose tolerance is insensitive to early defects in glucose homeostasis. A recent study documented a several fold higher risk for type 2 diabetes with fasting blood glucose values at the high range of normal versus the low range ( >87 mg/dL vs <81 mg/dL), especially in the presence of obesity or hypertriglyceridemia (62).

The question of which defect occurs first, and which is dominant, remains. One related long-standing argument is whether prolonged insulin resistance causes ß-cell failure through “exhaustion” (i.e., continued stimulation of otherwise normal ß-cells resulting in permanent dysfunction). The available facts do not support this proposal.

Many morbidly obese highly insulin resistant subjects never develop diabetes. One thus assumes that it is necessary for the ß-cell compensatory ability to be compromised in some way for diabetes to develop. Stated another way, if one has healthy ß-cells, it appears to be virtually impossible to develop type 2 diabetes with the usual lifestyle and environmental influences. Thus, the key to a better understanding of type 2 diabetes is to define what constitutes “susceptible” ß-cells.

To summarize, both beta-cell dysfunction and insulin resistance occur long before blood glucose values reach prediabetes. One has not been shown to precede, or cause, the other. As such, type 2 diabetes is considered a “dual-defect disease” with both defects of equal importance (63). However, it is unclear from this understanding whether early intervention for diabetes prevention would be most effective focusing on improving ß-cell function or insulin resistance. To date, the most effective treatment found for prevention of type 2 diabetes is diet and exercise (2,31,32), which improves insulin sensitivity. Alternatively, viewing one defect as independent from the other may be overly simplistic, as ß-cell function and insulin resistance are linked by the disposition index. The best example of this is a series of studies of Hispanic women in Los Angeles with prior gestational diabetes (an extremely high risk group for type 2 diabetes) who were treated with the insulin sensitizers Troglitazone or Pioglitazone. Both drugs markedly decreased progression to permanent type 2 diabetes (64,65). One might expect that the conclusion of these studies would highlight the importance of insulin resistance. Instead, the main reported benefit was prevention of ß-cell dysfunction as shown by sequential analysis of ß-cell function and insulin sensitivity, believed secondary to “ß-cell rest”.

These refer to nongenetic defects in glucose homeostasis that occur as diabetes develops. This concept was first identified in studies that intensively treated glycemia in persons with type 2 diabetes, with resultant improvement in ß-cell function (33). Later studies showed some reversal of insulin resistance. This effect is unrelated to the type of treatment used to lower the glucose level (34), and is most effective early in the disease. Studies have shown long-term recovery of glucose tolerance in newly diagnosed patients with type 2 diabetes after a short-term insulin infusion or high dose sulfonylurea therapy (35,36).

A particularly interesting study was performed in subjects with an average duration of 8 years of type 2 diabetes who were placed on an insulin pump for 3 weeks, attaining excellent glucose control (37). The pump was stopped, and the subjects studied 2 days later, at which time large improvements in ß-cell function along with normalization of hepatic glucose production and some improvement in insulin resistance were seen. These three problems make up the classic triad of type 2 diabetes. A question posed at the beginning of this review is why patients invariably have this triad, when the organs involved lack a known physiological link. One answer is that, irrespective of the genetic and environmental factors in any given patient, as glucose intolerance develops the acquired organ abnormalities result in the “common phenotype” of the disease.

A substantial amount of research has focused on these acquired abnormalities, in particular ß-cell dysfunction. It was initially assumed the reversal of [1]-cell dysfunction noted above stemmed from the improvement in blood glucose level, and the term “glucose toxicity” was coined (38,39). Supporting that idea were studies showing that experimental hyperglycemia in rodents invariably caused similar ß-cell dysfunction to that occurring in diabetic humans (40), which was reversed by a glucose-lowering agent called phlorizin that restores normoglycemia by lowering the threshold for glucose clearance into urine, thus promoting glycosuria (41). Many mechanisms for hyperglycemia-associated ß-celldysfunctionhavebeendescribedfromin vitro cellsystemsandanimalmodels,andwillbediscussedlater.

Also discussed later is a more recent suggestion that relates to another component of the diabetes phenotype, high circulating levels of triglycerides and fatty acids, as a cause of acquired organ abnormalities: so-called “lipotoxicity” (42). There has also been interest in the combined effects of both elements, termed “glucolipotoxicity” (43,44).

The diabetes genotype causes a predisposition for glucose intolerance. The development of type 2 diabetes is influenced by environmental factors, some clearly defined, others less so. The Nurses Health Survey showed the expected positive associations between obesity and lack of physical activity in the development of type 2 diabetes, but also protection related to abstinence from smoking and to moderate alcohol use (24). The protective effect of alcohol has been found in other studies, and was confirmed in a recent meta-analysis (25). Also obscure is a reported association between type 2 diabetes and sleep deprivation (26), and a protective effect of caffeine (27).

More understandable are the numerous studies showing associations between risk of type 2 diabetes and high calorie diets, physical inactivity, and our modern lifestyle in general.

These predisposing factors share an ability to negatively impact glucose homeostasis through worsening of insulin resistance or impairment of ß-cell function. Superimposing these factors onto a genetically compromised system augments the risk of hyperglycemia. The rapid emergence of these disadvantageous environmental factors is causing the worldwide diabetes epidemic. This concept was highlighted many years ago by nomadic or farm-based populations that moved to urban environments, followed by an explosion of diabetes, typically with profound obesity: Pima Indians in the Southwest United States, Saharan nomadic tribes, and Australian Aborigines are well-known examples. Studies that show reversal of the diabetes after members of these populations return to their prior way of life are particularly notable (28). A recent example of the effect of population shifts is the rapidly rising incidence of type 2 diabetes in China and India as people flood to the cities; there is a 0.1–0.2% incidence of diabetes in rural parts of China as opposed to more than 5% for city dwellers. Perhaps the most distressing example of rapid change is the rising incidence of obesity in children in the US. As many as 20% of US children are now obese, and they are developing all of the elements of the metabolic syndrome: insulin resistance, hypertension, hyperlipidemia, and glucose intolerance (29). Additionally gestational diabetes has doubled in prevalence in the U.S. over the last decade (30).

An obvious conclusion is that a return to a healthier lifestyle should reverse the diabetes trend. Indeed, many studies have shown diet and exercise markedly decrease the onset of diabetes in persons with IGT (2,31,32). The difficulty, of course, is trying to get people to change their habits. In addition, we are lacking long term studies of lifestyle modification in terms of a protective effect against cardiovascular disease.

The fact that type 2 diabetes is a genetic disease was confirmed more than 2 decades ago by a famous study of identical twins in the U.K. that found essentially a 100% concordance rate (9). However, this kind of study provides no insight into the underlying genetic defect (s) either directly impairing the glucose homeostasis system or causing insulin resistance or another defect that exceeds the capacity of a normal glucose homeostasis system.

With the advent of molecular biology, the basis is now known for many monogenic forms of diabetes, such as mitochondrial genome defects and their association with diabetes and deafness (10), Wolfram’s syndrome (11), several syndromes of extreme insulin resistance (12), and most of the MODY syndromes (13). Still, these account for only a small proportion of diabetes cases.

In contrast, genetic insight into type 2 diabetes has been frustratingly slow. One identified gene is calpain 10, a member of a ubiquitously expressed family of cysteine proteases. In the mid 1990s, linkage analysis identified a locus on chromosome 2 that was calculated to account for about 30% of type 2 diabetes in Mexican-Americans (14).

The specific gene was later shown to be calpain 10 (15). However, the role of calpain 10 in glucose homeostasis remains unclear, with a current focus on a regulatory role in insulin exocytosis (16).

A recent study of isolated islets from humans with type 2 diabetes reported a 90% reduced mRNA expression of aryl hydrocarbon receptor nuclear translocator (ARNT), a transcription factor previously unknown to the diabetes field (17). Mice were created with a ß-cell specific knockout of the ARNT gene. These animals developed glucose intolerance and impaired glucose-induced insulin secretion, along with a ß-cell mRNA expression profile that closely matches the human type 2 diabetes islets. Considerable interest was generated by these findings, and a role for ARNT in type 2 diabetes is under investigation.

Other chromosomal “hot spots” have been identified in various populations, and looking for the specific genes is now much faster because of the human genome project. Also, many research groups have focused on various gene polymorphisms. To date, all have lacked a strong association with type 2 diabetes after rigorous study. An example is Insulin Receptor Substrate-1 (IRS-1), the first downstream intermediate from the insulin receptor in the insulin action cascade. A common IRS-1 polymorphism was proposed to influence the kinetics of insulin secretion (18), but a large study failed to show a link with type 2 diabetes (19). Current polymorphisms of interest are the transcription factors TCF7L2 (20) and KLF11 (21), and the Kir6.2 subunit of the ß-cell K+ ATP channel (22,23).

Finally the breakthrough occurred in 2007 with the advent of genome-wide association screens for common diseases including type 2 diabetes. Unlike prior genetic studies that often tested for genes that seemed plausible as causing a predisposition for type 2 diabetes (so-called candidate gene approach, but in reality guesses), this kind of study uses small nucleotide sequences that are spaced throughout the whole genome to search for patterns that track with a disease such as type 2 diabetes, and thus identify regions in which to look for a predisposition gene. And they have been amazingly successful. In less than a year, 6 genome-wide association studies that examined 7,200 cases of type 2 diabetes and 12,000 controls in several population groups have identified 11 predisposition genes (reviewed in 163). Plus the results are fascinating. Only one, PPAR, was ever proposed as a candidate gene, with most having no known physiologic role in glucose homeostasis. Still, its easy to envision how they might act as several of the factors likely influence beta-cell development, insulin secretion, or proinsulin biosynthesis. And thats the second interesting finding, in that most of the identified genes seem to be involved in beta-cell biology as opposed to the insulin signaling or glucose transport systems. Finally, the greatest impact of any of these genes is quite modest – a 20% to 30% increase in diabetes risk – so we still have lots to learn about how these different genes interact to produce the profound diabetes susceptibility in certain families and ethnic groups. And one guesses more susceptibility genes will be found. So we have finally entered the genetic era, and its likely to be a very exciting time that finally may answer some of the tough questions in type 2 diabetes.

The pathophysiology of type 2 diabetes is complex, with many different elements acting to cause the disease. This review proposes a sequence of events that is based on a careful analysis of the human and animal model literature. It seems certain that a genetic predisposition is needed although, until recently, little was known about specific genetic mutations. Whether the diabetes phenotype then occurs depends on a large number of environmental factors that share an ability to stress the glucose homeostasis system by promoting insulin resistance or worsening ß-cell function. We propose that a lowered ß-cell mass through genetic and/or ß-cell cytotoxic factors is an important predisposing factor for glucose intolerance. As the blood glucose level rises to a minor degree above normal, acquired defects in the glucose homeostasis system occur—a key early one is an impaired first phase insulin response to a meal —that cause the blood glucose level to rise further into the prediabetes range. This increase in glycemia, perhaps in concert with hyperlipidemia, causes additional deterioration in ß-cell function and, to a smaller extent, resistance, resulting in a blood glucose level that continues to rise to full blown diabetes. This sequence provides insight into prevention and treatment of type 2 diabetes. One can modify predisposing environmental factors, although that is not easily done. Alternatively, one expects that, as the molecular basis for the organ dysfunctions are discovered (ß-cell dysfunction and death, and muscle and hepatic insulin resistance), novel therapies will be developed that target those defects.

INTRODUCTION

Type 2 diabetes is a worldwide health crisis. In the U.S., 20.8 million are affected at a cost of $132 billion in 2002 (1), and the numbers will likely continue to increase. The Centers for Disease Control and Prevention estimates there are more than 40 million people in the U.S. with prediabetes. Given that the Diabetes Prevention Program showed an 11% yearly conversion rate of impaired glucose tolerance (IGT) to diabetes (2), there could be as many as 4 million new cases each year. Furthermore, the incidence of type 2 diabetes is rising around the world (3), with a recent prediction that the worldwide prevalence will increase from 2.8% in 2000 to 4.4% in 2030, resulting in 366 million affected people (4).

Much of the current crisis stems from our modern lifestyle. Furthermore, the global shift from an agrarian existence to city living, resulting in less physically demanding office and factory jobs, is taking its toll. In the U.S these changes have been most evident in children—numerous studies have reported the epidemic of childhood obesity (5) and its root causes of reduced physical activity and high caloric intake (6,7).

Although returning to healthy lifestyles likely would reverse the rising incidence of type 2 diabetes, this may be an impractical solution. Instead the current focus is to investigate the pathogenesis, hoping to develop pharmaceuticals that target the key pathogenic elements. We entered the 1990s knowing that type 2 diabetes was characterized by the triad of ß-cell dysfunction, excess glucose production from the liver, and insulin resistance, defined as impaired insulin-mediated glucose clearance into skeletal muscle (8). However, the link among these organs was unknown. Considerable insight has been gained over the last decade, although much remains to be learned. This review provides an overview of the current understanding of the pathogenesis of type 2 diabetes  (Fig. 1).

A major focus of the proposed sequence relates to defects in the mass and function of islet ß-cells, as they are known to be important elements in the early stages of the disease.