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).