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GLUCOSE TOXICITY

George Fantus, MD 

 

INTRODUCTION PANCREATIC β-CELLS Pathophysiology and Phases of Hyperglycemia-induced β-cell Dysfunction: Lipotoxicity: Genetic Contributions Time course of β- cell glucose toxicity: PERIPHERAL INSULIN TARGET TISSUES CELLULAR AND MOLECULAR MECHANISMS OF GLUCOSE TOXICITY Pancreatic β-cells: Peripheral Insulin Target Tissues Microvascular and other complications: CLINICAL IMPLICATIONS

 

 

INTRODUCTION

 The term “glucose toxicity” was originally coined to describe the adverse effects of chronic exposure of pancreatic β-cells to high concentrations of glucose (1,2). First suggested in β-cells by the observations of Haist in 1940 (3), the notion that high glucose exerts multiple pathological effects on many cells and tissues has been established by abundant evidence for its causative role in the chronic microvascular complications of diabetes (4-6), its effects on insulin action in metabolic target tissues (7,8), and in several other adverse outcomes noted in people with diabetes, such as frequent fungal infections (9) and an increased frequency of congenital birth defects (10). This chapter will review the contribution of high glucose to β-cell dysfunction and to insulin resistance, two key components in the pathogenesis of type 2 diabetes. The cellular and molecular mechanisms will be outlined, and these mechanisms compared to those thought to contribute to the chronic complications of diabetes. Since these mechanisms appear to overlap to a considerable extent, the term “glucose toxicity” can be, and is currently used more broadly, to describe the pathogenic role of high glucose on multiple organ systems (Table 1).

 

 

 

PANCREATIC β-CELLS

The hypothesis that hyperglycemia may contribute to defects in insulin secretion and potentially, to the well documented deterioration of β-cell function in type 2 diabetes (T2DM) commonly referred to as β-cell exhaustion, resulted from clinical observations. Thus, it was noted that improvement of glycemic control in subjects with T2DM by either lifestyle changes (diet and exercise) (11), oral hypoglycemic agents (eg. sulfonylurea) (12), or even short term insulin therapy (13), resulted in an improvement in insulin secretion. The effects of high glucose on β-cell function, gene expression and survival have now been extensively investigated both in animal models and cell culture.

One very useful rodent model in the study of glucose toxicity has been the partial pancreatectomized rat (14,15). After 90% pancreatectomy rats develop mild fasting hyperglycemia and glucose intolerance. Insulin secretion in response to glucose becomes severely impaired. Treatment of these rats with phloloridzin, an inhibitor of renal tubular glucose reabsorption which results in normoglycemia, completely restored normal insulin secretion expressed per gram of pancreatic mass (16) (Fig. 1).

 

 

 

Apart from its direct stimulatory action to release insulin, glucose potentiates β-cell insulin secretion in response to nonglucose stimuli. The amino acid arginine (Arg) has been frequently used to study this phenomenon. In the 90% pancreatectomized hyperglycemic rat, early on, the response to Arg was increased, i.e. potentiated as expected, but returned to normal upon treatment with phloridzin. These observations have been confirmed in rats subjected to neonatal streptozotocin+ injection which causes mild to moderate insulin deficiency and hyperglycemia at 6-8 weeks of age. In this model maintenance or restoration of normal glucose levels by insulin supplementation improved both the acute insulin response to glucose and the potentiated insulin response to nonglucose stimuli (17,18).

 

 

+Streptozotocin is a β-cell toxin

In 60% pancreatectomized rats, glucose levels remain normal (19), confirming the observation that > 85 – 90% of pancreatic islet function must be lost in a “normal” rat or human before diabetes is observed. However, feeding these rats 10% sucrose in their water caused mild hyperglycemia and a subsequent 75% reduction in insulin secretion (19). The minimal elevation (average 0.83 mmol/L, 15 mg/dl) of glucose required to produce this response demonstrates the marked sensitivity of the β-cell to desensitization, at least in the presence of reduced β-cell mass. In intact rats, as short as 48 h exposure to high glucose, average 14 mM, by glucose infusion, impaired subsequent glucose-induced insulin release (20). Thus sustained hyperglycemia, namely elevation longer than that used to stimulate an acute insulin response, over a relatively short period of time, 2 days to ~ 6 weeks, results in impaired, but reversible, insulin secretion. However, in the absence of glucose normalization irreversible changes in β-cell function follow. For example, 50 – 80% partially pancreatectomized dogs did not develop diabetes over 8 – 9 months. Additional exposure to 2 weeks of glucose infusion irreversibly impaired insulin secretion and induced diabetes (21). This model is reminiscent of the progressive decline in β-cell function seen in human T2DM.

 

 

 

Pathophysiology and Phases of Hyperglycemia-induced β-cell Dysfunction:

To characterize the physiology and molecular changes caused by long term exposure to high glucose, Weir and colleagues have used the partial pancreatectomized rat. Based on studies of: 1) glucose-stimulated insulin secretion (GSIS), 2) gene expression of transcription factors, metabolic enzymes and proteins involved in the stress response and apoptosis (programmed cell death), and 3) morphological changes, it has been proposed that the β-cell response to increased insulin demand and hyperglycemia can be classified into 4 phases of adaptation (Fig.2) (22). Although these phases exist as a continuum with overlapping features and a variable time course, the classification is useful to investigate and identify the cellular and molecular basis of β-cell dysfunction. Briefly, phase 1 is characterized by successful adaptation to increased demand which appears to be mediated by increasing the residual β-cell mass by hypertrophy and to a lesser extent, hyperplasia and by a lower set point of insulin secretion in response to glucose. β-cell function otherwise remains normal. Phase 2 is classified as mild decompensation, in which GSIS is impaired while response to other secretagogues is relatively preserved. This phase is observed at minimally elevated glucose, eg. fasting glucose of 5.6 mmol/L (100 mg/dl), i.e. before the development of overt diabetes. Insulin stores and insulin mRNA levels are preserved, suggesting that the defect is at the level of secretion.

 

 

 

 

 

The mechanism by which high glucose desensitizes the β-cell to subsequent stimulation by glucose in phase 2 is not clear. Most studies have documented that glucose uptake and metabolism/oxidation by the β-cell, at least in the earliest phases of desensitization, remain normal (2,23). Since insulin stores are maintained, it has been proposed that coupling between glucose signaling and the secretory process is impaired. A key element here is the ATP-dependent K+ channel which is regulated by the cellular ratio of ATP/ADP. Thus, closure of the channel in response to glucose is dependent on ATP generation (see Chapter 3), which is regulated not only by the flux of glucose through the oxidative pathway but also by the efficiency of the electron transport chain and the coupling of oxidative phosphorylation. It has been found that uncoupling protein-2 (UCP-2) is upregulated in β-cells in rodent models of diabetes (24), and that elevated UCP-2 impairs GSIS (25). Another proposed mechanism of impairment of oxidative phosphorylation is the relative depletion of NADH by increased conversion of pyruvate to lactate (22,26). This may result from upregulation of LDH (lactate dehydrogenase) as part of a generalized β-cell dedifferentiation program initiated by exposure to high glucose (see below) (27). Oxidation of NADH would reduce electron transport and ATP generation. Another possibility is that rather than (or in addition to) decreased generation of ATP, increased consumption of ATP is a consequence of hyperglycemia. In the GK (Goto-Kakizaki) diabetic rat, a model of type 2 diabetes, glucose oxidation is elevated in the presence of diminished insulin release (28). It has been found that glucose cycling, i.e. the phosphorylation of glucose to glucose-6-phosphate (G6P) by glucokinase followed by dephosphorylation back to glucose by glucose-6-phosphatase (G6Pase), is significantly elevated in the islet of GK rats. Glucose cycling which has been termed a “futile cycle”, consumes one molecule of ATP with each cycle. This increase of glucose cycling was associated with elevated G6Pase activity. Correction of hyperglycemia with phloridzin in the GK rat normalized G6Pase activity associated with significantly improved insulin secretion (29).

 

Two other hypotheses previously proposed to explain high glucose – mediated desensitization are impaired membrane phosphoinositide hydrolysis (30), now considered unlikely (31), and elevated PGE2 (prostaglandin E2) which has been documented to inhibit insulin secretion (33). The contribution of this mechanism requires further investigation.

 

Phase 3 occurs in response to a longer duration of high glucose exposure and is termed severe decompensation. At this stage not only is GSIS impaired, but responses to nonglucose stimuli are also decreased. β-cell insulin mRNA is now decreased, degranulation is evident and a host of changes in gene expression are observed. Some of these changes are postulated to reflect β-cell dedifferentiation since a large subset of the downregulated genes code for proteins which regulate the specific insulin secretory function of the β-cell, e.g. the uptake, metabolism and response to glucose and the synthesis of insulin (Table 2) (34). Another set of genes which are affected in this setting are classified as “stress” genes. These include genes coding for pro- and anti-oxidant enzymes and pro- and anti-apoptotic proteins. The relevance of the stress genes relates to the known induction of cellular oxidative stress by high glucose which likely contributes to the pathogenesis of glucose toxicity (see below). In the final or 4th phase, β-cell decompensation is accompanied by structural damage. Microscopic changes may include amyloid deposits, glycogen and/or lipid deposits, and in some cases, fibrosis. The relative importance of apoptosis versus loss of β-cells by limited replication to the loss of β-cell mass is not clear. The role of amyloid as a mediator or consequence of β-cell dysfunction remains controversial (35). The frequent observation of lipid (triglyceride) accumulation in β-cells in rodent models of obesity-associated T2DM, along with the documentation of a β-cell “toxic” effect of excess circulating FFA (free fatty acids), has given rise to the term lipotoxicity (36).

 

 

 

 

 

Lipotoxicity:

Since it is beyond the scope of this chapter to discuss lipotoxicity in detail the reader is referred to recent reviews (36–38). It is important to mention that elevated FFA, in the short term, appears to stimulate insulin secretion (39), while in the longer term impairs GSIS, both in vivo and in vitro (40-42). This has been termed lipotoxicity and appears to relate to an impairment of lipid oxidation and shunting of FFA to the esterification or synthesis pathway. Thus, Prentki and Corkey have proposed that the LC-CoA (long chain fatty acyl Co-enzyme A) and/or one of its metabolic products, e.g. DAG (diacylglycerol), generate signals to impair insulin secretion (36,38). Although these intermediates are known to activate PKC (protein kinase C), the precise mechanism of lipotoxicity is not clear. Another metabolic product of FFA, specifically of the saturated FA, palmitate, is ceramide, an activator of phosphatases, kinases and a pro-apoptotic factor (42,43). It is significant that in the gene expression studies of Weir, high glucose resulted in increased β-cell expression of PPARg (peroxisome proliferator-activated receptor-g), which is a transcription factor which stimulates the expression of genes involved in fatty acid and triglyceride synthesis and storage, while downregulating PPARa, a transcription factor which promotes expression of genes encoding proteins involved in FFA oxidation. The net result would be an intra-islet metabolic state consistent with lipotoxicity and enhanced FA esterification. In this context treatment of islets with leptin, the adipocyte-derived satiety hormone, stimulated FA oxidation, reduced β-cell triglyceride content and improved insulin secretion in rodent models of obesity and diabetes (44).

The relative importance of glucotoxicity versus lipotoxicity in T2DM remains controversial. In a recent attempt to resolve this issue Robertson treated ZDF (Zucker diabetic fatty) rats, which are mildly hyperglycemic and hyperlipidemic and develop overt diabetes between 6 and 12 weeks of age, with either phloridzin to restore and maintain euglycemia or bezofibrate to treat the hyperlipidemia. Despite remaining hyperlipidemic, the phloridzin-treated rats were protected from β-cell decompensation, while the fibrate treatment, which did not alter glucose levels, was without effect (45). These results indicate that β-cell toxicity is mediated primarily by glucose but can be exacerbated by increased FFA or high fat diet. However, elevated FFA in the presence of low glucose (such as might occur during starvation) would not result in β-cell toxicity, presumably because FA oxidation remains active (46). In humans, hyperlipidemia, specifically hypertriglyceridemia and low HDL, is commonly associated with T2DM. Thus, the extent of the contribution of FFA to β-cell dysfunction and failure in the common form of T2DM in humans is not completely defined.

 

 

 

Genetic Contributions

It has long been appreciated that not all individuals with obesity and insulin resistance develop diabetes and that a positive family history of the disease indicates a genetic predisposition. A role for β-cell genetic defects in the etiology of diabetes is best demonstrated by the MODY (maturity-onset diabetes of the young) syndromes (47) (see Chapter 10). Although the most common of these is caused by a mutation in the gene encoding glucokinase (MODY-2), the major regulator of glucose metabolic rate in the β-cell, a number of the other forms are associated with mutation of transcription factors. One such transcription factor is IPF1/PDX-1 (pancreatic duodenal homeobox-1) which is a key regulator of insulin gene expression (48), and associated with MODY4 (49). In an animal model of type 2 diabetes, the gerbil or sand rat, Psammomys obesus, diabetes develops rapidly when the animal is switched from its native low energy diet to standard rat show (50,51). The time course of the phases of adaptation outlined above is greatly contracted in this model, and recent studies indicate that an absence of PDX-1 is responsible (52). When exposed to acute hyperglycemia, P. obesus islets do not increase insulin gene expression and insulin depletion follows fairly rapidly (50-52). In addition, these defects are reversed in isolated islets by PDX-1 gene transfer (52). The relevance of this exaggerated model, namely, total lack of PDX-1 and marked sensitivity to hyperglycemia-induced β-cell failure, is illustrated by the diminished activity of PDX-1 observed in other rodent models, e.g. exposure to FFAs (53), partial pancreatectomy (54), and in vitro in cultured insulin-secreting HIT-T15 cells exposed to chronic high glucose (55).

 

In human subjects, the best illustration of the influence of genetic predisposition to pancreatic β-cell decompensation has been documented in the population of Oji-Cree natives in Northern Ontario, Canada (56). In this genetically relatively homogeneous population, T2DM has increased over the past 2 generations from < 5% to almost 50%. This has been associated with obesity, high fat diet and sedentary lifestyle (57). A search for inherited traits revealed that a proportion of the population were heterozygous or homozgous for a mutation in the HNF-1a gene, the same gene associated with MODY-3 in other populations (47). The effect of harbouring the mutation was not to cause, but rather to accelerate the onset of diabetes. Thus heterozygotes developed T2DM about 7 years earlier than those without a mutation, while homozygotes did so ~ 14 years earlier (Fig.3) (56). Similar to PDX-1, HNFs are downregulated by hyperglycemia in the partial pancreatectomized rat (Table 2). These data support the concept that the sensitivity of the β-cell to decompensation in the presence of chronic mild hyperglycemia, for example in a state of impaired glucose tolerance (IGT), is influenced by genetic and likely, environmental factors. Unraveling these will ultimately lead to new therapeutic targets to prevent β-cell decompensation.

 

 

 

 

 

Time course of β- cell glucose toxicity:

As outlined above there are few formal studies of the time course of hyperglycemia-induced β-cell toxicity. In vivo models differ, with progression to β-cell failure in the partial pancreatectomy model occurring over weeks to months, in the ZDF rat over 1 –2 months (58) and in P. obesus over days. In vitro studies of cultured HIT-T15 cells (a hamster insulin-secreting cell line) revealed a continuous, slow and progressive diminution of insulin secretion and insulin content over months upon exposure to high glucose. This deterioration was reversible until passage 92 but became irreversible at passage 99 (59). (Passaging of cells refers to the subculturing of a confluent dish or flask of cells into equal aliquots, e.g. 1:4, and then feeding these subcultures to allow growth and proliferation until each again reaches confluence. This can generally vary from 3 – 10 days). The precise signal which determines irreversible damage is not clear but likely involves failure of β-cell replication and/or the induction β-cell death. In humans, the UKPDS (United Kingdom Prospective Diabetes Study), which followed subjects with T2DM over 12-14 years clearly demonstrated deterioration of β-cell function over time, and apparently, this was similar in the intensively managed and conventionally treated groups (60). The fact that the HbA1C was lower by 0.12% in the intensively treated group could be interpreted to mean that glucose toxicity did not contribute to this progressive β-cell failure. However, this difference in HbA1C hides the fact that the absolute HbA1C values in both groups were still significantly elevated. The median HbA1C rose above 7% after approximately 3 years follow-up in the conventional group and after 5 years in the intensively treated group. Furthermore, these values continued to increase over time. As discussed above, the data in rodents and humans indicate that the β-cell is extremely sensitive to very small, sustained increases in circulating glucose concentrations. Thus, one would speculate that the dose-response of β-cell glucose toxicity is shifted to the left (i.e. more sensitive), when compared to that demonstrated for the microvascular complications (see the Diabetes Control and Complications Trial) (5).

 

 

 

PERIPHERAL INSULIN TARGET TISSUES

Insulin resistance is a well described characteristic of individuals with T2DM and is present prior to the onset of overt hyperglycemia (1,2 and see Chapter 9). However, it was reported in 1977 that induction of diabetes in the dog model with alloxan, a β-cell toxin, resulted in insulin resistance (61). Over the next decade it became apparent that insulin resistance was not only a precursor, but also a consequence of diabetes (62, 63). A significant observation was that correction of hyperglycemia with phloridzin (an inhibitor of renal tubular glucose reabsorption) which does not alter insulin levels, normalized insulin action in 90% pancreatectomized rats (Fig. 4) (7). In addition, human subjects with poorly controlled T1DM were also found to be insulin resistant, and institution of intensive insulin therapy to normalize glucose concentrations restored insulin sensitivity (64). Together, these data demonstrated that peripheral insulin target tissues in vivo could be desensitized to insulin by high glucose. These studies utilized the hyperinsulinemic-euglycemic clamp technique, a highly sensitive measure of in vivo insulin-stimulated muscle glucose uptake (65). This idea has been supported by other in vivo reports and in vitro studies in muscle tissue and adipocytes. Significantly, incubation of skeletal muscle tissue (66-68) and adipocytes (8) in the presence of increasing medium glucose concentrations leads to insulin resistance of glucose uptake. This has been associated with a decrease in both the total number of glucose transporters and the ability of insulin to stimulate their translocation (68,69). Since the ability of glucose to enhance its own uptake independent of insulin, i.e. by mass action, has also been found to be downregulated, and the fact that this phenomenon has been observed in non-insulin target tissues such as brain (70,71), implies that basal glucose transporters such as GLUT1, as well as GLUT4 (the insulin-sensitive glucose transporter) are downregulated. Recent data support these concepts.

 

 

 

 

 

It is important to note that high glucose-induced insulin resistance requires increased glucose uptake and metabolism, and therefore, in the peripheral insulin target tissues muscle and fat, is accentuated by increasing amounts of insulin (72, 81). In the in vivo studies of pancreatectomized rats, liver insulin responsiveness was unaffected (7). Under the clamp conditions employed hepatic glucose production was nearly completely suppressed by insulin. Thus, whether the hepatic insulin resistance found in T2DM can be exacerbated by hyperglycemia was not adequately addressed. In more recent studies, evidence has been provided that insulin signaling is impaired by hyperglycemia in the liver (73). So, although clearly not all hepatic metabolic abnormalities observed in T2DM are caused by high glucose, there is likely a substantial contribution of hyperglycemia to insulin resistance, as observed in muscle and adipose tissue.

 

 

CELLULAR AND MOLECULAR MECHANISMS OF GLUCOSE TOXICITY

A number of cellular abnormalities resulting from exposure to high glucose have been observed in both pancreatic β-cells and peripheral insulin target tissues: These include: 1) enhanced oxidative stress, 2) increased flux of glucose through the hexosamine biosynthesis pathway (HBP), 3) activation of ser/thr kinases such as PKC (protein kinase C) and 4) altered gene expression (Table 3). It is noteworthy that many of these same signaling pathways and/or pathophysiological changes have been documented to occur in non-insulin target tissues which are subject to the long term complications of diabetes, namely vascular endothelial cells, and cells of the kidney, retina and peripheral nerves (74-76). Thus, the data suggest that there is overlap among the mechanisms of “glucose toxicity” of β-cells, insulin metabolic target tissues, and that of other organs and tissues which manifest the chronic complications of diabetes. In addition, these mechanisms have been reported in tissues subject to pathological responses to high glucose such as the developing embryo (77,78).

 

 

 

 

 

Pancreatic β-cells:

In discussing the β-cell, it should be noted that there are differences among authors in the definition of “glucose toxicity”. In most reports, although it is recognized that changes in insulin secretory responses, pancreatic insulin content, gene expression and irreversible β-cell injury occur in a continuum reflective of the duration of high glucose exposure, all of these processes are encompassed by the term “glucotoxicity”. However, Poitout and Robertson (79) have emphasized that early β-cell secretory refractoriness after short term exposure to high glucose may be viewed as a physiological adaptation and should be termed “glucose desensitization”. Medium term exposure results in “β-cell exhaustion” in which releasable insulin is depleted. The term “glucose toxicity” was reserved for progressive and irreversible β-cell failure resulting from long term exposure. In this chapter the term is used to describe the widespread effects of exposure to high glucose, recognizing the varied mechanisms, tissue responses and time courses of these effects.

 

As alluded to above, one mechanism for which substantial evidence has been provided is “oxidative stress”. Oxidative stress refers to the presence of an excess amount of reactive oxygen intermediates (ROIs), due to an imbalance between their formation and degradation. The presence of oxidative stress caused by high glucose is well documented in many tissues subject to complications, e.g. endothelial cells (80), nerve cells (81), proximal renal epthelial cells (82), as well as β-cells (83). Recent studies support the important role of oxidative stress in β-cell failure. First, treatment of ZDF (Zucker Diabetic Fatty) rats with the antioxidant, NAC (N-acetylcysteine) between 6 and 12 weeks of age, prevented β-cell failure and ameliorated hyperglycemia (84). In vitro, similar treatment of HIT-T15 cells or isolated islets exposed to high glucose for several months preserved insulin gene expression and glucose stimulated insulin secretion (84,85). These data were reproduced in another rodent model of type 2 diabetes, the db/db mouse (86). Other in vitro studies demonstrated that while inhibition of GSH (glutathione) synthesis in islets augmented ribose-induced oxidative stress and decreased insulin mRNA and secretion, overexpression of the antioxidant enzyme, glutathione peroxidase, using adenovirus-mediated gene transfer, protected human islets from ribose toxicity (87). Notably, part of the β-cell response to hyperglycemia is an increase in gene expression of various antioxidant enzymes, namely heme oxygenase-1, glutathione peroxidase, Cu/Zn SOD (superoxide dismutase) and Mn-SOD (Table 2) (22). This observation in the rat partial pancreatectomy model suggests that a compensatory protective response to the increased oxidative stress may occur and contribute to the prolonged time course observed for “glucose toxicity” to become irreversible (59).

 

There have been few studies investigating of the role of increased flux via the HBP in the induction of β-cell glucose toxicity. Overexpression of GFA (glutamine fructose-6-phosphate amidotransferase), the rate-limiting enzyme of this pathway (Fig.5) in a β-cell specific manner in mice resulted in hyperinsulinemia, insulin resistance and (in male mice) the eventual development of mild type 2 diabetes (88). Furthermore, GFA overexpression in rat islets using adenovirus, or exposure of islets to glucosamine which bypasses GFA and greatly augments flux through the HBP, impaired glucose-stimulated insulin secretion and reduced β-cell gene expression (e.g. insulin, GLUT 2) (89). Interestingly, it was found that these maneuvers increased oxidative stress and that the antioxidant, NAC, was able to block these effects (89). Thus, although enhanced protein O-GlcNAcylation (the covalent and reversible modification of proteins on Ser and/or Thr residues by addition of single moiety of N-Acetylglucosamine has been suggested to mediate the effects of increased HBP flux (90, 91), its exact role remains to be defined.

 

 

 

 

 

In the pathogenesis of diabetes complications, the activation of the b isoform of PKC appears to be particularly important (92), and currently, clinical trials in humans of a PKC-b inhibitor are underway (93). It has now been documented that the PKC-b isoform is also activated by high glucose in β-cells, and that expression of a PKC b2-isoform specific dominant-negative mutant in islets blocked the induction of c-myc (94). The transcription factor c-myc is known to be upregulated by high glucose in various diabetic models and appears to mediate a number of the changes in β-cell gene expression seen in high glucose (22, 79, 95). A recent study demonstrated that enhanced HBP flux activates PKC-b in glomerular mesangial cells (96). While this has not yet been demonstrated in β-cells, these data suggest a close interaction amongst the 3 mechanisms discussed above, namely oxidative stress, flux through the HBP and activation of PKC.

 

 

Peripheral Insulin Target Tissues

Similar to the β-cell, in vitro and in vivo studies of muscle and adipose tissue have implicated oxidative stress, the HBP, and PKC. Incubation of rat adipocytes in the presence of high glucose and insulin induced insulin resistance which could be blocked by GFA inhibitors (97). Furthermore, exposure to glucosamine in the presence of low concentrations of insulin reproduced the insulin resistance (97). There has been some controversy about the mechanism and relevance of the glucosamine effect (98), however more recent data demonstrate that insulin resistance can be induced by PUGNAc [O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino-N-phenylcarbamate], an inhibitor of N-acetylglucosaminidase, which blocks deglycosylation and thus turnover of the O-GlcNAc modified proteins (99). In vivo, overexpression of GFA in transgenic mice causes insulin resistance (100). Infusion of glucosamine into rats increases flux through the HBP, induces insulin resistance and has been proposed to increase modification of IRS-1 by O-GlcNAc (101,102). As described in detail in Chapter 2, insulin signals its metabolic effects by stimulating autophosphorylation of its cell surface receptor and its subsequent activation as a tyrosine protein kinase. The IRK (insulin receptor kinase) phosphorylates IRS (insulin receptor substrate) proteins 1 and 2 on tyrosine residues which results in the recruitment of signaling proteins with SH2 (Src homology 2) domains. One of the most important of these is the lipid kinase, phosphatidylinositol-3-kinase (PI3K), which phosphorylates the 3 position of phosphatidylinositols such as PI(4,5) P2 to generate PI (3,4,5) P3. The latter phospholipid recruits other proteins to membranes via their PH (pleckstrin homology) domains. Ultimately serine kinases such as akt/PKB (protein kinase B) and PKC-z are activated. This pathway, responsible for glucose transport and glycogen synthesis, may be inhibited at multiple steps in various states of insulin resistance. The effects of hyperglycemia have been reported to involve IRS-1 and/or akt/PKB (67,69,102-105). It is not clear whether O-GlcNAc on IRS-1 is a direct cause of either its decreased tyrosine phosphorylation or decreased binding of the p85 regulatory subunit of PI3-kinase.

 

There are several studies in cultured cells as well as in muscle and adipose tissue implicating activation of the Ser kinase, PKC, by high glucose as a contributor to insulin resistance (68,103, 106-108). The resistance appears to be mediated at the level of the insulin receptor (106,107,109) and/or IRS-1 (110). Ser phosphylation of IRS-1 has been demonstrated to impair its tyrosine phosphorylation and, in some cases, convert IRS-1 from an effective substrate to an inhibitor of the IRK (111). Cotransfection of various conventional PKC (cPKC) and novel PKC (nPKC) iosforms with the insulin receptor and IRS-1 demonstrated that most isoforms, particularly the novel PKC-d and -e, inhibit the IRK only in the presence of IRS-1 (112).

 

The activation of the cPKCs and nPKCs is dependent on DAG (diacylglycerol) (113). It has been found that DAG levels are elevated in muscle tissue in insulin-resistant hyperglycemic rodent models (114). It has been proposed that excess glucose flux to glyceraldehyde-3-phosphate is shunted towards DHAP (dihydroxyacetone phosphate) and DAG synthesis. At the same time, it has been found that high glucose also increases malonyl-CoA, thus inhibiting CPT-1 (carnitine palmitoyl transferase-1) and fatty acid oxidation. The resultant increase in LCFA (long chain fatty acyl)-CoA will also promote the generation of DAG. Exposure of rat adipocytes to high glucose (30 mM) has been associated with an impairment of IRK activity which was blocked by PKC inhibitors (107).

While IRS-1 is a key signaling intermediate subject to multiple forms of regulation, e.g. phosphorylation, O-glycosylation and enhanced degradation (99,102,109-112, 115), it should be noted that in some studies exposure of target tissues to high glucose has been found to be associated with insulin resistance at a step distal to IRS-1 and PI3K, namely akt/PKB (67,73,105,115). The mechanism of inhibiton of akt/PKB in the context of normal PI3 kinase activation is not clear (68), but in the case of exposure to the fatty acid palmitate, an increase in akt/PKB dephosphorylation has been reported (116). This has been attributed to increased ceramide synthesis in response to excess palmitate (117). Exposure to high glucose alone has not been associated with elevated ceramide (68). However, a recent report suggests that in human subjects with diabetes, elevated ceramide concentrations may exist in peripheral insulin target tissues (118). Further work is necessary to sort out the varied signaling defects contributing to the insulin resistance induced by hyperglycemia.

 

Several studies suggest that oxidative stress may play a role in the pathogenesis of insulin resistance (119-122). First, exposure of cells directly to H2O2 causes insulin resistance (121,122). Second, exposure to high glucose has been associated with oxidative stress in many tissues and cell types (123,124) and recently, in adipocytes (125). Third, antioxidant treatment of rats exposed to short term hyperglycemia was able to prevent insulin resistance (126). Finally, several small studies in human subjects with T2DM showed improvement in insulin resistance with oral vitamin E (127) or intravenous vitamin C treatment (128). ROIs have been associated with activation of multiple signaling pathways (129-133), including activation of PKCs (132), and altered gene expression (133). Thus, it is plausible that oxidative stress contributes to “glucose toxicity” in insulin target tissues but its precise role remains to be defined.

 

 

 

Microvascular and other complications:

It has been well documented that the chronic microvascular complications of diabetes along with fetal malformations are caused by hyperglycemia. The clinical and pathological features of diabetic retinopathy, nephropathy and neuropathy are described in chapters 33, 34 and 35, respectively. The current views of the molecular mechanisms of these complications share similarities with those of “glucose toxicity” discussed above. Thus the major contributors appear to be: 1) increased flux of glucose through the aldose reductase (polyol) pathway, 2) the DAG-PKC activation pathway, 3) the AGE (advanced glycation endproduct) formation pathway and 4) the hexosamine biosynthesis pathway (reviewed in 76). In addition, the importance of high glucose-induced oxidative stress has been emphasized. Various sources of increased ROS include, 1) glucose autoxidation in the presence of metal ions (134), 2) a byproduct of formation of AGEs via 2-deoxyglucosone and/or methylglyoxal (135), 3) increased glucose oxidation in mitochondria (136), and 4) depletion of NADPH via the polyol pathway (137) and by inhibition of glucose-6 phosphate dehydroxygenase (G6PDH) by HBP flux, which increases the concentration of the G6PDH inhibitor, glucosamine - 6- P (138). Decreased NADPH limits the regeneration of the intracellular antioxidant reduced glutathione (GSH) by glutathione (GSSG) reductase (139). In some subjects additional susceptibility to oxidative stress may result from a genetically determined decrease in endogenous antioxidant enzymes, e.g. catalase, SOD (superoxide dismutase) (140).

 

Recently, the role of oxidative stress has been proposed to be primary. Thus increased mitochondrial glucose metabolism leads to increased ROS which inhibit glyceraldehyde phosphate dehydrogenase (GAPDH) blocking the glycolytic pathway (136). The increase in upstream metabolites redirects metabolic flux through the pathways mentioned above (Fig.6), which in turn lead to alterations in cell signaling and gene expression associated with complications. Evidence supporting this concept includes the finding that normalization of increased flux and reversal of several outcomes is achieved by inhibition of mitochondrial ROS generation (78,91,136), as well as by shunting of metabolites to the pentose phosphate pathway by treatment with benfotiamine (lipid soluble thiamine) (141) (Fig.6). Furthermore, antioxidant treatment has been demonstrated to have salutory effects on high glucose induced β-cell dysfunction (84-87), insulin resistance (126-128), neuropathy (142,143), retinopathy (143,144), and congenital malformations (145,146). Whether these novel observations will lead to a better treatment and improved outcomes remains to be determined

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CLINICAL IMPLICATIONS

The clinical implications of “glucose toxicity” are clear. Since hyperglycemia leads to insulin resistance, and in subjects with pre-existing insulin resistance, is an exacerbating factor, and because high glucose also leads to impaired insulin secretion and contributes to β-cell failure, it is evident that meticulous control of glycemia should be an early goal of therapy in diabetes. Whether better glucose control is able to preserve endogenous insulin secretion in type 1 or type 2 diabetes in humans is not proved. However, insulin resistance, which presents a stress on the β-cell, has been associated with more rapid deterioration of β-cell function in type 1 diabetes (147). On the other hand, in the UKPDS study, apparent β-cell function deteriorated at similar rates in conventional and more intensively controlled groups in type 2 diabetes. Since the β-cell is sensitive to even small elevations in circulating glucose concentrations, it is possible that the glycemic control achieved in the intensively treated group in the UKPDS was not adequate to prevent glucose toxicity.

 

In the case of microvascular complications, it has been well documented that intensive therapy to lower glucose in both types of diabetes and importantly, by any manoeuver in T2DM, i.e. diet/exercise/oral hypoglycemic agents/insulin, will lead to improved outcomes. In summary, glucose toxicity may be viewed as a general pathophysiological process in diabetes, encompassing adverse effects on multiple tissues, with at least overlapping, if not identical molecular mechanisms. Current and future treatment strategies (Table 4) are based on these concepts.

 

 

 

 

 

 

 

1. DeFronzo RA. The triumvirate: (-cell muscle, liver: a collusion responsible for NIDDM. Diabetes 37:677-687, 1988.

2. Rossetti L, Giaccari A, DeFronzo RA. Glucose toxicity. Diabetes Care 13: 610-630, 1990.

3. Haist RE, Campbell J. Best CH: The prevention of diabetes. N. Engl. J. Med 223: 607-615, 1940.

4. Nathan DM, Long-term complications of diabetes mellitus. N. Engl. J. Med. 328:1676-1685, 1993.

5. The Diabetes Control and Complications Trial Research Group. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. N. Engl. J. Med. 329, 977-986, 1993.

6. UK Prospective Diabetes Study (UKPDS) Group. Intensive blood-glucose control with

sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS 33). Lancet 352, 837-853, 1998.

7. Rossetti L, Smith D, Shulman GI, Papachristou D, DeFronzo RA. Correction of hyperglycemia with phlorizin normalizes tissue sensitivity to insulin in diabetic rats. J. Clin. Invest. 79: 1510-1515, 1987,

8. Garvey WT, Olefsky JM, Matthaei S, Marshall S: Glucose and insulin co-regulate the glucose transport system in primary cultured adipocytes. J. Biol. Chem 262: 189-197, 1987.

9. Rayfield EJ, Ault MJ, Keusch GT, Brothers MJ, Nechemias C, Smith H: Infection and diabetes: the case for glucose control. Am J. Med 72: 438-450, 1982.

10. Boulot P, Chabbert-Buffet N, d'Ercole C, Floriot M, Fontaine P, Fournier A, Gillet JY, Grandperret-Vauthier S, Geudj AM, Guionnet B, Hauguel-de-Mouzon S, Hieronimus Hoffet M, Jullien D, Lamotte MF, Lejeune V, Lepercq J, Lorenzi F, Mares P, Miton A, Penfornis A, Pfister B, Renard E, Rodier M, Roth P, Sery GA, Timsit J, Valkat S, Van A, Verier-Mine O; Diabetes and Pregnancy Group. French milticentric survey of outcome of pregnancy in women with pregestational diabetes. Diabetes Care 26: 2990-1993, 2003.

11. Savage PJ, Bennion LO, Flock EV, Nagulesparan M, Mott D, Roth J, Unger RH, Bennett PH: Diet-induced improvement of abnormalities in insulin and glucagon secretion and in insulin receptor binding in diabetes mellitus. J. Clin. Endocrinol. Metab. 48: 999-1007, 1979.

12. Kolterman OG, Gray RS, Shapiro G, Scarlett JA, Griffin J, Olefsky JM. The acute and chronic effects of sulfonylurea therapy in type II diabetes. Diabetes 33: 346-354, 1984.

13. Garvey WT, Olefsky JM, Griffin J, Hamman RF, Kolterman OG: The effect of insulin

treatment on insulin secretion and insulin action in type II diabetes mellitus. Diabetes 34: 222-234, 1985.

14. Weir GC, Leahy JL, Bonner-Weir S: experimental reduction of beta-cell mass: implications

for the pathogenesis of diabetes. Diabetes Metab. Rev. 2: 125-161, 1986.

15. Weir GC, Clore ET, Zmachinski CJ, Bonner-Weir S: Islet secretion in a new experimental

model for non-insulin-dependent diabetes. Diabetes 30: 590-595, 1981.

16. Rossetti L, Shulman GI, Zawalich W, DeFronzo RA: Effect of chronic hyperglycemia on in

vivo insulin secretion in partially pancreatectomized rats. J. Clin. Invest. 80: 1037-1044, 1987.

17. Leahy JL, Bonner-Weir S, Weir GC: Abnormal insulin secretion in a streptozocin model of

diabetes: effects of insulin treatment. Diabetes 34: 660-666, 1985.

18. Kergoat M, Bailbe D, Portha B: Insulin treatment improves glucose-induced insulin release in rats with NIDDM induced by streptozocin. Diabetes 36: 971-977, 1987.

19. Leahy JL, Bonner-Weir S, Weir GC: Minimal chronic hyperglycemia is a critical determinant of impaired insulin secretion after an incomplete pancreatectomy. J. Clin. Invest. 81: 1407-1414, 1988.

20. Leahy JL, Cooper HE, Weir GC: Impaired insulin secretion associated with near

normoglycemia: study in normal rats with 96-h in vivo glucose infusions. Diabetes 36: 459-464, 1987.

21. Imamura T, Koffler M, Helderman JH, Prince D, Thirlby R, Inman L, Unger RH: Severe

diabetes induced in subtotally depancreatized dogs by sustained hyperglycemia. Diabetes 37: 600-609, 1988.

22. Weir GC, Laybutt DR, Kaneto H, Bonner-Weir S, Sharma A. (-cell adaptation and decompensation during the progression of diabetes. Diabetes 50 (Suppl.1) S154-S159, 2001.

23. Colella RM, May JM, Bonner-Weir S, Leahy JL, Weir GC: Glucose utilization in islets of

hyperglycemic rat models with impaired glucose-induced insulin secretion. Metabolism 36: 335-337, 1987.

24. Chan CB, MacDonald PE, Saleh MC, Johns DC, Marban E Wheeler MB. Over-expression of uncoupling protein-2 inhibits glucose-stimulated insulin secsretion from rat islets. Diabetes 48: 1482-1986, 1999.

25. Zhang CY, Baffy G, Perret P, Krauss S, Peroni O, Grujic D, Hagen T, Vidal-Puig AJ, Boss O, Kim YB, Zheng XX, Wheeler MB, Shulman GI, Chan CB, Lowell BB. Uncoupling protein-2 negatively regulates insulin secretion and is a major link between obesity, beta cell dysfunciton, and type 2 diabetes. Cell 105: 745-755, 2001

26. Zhao, C, Rutter GA. Overexpression of lactate dehydrogenase A attenuates glucose-induced insulin secretion in stable MIN-6 beta-cell lines. FEBS. Lett. 4300:213-216, 1998.

27. Jonas J-C, Sharma A, Hasenkamp W, Ilkova H, Patane G, Laybutt R, Bonner-Weir S, Weir G. Chronic hyperglycemia triggers loss of pancreatic B-cell differentiation in an animal model of diabetes. J. Biol. Chem. 274: 14112-14121, 1999.

28. Ostenson C-G, Khan A, Abdel-Halim SM, Guenifi A, Suzuki K, Goto Y, Efendic S. Abnormal insulin secretion and glucose metabolism in pancreatic islets from the spontaneously diabetic GK rat. Diabetologia 36: 3-8, 1993,

29. Ling Z-C, Hong-Lie C, Ostenson C-G, Efendic S, Khan A. Hyperglycemia contributes to impaired insulin response in GK rat islets. Diabetes 50 (Suppl.1) S108-S112, 2001.

30. Zawalich WS, Zawalich KC, Shulman GI, Rossetti L: Chronic in vivo hyperglycemia impairs phosphoinositide hydrolysis and insulin release in isolated perfused rat islets. Endocrinology 126: 253-260, 1990.

31. Metz SA, Meredith M, Vadakekalam J, Rabaglia ME, Kowluru A. A defect late in stimulus-secretion coupling impairs insulin secretion in (Kakizaki diabetic rats). Diabetes 48: 1754-1762, 1999.

32. Robertson RP, Type II diabetes, glucose "non-sense" and islet desensitization. Diabetes 38:

1501-1505, 1989.

33. Robertson RP, Eicosanoids asvpluripotential modulators of pancreatic islet function.

Diabetes 37: 367-379, 1988.

34. Laybutt DR, Sharma A, Sgroi DC, Gaudet J, Bonner-Weir S, Weir GC. Genetic regulation of metabolic pathways in (-cells disrupted by hyperglycemia. J. Biol. Chem. 277:10912-10921.

35. Hoppener JW, Nieuwenhuis MG, Vroom TM, Ahren B, Lips CJ. Role of islet amyloid in type 2 diabetes mellitus: consequence or cause? Mol. Cell. Endocrinol. 197: 205-212, 2002.

36. McGarrry JD, Dobbins RL. Fatty acids, lipotoxicity and insulin secretion. Diabetologia 42: 128-138, 1999.

37. Prentki M, Corkey BE. Are the (-cell signaling molecules malonyl-CoA and cytosolic long-chain acyl-CoA implicated in multiple tissue defects of obesity and NIDDM? Diabetes 45: 273-283, 1996.

38. Prentki M, Roduit R, Lameloise N, Corkey BE, Assimacopoulos-Jeannet F. Glucotoxicity, lipotoxicity and pancreatic B-cell failure: a role for malonyl-CoA, PPAR( and altered lipid partitioning. Can. J. Diabetes Care 25:36-46, 2001.

39. Prentki M, Vischer S, Glennon MC, Regazzi R, Deeney JT, Corkey BE: Malonyl-CoA and long chain acyl-CoA esters as metabolic coupling factors in nutrient-induced insulin secretion. J. Biol. Chem. 267: 5802-5810, 1992.

40. Sako Y, Grill VE. A 48-hour lipid infusion in the rat time-dependently inhibits glucose-induced insulin secretion and (-cell oxidation through a process likely coupled to fatty acid oxidation. Endocrinology 127: 1580-1589, 1990.

41. Bollheimer LC, Skelly RH, Chester MW, McGarry JD, Rhodes CJ. Chronic exposure to free fatty acid reduces pancreatic (-cell insulin content by increasing basal insulin secretion that is not compensated for by a corresponding increase in proinsulin biosynthesis and translation. J. Clin. Invest. 101: 1094-1101, 1998.

42. Maedler K, Spinas GA, Dyntar D, Moritz W, Kaiser N, Donath MY. Distinct effects of saturated and monounsaturated fatty acids on beta-cell turnover and function. Diabetes 50: 69-76, 2001.

43. Shimabukuro M, Higi M, Zhou YT, Wang MY, Newgard CB, Unger RH. Lipoapoptosis in (-cells of obese prediabetic fa/fa rats. Role of serine palmitoyltransferance overexpression. J. Biol. Chem. 273: 32487-32490.

44. Wang M-Y, Koyama K, Shimabukuro M, Newgard CB, Unger RH. Overexpression of leptin receptors in pancreatic islets of Zucker diabetic fatty rats restores GLUT-2, glucokinase and glucose-stimulated insulin secretion. Proc. Natl. Acad. Sci. USA 95:11921-11926, 1998.

45. Harmon JS, Gleason CE, Tanaka Y, Poitout V, Robertson RP. Antecedent hyperglycemia, not hyperlipidemia, is associated with increased islet triacylglycerol content and decreased insulin gene mRNA level in Zucker diabetic fatty rats. Diabetes 50: 2481-2486, 2001.

46. Dobbins RL, Chester MW, Daniels MB, McGarry JD, Stein DT. Circulating fatty acids are essential for efficient glucose-stimulated insulin secretion after prolonged fasting in humans. Diabetes 47: 1613-1618, 1998.

47. Fajans SS, Bell GI, Polonsky KS. Molecular mechanisms and clinical pathophysiology of maturity-onset diabetes of the young. N. Engl. J. Med. 345:971-980, 2001.

48. Ahlgren U, Jonsson J, Jonsson L, Simu K, Edlund H. Beta-cell-specific inactivation of the mouse Ipf1/Pdx1 gene results in loss of the beta-cell phenotype and maturity onset diabetes. Genes Dev . 15: 1763-1768, 1998.

49. Stoffers DA, Ferrer J, Clarke WL, Habener JF. Early-onset type II diabetes mellitus (MODY4) linked to IPF1. Nat. Genet 17: 138-13l9, 1997.

50. Gadot M, Leibowitz G, Shafrir E, Cerasi E, Gross D, Kaiser N. Hyperproinsulinemia and insulin deficiency in the diabetic Psammomys obesus. Endocrinology 135: 610-616, 1994.

51. Nesher R, Gross DJ, Donath MY, Cerasi E, Kaiser N. Interaction between genetic and dietary factors determines (-cell function in Psammomys obesus, an animal model of type 2 diabetes. Diabetes 48: 731-737, 1999.

52. Leibowitz G. Ferber S, Apelqvist A, Edlund H, Gross DJ, Cerasi E, Melloul D, Kaiser N. IPF1/PDX1 deficiency and (-Cell dysfunction in Psammomys obesus, an animal with type 2 diabetes. Diabetes 50:17989-1806, 2001.

53. Gremlich S, Bonny C, Waeber G, Thorens B. Fatty acids decrease IDX-1 expression in rat pancreatic islets and reduce GLUT2, glucokinase, insulin, and somatostatin levels. J. Biol. Chem. 272: 30262-30269, 1997.

54. Zangen DH, Bonner-Weir S, Lee CH, Latimer JB, Miller CP, Habener JF, Weir GC. Reduced insulin, GLUT2 and IDX-1 in beta cells after partial pancreatectomy. Diabetes 46: 258-264, 1997.

55. Sharma A, Olson LK, Robertson RP, Stein R. The reduction of insulin gene transcription in HIT-T15 B cells chronically exposed to high glucose concentration is associated with the loss of RIPE3b1 and STF-1 transcription factor expression. Mol. Endocrinol. 9:1127-1134, 1995.

56. Triggs-Raine BL, Kirkpatrick RD, Kelly SL, Norquay LD, Cattini PA, Yamagata K, Hanley AJG, Zinman B, Harris SB, Barrett PH, Hegele RA, HNF1( G319S, a transactivation-deficient mutant, is associated with altered dynamics of diabetes onset in an Oji-Cree community. Proc. Natl. Acad. Sci. USA 99: 4614-4619, 2002.

57. Harris SB, Gittelsohn J, Hanley A, Barnie A, Wolever TM, Gao J, Logan A, Zinman B. The prevalence of NIDDM and associated risk factors in native Canadians. Diabetes Care 20: 185-187, 1997.

58. Harmon JS, Gleason CE, Tanaka Y, Oseid EA, Hunter-Berger KK, Robertson RP. In vivo prevention of hyperglycemia also prevents glucotoxic effects on PDX-1 and insulin gene expression. Diabetes 48: 1995-2000, 1999.

59. Gleason CE, Gonzalez M, Harmon JS, Robertson RP. Determinants of glucose toxicity and its reversibility in the pancreatic islet (-cell line, HIT-T15. Am. J. Physiol. Endocrinol. Metab. 279: E997-E1002, 2000.

60. Turner RC. The UK Prospective Diabetes Study. A review. Diabetes Care. 21: Suppl. 3. C35-C38, 1998.

61. Reaven GM, Sageman WS, Swenson RS. Development of insulin resistance in normal dogs following alloxan-induced insulin deficiency. Diabetologia 13: 459-462, 1977.

62. Dall'Aglio E. Chang H, Hollenbeck CB, Mondon CE, Sims C, Reaven GM. In vivo and in vitro resistance to maximal insulin stimulated glucose disposal in insulin deficiency. Am. J. Physiol. 249: E312-E316, 1985.

63. Unger RH, Grundy S. Hyperglycemia as an inducer as well as a consequence of impaired islet cell function and insulin resistance: implications for the management of diabetes. Diabetologia 28: 119-121, 1985.

64. Yki-Jarvinen H, Koivisto VA. Continuous subcutaneous insulin infusion therapy decreases insulin resistance in type 1 diabetes. J. Clin. Endocrinol. Metab. 58: 659-666, 1984.

65. DeFronzo RA, Tobin JD, Andres R. Glucose clamp technique: a method of quantifying insulin secretion and resistance. Am. J. Physiol. 232: E214-E223, 1979.

66. Richter EA, Hansen BF, Hansen SA. Glucose-induced insulin resistance of skeletal-muscle glucose transport and uptake. Biochem. J. 252:733-737, 1988.

67. Kurowski TG, Lin Y, Luo Z, Tsichlis PN, Buse MG, Heydrick SJ, Ruderman NB. Hyperglycemia inhibits insulin activation of Akt/protein kinase B, but not phosphatidylinositol 3-kinase in rat skeletal muscle. Diabetes 48: 658-663.

68. Tomas E, Lin Y-S, Dagher Z, Saha A, Luo Z, Ido Y, Ruderman NB. Hyperglycemia and insulin resistance: Possible mechanisms. Ann. N.Y. Acad. Sci. 967: 43-51, 2002.

69. Shepherd PR, Kahn BB. Glucose transporters and insulin action. N. Engl. J. Med. 341: 248-257, 1999.

70. Matthaei S, Horuk R, Olefsky JM: Blood-brain glucose transfer in diabetes mellitus decreased number of glucose transporters at blood-brain barrier. Diabetes 35:1181-1184, 1986.

71. McCall AL, Fixman LB, Fleming N, Tornheim K, Chick W, Ruderman NB: Chronic hypoglycemia increases brain glucose transport. Am J. Physiol. 251: E442-E447, 1986.

72. Traxinger RR, Marshall S: Recovery of maximal insulin responsiveness and insulin sensitivity after induction of insulin resistance in primary cultured adipocytes. J. Biol. Chem. 264:8156-8163, 1989.

73. Nawano M, Ueta K, Oku A, Arakawa K, Saito A, Funaki M, Anai M, Kikuchi M, Oka Y, Asano T. Hyperglycemia impairs the insulin signaling step between PI 3-kinase and Akt/PKB activation in ZDF rat liver. Biochem. Biophys. Res. Commun. 9:252-256, 1999.

74. Koya D, King GL. Protein kinase C activation and the development of diabetic complications. Diabetes 47: 859-866, 1998.

75. Giugliano D, Ceriello A, Paolisso G. Oxidative stress and diabetic vascular complications. Diabetes Care 19: 257-267, 1996.

76. Brownlee M. Biochemistry and molecular cell biology of diabetic complications. Nature 414: 813-820, 2001.

77. Fine E, Horal M, Chang T, Fortin G. Loeken M. Hyperglycemia is responsible for altered gene expression, apoptosis, and neural tube defects associated with diabetic pregnancy. Diabetes 48: 2454-2462, 1999.

78. Garcia-Patterson A, Erdozain L, Ginovart G, Adelantado JM, Cubero JM, Gallo G, deLeiva A, Corcoy R. In human gestational diabetes mellitus congenital malformations are related to pre-pregnancy body mass index and to severity of diabetes. Diabetologia 509-514, 2004.

79. Poitout V, Robertson RP. Minireview: Secondary (-cell failure in type 2 diabetes - A convergence of glucotoxicity and lipotoxicity. Endocrinology 143: 339-342, 2002.

80. Du Y, Miller CM, Kern TS. Hyperglycemia increases mitochondrial superoxide in retina and retinal cells. Free Radic. Biol. Med. 35: 1491-1499, 2003.

81. Purves T, Middlemas A, Agthong S, Jude EF, Bouton AJ, Fernyhough P, Tomlinson D, A role for mitogen-activated protein kinasesx in the etiology of diabetic neuropathy. FASEB J. 15: 2508-2514, 2001.

82. Hsieh TJ, Zhang SL, Filep JG, Tang SS, Ingelfinger JR, Chan JS. High glucose stimulates angiotensinogen gene expression via reactive oxygen spe4cies generation in rat kidney proximal tubular cells. Endocrinol. 143: 2975-2985, 2002.

83. Ihara Y, Toyokuni S, Uchida K, Odaka H, Tanaka T, Ikeda H, Hiai H, Seino Y, Yamada Y. Hyperglycemia causes oxidative stress in pancreatic beta-cells of GK rats, a model of type 2 diabetes. Diabetes 48: 927-932, 1999.

84. Tanaka Y, Gleason CE, Tran POT, Harmon JS, Robertson RP. Prevention of glucose toxicity in HIT-T15 cells and Zucker diabetic fatty rats by antioxidants. Proc. Natl. Acad. Sci. USA 96: 10857-10862, 1999.

85. Kaneto H, Fujii J, Myint T, Miyazawa N, Islam KN, Kawasaki Y, Suzuki K, Nakamura M, Tatsumi H, Yamasaki Y, Taniguchi N. Reducing sugars trigger oxidative modification and apoptosis in pancreatic beta-cells by provoking oxidative stress through the glycation reaction. Biochem. J. 320: 855-863, 1996.

86. Kaneto H, Kajimoto Y, Miyagawa J, Matsuoka T, Fujitani Y, Umayahara Y, Hanafusa T, Matsuzawa Y, Yamasaki Y, Hori M. Beneficial effects of antioxidants in diabetes: possible protection of pancreatic beta-cells against glucose toxicity. Diabetes 48: 2398-2406, 1999.

87. Tanaka Y, Tran PO, Harmon J, Robertson RP. A role for glutathione peroxidase in protecting pancreatic beta cells against oxidative stress in a model of glucose toxicity. Proc. Natl. Acad. Sci. USA 99: 12363-12368, 2002.

88. Tang J, Neidigh JL, Cooksey RC, McClain DA. Transgenic mice with increasesd hexosamine flux specifically targeted to beta-cells exhibit hyperinsulinemia and peripheral insulin resistance. Diabetes 49: 1492 - 1499, 2000.

89. Kaneto H, Xu G, Song KH, Suzuma K, Bonner-Weir S, Sharma A, Weir GC. Activation of the hexosamine pathway leads to deterioration of pancreatic beta-cell function through the induction of oxidative stress. J. Biol. Chem. 276: 31099-31104, 2001.

90. Konrad RJ, Kudlow JE. The role of O-linked protein glycosylation in beta cell dysfunction. Int. J. Mol. Med. 10: 535-539, 2002.

91. Du XL, Edelstein D, Rossetti L, Fantus IG, Goldberg H, Ziyadeh F, Wu J, Browlee M. Hyperglycemia-induced mitochondrial superoxide overproduction activates the hexosamine pathway and induces plasminogen activator inhibitor-1 expression by increasing Sp1 glycosylation. Proc. Natl. Acad. Sci. USA 97: 12222-12226, 2000.

92. Koya D, Haneda M, Nakagawa H, Isshiki K, Sato H, Maeda S, Sugimoto T, Yasuda H, Kashiwagi A, Ways DK, King GI, Kikkawa R. Amelioration of accelerated diabetic mesangial expansion by treatment with a PKC beta inhibitor in diabetic db/db mice, a rodent model for type 2 diabetes. FASEB J. 14: 439-447, 2000.

93. Aiello LP, The potential role of PKC beta in diabetic retinopathy and macular edema. Surv. Ophthalmol. 47: Suppl. 2, S263-S269, 2002.

94. Kaneto H, Suzuma K, Sharma A, Bonner-Weir S, King GL, Weir GC. Involvement of protein kinase C (2 in c-myc induction by high glucose in pancreatic (-cells. J. Biol. Chem. 277: 3680-3685, 2002.

95. Kaneto H, Sharma A, Suzuma K Laybutt DR, Xu G, Bonner-Weir S, Weir GC. Induction of c-Myc expression suppresses insulin gene transcription by inhibiting neuroD/BETA2-mediated transcriptional activation. J. Biol. Chem. 277:12998-13006, 2002.

96. Goldberg HJ, Whiteside CI, Fantus IG. The hexosamine pathway regulates the plasminogen activator inhibitor-1 gene promoter and Sp1 transcriptional activation through protein kinase C-(I and -(. J. Biol. Chem. 277: 33833-33841, 2002.

97. Marshall S, Garvey WT, Taxinger RR. New insights into the metabolic regulation of insulin action and insulin resistance: role of glucose and amino acids. FASEB J. 5: 3031-3036, 1991.

98. Hresko RC, Heimberg H, Chi MM, Mueckler M. Glucosamine-induced insulin resistance in 3T3-L1 adipocytes is caused by depletion of intracellular ATP. J. Biol. Chem. 273: 20658-20668, 1998.

99. Vosseller K, Wells L, Lane MD, Hart GW. Elevated nucleocytoplasmic glycosylation by O-GlcNAc results in insulin resistance assocaited with defects in Akt activation in 3T3-L1 adipocytes. Proc. Natl. Acad. Sci. USA 99: 5313-5318, 2002.

100. Herbert LF, Daniels MC, Zhou J, Crook ED, Turner RL, Simmons ST, Neidigh JL, Zhu J-S, Baron AD, McClain DA. Overexpression of glutamine: fructose-6-phosphate amidotransferase in transgenic mice leads to insulin resistance. J. Clin. Invest. 98: 930-936, 1996.

101. Rossetti L, Hawkins M, Chen W, Gindi J, Barzilai N. In vivo glucosamine infusion induces insulin resistance in normoglycemic but not in hyperglycemic conscious rats. J. Clin. Invest. 96: 132-140, 1995.

102. Patti ME, Virkamaki A, Landaker EJ, Kahn CR, Yki-Jarvinen H. Activation of the hexosamine pathway by glucosamine in vivo induces insulin resistance of early postreceptor insulin signaling events in skeletal muscle. Diabetes 48: 1562-1571, 1999.

103. Virkamaki A, Ueki K, Kahn CR. Protein-protein interaction in insulin signaling and the molecular mechanisms of insulin resistance. J. Clin. Invest. 103: 931-943, 1999.

104. Carvalho E, Eliasson B, Wesslan C, Smith U. Impaired phosphorylation and insulin-stimulated translocation to the plasma membrane of protein kinase B/Akt in adipocytes from type II diabetic subjects. Diabetologia 43: 1107-1115, 2000.

105. Oku A, Nawano M, Ueta K, Fuijita T, Umebayashi I, Arakawa K, Kano-Ishihara T, Saito A, Anai M, Funaki M, Kikuchi M, Oka Y, Asano T. Inhibitory effect of hyperglycemia on insulin-induced Akt/protein kinase B activation in skeletal muscle. Am. J. Physiol. Endocrinol. Metab. 280: E816-E824, 2001.

106. Pillay TS, Xiao S, Olefsky JM. Glucose-induced phosphorylation of the insulin receptor: functional effects and characterization of phosphorylation sites. J. Clin. Invest. 97: 613-620, 1996.

107. Muller HK, Kellerer M, Ermel B, Muhlhofer A, Obermaier-Kusser B, Vogt B, Haring HU. Prevention by protein kinase C inhibitors of glucose-induced insulin-receptor tyrosine kinase resistance in rat fat cells. Diabetes 40: 1440-1448, 1991.

108. Avignon A, Yamada K, Zhou X, Spencer B, Cardona O, Saba-Siddique S, Galloway L, Standaert ML, Farese RV. Chronic activation of protein kinase C in soleus muscles and other tissues of insulin-resistant type II diabetic Goto-Kakizaki (GK), obese/aged, and obese/Zucker rats. A mechanism for inhibiting glycogen synthesis. Diabetes 45: 1396-1404, 1996.

109. Bollag GE, Roth RA, Beaudoin J, Mochly-Rosen D, Koshland DEJ. Protein kinase C directly phosphorylates the insulin receptor in vivo and reduces its protein-tyrosine kinase activity. Proc. Natl. Acad. Sci. USA 83: 5822-5824, 1987.

110. Kroder G, Bossenmaier B, Kellerer M, Capp E, Stoyanov B, Muhlhofer A, Berti L, Horikoshi H, Ullrich A, Haring H. Tumor necrosis factor-alpha- and hyperglycemia-induced insulin resistance. Evidence for different mechanisms and different effects on insulin signaling. J. Clin. Invest. 97: 1471-1477, 1996.

111. Peraldi P, Spiegelman B. TNF-alpha and insulin resistance: summary and future prospects. Mol. Cell. Biochem. 182: 169-175, 1998.

112. Kellerer M, Mushack J, Seffer E, Mischak H, Ullrich A, Haring HU. Protein kinase C isoforms (, ( and ( require insulin receptor substrate-1 to inhibit the tyrosine kinase activity of the insulin receptor in human kidney embryonic cells (HEK 293 cells). Diabetologia 41: 833-838,1998.

113. Newton AC. Protein kinase C: structure, function and regulation. J. Biol. Chem. 270: 28495-28498, 1995.

114. Chen KS, Heydrick SJ, Brown ML, Friel JC, Ruderman NB. Insulin increases a biochemically distinct pool of diacylglycerol in the rat soleus muscle. Am. J. Physiol. 266: E479-E485, 1994.

115. Rui L, Yuan M, Frantz D, Shoelson S, White MF. SOCS-1 and SOCS-3 block insulin signaling by ubiquitin-mediated degradation of IRS1 and IRS2. J. Biol. Chem. 277:42394-42398, 2002.

116. Cazzolli R, Carpenter L, Biden TJ, Schmitz-Peiffer C. A role for protein phosphatase 2A-like activity, but not atypical protein kinase Czeta, in the inhibition of protein kinase B/Akt and glycogen synthesis by palmitate. Diabetes 50: 2210-2218, 2001.

117. Schmitz-Peiffer C, Craig DL, Biden TJ. Ceramide generation is sufficient to account for the inhibition of the insulin stimulted PKB pathway in C2C12 skeletal muscle cells pretreated with palmitate. J. Biol. Chem. 274: 24202 - 24210, 1999.

118. Straczkowski M, Kowalska I, Kikolajuk A, Dzienis-Straczkowska S, Kinalska I, Baran M. Zendzian-Piotrowska M, Brzezinska Z, Gorski J. Relationship between insulin sensitivity and sphingomyelin signaling pathway in human skeletal muscle. Diabetes 53: 1215-1221, 2004.

119. Paolisso G, Giugliano D. Oxidative stress and insulin action: is there a relationship? Diabetologia 39: 357-363, 1996.

120. Paolisso G, D'Amore A, Volpe C, Balbi V, Saccomanno F, Galzerano D, Giugliano D, Varricchio M, D'Onofrio F. Evidence for a relationship between oxidative stress and insulin action in non-insulin dependent (type 2) diabetic patients. Metabolism 43: 1426-1429, 1994.

121. Rudich A, Tirosh A, Potashnik R, Hemi R, Kanety H, Bashan N. Prolonged oxidative stress impairs insulin-induced GLUT4 translocation in 3T3-L1 adipocytes. Diabetes 47: 1562-1569, 1998.

122. Tirosh A, Potashnik R, Bashan N, Rudich A. Oxidative stress disrupts insulin-induced cellular redistribution of inuslin receptor substrate-1 and phosphatidylinositol 3-kinase in 3T3-L1 adipocytes. A putative cellular mechanism for impaired protein kinase B activation and GLUT4 translocation. J. Biol. Chem. 274: 10595-10602, 1999.

123. Jain SK, Levine SN, Duett J, Hollier B. Elevated lipid peroxidation levels in red blood cells of streptozotocin-treated diabetic rats. Metabolism 39: 971-975, 1990.

124. Baynes JW, Thorpe SR. Role of oxidative stress in diabetic complications: a new perspective on an old paradigm. Diabetes 48: 1-9, 1999.

125. Lu B, Ennis D, Lai R, Bogdanovic E, Nikolov R, Salamon L, Fantus C, Le-Tien H, Fantus IG. Enhanced sensitivity of insulin-resistant adipocytes to vanadate is associated with oxidative stress and decreased reduction of vanadate (+5) to vanadyl (+4). J. Biol. Chem. 276: 35589-35598, 1001.

126. Haber CA, Lam TKT, Yu Z, Gupta N, Goh T, Bogdanovic E, Giacca A, Fantus IG. N-acetylcysteine (NAC) and taurine prevent hyperglycemia-induced insulin resistance in vivo: possible role of oxidative stess. Am J. Physiol. Endocrinol. Metab. 285:E744-E753, 2003.

127. Paolisso G, D'Amore A, Giugliano D, Ceriello A, Varricchio M, D'Onofrio F. Pharmacologic doses of vitamin E improve insulin action in healthy subjects and non-insulin-dependent diabetic patients. Am J. Clin. Nutr. 57: 650-656, 1993.

128. Paolisso G, D'Amore A, Balbi V, Volpe C, Galzerano D, Giuliano D, Sgambato S, Varricchio M, D'Onofrio F. Plasma vitamin C affects glucose homeostasis in healthy subjects and in non-insulin-dependent diabetics. Am. J. Physiol. 266: E261-E268, 1994.

129. Powis G, Gasdaska JR, Baker A. Redox signaling and the control of cell growth and death. Adv. Pharmacol. 38: 329-359, 1997.

130. Guyton KZ, Liu Y, Gorospe M, Xu Q, Holbrook NJ. Activation of mitogen-activated protein kinase by H2O2: Role in cell survival following oxidant injury. J. Biol. Chem. 271: 4138-4142, 1996.

131. Blair AS, Hajduch E, Litherland GJ, Hundal HS. Regulation of glucose transport and glycogen synthesis in L6 muscle cells during oxidative stress. Evidence for cross-talk between the insulin and SAPK2/p38 mitogen-activated protein kinase signaling pathways. J. Biol. Chem. 274: 36293-36299, 1999.

132. Konishi H, Tanaka M, Takemura Y, Matsuzaki H, Ono Y, Kikkawa U, Nichizuka Y. Activation of protein kinase C by tyrosine phosphorylation in response to H2O2 . Proc. Natl. Acad. Sci. USA 94: 11223-11237, 1997.

133. Palmer HJ, Paulson KE. Reactive oxygen species and antioxidants in signal transduction and gene expression. Nutr. Rev. 55: 353-361, 1997.

134. Hunt J.V., Dean RT, Wolff SP. Hydroxyl radical production and autoxidative glycosylation. Glucose autoxidation as the cause of protein damage in the experimental glycation model of diabetes mellitus and aging. Biochem. J. 256: 205-212, 1988.

135. Thornalley PJ, Langborg A, Minhas HS. Formation of glyoxal, methylglyoxal and deoxyglucosone in the glycation of proteins by glucose. Biochem. J. 344: 109-116, 1999.

136. Nishikawa T, Edelstein D, Du XL, Yamagishi S-I, Matsumura T, Kaneda Y, Yorek MA, Beebe D, Oates PJ, Hammes H-P, Giardino I, Brownlee M. Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage. Nature 404: 787-790, 2000.

137. Lee AY, Chung SS. Contributions of polyol pathway to oxidative stress in diabetic cataract. FASEB J. 13: 23-30, 1999.

138. Wu G, Haynes TE, Li H, Yan W, Meininger CJ. Gutamine metabolism to glucosamine is necessary for glutamine inhibition of endothelial nitric oxide synthesis. Biochem. J. 353: 245-252, 2001.

139. Halliwell B, Gutteridge JM. Role of free radicals and catalytic metal ions in human disease: an overview. Methods Enzymol.186: 1-85, 1990

140. Hodgkinson AD, Bartlett T, Oates PJ, Millward BA, Demaine AG. The response of antioxidant genes to hyperglycemia is abnormal in patients with type 1 diabetes and diabetic nephropathy. Diabetes 52: 846-851, 2003.

141. Hammes H-P, Du X, Edelstein D, Taguchi T, Matsumura T, Ju Q, Lin J, Bierhaus

A, Nawroth P, Hannak D, Neumaier M, Bergfeld R, Giardino I, Brownlee M. Benfotiamine blocks three major pathways of hyperglycemic damage and prevents experimental diabetic retinopathy. Nature Med.1 - 6, 2003.

142. Cowell RM, Russell JW. Nitrosative injury and antioxidant therapy in the management of diabetic neuropathy. J. Investig. Med. 52: 33-44, 2004.

143. Van Dam PS, Van Asbeck S, Erkelens DW, Marx JJ, Gispen WH, Bravenboer B. The role of oxidative stress in neuropathy and other diabetic complications. Diabets Metab. Rev. 11: 181-192, 1995.

144. Kowlura RA, Tang J, Kern TS. Abnormalities of retinal metabolism in diabetes and experimental galactosemia. VII. Effect of long-term administration of antioxidants on the development of retinopathy. Diabetes 50: 1938-1942, 2001.

145. Hugay ZJ, Weiss Y, Zusman I, Peled-Kamar M, Reece EA, Eriksson UJ, Groner Y. Prevention of diabetes-associated embryopathy by overexpression of the free radical scavenger copper zinc superoxide disminutase in transgenic mouse embryos. Am. J. Obstet. Gynecol. 173: 1036-1041, 1995.

146. Wentzel P, Eriksson UJ. Antioxidants diminish developmental damage induced by high glucose and cyclooxygenase inhibitors in rat embryos in vitro. Diabetes 47: 677-684, 1998.

147. Yki Jarvinen H, Koivisto VA. Natural course of insulin resistance in type 1 diabetes. N. Engl. J. Med. 315: 224-230, 1986.