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Oxidative stress and impaired insulin secretion in type 2 diabetes
Oxidative stress and impaired insulin secretion in type 2 diabetes R Paul Robertson Molecular mechanisms involving oxidative stress have been increasingly implicated in the pathogenesis of type 2 diabetes. These implications have arisen from reports that glucolipotoxicity of the pancreatic islet and non-islet tissues can lead to deterioration of islet function and insulin sensitivity, as well as structural abnormalities in tissues adversely affected by diabetes. Co-incident with these changes are profound alterations in insulin gene expression, which involve greatly diminished levels of two transcription factors, MafA and Pdx-1. Addresses Pacific Northwest Research Institute and the Departments of Medicine and Pharmacology, University of Washington, 720 Broadway, Seattle, WA 98122, USA Corresponding author: Robertson, R Paul ([email protected])
Current Opinion in Pharmacology 2006, 6:615–619 This review comes from a themed issue on Endocrine and metabolic diseases Edited by Antoine Bril and Alain Ktorza Available online 10th October 2006 1471-4892/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coph.2006.09.002
Introduction This brief review provides a short historical retrospective on the concept that oxidative stress might adversely affect pancreatic islet function and insulin action, and focuses more intensely on recent studies suggesting molecular mechanisms by which excessive concentrations of reactive oxygen species (ROS) can cause pancreatic islet b cell dysfunction and impair insulin action. The rationale for examining oxidative stress in diabetes stems from the large number of reports documenting excess levels of biochemical markers in the blood, urine and pancreas of type 2 diabetic patients. The importance of a better understanding of these phenomena lies in the possibility that ancillary antioxidant therapy in such patients could provide a layer of protection against incomplete normalization of glycemia by conventional drugs, such as sulfonylureas, insulin sensitizers and exogenous insulin.
Pathogenesis of type 2 diabetes: b cell dysfunction and insulin resistance Type 2 diabetes is usually the product of two distinct abnormalities: abnormal b cell function and decreased insulin sensitivity. It appears that type 2 diabetes is www.sciencedirect.com
primarily a genetic disease, based on its strong familial association and high concordance rates in identical twins [1]. However, no single gene has been identified that is common to a general population of type 2 diabetic patients, leading to the conclusion that this must be a polygenic disease [2–4]. Most type 2 diabetic patients are obese, and obese patients generally have resistance to the actions of insulin on liver, muscle and fat tissues — the major targets for the beneficial effects of insulin. Environmental influences also play a major role by enhancing the phenotypic expression of genes that place individuals at risk for diabetes. This is becoming increasingly apparent as witnessed by the recent epidemic proportions of new-onset type 2 diabetes in cultures such as American Indian, African American, Latino, and Alaskan American. Environmental precipitants that are common to these cultures include obesity, insufficient physical activity, and excessive carbohydrate intake. However, only a minority of obese patients develops diabetes, and 20% of type 2 diabetic patients are not obese, emphasizing that obesity does not cause diabetes; rather, it contributes to the phenotypic expression of genes that predispose the individual to type 2 diabetes. These clinical facts point to the conclusion that the initial lesion in type 2 diabetes probably involves genetically determined diminution of intrinsic b cell function, which is thus unable to adequately meet the challenge of states of insulin resistance, such as obesity. Consequently, the b cell is continually called upon to secrete insulin because of unresolved hyperglycemia, and this stress gradually causes b cell deterioration and accelerated apoptosis [5]. Both b cell dysfunction and insulin resistance work in concert to cause further deterioration of insulin secretion and increase insulin resistance (Figure 1). Nonetheless, it is interesting to consider that not all lean type 2 diabetic patients are insulin resistant, and that patients with cystic fibrosis and type 2 diabetes are characteristically insulin sensitive [6].
Reactive oxygen species: physiology versus stress Many forms of ROS exist, which in physiologic concentrations are thought to support physiologic functions such as gene transcription, mitochondrial function and leukocyte function. However, when concentrations of ROS reach excessive levels, they can cause structural and functional damage to proteins, lipids and DNA. With regard to the pancreatic islet, reports from studies of rodent islets have emphasized the low levels of intrinsic antioxidant enzyme mRNA, proteins and activities for superoxide dismutases, catalase and glutathione peroxidase [7,8]. This scenario sets the islet at greater risk of Current Opinion in Pharmacology 2006, 6:615–619
616 Endocrine and metabolic diseases
Figure 1
The inter-relationship between defective b cell function and insulin resistance that causes type 2 diabetes. Resultant hyperglycemia and increased lipogenesis create a lipotoxic milieu that worsens both b cell function and insulin resistance.
oxidative damage by ROS that are generated locally and reach abnormally high concentrations. A caveat regarding this line of reasoning is that most published studies have involved male animals only, and only rarely have human islets been examined [9]. This is relevant because female rodents [10,11] are characteristically more protected against development of type 2 diabetes, with evidence suggesting that estrogens are protective against this disease [12]. Thus, more information is needed regarding islet antioxidant enzyme levels in male rodents and in human islets before it can be concluded convincingly that all pancreatic islets are disproportionately at risk for oxidative damage.
Glucolipotoxicity in the b cell and oxidative stress The common findings of elevated glucose and lipid levels in the blood of diabetic patients led to the hypotheses of glucose toxicity [13] and lipotoxicity [14]. Relatively more information has been published about biochemical pathways through which elevated glucose concentrations can generate excessive levels of ROS (reviewed in [15]). These include glycolysis and oxidative phosphorylation; methylglyoxal formation and glycation; enediol and aketoaldehyde formation (glucoxidation); diacylglycerol formation and protein kinase C activation; glucosamine formation and hexosamine metablolism; and sorbitol metabolism. Conceptually, as b cells are exposed to high glucose concentrations for increasingly prolonged periods of time, glucose saturates the normal route of glycolysis and increasingly is shunted to alternate pathways, such that ROS are generated from distinct metabolic processes within and outside the mitochondria. Reports also indicate that excessive levels of palmitate are associated with abnormal islet function (especially in the presence of high glucose concentrations), which leads to excessive lipid esterification that, in turn, can generate ceramide, thereby increasing oxidative stress [14,16,17]. It seems unlikely, Current Opinion in Pharmacology 2006, 6:615–619
however, that circulating lipid itself, such as triglyceride or cholesterol, would be responsible for damaging islet tissue. It seems more likely that excessive circulating glucose levels lead to accelerated de novo synthesis of islet lipid. One mechanism by which glucose might contribute to liptoxicity is by virtue of its ability to drive synthesis of malonyl CoA, which inhibits b-oxidation of free fatty acids. This in turn shunts free fatty acids towards esterification pathways, thereby forming triglyceride, ceramide and other esterification products [17,18]. The relationship between glucose toxicity and lipotoxicity was examined in a study of isolated islets and in diabetic rodents who had hyperglycemia and hypertriglyceridemia. In the islet study, it was demonstrated that high concentrations of palmitate do not by themselves harm islet function whereas, in the combined presence of high glucose and high palmitate, insulin gene expression and insulin secretion was decreased [16]. Similarly, it was found in the animal studies that treatment with a lipidlowering drug (bezafibrate) did not protect the animals from worsening of islet function and diabetes, whereas a glucose-lowering drug (phlorizin) was successful [19]. This suggested that lipotoxicity requires concomitant hyperglycemia to damage islet function, whereas glucose toxicity can exert harmful effects on the islet in the absence of elevated circulating triglyceride. One molecular mechanism of action through which chronic hyperglycemia can cause worsening b cell function is via decreased protein expression of two important transcription factors: Pdx-1 and MafA ([15]; Figure 2). Both proteins are critical for normal insulin gene expression, as their absence or mutation of their DNA binding sites on the insulin promoter leads to decreased insulin Figure 2
Sequence of events that begins with chronic exposure to hyperglycemia and leads to progressive b cell failure. In a model of glucose toxicity — the b cell line HIT-T15 — chronic passaging of cells for many months in supraphysiologic glucose concentrations causes gradual loss of important transcription factors (Pdx-1 and MafA), which results in decreased insulin promoter activity, decreased insulin synthesis and content, and decreased insulin secretion. www.sciencedirect.com
Oxidative stress and impaired insulin secretion in type 2 diabetes Robertson 617
mRNA levels, insulin content and insulin secretion [20,21]. Studies in the b cell line HIT-T15 demonstrated normal pdx-1 gene transcription but not expression [20], along with normal mafA gene expression at the mRNA level but not at the protein level [22]. Thus, the molecular defects leading to decreased b cell Pdx-1 levels is post-transcriptional in nature, whereas the mechanism leading to disappearance of normal MafA levels is posttranslational. The possibility that oxidative stress is responsible for the decreased levels of Pdx-1 and MafA was highlighted by studies showing that antioxidant treatment of b cell lines and rodent models of type 2 diabetes protected against deterioration of insulin gene expression induced by exposure to high glucose concentrations [23–25]. N-acetylcysteine (NAC), a drug commonly used in these studies, provides direct antioxidant actions, as well as supplying the limiting substrate cysteine for glutathione synthesis via the rate-limiting enzyme g-cysteine ligase. Treatment with NAC protected against decreases in Pdx-1 and MafA caused by high glucose concentrations in vitro, and also protected against loss of insulin gene expression and insulin secretion in vivo.
Glucolipotoxicity in non-b cells, insulin resistance and oxidative stress As this is a short review that centers primarily on the islet and the number of references is limited, only brief mention can be given to well-developed research areas involving interplay among oxidative stress, insulin resistance and the secondary complications of diabetes. Insulin resistance accompanies the development of obesity, pregnancy, excess growth hormone and glucocorticoid levels, and lack of exercise. Although clinical and laboratory methods for quantifying insulin resistance are well established, and molecular mechanisms for insulin action are being identified, explanations at the molecular level for resistance to the actions of insulin are generally less well developed. Nonetheless, ample evidence exists that oxidative stress plays a role in insulin resistance and in the cellular damage of tissues that leads to the late complications of diabetes (for review, see [26]). Abnormal levels of free fatty acids, tumour necrosis factor-a, leptin and resistin are frequently found in obese individuals and are prominently mentioned as potential mediators of insulin resistance [26]. Of these, free fatty acids have been reported to impair insulin action via oxidative stressinduced activation of nuclear factor-kB [26]. Secondary complications of diabetes involve microvascular and macrovascular changes that lead to retinopathy, nephropathy, neuropathy and damage to critical blood vessels, such as the coronary arteries. Stress-activated signaling pathways that might play a role in these phenomena are those involving protein kinase C, nuclear factor-kB, p38 mitogen-activated protein kinase, advanced glycosylation end-products and their receptors, and amino-terminal www.sciencedirect.com
JUN kinases, among others [26]. Antioxidant agents that have been reported to reduce insulin resistance, as well as secondary complications of diabetes, include lipoic acid, NAC, aminoguanidine, vitamine C, vitamin E, resveratrol, silymarin and curcumin [26]. Vascular endothelial growth factor has been proposed as an initiator of diabetic complications, whereas antioxidants have been reported to inhibit advanced glycosylation end-product-induced expression of vascular endothelial growth factor [26]. Thus, there is ample reason to consider that glucoliptoxicity via chronic oxidative stress contributes to both insulin resistance and secondary complications of diabetes involving multiple target tissues.
Antioxidant therapy and the islet Mention has already been made of several antioxidants that have been used in clinical studies which protected against b cell dysfunction and insulin resistance associated with type 2 diabetes. A careful distinction must be made, however, between prevention of the disease and protection against its complications. There is little, if any, evidence that oxidative stress causes type 2 diabetes, which appears to be primarily a polygenic disease involving intrinsic defects in islet function. Rather, the role for oxidative stress appears to be as one mechanism in the second wave of pathogenic forces that arise from chronic hyperglycemia and increased lipogenesis after type 2 diabetes is established. The hypothesis arising from this consideration is that any treatment that provided truly perfect control of glucose and lipid metabolism would Figure 3
Protective effects of antioxidants against b cell deterioration in an animal model of hyperglycemia. The natural history of the development of marked hyperglycemia in Zucker Diabetic Fatty (ZDF) rats can be ameliorated by treating the rats at 6 weeks of age with the antioxidants aminoguanidine (AG) or n-acetylcysteine (NAC) [23]. Control glucose data are shown from Zucker Lean Control (ZLC) rats that do not develop diabetes. Data adapted from [23]. Current Opinion in Pharmacology 2006, 6:615–619
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prevent the complications of diabetes, as suggested by several therapeutic trials with conventional drugs used for treatment of hyperglycemia [27,28]. However, as it is not yet possible to establish constant normoglycemia with available drugs, there could be a role for antioxidants as ancillary treatment to provide protection against residual hyperglycemia during conventional therapy. In this manner, the toxic effects of residual hyperglycemia, especially post-prandially, might be neutralized, and thereby the secondary adverse effects of residual hyperglycemia on islet function, as well as other secondary complications of diabetes, might be avoided. As mentioned above, several studies in animals lend credence to this approach; for example, in the Zucker Diabetic Fatty rat [24], as well as the db/db mouse [25], treatment with NAC ameliorated the course of type 2 diabetes such that abnormal changes in glycemia and insulin gene expression were not as severe (Figure 3). To date, no trials have been reported in humans to test this hypothesis. Preliminary data from a study of NAC in combination with oral agents [29] suggests that such trials are warranted.
Conclusions Although ROS in small concentrations support physiologic functions, at abnormally high concentrations they cause both biochemical abnormalities and structural damage to tissue. Chronic hyperglycemia increases local concentrations of ROS, and thereby induces chronic oxidative stress. This sequence of events provides one explanation for many of the secondary complications of hyperglycemia, including ever-worsening abnormalities in the pancreatic b cell in type 2 diabetes. Other important defects include decreased levels of the transcription factors Pdx-1 and MafA, which are essential to normal insulin gene expression and, therefore, insulin synthesis, insulin content and insulin secretion. Interventional trials using antioxidants in animal models partially prevent the adverse consequences of hyperglycemia on insulin gene expression and islet function, suggesting that human trials of antioxidants in patients with impaired glucose tolerance and postprandial hyperglycemia might offer protective effects against unrelenting b cell deterioration in type 2 diabetes.
Acknowledgements Supported in part by NIH grant NIDDK RO-1 38325.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
2.
Nelson PG, Pyke DA, Cudworth AG, Woodrow JC, Batchelor JR: Histocompatibility antigens in diabetic identical twins. Lancet 1975, 2:193-194. Groop LC, Kankuri M, Schalin-Jantti C, Ekstrand A, Nikula-Ijas P, Widen E, Kuismanen E, Eriksson J, Franssila-Kallunki A, Saloranta C et al.: Association between polymorphism of the
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glycogen synthase gene and non-insulin-dependent diabetes mellitus. N Engl J Med 1993, 328:10-14. 3.
Froguel P, Zouali H, Vionnet N, Velho G, Vaxillaire M, Sun F, Lesage S, Stoffel M, Takeda J, Passa P et al.: Familial hyperglycemia due to mutations in glucokinase. Definition of a subtype of diabetes mellitus. N Engl J Med 1993, 328:697-702.
4.
Byrne MM, Sturis J, Clement K, Vionnet N, Pueyo ME, Stoffel M, Takeda J, Passa P, Cohen D, Bell GI et al.: Insulin secretory abnormalities in subjects with hyperglycemia due to glucokinase mutations. J Clin Invest 1994, 93:1120-1130.
5.
Butler AE, Janson J, Bonner-Weir S, Ritzel R, Rizza RA, Butler PC: Beta-cell deficit and increased beta-cell apoptosis in humans with type 2 diabetes. Diabetes 2003, 52:102-110.
6.
Moran A, Diem P, Klein DJ, Levitt MD, Robertson RP: Pancreatic endocrine function in cystic fibrosis. J Pediatr 1991, 118:715-723.
7.
Lenzen S, Drinkgern J, Tiedge M: Low antioxidant enzyme gene expression in pancreatic islets compared with various other mouse tissues. Free Radic Biol Med 1996, 20:463-466.
8.
Sigfrid LA, Cunningham JM, Beeharry N, Hakan Borg LA, Rosales Hernandez AL, Carlsson C, Bone AJ, Green IC: Antioxidant enzyme activity and mRNA expression in the islets of Langerhans from the BB/S rat model of type 1 diabetes and an insulin-producing cell line. J Mol Med 2004, 82:325-335.
9.
Welsh N, Margulis B, Borg LA, Wiklund HJ, Saldeen J, Flodstrom M, Mello MA, Andersson A, Pipeleers DG, Hellerstrom C et al.: Differences in the expression of heat-shock proteins and antioxidant enzymes between human and rodent pancreatic islets: implications for the pathogenesis of insulin-dependent diabetes mellitus. Mol Med 1995, 1:806-820.
10. Friesen NT, Buchau AS, Schott-Ohly P, Lgssiar A, Gleichmann H: Generation of hydrogen peroxide and failure of antioxidative responses in pancreatic islets of male C57BL/6 mice are associated with diabetes induced by multiple low doses of streptozotocin. Diabetologia 2004, 47:676-685. 11. Roza AM, Pieper GM, Johnson CP, Adams MB: Pancreatic antioxidant enzyme activity in normoglycemic diabetic prone BB rats. Pancreas 1995, 10:53-58. 12. Le May C, Chu K, Hu M, Ortega C, Simpson E, Korach K, Tsai M, Mauvais-Jarvis F: Estrogens protect pancreatic beta-cells from apoptosis and prevent insulin-deficient diabetes mellitus in mice. Proc Natl Acad Sci USA 2006, 103:9232-9237. The authors examined b cell apoptosis after exposure to acute oxidative stress induced by streptozotocin in mice that were estradiol-deficient or estradiol-resistant. They observed that estradiol protection against apoptosis was lost in b cells treated with an estrogen receptor antagonist, and in islets with targeted disruption of the estrogen receptor. 13. Unger RH, Grundy S: Hyperglycaemia as an inducer as well as a consequence of impaired islet cell function and insulin resistance: implications for the management of diabetes. Diabetologia 1985, 28:119-121. 14. Unger RH: Lipotoxicity. In Diabetes Mellitus: A fundamental and Clinical Text - 3rd Edition. Edited by LeRoith D OJ, Taylor S: Lippincott Williams & Wilkins, Inc.; 2004:141-149. 15. Robertson RP: Chronic oxidative stress as a central mechanism for glucose toxicity in pancreatic islet beta cells in diabetes. J Biol Chem 2004, 279:42351-42354. 16. Briaud I, Harmon JS, Kelpe CL, Segu VB, Poitout V: Lipotoxicity of the pancreatic beta-cell is associated with glucosedependent esterification of fatty acids into neutral lipids. Diabetes 2001, 50:315-321. 17. Prentki M, Corkey BE: Are the beta-cell signaling molecules malonyl-CoA and cystolic long-chain acyl-CoA implicated in multiple tissue defects of obesity and NIDDM? Diabetes 1996, 45:273-283. 18. Poitout V, Robertson RP: Minireview: secondary beta-cell failure in type 2 diabetes–a convergence of glucotoxicity and lipotoxicity. Endocrinology 2002, 143:339-342. 19. Harmon JS, Gleason CE, Tanaka Y, Poitout V, Robertson RP: Antecedent hyperglycemia, not hyperlipidemia, is associated www.sciencedirect.com
Oxidative stress and impaired insulin secretion in type 2 diabetes Robertson 619
with increased islet triacylglycerol content and decreased insulin gene mRNA level in Zucker diabetic fatty rats. Diabetes 2001, 50:2481-2486. 20. Olson LK, Sharma A, Peshavaria M, Wright CV, Towle HC, Robertson RP, Stein R: Reduction of insulin gene transcription in HIT-T15 beta cells chronically exposed to a supraphysiologic glucose concentration is associated with loss of STF-1 transcription factor expression. Proc Natl Acad Sci USA 1995, 92:9127-9131 [published erratum appears in Proc Natl Acad Sci U S A 1995 Nov 21;92(24):11322]. 21. Poitout V, Olson LK, Robertson RP: Chronic exposure of betaTC-6 cells to supraphysiologic concentrations of glucose decreases binding of the RIPE3b1 insulin gene transcription activator. J Clin Invest 1996, 97:1041-1046. 22. Harmon JS, Stein R, Robertson RP: Oxidative stress-mediated, post-translational loss of MafA protein as a contributing mechanism to loss of insulin gene expression in glucotoxic beta cells. J Biol Chem 2005, 280:11107-11113. The authors report that the loss of MafA is post-translational in a b cell line continuously cultured under glucotoxic conditions. This loss is prevented by an antioxidant. 23. Tanaka Y, Gleason CE, Tran PO, Harmon JS, Robertson RP: Prevention of glucose toxicity in HIT-T15 cells and Zucker diabetic fatty rats by antioxidants. Proc Natl Acad Sci USA 1999, 96:10857-10862.
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24. 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 2002, 99:12363-12368. 25. 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 1999, 48:2398-2406. 26. Evans JL, Goldfine ID, Maddux BA, Grodsky GM: Oxidative stress and stress-activated signaling pathways: a unifying hypothesis of type 2 diabetes. Endocr Rev 2002, 23:599-622. 27. DCCT RG: 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 1993, 329:977-985. 28. Ohkubo Y, Kishikawa H, Araki E, Miyata T, Isami S, Motoyoshi S, Kojima Y, Furuyoshi N, Shichiri M: Intensive insulin therapy prevents the progression of diabetic microvascular complications in Japanese patients with non-insulindependent diabetes mellitus: a randomized prospective 6-year study. Diabetes Res Clin Pract 1995, 28:103-117. 29. Robertson R, Zhou H, Zhang T, Harmon J: Chronic oxidative stress as a mechanism for glucose toxicity of the beta cell in type 2 diabetes. Cell Biochem Biophys 2006, in press.
Current Opinion in Pharmacology 2006, 6:615–619
Current Opinion in Pharmacology 2006, 6:615–619 This review comes from a themed issue on Endocrine and metabolic diseases Edited by Antoine Bril and Alain Ktorza Available online 10th October 2006 1471-4892/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coph.2006.09.002
Introduction This brief review provides a short historical retrospective on the concept that oxidative stress might adversely affect pancreatic islet function and insulin action, and focuses more intensely on recent studies suggesting molecular mechanisms by which excessive concentrations of reactive oxygen species (ROS) can cause pancreatic islet b cell dysfunction and impair insulin action. The rationale for examining oxidative stress in diabetes stems from the large number of reports documenting excess levels of biochemical markers in the blood, urine and pancreas of type 2 diabetic patients. The importance of a better understanding of these phenomena lies in the possibility that ancillary antioxidant therapy in such patients could provide a layer of protection against incomplete normalization of glycemia by conventional drugs, such as sulfonylureas, insulin sensitizers and exogenous insulin.
Pathogenesis of type 2 diabetes: b cell dysfunction and insulin resistance Type 2 diabetes is usually the product of two distinct abnormalities: abnormal b cell function and decreased insulin sensitivity. It appears that type 2 diabetes is www.sciencedirect.com
primarily a genetic disease, based on its strong familial association and high concordance rates in identical twins [1]. However, no single gene has been identified that is common to a general population of type 2 diabetic patients, leading to the conclusion that this must be a polygenic disease [2–4]. Most type 2 diabetic patients are obese, and obese patients generally have resistance to the actions of insulin on liver, muscle and fat tissues — the major targets for the beneficial effects of insulin. Environmental influences also play a major role by enhancing the phenotypic expression of genes that place individuals at risk for diabetes. This is becoming increasingly apparent as witnessed by the recent epidemic proportions of new-onset type 2 diabetes in cultures such as American Indian, African American, Latino, and Alaskan American. Environmental precipitants that are common to these cultures include obesity, insufficient physical activity, and excessive carbohydrate intake. However, only a minority of obese patients develops diabetes, and 20% of type 2 diabetic patients are not obese, emphasizing that obesity does not cause diabetes; rather, it contributes to the phenotypic expression of genes that predispose the individual to type 2 diabetes. These clinical facts point to the conclusion that the initial lesion in type 2 diabetes probably involves genetically determined diminution of intrinsic b cell function, which is thus unable to adequately meet the challenge of states of insulin resistance, such as obesity. Consequently, the b cell is continually called upon to secrete insulin because of unresolved hyperglycemia, and this stress gradually causes b cell deterioration and accelerated apoptosis [5]. Both b cell dysfunction and insulin resistance work in concert to cause further deterioration of insulin secretion and increase insulin resistance (Figure 1). Nonetheless, it is interesting to consider that not all lean type 2 diabetic patients are insulin resistant, and that patients with cystic fibrosis and type 2 diabetes are characteristically insulin sensitive [6].
Reactive oxygen species: physiology versus stress Many forms of ROS exist, which in physiologic concentrations are thought to support physiologic functions such as gene transcription, mitochondrial function and leukocyte function. However, when concentrations of ROS reach excessive levels, they can cause structural and functional damage to proteins, lipids and DNA. With regard to the pancreatic islet, reports from studies of rodent islets have emphasized the low levels of intrinsic antioxidant enzyme mRNA, proteins and activities for superoxide dismutases, catalase and glutathione peroxidase [7,8]. This scenario sets the islet at greater risk of Current Opinion in Pharmacology 2006, 6:615–619
616 Endocrine and metabolic diseases
Figure 1
The inter-relationship between defective b cell function and insulin resistance that causes type 2 diabetes. Resultant hyperglycemia and increased lipogenesis create a lipotoxic milieu that worsens both b cell function and insulin resistance.
oxidative damage by ROS that are generated locally and reach abnormally high concentrations. A caveat regarding this line of reasoning is that most published studies have involved male animals only, and only rarely have human islets been examined [9]. This is relevant because female rodents [10,11] are characteristically more protected against development of type 2 diabetes, with evidence suggesting that estrogens are protective against this disease [12]. Thus, more information is needed regarding islet antioxidant enzyme levels in male rodents and in human islets before it can be concluded convincingly that all pancreatic islets are disproportionately at risk for oxidative damage.
Glucolipotoxicity in the b cell and oxidative stress The common findings of elevated glucose and lipid levels in the blood of diabetic patients led to the hypotheses of glucose toxicity [13] and lipotoxicity [14]. Relatively more information has been published about biochemical pathways through which elevated glucose concentrations can generate excessive levels of ROS (reviewed in [15]). These include glycolysis and oxidative phosphorylation; methylglyoxal formation and glycation; enediol and aketoaldehyde formation (glucoxidation); diacylglycerol formation and protein kinase C activation; glucosamine formation and hexosamine metablolism; and sorbitol metabolism. Conceptually, as b cells are exposed to high glucose concentrations for increasingly prolonged periods of time, glucose saturates the normal route of glycolysis and increasingly is shunted to alternate pathways, such that ROS are generated from distinct metabolic processes within and outside the mitochondria. Reports also indicate that excessive levels of palmitate are associated with abnormal islet function (especially in the presence of high glucose concentrations), which leads to excessive lipid esterification that, in turn, can generate ceramide, thereby increasing oxidative stress [14,16,17]. It seems unlikely, Current Opinion in Pharmacology 2006, 6:615–619
however, that circulating lipid itself, such as triglyceride or cholesterol, would be responsible for damaging islet tissue. It seems more likely that excessive circulating glucose levels lead to accelerated de novo synthesis of islet lipid. One mechanism by which glucose might contribute to liptoxicity is by virtue of its ability to drive synthesis of malonyl CoA, which inhibits b-oxidation of free fatty acids. This in turn shunts free fatty acids towards esterification pathways, thereby forming triglyceride, ceramide and other esterification products [17,18]. The relationship between glucose toxicity and lipotoxicity was examined in a study of isolated islets and in diabetic rodents who had hyperglycemia and hypertriglyceridemia. In the islet study, it was demonstrated that high concentrations of palmitate do not by themselves harm islet function whereas, in the combined presence of high glucose and high palmitate, insulin gene expression and insulin secretion was decreased [16]. Similarly, it was found in the animal studies that treatment with a lipidlowering drug (bezafibrate) did not protect the animals from worsening of islet function and diabetes, whereas a glucose-lowering drug (phlorizin) was successful [19]. This suggested that lipotoxicity requires concomitant hyperglycemia to damage islet function, whereas glucose toxicity can exert harmful effects on the islet in the absence of elevated circulating triglyceride. One molecular mechanism of action through which chronic hyperglycemia can cause worsening b cell function is via decreased protein expression of two important transcription factors: Pdx-1 and MafA ([15]; Figure 2). Both proteins are critical for normal insulin gene expression, as their absence or mutation of their DNA binding sites on the insulin promoter leads to decreased insulin Figure 2
Sequence of events that begins with chronic exposure to hyperglycemia and leads to progressive b cell failure. In a model of glucose toxicity — the b cell line HIT-T15 — chronic passaging of cells for many months in supraphysiologic glucose concentrations causes gradual loss of important transcription factors (Pdx-1 and MafA), which results in decreased insulin promoter activity, decreased insulin synthesis and content, and decreased insulin secretion. www.sciencedirect.com
Oxidative stress and impaired insulin secretion in type 2 diabetes Robertson 617
mRNA levels, insulin content and insulin secretion [20,21]. Studies in the b cell line HIT-T15 demonstrated normal pdx-1 gene transcription but not expression [20], along with normal mafA gene expression at the mRNA level but not at the protein level [22]. Thus, the molecular defects leading to decreased b cell Pdx-1 levels is post-transcriptional in nature, whereas the mechanism leading to disappearance of normal MafA levels is posttranslational. The possibility that oxidative stress is responsible for the decreased levels of Pdx-1 and MafA was highlighted by studies showing that antioxidant treatment of b cell lines and rodent models of type 2 diabetes protected against deterioration of insulin gene expression induced by exposure to high glucose concentrations [23–25]. N-acetylcysteine (NAC), a drug commonly used in these studies, provides direct antioxidant actions, as well as supplying the limiting substrate cysteine for glutathione synthesis via the rate-limiting enzyme g-cysteine ligase. Treatment with NAC protected against decreases in Pdx-1 and MafA caused by high glucose concentrations in vitro, and also protected against loss of insulin gene expression and insulin secretion in vivo.
Glucolipotoxicity in non-b cells, insulin resistance and oxidative stress As this is a short review that centers primarily on the islet and the number of references is limited, only brief mention can be given to well-developed research areas involving interplay among oxidative stress, insulin resistance and the secondary complications of diabetes. Insulin resistance accompanies the development of obesity, pregnancy, excess growth hormone and glucocorticoid levels, and lack of exercise. Although clinical and laboratory methods for quantifying insulin resistance are well established, and molecular mechanisms for insulin action are being identified, explanations at the molecular level for resistance to the actions of insulin are generally less well developed. Nonetheless, ample evidence exists that oxidative stress plays a role in insulin resistance and in the cellular damage of tissues that leads to the late complications of diabetes (for review, see [26]). Abnormal levels of free fatty acids, tumour necrosis factor-a, leptin and resistin are frequently found in obese individuals and are prominently mentioned as potential mediators of insulin resistance [26]. Of these, free fatty acids have been reported to impair insulin action via oxidative stressinduced activation of nuclear factor-kB [26]. Secondary complications of diabetes involve microvascular and macrovascular changes that lead to retinopathy, nephropathy, neuropathy and damage to critical blood vessels, such as the coronary arteries. Stress-activated signaling pathways that might play a role in these phenomena are those involving protein kinase C, nuclear factor-kB, p38 mitogen-activated protein kinase, advanced glycosylation end-products and their receptors, and amino-terminal www.sciencedirect.com
JUN kinases, among others [26]. Antioxidant agents that have been reported to reduce insulin resistance, as well as secondary complications of diabetes, include lipoic acid, NAC, aminoguanidine, vitamine C, vitamin E, resveratrol, silymarin and curcumin [26]. Vascular endothelial growth factor has been proposed as an initiator of diabetic complications, whereas antioxidants have been reported to inhibit advanced glycosylation end-product-induced expression of vascular endothelial growth factor [26]. Thus, there is ample reason to consider that glucoliptoxicity via chronic oxidative stress contributes to both insulin resistance and secondary complications of diabetes involving multiple target tissues.
Antioxidant therapy and the islet Mention has already been made of several antioxidants that have been used in clinical studies which protected against b cell dysfunction and insulin resistance associated with type 2 diabetes. A careful distinction must be made, however, between prevention of the disease and protection against its complications. There is little, if any, evidence that oxidative stress causes type 2 diabetes, which appears to be primarily a polygenic disease involving intrinsic defects in islet function. Rather, the role for oxidative stress appears to be as one mechanism in the second wave of pathogenic forces that arise from chronic hyperglycemia and increased lipogenesis after type 2 diabetes is established. The hypothesis arising from this consideration is that any treatment that provided truly perfect control of glucose and lipid metabolism would Figure 3
Protective effects of antioxidants against b cell deterioration in an animal model of hyperglycemia. The natural history of the development of marked hyperglycemia in Zucker Diabetic Fatty (ZDF) rats can be ameliorated by treating the rats at 6 weeks of age with the antioxidants aminoguanidine (AG) or n-acetylcysteine (NAC) [23]. Control glucose data are shown from Zucker Lean Control (ZLC) rats that do not develop diabetes. Data adapted from [23]. Current Opinion in Pharmacology 2006, 6:615–619
618 Endocrine and metabolic diseases
prevent the complications of diabetes, as suggested by several therapeutic trials with conventional drugs used for treatment of hyperglycemia [27,28]. However, as it is not yet possible to establish constant normoglycemia with available drugs, there could be a role for antioxidants as ancillary treatment to provide protection against residual hyperglycemia during conventional therapy. In this manner, the toxic effects of residual hyperglycemia, especially post-prandially, might be neutralized, and thereby the secondary adverse effects of residual hyperglycemia on islet function, as well as other secondary complications of diabetes, might be avoided. As mentioned above, several studies in animals lend credence to this approach; for example, in the Zucker Diabetic Fatty rat [24], as well as the db/db mouse [25], treatment with NAC ameliorated the course of type 2 diabetes such that abnormal changes in glycemia and insulin gene expression were not as severe (Figure 3). To date, no trials have been reported in humans to test this hypothesis. Preliminary data from a study of NAC in combination with oral agents [29] suggests that such trials are warranted.
Conclusions Although ROS in small concentrations support physiologic functions, at abnormally high concentrations they cause both biochemical abnormalities and structural damage to tissue. Chronic hyperglycemia increases local concentrations of ROS, and thereby induces chronic oxidative stress. This sequence of events provides one explanation for many of the secondary complications of hyperglycemia, including ever-worsening abnormalities in the pancreatic b cell in type 2 diabetes. Other important defects include decreased levels of the transcription factors Pdx-1 and MafA, which are essential to normal insulin gene expression and, therefore, insulin synthesis, insulin content and insulin secretion. Interventional trials using antioxidants in animal models partially prevent the adverse consequences of hyperglycemia on insulin gene expression and islet function, suggesting that human trials of antioxidants in patients with impaired glucose tolerance and postprandial hyperglycemia might offer protective effects against unrelenting b cell deterioration in type 2 diabetes.
Acknowledgements Supported in part by NIH grant NIDDK RO-1 38325.
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