Abstract: Mainstays of therapy for type 2 diabetes involve drugs that are insulin-centric, i.e., they are designed to increase insulin secretion and decrease insulin resistance. The usual clinical course for people so treated is to have initially improved glycemic control but over time a need for intensification of drug-based treatment of hyperglycemia. The mechanism for this unrelenting deterioration of β-cell function is related to chronic oxidative stress. This suggests that drug discovery should not exclusively focus on insulin-centric targets, but also include glucose-centric strategies, such as antioxidant protection of the β-cell. This may facilitate repair of β-cells undergoing damage by oxidative stress secondary to chronic hyperglycemia.

Limitations to Conventional Insulin-centric Treatment

The search for new drugs for managing hyperglycemia in patients with diabetes is intense, and it should be. We are in the midst of an international diabetes epidemic even though diabetes is in large part preventable (Gerstein et al., 2002; Knowler et al., 2009; Li et al., 2008; Lindstrom et al., 2006; Ramachandran et al., 2006; Robertson et al., 2010). All nations recognize that the secondary complications of diabetes involving eyes, kidneys, nerves, and blood vessels are difficult and costly to manage. The damage diabetes causes can be devastating and is the leading cause of blindness, kidney failure, non-traumatic amputation, and heart disease. The villain responsible for all the damage is chronic hyperglycemia which is not preventable despite a panoply of available drugs designed to normalize blood glucose levels. To be certain, they are partially successful in lowering glucose levels with improved levels of hemoglobin A1c (HbA1c), a fraction of hemoglobin that reflects the average level of blood glucose over the past 90 days. But almost normal is not completely normal. No drug, including insulin, is capable of keeping glucose levels in the normal range all the time. No drug can replace the exquisitely tuned rates of insulin secretion from the native β-cell that finely control glucose levels. Attempts with drugs to control blood glucose levels within the normoglycemic range often force it into the hypoglycemic range, which is associated with severe problems of decreased mentation and sometimes frank coma. Yet, the pharmaceutical industry persists in its efforts to create newer sulfonylureas (drugs that stimulate insulin secretion), insulin analogs, insulin-sensitizers, and most recently drugs based on the actions of glucagon-like peptide-1 (GLP-1), a hormone that enhances glucose-induced insulin secretion from pancreatic islet β-cells. This approach seems rational because the β-cell is at the center stage in the pathogenesis of all forms of diabetes. Currently, the general goal is to create drugs that either stimulate β-cells to secrete more insulin, to increase sensitivity of targets to insulin action (liver, fat, muscle), or to replace insulin by giving exogenous insulin injections. These therapeutic approaches are in essence insulin-centric.

Glucose-centric Management of Diabetes Is Underdeveloped

The problem with insulin-centric therapy is that it is incomplete. For nearly 90 years this has been the sole approach to discovering new drugs for diabetes. It has garnered results that have been life-saving and excellent in many ways, but these drugs appear to have reached their limit of efficacy without successfully normalizing glucose levels. Not all patients who are treated achieve HbA1c levels in an acceptable range. After initial impressive responses, secondary failure to these drugs within a few years is the typical outcome (UK Prospective Diabetes Study Group, 1998). Why is this? The view I favor is that our therapeutic approach does not take the main villain into consideration. The villain is hyperglycemia itself, the primary consequence of the disease.

What do I mean by glucose-centric drugs? Glucose excess, not insulin deficiency, is what causes the secondary complications of diabetes. Certainly it is insulin deficiency that causes hyperglycemia, but hyperglycemia causes the complications. I believe it is time to accept that we may never completely avoid hyperglycemia by insulin-centric drugs and to refocus our energies on combating the adverse effects of residual hyperglycemia after conventional drug treatment has reached maximal benefits. Which leads to the question: what is the mechanism of action of hyperglycemia-driven tissue damage? One hypothesis for the mechanism of glucose toxicity is chronic oxidative stress (Robertson, 2004). Glucose normally forms reactive oxygen species (ROS) via oxidative phosphorylation. This pathway spins off physiologic levels of ROS which in normal levels support important physiologic functions. However, when glucose levels become excessive in blood, ROS accumulate in excessive levels that are capable of causing tissue damage. As the oxidative phosphorylation pathway becomes saturated, ROS are also formed by other alternative pathways of glucose metabolism. At this point, ROS create states of toxicity. It is a matter of good radicals gone bad. This challenges us to devise new strategies to combat chronic oxidative stress in diabetic patients as an ancillary approach to protect them from residual hyperglycemia that is not prevented by conventional treatment.

The β-cell and Chronic Oxidative Stress

The β-cell is an unwitting participant in this web of events. For unknown reasons, this cell has the lowest intrinsic antioxidant enzyme expression of any cell in the body. One can only speculate why this is so. Perhaps physiologic β-cell function requires a higher intrinsic level of ROS than most cells (Leloup et al., 2009; Pi et al., 2007) and has unknown means other than antioxidant enzymes to regulate ROS levels. The only antioxidant enzymes appreciably expressed in β-cells are superoxide dismutases (Grankvist et al., 1981; Tiedge et al., 1997), which actually form the oxidant hydrogen peroxide. Missing are catalase and glutathione peroxidase (GPx), which serve to catabolize hydrogen peroxide and lipid peroxides to form water and oxygen. Clearly, the islet β-cell was not designed to withstand excessive levels of ROS. This sets up a curious conspiracy. The pathogenesis of diabetes involves genomic as well as environmental forces outside of the β-cell. As the β-cell begins to fail, hyperglycemia worsens, and elevated ROS formation occurs, leading to increased damage of tissues, including β-cells. Once this vicious cycle is in play, relentless deterioration of β-cell function is inevitable (Robertson, 2009). This is where glucose-centric treatment becomes essential to protect β-cells and is ripe for pharmaceutical intervention.

Management of Glucose Toxicity by Drugs That Combat Oxidative Stress

A plethora of scientific reports exist that demonstrate protection of β-cells against oxidative stress by antioxidant drugs in β-cell lines and isolated pancreatic islets, rodents and humans (Hussain, 2007; Kaneto et al., 1999; Lortz et al., 2003; Tanaka et al., 1999; Yamamoto et al., 2008). These experiments usually involve antioxidants that are far more potent than commonly available vitamins taken in conventional doses. Some scientists eschew the use of antioxidants for protection against diabetes because of well known but hardly relevant reports that vitamins given in conventional doses for treating heart disease do not favorably affect glucose levels in diabetic participants. This is not surprising since vitamins are not potent antioxidants and have never been used in trials designed to manage glycemia as the primary endpoint. Potent antioxidants, such as N-acetylcysteine and aminoguanine, have been clearly demonstrated in vitro to provide protection of β-cells against potent oxidants, such as ribose and prolonged exposure to supraphysiologic concentrations of glucose.

Figure 1. Comparison of glycemic courses in db/db animals with and without β-cell-specific overexpression of glutathione peroxidase (GPx) and with wild type C57 non-diabetic animals. Overexpression of GPx delayed the initial onset and decreased the peak level of hyperglycemia in db/db animals. Thereafter, at 20 weeks of age, GPx overexpression resulted in return of glucose levels to values not significantly different from the 4 week values. Numbers in parentheses indicate numbers of animals in the three groups. Adapted from Harmon et al., 2009.

Recently, our laboratory has overexpressed GPx specifically in pancreatic islet β-cells in db/db mice (Harmon et al., 2009). This rodent is a classic genetic model of type 2 diabetes that almost inevitably develops profound hyperglycemia in a matter of weeks after birth, usually beginning at the 5th week and maximally by the 10^th week of age. This time course was dramatically different in animals we generated to overexpress GPx in β-cells specifically (Figure 1). They initially became hyperglycemic, although in a delayed manner and with a lower maximal level of glycemia compared with wild type animals. By 10-20 weeks of age the transgenic animals gradually auto-regulated their glucose levels so that they reached levels not significantly greater than the levels observed at 4 weeks of age. Islet imaging revealed intranuclear absence of a critical insulin gene transcription regulator, termed MafA, in the wild type db/db animals at 20 weeks of age (Figure 2). In contrast, the GPx-overexpressing animals had abundant intranuclear MafA at 20 weeks. No differences were observed in staining for PDX-1, another important regulator of insulin gene expression. These changes in MafA are highly relevant to the loss of insulin synthesis and content in the db/db animals. Whether primary loss of MafA expression caused loss of insulin content and hyperglycemia, or whether hyperglycemia caused loss of MafA expression and loss of insulin content and secretion, are unresolved questions. That the former is a possibility is supported by our observation that mutation of MafA binding sites on the insulin promoter severely inhibits insulin gene reporter expression (Figure 3). That the latter is a possibility is supported by our observation that β-cell lines chronically cultured in supraphysiologic glucose concentrations undergo loss of MafA protein.

Figure 2. Immunoflorescence microscopic merged images of insulin and MafA staining. Left panel: Presence of intranuclear MafA in transgenic db/db-GPx animals at 20 weeks of age. Right panel: Absence of intranuclear MafA in non-transgenic db/db animals of 20 weeks of age. Adapted from Harmon et al., 2009.

The recovery of nearly normal glucose levels in db/db-GPx animals at 20 weeks was surprising because no insulin-centric drugs were used; no sulfonylureas, no GLP-1 mimetics, no insulin sensitizers, and no exogenous insulin. Moreover, no evidence was found for differences between the wild type and GPx-overexpressing animals at 20 weeks in body weight, β-cell apoptosis, or β-cell regeneration. This leads to the conclusion that the antioxidant action of GPx expression probably protected β-cells that were foundering at 10 weeks by mopping up harmful ROS caused by the initial hyperglycemia. Not unexpectedly, GPx did not prevent onset of diabetes. The db/db mouse is a genetic model of type 2 diabetes for which there is no evidence that oxidative stress contributes to the onset of the genetic disease. It may seem curious that there was a delay in the beneficial effects of GPx, which did not take full effect until after 10 weeks of age. However, this makes sense because the GPx transgene we used was driven by the insulin promoter, which is stimulated by glucose. Our thesis is that as hyperglycemia developed, the insulin promoter was increasingly turned on, which increased GPx expression in the β-cell of the animals. This resulted in amelioration of oxidative stress and repair of existing β-cells with a return to full function, providing insulin secretion, and normalization of blood glucose levels.

Figure 3. Molecular mechanisms of action for glucose toxicity involve abnormalities of intranuclear PDX-1 and MafA binding. Left panel: normal entry of PDX-1 and MafA proteins into the nucleus of a β-cell where they bind to A and C sites, respectively, of the promoter region of the insulin gene to support synthesis of insulin mRNA. Right panel: Glucose toxicity of β-cells via chronic oxidative stress is initially associated with absence of intranuclear MafA (as in Figure 2) and diminished insulin synthesis. Later defects include absence of intranuclear PDX-1 as well as decreased cytoplasmic levels of MafA and PDX-1. Adapted from Robertson, 2009.

Lessons from the db/db-GPx β-cell-specific Overexpressing Mouse

The experiment described above ratifies and extends earlier in vitro and in vivo data demonstrating that antioxidant drugs protect islet β-cells against oxidative stress caused by prolonged supraphysiologic glucose levels. This line of research opens the door for the pharmaceutical industry to think seriously about designing drugs that will protect chronically hyperglycemic patients against glucose toxicity mediated by oxidative stress. This approach would finesse the need to keep searching for the next generation of pharmaceutical insulin-centric agents, which may not be any more effective than the current generation. Addition of potent antioxidants, ideally specific for β-cells, to conventional drug treatment for diabetes might provide protection as well as repair of β-cells in diabetic patients. As with the db/db mouse, this might even allow sufficient return of β-cell function so that conventional treatment could be decreased or even discontinued. That this is a reasonable hope follows from the evidence that β-cell neogenesis occurs in diabetic humans and is seriously offset by accelerated apoptosis (Butler et al., 2003). Amelioration of oxidative stress might slow down apoptosis at the same time it repairs existing β-cells, which could lead to improvement of insulin secretion independently from conventional therapy.

(Correspondence should be addressed to R. Paul Robertson, M.D., Pacific Northwest Diabetes Research Institute, 720 Broadway, Seattle, WA 98122, USA.)

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