From the Department of Medicine, University of Chicago, Chicago, Illinois
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ABSTRACT |
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Insulin-secreting ß-cells are subject to injury from oxidative stress. Formation of reactive oxygen species (ROS) such as superoxide anion (O2), hydrogen peroxide, hydroxyl radicals, and the concomitant generation of nitric oxide have been implicated in ß-cell dysfunction or cell death caused by autoimmune attack and actions of cytokines in type 1 diabetes. ROS have also been associated with the impairment of ß-cell function in type 2 diabetes (14). Compared with many other cell types, the ß-cell may be uniquely at high risk of oxidative damage and has an increased sensitivity for apoptosis (2,3,5).
Investigations (13,6) implicating ROS in ß-cell death or damage have, for the most part, relied on the protective effects of antioxidants, scavengers, and overexpression of antioxidant enzymes in islets or transgenic mice to reduce the destructive influence of some oxyradicals. However, elevated glucose concentrations are thought to alter metabolism, create oxidative stress, and induce apoptosis in many cell types in addition to glucose-responsive ß-cells (2,5). Why should ROS generation in ß-cells be more dangerous than in other cell types?
We have analyzed the existing data on mechanisms of glucose-dependent insulin secretion (GDIS) in ß-cells, ROS production, oxidative stress, and apoptosis and propose that the same pathways can dramatically influence oxidative stress, apoptosis, and insulin production.
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GDIS and adenine nucleotide regulation. |
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We have recently developed (10) a computer model of regulation Ca2+ and ATP concentrations in pancreatic ß-cells. However, our modeling studies suggest that the current understanding of adenine nucleotide regulation in ß-cells is incomplete (L.E.F., L.H.P., unpublished observations). In particular, the effect of substrates that markedly enhance insulin secretion, including glucose, on ATP concentration is small, but the ratio of total ATP to total ADP increases considerably in most studies (1113).
In contrast to ATP, only a small fraction of total cellular ADP is free (11,14). Several measurements of free ADP have been performed in ß-cells. Ghosh et al. (15) found in ß-cellrich rat pancreatic islet cores that an increase of glucose from 4 to 8 mmol/l led to a decrease of free ADP from 44 to
31 µmol/l (pooled data from Table 5 in Ghosh et al. [15]). ATP concentration increased only insignificantly following glucose challenge in these experiments. Ronner et al. (12) found (in clonal ßHC9 insulin-secreting cells) that increased glucose concentration was associated with an exponential decline in the concentration of free ADP from
50 µmol/l at 0 mmol/l glucose to
5 µmol/l at 30 mmol/l glucose, whereas the concentration of ATP remained nearly constant. These data suggest that a sharp decrease of free ADP with only a relatively small change in ATP concentrations is the characteristic feature of the response of ß-cells to glucose stimulation. The functional necessity of this change in the ATP-to-ADP ratio can be understood from a consideration of KATP regulation (for details, see the review in ref. 9).
The activity of KATP channels decreases when pancreatic ß-cells are exposed to increasing concentrations of glucose. However, as discussed above, only small changes in ATP concentrations were found following exposure to increased glucose concentrations. This cannot by itself induce sufficient closure of KATP channels. However, decreased free ADP concentrations in the physiological range can close KATP channels at constant ATP concentration (7,16,17). This means that a considerable increase in glycolytic flux and a sharp decrease in free ADP levels could be the necessary conditions leading to closure of KATP channels following glucose challenge and to insulin secretion.
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Dependence of ROS production in ß-cells on the GDIS mechanism. |
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The principal source of ROS in most mammalian tissues is the ETC itself (2022). Superoxide anions are generated by single-electron reduction of molecular oxygen in complexes of the mitochondrial ETC. ROS production depends on the concentration of the intermediate metabolites of these complexes because the ETC carriers in a more reduced state have the property of donating electrons to oxygen (2022). The reduced state of the ETC carriers can be achieved by increased production of reducing equivalents in mitochondria or by decreased electron transfer capability on (or after) these carriers (2022).
ß-Cells have a sensitive system, starting with glucokinase, for initiating the response to physiological changes in glucose concentration. Therefore, in contrast to most other mammalian cell types, increased glucose concentration stimulates a steeply increased glycolytic flux in ß-cells, followed by a sharp stimulation in the production of reducing equivalents (7,23). This means that this part of the GDIS mechanism could lead itself to an enhancement of ROS production in pancreatic ß-cells following glucose challenge. Increased fatty acid oxidation and the addition of some intermediate metabolites could also lead to additional production of reducing equivalents.
However, decreased electron transfer capability in the ETC can also be an important mechanism affecting ROS production. Because ETC is coupled to ATP synthesis through membrane potential (), the electron transport rate and, consequently, the rate of superoxide production will also depend on
. The increased
decreases electron transport capability in ETC, leading to a reduced state of the carriers and increased ROS production (2022). It was shown experimentally (2426) and confirmed by simulation with the corresponding mathematical model (27) that the rate of superoxide production increases dramatically with increased
>140 mV, when the rate of electron transport is restricted by increased
.
Since is used to make ATP from ADP and Pi, driven by proton movement back through the ATP synthase complex, its value also depends on the ATP production rate and, in particular, on free ADP concentration. The mitochondrial oxidative phosphorylation rate increases with increased free ADP concentration, with an apparent half-saturated concentration of
2045 µmol/l (2830). Therefore, a decrease in free ADP concentration leads to decreased ATP production, which in turn increases
and, correspondingly, ROS production. Results of mathematical modeling of coupled mitochondria show that
can increase from 120 to
200 mV as ADP decreases from 40 to 15 µmol/l (Fig. 3A from Demin, Westerhoff, and Kholodenko [27]). This explains the sharp increase in ROS production with decreased ADP concentration (Fig. 2). Modeling data were supported by the finding that a decrease in steady-state
level and a corresponding fall in H2O2 generation rate were both obtained after the addition of progressively increasing amounts of uncoupler (SF6847) or ADP into a mitochondrial suspension (25). These data lead to the conclusion that decreased ADP concentration can cause a considerable increase in ROS production (20,27). This idea was recently confirmed for ß-cells by the demonstration that ADP inhibited ROS generation in permeabilized MIN6 cells (31).
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To make matters worse, ß-cells have relatively low levels of free radicaldetoxifying and redox-regulating enzymes such as superoxide dismutase, glutathione peroxidase, catalase (2,3,32), and thioredoxin (1). The reasons for this are unclear. Because ROS are involved in different physiological processes as mediators in signal transduction pathways (33), it was hypothesized that ROS are involved in some signaling pathways that take part in the insulin-secretion mechanism (18). In any case, the limited scavenging systems suggest that enhanced ROS concentrations in ß-cells may occur due to both decreased scavenging systems and ROS overproduction.
In support of this hypothesis we recently reported an estimation of ROS using an optical method. We found that stimulation with 10 mmol/l glucose (from an initial 2 mmol/l) increased nearly twofold the O2 production rate in pancreatic ß-cells from Zucker lean rats, confirming the possibility of abrupt increases in O2 production with increased glucose (18). A similar increased ROS production rate was obtained by Sakai et al. (19) at increased glucose concentrations in a pancreatic ß-cell line (MIN6) and in human islets. These studies are the first to measure the production of ROS in response to glucose in the ß-cell.
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Manifestations of oxidative stress and apoptosis. |
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Free radicals in cells (including the ß-cell) may directly damage proteins, lipids, and nucleic acids, leading to mitochondria and cell dysfunction and death (22,3335). In our experiments, the mitochondria were generally short and swollen in islets with the highest ROS production from the Zucker diabetic fatty (ZDF) rat, in contrast to Zucker lean control (ZLC) rat islets (18).
In addition to their ability to directly damage cellular macromolecules, ROS may also activate intracellular signaling pathways that lead to cell dysfunction and apoptosis (4,5,33,34,36). Two principal apoptotic pathways exist in ß-cells: the "intrinsic" pathway initiated by the mitochondria and the "extrinsic" pathway initiated by cell-surface receptors.
The "intrinsic" pathway includes the activation of nuclear factor (NF)-B and additional stress-sensitive targets (5,34). There is some evidence that activation of NF-
B is mostly a proapoptotic event in ß-cells (36). However, in vascular endothelial cells, normalizing mitochondrial superoxide production blocks several major pathways leading to hyperglycemic damage (including NF-
B activation), and it was suggested that ROS production in mitochondria is a causal link between elevated glucose and the main pathways responsible for hyperglycemic damage (37). It would appear reasonable that these pathways are also activated by ROS in the ß-cell (2), but this has not been directly confirmed.
The "extrinsic" pathway includes cytokine signaling and is considered in detail in a recent review by Donath et al. (4). However, the question "What makes the ß-cell so sensitive to proinflammatory cytokines?" remains open (4). It has been suggested that glucose-induced ß-cell apoptosis involves the induction of both free oxygen radicals and the synthesis of proinflammatory cytokines, especially interleukin-1, activating proapoptotic pathways (5). Apoptosis may also be induced by a combination of macromolecular and mitochondrial damage, mainly due to ROS action (35). Altered mitochondria function plays a prominent role in the induction of apoptosis in several cellular models (33,35) as well as in the ß-cell line Ins-1 (38). If this is the case in the ß-cell, then the specific ß-cell sensitivity to proinflammatory cytokines may be explained by the combination of ROS overproduction and insufficient scavenging systems.
Interestingly, transfection of a glucagon-producing rat cell line with the pancreatic duodenal homeobox transcription factor leading to an insulin-producing ß-cell phenotype resulted in a higher sensitivity to cytokine toxicity (39). In this case, the development of insulin-secretion mechanisms led to enhanced in vitro sensitivity to cytokines.
An elevation of intracellular Ca2+ through voltage-gated Ca2+ channels is an integral part of the GDIS mechanism (see GDIS AND ADENINE NUCLEOTIDE REGULATION) (Fig. 1). However, increased intracellular Ca2+ is also believed to stimulate mitochondrial generation of ROS (26). Voltage-gated Ca2+ channels are also likely to play an activating role in ß-cell apoptosis, although the molecular mechanisms remain to be described (4). Hence, an increase in cytoplasmic Ca2+ concentration and an activation of voltage-gated Ca2+ channels are additional specific stages in GDIS, which may share responsibility for an increase of oxidative stress and/or for a mediation of apoptosis.
We can conclude that at least three stages of the GDIS mechanism (increased glycolytic flux, decreased ADP concentration, and increased intracellular Ca2+concentration) could lead to a dramatic increase in the development of oxidative stress and apoptosis in pancreatic ß-cells. We can name this connection the GDISROS hypothesis. This GDIS
ROS hypothesis provides a testable framework to explain how ß-cells may be uniquely at high risk for oxidative damage and apoptosis.
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Dual role of the GDIS mechanism. |
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This dual role of the GDIS mechanism might hinder investigations since an initial increase in the insulin secretion rate can at first mask eventual detrimental effects of oxidative stress on insulin production. However, there is an essential difference in the temporal development of these processes. Insulin secretion changes relatively quickly and oxidative stress seems to develop more gradually and may be revealed only after several days of exposure to metabolic secretagogues (2,40). Therefore, progressive injury of ß-cell function by the same effectors that increase GDIS quickly could be considered a characteristic feature of oxidative stress activated by the GDIS mechanism itself. For example, chronic exposure to elevated glucose concentrations may cause damage to ß-cells through mechanisms involving oxidative stress (2,3,34,40). This reinforces the idea that glucose initially activating insulin secretion can also injure ß-cell function with time. However, the idea that glucose-induced ROS generation is responsible for ß-cell glucose toxicity remains a testable speculation because glucose-induced ROS generation does occur with a brief exposure to physiologically relevant elevated glucose concentration (18), whereas glucose toxicity does not.
Lipotoxicity can also develop in ß-cells in a similar fashion to oxidative stress at elevated glucose. On a short-term basis (<24 h), fatty acids stimulate GDIS in part by causing an increase in the production of reducing equivalents due to ß-oxidation and additional acyl-CoA mitochondrial oxidation (7). Fatty acids may also increase Ca2+ mobilization from the endoplasmic reticulum (41). This can lead to decreased ADP levels, increased cytoplasmic Ca2+, and increased insulin production. In contrast, chronic exposure (>24 h) of ß-cells to fatty acids leads to a reduction in GDIS (2,40). Current explanations (2,42) of this lipid-induced toxicity in ß-cells certainly involve the effects of oxidative stress. Hence, lipotoxicity appears to be at least partly a manifestation of supplementary ROS production induced by additional production of reducing equivalents in mitochondria pari pasu with fatty acid metabolism.
Direct data on mitochondrial O2 production rates obtained in our laboratory also confirms the possibility that ß-cells are subject to oxidative stress at increased concentrations of fatty acids. Superoxide production in ZDF rat islets was significantly higher than in ZLC rat islets under resting conditions (with 2 mmol/l glucose), and the overproduction of superoxide was associated with perturbed mitochondrial morphology in ZDF rat islets (18). Abnormal mitochondrial morphology in ZDF rat islets and its reversal by systemic treatment with troglitazone were also observed by Higa et al. (43). Because ZDF rat islets accumulate triglycerides (43), these changes can be explained by increased ROS production as a result of increased content of free fatty acids in these ß-cells.
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Dual role of ß-cell mitochondria uncoupling. |
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This dual role of uncoupling can be illustrated by considering the principal ß-cell uncoupling protein (UCP)2, which catalyzes a regulated proton leak across the mitochondrial inner membrane (Fig. 1) (see Saleh, Wheeler, and Chan [44] for review). Indeed, islets from UCP2-deficient mice have an increased ATP level and an enhanced glucose-stimulated insulin secretion compared with control animals (45). On the other hand, overexpression of UCP2 in isolated pancreatic islets results in decreased ATP content, reduced , and blunted glucose-stimulated insulin secretion (46). However, in line with the suggested dual role of mitochondrial membrane uncoupling, overexpression of UCP2 enhanced the resistance of ß-cells toward H2O2 toxicity (47).
The inner mitochondrial membrane KATP channel is another mechanism through which could be regulated. Enhanced K+ uptake through mitochondrial KATP channels would lead to a lower
. This effect could promote a decline in mitochondrial ROS production (48,49). There is as yet no direct data on the role of mitochondrial KATP channels in pancreatic ß-cells; however, one would expect that the openers of mitochondrial KATP will act similarly to UCP2 activation.
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Intracellular inhibition of ROS production can lead to a decrease in GDIS. |
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It also seems likely that ß-cells can increase UCP2 expression to decrease oxidative stress. For example, superoxide increases proton conductance in mitochondria from pancreatic ß-cells, probably via activation of UCP2 (53). Increased glucose induces expression of UCP2 in isolated human islets (54). Chronic exposure of pancreatic islets to free fatty acids, blunting GDIS, is accompanied by increased synthesis of UCP2 (55). These mechanisms of protection from oxidative stress would decrease the rate of ATP production and the corresponding ATP-to-ADP ratio, leading to impaired ß-cell sensitivity to glucose simulation, a characteristic feature of type 2 diabetes.
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Preservation of ß-cell function and islet mass versus enhancement of GDIS: mutually exclusive goals? |
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Because the activation of initial stages of GDIS or increased immediately leads to increased insulin secretion, it is not surprising that these stages are targets for therapeutic intervention. For example, glucokinase plays a key role in initial GDIS stages by catalyzing the phosphorylation of glucose in ß-cells. A new class of antidiabetic agents, mixed-type glucokinase activators that increased both the affinity for glucose and the Vmax, was shown to stimulate GDIS (8,57). A reduction in UCP2 activity was also suggested as a mechanism for significant improvement in insulin secretion (58). We can also suggest that a reduction in KATP activity in the inner mitochondrial membrane should lead to increased insulin secretion rate, as does decreased UCP2 activity. However, such a therapeutic strategy should be used with caution, since according to our proposal an increase in insulin secretion achieved by these approaches could also considerably increase ROS production, leading to oxidative stress.
The concept of "ß-cell rest" as originally developed, perhaps more for amelioration of type 1 than type 2 diabetes, argued that decreased demand on ß-cell function can lead to improvements in insulin secretion and ß-cell viability (2,59,60). Such agents as diazoxide and calcium channel blockers, which reversibly inhibit insulin secretion, have improved ß-cell function both in rodent models of diabetes (61,62) and in humans (63). This beneficial effect could be explained by the decreased ROS production during "ß-cell rest" associated with decreased GDIS activity.
Inhibition of the early stages of GDIS or decreased should also lead to decreased ROS production. Any inhibitor of glycolytic flux, the tricarboxylic acid cycle, fatty acid oxidation, or mitochondrial membrane uncoupling could result in decreased ROS production. For example, this could be accomplished by specific inhibitors of ß-cell glucokinase, by an increase in UCP2 expression, or by openers of mitochondrial KATP channels. However, a decreased insulin secretion rate is the necessary price to pay for these approaches to increasing ß-cell function and survival. For this reason, such methods can predominantly be used when the GDIS mechanism is not the main source of insulin production. This of course can occur following treatment by plasma membrane KATP channel blockers, such as sulfonylureas and meglitinides, which can compensate for the inadequate closure of these KATP channels at reduced ATP/ADP levels, or simply by insulin therapy.
However, plasma membrane KATP channel blockade is accompanied by increased Ca2+ levels in ß-cells, which can itself increase oxidative stress (see DEPENDENCE OF ROS PRODUCTION IN ß-CELLS ON THE GDIS MECHANISM). For this reason, the simplest and potentially most beneficial method to decrease oxidative stress in ß-cells may be that of early use of the above-mentioned GDIS inhibitors, with insulin as necessary. This approach would decrease both glucose levels and the corresponding ROS production. Although this approach has not always been used to an advantage (59), recent studies (60,64) have suggested that early insulin treatment in type 2 diabetes indeed preserved endogenous insulin secretion. Additional intervention with GDIS inhibitors could improve the "ß-cell rest" approach to treatment.
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CONCLUSIONS |
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ACKNOWLEDGMENTS |
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We thank N. Tamarina and D. Jacobson for helpful discussions.
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FOOTNOTES |
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Address correspondence and reprint requests to Louis H. Philipson, Department of Medicine, MC 1027, University of Chicago, 5841 S. Maryland Ave., Chicago, IL 60637. E-mail: l-philipson{at}uchicago.edu
Received for publication October 16, 2003 and accepted in revised form March 15, 2004
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