This study demonstrates that the enzyme Ogg1, which is involved in the repair of oxidative DNA damage, is up-regulated in the islets of patients with type 2 diabetes. This is consistent with the hypothesis that oxidative DNA damage is an important factor in the β-cell failure that plays a role in the pathogenesis of human type 2 diabetes.
We interpret the upregulation of Ogg1 as a response to an increased level of 8-OH-dG DNA, the lesion that it repairs. Increases in Ogg1 activity and expression have been found by a number of investigators [35
]. Although antibodies to 8-OH-dG exist [38
], Ogg1 was used as a surrogate for the amount of oxidative DNA damage. The reason for this is that we found high levels of background staining with the anti-8-OH-dG antibodies, which is not unusual when using streptavidin/biotin detection systems in immunohistochemistry [39
]. Unfortunately, secondary antibodies directly linked to a fluorophore or a detection enzyme were not sensitive enough to detect the 8-OH-dG adduct in our hands.
The mechanism by which Ogg1 protein is increased in type 2 diabetic islets is unknown. The promoter of the Ogg1 gene does not have a TATA box [40
], but Ogg1 is differentially expressed in the rat CNS, and its expression is increased after mouse forebrain ischemia-reperfusion [35
], suggesting that regulation occurs, either at the transcriptional or post-transcriptional level. Unfortunately, there have been no studies of the effects of long-term chronic increases in oxidative DNA damage on Ogg1 expression. The association between the duration of diabetes and Ogg1 levels (Fig. ) suggests that such long-term exposure may be important for Ogg1 up-regulation.
Most of the staining observed was detected in the cytoplasm of the islet cells. As Ogg1 either is sublocalized into the nucleus or the mitochondria, this indicates that βOgg1, the mitochondrial form of Ogg1, accounts for the observed increase. βOgg1 is an alternatively spliced form of Ogg1 that contains a mitochondrial targeting signal in the C-terminal portion of the protein [41
]. Specific upregulation of mitochondrial but not nuclear Ogg1 occurs during aging in rodents [42
]. A specific increase of Ogg1 in islet cell mitochondria is consistent with the idea that increased β-cell mitochondrial oxidative metabolism due to hyperglycemia is a major factor behind the DNA damage. In a study published after this report was submitted, an increased number of 8-OH-dG positive islet cells in human type 2 diabetic subjects was observed [32
]. The staining was found to be mainly nuclear in contrast to the cytoplasmic Ogg1 staining found in the present report. Unfortunately, we were unable to obtain specific staining with antibodies to 8-OH-dG. However, the nuclear localization of 8-OH-dG [32
] and the increased Ogg1 reported here are consistent with a model in which mitochondrial Ogg1 is up-regulated but nuclear Ogg1 is not, leading to an increase in nuclear but not mitochondrial 8-OH-dG adducts [32
]. Further studies will be required to resolve this issue, but β-cells in type 2 diabetes may well suffer from both nuclear and mitochondrial DNA damage, leading to decreased β-cell function and eventual loss of β-cell mass.
In this study, both the area and the intensity of Ogg1 staining were assessed. Since it was not practical to collect specimens in a controlled manner, it could be argued that differences in fixation technique could affect the fluorescence intensity of the immunostaining. However, as we also detected a significant increase in the islet area stained for Ogg1, we are confident that islet cell Ogg1 is actually upregulated in type 2 diabetes. The direct correlation between the fluorescence intensity and the duration of diabetes that held true for samples collected from disparate locations also argues in favor of the hypothesis. It is interesting that the Ogg1 positive area did not show the same increase with increasing duration of diabetes as the Ogg1 staining intensity. We have observed that the number of β-cells varies considerably in the islets from type 2 diabetic subjects and that large areas of the islets are sometimes replaced by amorphous material (data not shown), probably reflecting fibrosis or amyloidosis. Such a variation in cell number per islet would bias a correlation of Ogg1 positive cells with duration of diabetes. Moreover, it is possible that β-cells from patients with longstanding diabetes have a higher threshold for an apoptotic response to DNA-damage than β-cells in normal subjects, as it has been shown that this threshold increases with age [43
]. Consequently, β-cells with high levels of DNA damage from younger individuals with a shorter duration of diabetes might be eliminated by apoptosis, while similarly damaged β-cells from older individuals, who are likely to have had diabetes for a longer time may be resistant to apoptosis and so remain. Such an apoptotic defect could account for the increasing intensity of Ogg1 staining, following accumulated levels of 8-OH-dG adducts with longer duration of diabetes. Further epidemiological studies in which age was specifically studied would be required to address this issue more definitively.
We noted that the variability in the amount of Ogg1 staining is higher in the control group than in the samples from the diabetic patients. It is well known that there is a high incidence of undiagnosed diabetes in the general population. Since detailed patient records were not available, it is possible that undiagnosed diabetes in the control group could account for the increased variability.
Ogg1 knockout mice possess increased levels of 8-OH-dG in their genome, particularly in the mitochondrial genome, but do not seem to exhibit an increased frequency of malignancies [44
]. It was postulated that the consequences of 8-OH-dG adducts and Ogg1 deficiency affects mainly cells with high oxygen metabolism and slow proliferation, such as liver cells [45
]. While β-cells were not examined in that study, they certainly exhibit both high oxidative metabolism and a low rate of proliferation, consistent with them being a target for the effects of 8-OH-dG adducts. It would be of great interest to study the Ogg1 mutant mice to determine whether an increase level of 8-OH-dG adducts predisposes to diabetes. This would be important in establishing oxidative DNA damage as a causal mechanism in diabetes as opposed to merely being an epiphenomenon in which the important targets for diabetes pathogenesis lie elsewhere in the β-cell. Further epidemiological studies of Ogg1 expression as well as studies of the expression of other genes and proteins that are involved in DNA damage responses should also be performed to further test the hypothesis that DNA damage is involved in the pathogenesis of type 2 diabetes.