Mechanisms Of Poly (ADP-Ribose) Polymerase 1 Involved In Glucose Toxicity In HepG2 Hepatocytes

BACKGROUND:Diabetes mellitus is a chronic metabolic disorder characterized by sustained extracellular high glucose state (hyperglycemia). The deleterious effects of chronic hyperglycemia can lead to a change of intracellular homeostasis and certain components. These changes are closely linked to cellular responses to its environment and correlated with its function maintenance. One of the most common changes is the increase of reactive oxygen species (ROS) production and the direct oxidative damage of biological macromolecules by ROS. More and more evidence indicated that high glucose induced negative effects not only related to direct damage of biological macromolecules by ROS, including damage of lipids, proteins and DNA, etc, but also directly related to cellular stress regulation to ROS damage. From cell self-reaction and cell function modulation perspective, we discussed the relationship between DNA repair system activation, as a cell normal response to high glucose-induced DNA damage, and insulin signal pathway. We hypothesis that DNA repair enzyme poly(ADP-ribose) polymerase 1 (PARP1) activated by high glucose concentration initiated a series of negative reactions including decrease of insulin sensitivity, which may elucidate a novel pathway involved in glucose toxicity.METHODS:HepG2 cells were cultured in 5.5mM glucose (control) and 30mM glucose (high glucose state) respectively. ROS generation and DNA damage of HepG2 cells were examined by flow cytometry and comet assay. Protein expression and activity levels were measured by Western blotting. Intracellular concentration of NAD was determined by NAD/NADH quantification kit. PJ34, PARP inhibitor, was used to observe the effect of PARP 1 activation.RESULTS:1) High glucose concentration induced an accumulation of ROS generation in cells from 1d (1.7-fold increase) to 4d (3-fold increase) compared to that in cells of control group. However, ROS production was significantly inhibited in treated cells with NAC (p<0.05). No significant differences in ROS production between cells incubated with mannitol and 5.5mM glucose were observed. ROS formation induced by Rosup increased markedly, which was differed from high glucose effect. 2) High glucose concentration-induced oxidative stress increased DNA damage and activated DNA excision repair enzymes. DNA damage in HepG2 cells incubated in 30mM glucose for 2d and 4d was much higher than that in cells of control group with 5.5mM glucose (1.7-fold and 2.7-fold increase respectively, p<0.05). DNA repair enzyme PARP1 activity increased gradually in response to 30mM glucose; however, ROS scavenger NAC inhibited PARP1 activation.3) PARP1 activation decreased intracellular NAD content and NAD biosynthesis enzyme NMNAT1 protein level. The PARP1 inhibitor, PJ34, suppressed high glucose-induced activation of PARP1, was paralleling its effect on the repletion of NAD levels.4) PARP1 activation decreased NAD-dependent protein SIRT1 and AMPK protein levels and activity. PJ34 reversed it.5) PARP1 activation down-regulated the phosphorylation level of insulin receptor in response to insulin stimulation. PJ34 could reverse it.6) PARP1 activation slightly affected HepG2 cells viability. PJ34 could partially reverse it.CONCLUSIONS:1) Sustained extracellular high glucose concentration in vitro could induce an accumulation of ROS generation in HepG2 cells, accompanied by the increase of DNA damage and DNA repair enzyme PAPR1 activity.2) PARP1 activated by high glucose concentration reduced intracellular NAD content through NAD depletion and NAD biosynthesis enzyme NMNAT1 decrease, resulting in down-regulation of SIRT1 and AMPK expression and activity levels, thus affecting cellular insulin sensitivity.3) PARP1 inhibitor PJ34 could reversed a series of negative reactions initiated by PARP1 activation, proved that high glucose-induced PARP1 activation might involve in glucose toxicity.4) These could provide an experiment evidence for recognizing the relationship between cell self-reaction and cell function modulation, which may invovled in disease development.