Relationship between the strength of the hip and knee stabilizer muscles and the Y balance test performance in adolescent volleyball athletes.

BACKGROUND: Due to the large number of jumping and landing movements, volleyball exerts a high demand on lower limbs, causing a high incidence of injuries. On average, 52% of volleyball injuries occur in lower limbs. Y Balance-test (YBT) is widely used to predict lower limb injury in athletes who jump, so it is important to identify whether the hip and knee stabilizer muscle strength is related to the performance of this test, since muscle strength is important for injury prevention, given the possibility of modifying these factors. The aim of this cross-sectional study was to correlate the strength of the knee and hip stabilizing muscles with YBT in adolescent volleyball athletes. METHODS: A study including 25 young volleyball players (aged 15.3 [0.85] years), was evaluated. The isometric strength of knee extensor muscles, abductors, extensors and lateral hip rotators were assessed by using the manual isometric dynamometer (Lafaytte Instrument Company®, Lafayette, IN, USA). YBT was performed three times on each posterolateral (PL), anterior (ANT) and posteromedial (PM) direction to evaluate the dominant leg. The average of 3 repetitions of isometric strength tests of tested muscles and YBT was analyzed. Pearson's Correlation test was used for the correlation of the variables, considering as strong correlation values (0.5≤r<1), with P≤0.05. RESULTS: A moderate correlation was observed between the knee extensor strength and the ANT direction, and a moderate correlation between the hip extensor strength and the PL direction in YBT. CONCLUSIONS: The strength of knee and hip extensors may be one of the factors that influence the performance of YBT in adolescent volleyball players.

The effect of BPA exposure on insulin resistance and type 2 diabetes - The impact of muscle contraction.

Type 2 diabetes (T2D) is considered one of the leading causes of death worldwide. In addition to physical inactivity and obesity, established risk factors for T2D, chemical contaminants consumed in industrialized food such as BPA might also be a contributor to the development of T2D. Epidemiological studies have shown that BPA concentrations are higher in human specimens of T2D when compared to healthy subjects, while experimental studies suggested that bisphenol A (BPA) impairs the pathway by which insulin stimulates glucose uptake. In skeletal muscle and adipocytes, insulin resistance is developed by the impairment of the insulin pathway to stimulate the translocation of glucose transporter, GLUT4, to the cell membrane. Recent results demonstrated that BPA impairs several components of insulin-induced glucose uptake pathway and affect the expression of GLUT4. Regular physical exercise delays or inhibits the development of T2D due to the physiologic processes taking place during muscle contraction, and the fact that skeletal muscle is the site for almost 80% of the glucose transported under insulin stimulation. In fact, the mechanism by which contraction induces glucose uptake in skeletal muscle is partially independent of the insulin pathway, therefore, the effect of BPA on this mechanism is unknown. We hypothesize that during the development of insulin resistance, BPA contributes to the impairment of the molecular pathway by which insulin induces glucose uptake while contraction-induced glucose uptake is not impaired. At the late stages of T2D, BPA may affect GLUT4 expression that will decrease the ability of muscle contraction to induce glucose uptake.

The role of soluble epoxide hydrolase in preeclampsia.

Preeclampsia is a serious complication of pregnancy characterized by the development of vasospasm, hypertension and often associated with proteinuria after the 20th week of gestation. Because termination of pregnancy results in the most efficacious resolution of preeclampsia, it is a leading cause of premature delivery worldwide. In pregnancy, 14,15-epoxyeicosatrienoic acids (EETs) have been shown to facilitate uterine blood flow during preeclampsia, in which the classic vasodilator agents such as nitric oxide and prostacyclin are reduced. EETs are converted to dihydroxyeicosatrienoic acids (DHETs) by the activity of soluble epoxide hydrolase (sEH). We tested the hypothesis that sEH activity is increased in preeclampsia by measuring urinary 14,15-DHET in healthy and preeclamptic pregnant women. Urine samples were collected and incubated with or without ß-glucuronidase to enable the measurement of both the glucuronidated and free forms of 14,15-DHET, which were quantified using a 14,15-DHET ELISA. Levels of total (free+glucuronidated) 14,15-DHET, which is a measurement of EET-dependent sEH activity, were higher in urine samples obtained from preeclamptic women compared to healthy pregnant women. Considering the fact that free+glucuronidated 14,15-DHET levels are increased in urine of preeclamptic women, we hypothesize that sEH expression or activity is augmented in these patients, reducing EET and increasing blood pressure. Moreover we suggest that novel anti-hypertensive agents that target sEH might be developed as therapeutics to control high blood pressure in women with preeclampsia.

Differential BPA levels in sewage wastewater effluents from metro Detroit communities.

The endocrine disruptor Bisphenol A (BPA) is ubiquitous in both aquatic and surface sediment environments because it is continuously released into sewage wastewater effluent. The measurement of BPA at wastewater treatment plants is rarely performed even though the United States Environmental Protection Agency (EPA) states that current levels of environmental BPA could be a threat to aquatic organisms. Therefore, the aims of this study were to measure BPA levels in sewage wastewater at different collection points over a 1-year period and to compare the levels of BPA to 8-isoprostane, a human derived fatty acid, found in sewage wastewater. We analyzed pre-treated sewage samples collected from three source points located in different communities in the metropolitan Detroit area provided by the Detroit Water and Sewerage Department. Human urine samples were also used in the study. BPA and 8-isoprostane were measured using ELISA kits from Detroit R&D, Inc. BPA levels from the same collection point oscillated more than 10-fold over 1 year. Also, BPA levels fluctuated differentially at each collection point. Highly fluctuating BPA values were confirmed by LC/MS/MS. The concentration of BPA in sewage wastewater was ~100-fold higher than the concentration of 8-isoprostane, while urinary concentration was ~20-fold higher. Thus, BPA levels discharged into the sewage network vary among communities, and differences are also observed within communities over time. The difference in BPA and 8-isoprostane levels suggest that most of the BPA discharged to sewage wastewater might be derived from industries rather than from human urine. Therefore, the continuous monitoring of BPA could account for a better regulation of BPA release into a sewage network.

Could sewage epidemiology be a strategy to assess lifestyle and wellness of a large scale population?

The use of sewage epidemiology to estimate the behavior of a large scale population has mainly been used to assess illicit drug use within a community. The systemic oxidative stress marker, 8-isoprostane, is a wildly accepted biomarker for various diseases such as diabetes, and cardiovascular and renal diseases. 8-Isoprostane is detected in urine and, as with illicit drugs, is excreted into urban sewer networks. Initially, we tested the hypothesis that differential 8-isoprostane levels are detected in wastewater of different communities and that 8-isoprostane values adjusted for the flow rate and population size will remain constant over a 2 months period. Sewage samples were collected from three sewage collection points supplied by different communities located in the Detroit metropolitan area and concentration of 8-isoprostane and synthetic plastic component, bisphenol A (BPA), were measured. Levels of 8-isoprostane were constant during the two measured months at each collection point in oppose to BPA levels. When the levels were compared among communities, 8-isoprostane levels in 24h flow and their concentrations per capita in each community varied by more than 5-fold among them. Considering the fact that 8-isoprostane is a biomarker of several diseases, we hypothesize that measurement of 8-isoprostane levels in sewage may serve as a risk assessment tool of oxidative stress-related diseases in a large scale population. Thus, sewage epidemiology can be utilized to obtain an early warning in a community to facilitate intervention for improvement of the community health.

Elevation of brain allopregnanolone rather than 5-HT release by short term, low dose fluoxetine treatment prevents the estrous cycle-linked increase in stress sensitivity in female rats.

Withdrawal from long-term dosing with exogenous progesterone precipitates increased anxiety-linked changes in behavior in animal models due to the abrupt decrease in brain concentration of allopregnanolone (ALLO), a neuroactive metabolite of progesterone. We show that a withdrawal-like effect also occurs during the late diestrus phase (LD) of the natural ovarian cycle in rats, when plasma progesterone and ALLO are declining but estrogen secretion maintains a stable low level. This effect at LD was prevented by short-term treatment with low dose fluoxetine. During LD, but not at other stages of the estrous cycle, exposure to anxiogenic stress induced by whole body vibration at 4 Hz for 5 min evoked a significant decrease in tail flick latency (stress-induced hyperalgesia) and a decrease in the number of Fos-positive neurons present in the periaqueductal gray (PAG). The threshold to evoke fear-like behaviors in response to electrical stimulation of the dorsal PAG was lower in the LD phase, indicating an increase in the intrinsic excitability of the PAG circuitry. All these effects were blocked by short-term administration of fluoxetine (2 × 1.75 mg kg(-1) i.p.) during LD. This dosage increased the whole brain concentration of ALLO, as determined using gas chromatography-mass spectrometry, but was without effect on the extracellular concentration of 5-HT in the dorsal PAG, as measured by microdialysis. We suggest that fluoxetine-induced rise in brain ALLO concentration during LD offsets the sharp physiological decline, thus removing the trigger for the development of anxiogenic withdrawal effects.

Sirt1, a negative regulator of matrix metalloproteinase-9 in diabetic retinopathy.

PURPOSE: In the pathogenesis of diabetic retinopathy, matrix metalloproteinase (MMP)-9 damages retinal mitochondria, activating the apoptotic machinery. Transcription of MMP-9 is regulated by nuclear factor kappa B (NF-κB), and the activation of NF-κB is modulated by the acetylation of its p65 subunit. Sirtuin 1 (Sirt1), a deacetylase, plays an important role in the acetylation-deacetylation of p65. The goal of this study is to investigate the role of Sirt1 in the activation of MMP-9 in diabetic retinopathy. METHODS: The effect of hyperglycemia and Sirt1 activator, resveratrol, on acetylation of p65 and its binding at MMP-9 promoter-and mitochondrial damage and apoptosis-was assessed in the retinal endothelial cells. Role of oxidative stress in the regulation of Sirt1 was evaluated in the cells incubated in H2O2. The results were confirmed in the retina from diabetic mice with Sod2 or MMP-9 gene manipulated. RESULTS: High glucose decreased Sirt1 activity and increased p65 acetylation, and resveratrol prevented increase in p65 acetylation, binding of p65 at MMP-9 promoter and MMP-9 activation, mitochondria damage, and cell apoptosis. While Sirt1 was decreased by H2O2, MMP-9 was significantly increased. Retina from wild-type diabetic mice presented similar decrease in Sirt1, and diabetic mice with Sod2 overexpression or MMP-9 deletion had normal retinal Sirt1. Retinal microvasculature from human donors with established diabetic retinopathy also had decreased Sirt1. CONCLUSIONS: Thus, in diabetes, increase in oxidative stress inhibits Sirt1 and p65 is hyperacetylated, increasing the binding of p65 at MMP-9 promoter. Prevention of Sirt1 inhibition, via modulating acetylation of p65, should protect activation of MMP-9 and inhibit the development of diabetic retinopathy.

The effect of exercise on epigenetic modifications of PGC1: The impact on type 2 diabetes.

The worldwide prevalence of diabetes type 2 is increasing and intramuscular accumulation of fatty acid metabolites is gradually becoming recognized as core features of this condition as lipotoxicity induces insulin resistance. Emerging evidences suggest that defects in mitochondria, key organelle in lipid metabolism, play a central role on insulin resistance. Mitochondria homeostasis is tightly regulated by a nucleus-mitochondria signaling pathway and peroxisome proliferator-activated receptor γ coactivator-1α (PGC1) is the master regulator of important mitochondria process. PGC1 is down regulated in insulin resistant skeletal muscle and abnormal posttranslational modification at histone, epigenetic modifications, is an important factor. Studies have demonstrated the benefits of regular exercise on improving insulin sensitivity however the mechanism for this outcome is not entirely identified. Moreover evidences point out the increase in PGC1 expression induced by exercise as an important element for the improvement of insulin sensitivity in skeletal muscle via increase in mitochondria density and glucose transporter expression (GLUT4). Therefore, we here proposed that aerobic exercise attenuates epigenetic modifications at PGC1 induced by high-energy diets and reduced physical activity, and that leads to inhibition/delay of type 2 diabetic onset.

Posttranslational modification of mitochondrial transcription factor A in impaired mitochondria biogenesis: implications in diabetic retinopathy and metabolic memory phenomenon.

Mitochondrial transcription factor A (TFAM) is one of the key regulators of the transcription of mtDNA. In diabetes, despite increase in gene transcripts of TFAM, its protein levels in the mitochondria are decreased and mitochondria copy numbers become subnormal. The aim of this study is to investigate the mechanism(s) responsible for decreased mitochondrial TFAM in diabetes. Using retinal endothelial cells, we have investigated the effect of overexpression of cytosolic chaperone, Hsp70, and TFAM on glucose-induced decrease in mitochondrial TFAM levels, and the transcription of mtDNA-encoded genes, NADH dehydrogenase subunit 6 (ND6) and cytochrome b (Cytb). To investigate the role of posttranslational modifications in subnormal mitochondrial TFAM, ubiquitination of TFAM was assessed, and the results were confirmed in the retina from streptozotocin-induced diabetic rats. While overexpression of Hsp70 failed to prevent glucose-induced decrease in mitochondrial TFAM and transcripts of ND6 and Cytb, overexpression of TFAM ameliorated decrease in its mitochondrial protein levels and transcriptional activity. TFAM was ubiquitinated by high glucose, and PYR-41, an inhibitor of ubiquitination, prevented TFAM ubiquitination and restored the transcriptional activity. Similarly, TFAM was ubiquitinated in the retina from diabetic rats, and it continued to be modified after reinstitution of normal glycemia. Our results clearly imply that the ubiquitination of TFAM impedes its transport to the mitochondria resulting in subnormal mtDNA transcription and mitochondria dysfunction, and inhibition of ubiquitination restores mitochondrial homeostasis. Reversal of hyperglycemia does not provide any benefit to TFAM ubiquitination. Thus, strategies targeting posttranslational modification could provide an avenue to preserve mitochondrial homeostasis, and inhibit the development/progression of diabetic retinopathy.

TIAM1-RAC1 signalling axis-mediated activation of NADPH oxidase-2 initiates mitochondrial damage in the development of diabetic retinopathy.

AIMS/HYPOTHESIS: In diabetes, increased retinal oxidative stress is seen before the mitochondria are damaged. Phagocyte-like NADPH oxidase-2 (NOX2) is the predominant cytosolic source of reactive oxygen species (ROS). Activation of Ras-related C3 botulinum toxin substrate 1 (RAC1), a NOX2 holoenzyme member, is necessary for NOX2 activation and ROS generation. In this study we assessed the role of T cell lymphoma invasion and metastasis (TIAM1), a guanine nucleotide exchange factor for RAC1, in RAC1 and NOX2 activation and the onset of mitochondrial dysfunction in in vitro and in vivo models of glucotoxicity and diabetes. METHODS: RAC1 and NOX2 activation, ROS generation, mitochondrial damage and cell apoptosis were quantified in bovine retinal endothelial cells exposed to high glucose concentrations, in the retina from normal and streptozotocin-induced diabetic rats and mice, and the retina from human donors with diabetic retinopathy. RESULTS: High glucose activated RAC1 and NOX2 (expression and activity) and increased ROS in endothelial cells before increasing mitochondrial ROS and mitochondrial DNA (mtDNA) damage. N6-[2-[[4-(diethylamino)-1-methylbutyl]amino]-6-methyl-4-pyrimidinyl]-2-methyl-4,6-quinolinediamine, trihydrochloride (NSC23766), a known inhibitor of TIAM1-RAC1, markedly attenuated RAC1 activation, total and mitochondrial ROS, mtDNA damage and cell apoptosis. An increase in NOX2 expression and membrane association of RAC1 and p47(phox) were also seen in diabetic rat retina. Administration of NSC23766 to diabetic mice attenuated retinal RAC1 activation and ROS generation. RAC1 activation and p47(phox) expression were also increased in the retinal microvasculature from human donors with diabetic retinopathy. CONCLUSIONS/INTERPRETATION: The TIAM1-RAC1-NOX2 signalling axis is activated in the initial stages of diabetes to increase intracellular ROS leading to mitochondrial damage and accelerated capillary cell apoptosis. Strategies targeting TIAM1-RAC1 signalling could have the potential to halt the progression of diabetic retinopathy in the early stages of the disease.

Beneficial effects of the nutritional supplements on the development of diabetic retinopathy.

PURPOSE: Increased oxidative stress and inflammatory mediators are implicated in the development of diabetic retinopathy, and in rats, its development can be prevented by antioxidants. Carotenoids are some of the powerful antioxidants, and diabetes decreases lutein and zeaxanthin levels in the serum and retina. The aim of this study is to investigate the effect of carotenoid containing nutritional supplements (Nutr), which is in clinical trials for 'Diabetes Vision Function', on diabetic retinopathy. METHODS: Streptozotocin-induced diabetic rats (Wistar, male) were fed Purina 5001 supplemented with nutritional supplements containing zeaxanthin, lutein, lipoic acid, omega-3 fatty acids and other nutrients, or without any supplementation. Retinal function was analyzed at ~4 months of diabetes by electroretinography. After 11 months of diabetes, capillary cell apoptosis (TUNEL-staining) and histopathology (degenerative capillaries) were quantified in trypsin-digested retinal vasculature. Retina was also analyzed for mitochondrial damage (by quantifying gene expressions of mtDNA-encoded proteins of the electron transport chain), VEGF and inflammatory mediators, interleukin-1ß and NF-kB. RESULTS: Diabetes impaired retinal function decreasing the amplitudes of both a- and b-waves. In the same animals, retinal capillary cell apoptosis and degenerative capillaries were increased by 3-4 fold. Gene expressions of mtDNA encoded proteins were decreased, and VEGF, interleukin-1ß and NF-kB levels were elevated. Supplementation with the nutrients prevented increased capillary cell apoptosis and vascular pathology, and ameliorated these diabetes-induced retinal abnormalities. CONCLUSIONS: Nutritional supplementation prevents diabetic retinopathy, and also maintains normal retinal function, mitochondrial homeostasis and inflammatory mediators. Thus, this supplementation could represent an achievable and inexpensive adjunct therapy to also inhibit retinopathy, a slow progressing disease feared most by diabetic patients.

Epigenetic modifications and diabetic retinopathy.

Diabetic retinopathy remains one of the most debilitating chronic complications, but despite extensive research in the field, the exact mechanism(s) responsible for how retina is damaged in diabetes remains ambiguous. Many metabolic pathways have been implicated in its development, and genes associated with these pathways are altered. Diabetic environment also facilitates epigenetics modifications, which can alter the gene expression without permanent changes in DNA sequence. The role of epigenetics in diabetic retinopathy is now an emerging area, and recent work has shown that genes encoding mitochondrial superoxide dismutase (Sod2) and matrix metalloproteinase-9 (MMP-9) are epigenetically modified, activates of epigenetic modification enzymes, histone lysine demethylase 1 (LSD1), and DNA methyltransferase are increased, and the micro RNAs responsible for regulating nuclear transcriptional factor and VEGF are upregulated. With the growing evidence of epigenetic modifications in diabetic retinopathy, better understanding of these modifications has potential to identify novel targets to inhibit this devastating disease. Fortunately, the inhibitors and mimics targeted towards histone modification, DNA methylation, and miRNAs are now being tried for cancer and other chronic diseases, and better understanding of the role of epigenetics in diabetic retinopathy will open the door for their possible use in combating this blinding disease.

Interrelationship between activation of matrix metalloproteinases and mitochondrial dysfunction in the development of diabetic retinopathy.

Mitochondria dysfunction plays a significant role in the apoptosis of retinal cells. Diabetes activates retinal matrix metalloproteinases (MMP-9 and MMP-2), damages retinal mitochondria and activates the apoptotic machinery. This study is to investigate the temporal relationship between the activation of retinal MMPs and mitochondria damage in the development of diabetic retinopathy. Time course of activation of cytosolic MMP-9 and MMP-2 was investigated in the retinal endothelial cells incubated in high glucose for 6-96 h, and correlated with their mitochondrial accumulation and mitochondrial damage. This was confirmed in the retina from rats diabetic for 15 days to ~12 months (streptozotocin-induced). The results show that the activation of cytosolic MMP-9 and MMP-2 is an early event, which is followed by their accumulation in the mitochondria. Increased mitochondrial MMPs dysfunction them and begin to damage their DNA, which initiates a vicious cycle of reactive oxygen species. Thus, modulation of these gelatinase MMPs by pharmacological agents during the early stages of diabetes could provide a strategy to inhibit the development of diabetic retinopathy.

Impaired transport of mitochondrial transcription factor A (TFAM) and the metabolic memory phenomenon associated with the progression of diabetic retinopathy.

BACKGROUND: Diabetes damages retinal mitochondrial DNA (mtDNA) and compromises the mtDNA transcription. In the transcription and replication of mtDNA, nuclear-encoded mitochondrial transcription factor A (TFAM) is considered a key activator. We have shown that in diabetes, although retinal TFAM gene expression is increased, its mitochondrial levels are decreased. This study investigates the role of mitochondrial outer and inner membrane transport systems in the transfer of TFAM into the mitochondria in diabetes and how reversal of hyperglycaemia affects the ability of TFAM to reach the mitochondria. METHODS: Components of the membrane transport system, Tom70, Tom40, Tim23, and Tim44, were analysed in the retina from streptozotocin-induced diabetic rats maintained in poor control or in good control for 8 months, or in poor control for 4 months followed by in good control for 4 months. The binding of TFAM with Tom70 and Tim44 was determined by co-immunoprecipitation and that with mtDNA by chromatin immunoprecipitation. RESULTS: Retinal expressions of Tom70, Tom40, and Tim44 were significantly decreased in diabetes, and the binding of TFAM with Tom70, Tim44, and mtDNA was impaired. Reversal of hyperglycaemia had no beneficial effect on the decreased binding of TFAM to Tom proteins and mtDNA. CONCLUSIONS: Thus, subnormal membrane transport to systems in diabetes impair the transfer of TFAM into the mitochondria, and decreased TFAM-mtDNA binding that results in subnormal mitochondria transcription. These processes continue to be dysfunctional even after the hyperglycaemic insult is terminated. Strategies targeting mitochondrial membrane transport proteins could have the potential of improving mitochondrial biogenesis and slowing or halting the progression of diabetic retinopathy.

A compensatory mechanism protects retinal mitochondria from initial insult in diabetic retinopathy.

In the pathogenesis of diabetic retinopathy, an increase in retinal oxidative stress precedes mitochondrial dysfunction and capillary cell apoptosis. This study is designed to understand the mechanism responsible for the protection of mitochondria damage in the early stages of diabetic retinopathy. After 15 days-12 months of streptozotocin-induced diabetes in rats, retina was analyzed for mitochondria DNA (mtDNA) damage by extended length PCR. DNA repair enzyme and replication machinery were quantified in the mitochondria, and the binding of mitochondrial transcriptional factor A (TFAM) with mtDNA was analyzed by ChIP. Key parameters were confirmed in the retinal endothelial cells incubated in 20mM glucose for 6-96h. Although reactive oxygen species (ROS) were increased within 15 days of diabetes, mtDNA damage was observed at 6 months of diabetes. After 15 days of diabetes DNA repair/replication enzymes were significantly increased in the mitochondria, but at 2 months, their mitochondrial accumulation started to come down, and mtDNA copy number and binding of TFAM with mtDNA became significantly elevated. However, at 6 months of diabetes, the repair/replication machinery became subnormal and mtDNA copy number significantly decreased. A similar temporal relationship was observed in endothelial cells exposed to high glucose. Thus, in the early stages of diabetes, increased mtDNA biogenesis and repair compensates for the ROS-induced damage, but, with sustained insult, this mechanism is overwhelmed, and mtDNA and electron transport chain (ETC) are damaged. The compromised ETC propagates a vicious cycle of ROS and the dysfunctional mitochondria fuels loss of capillary cells by initiating their apoptosis.

Mitochondria DNA replication and DNA methylation in the metabolic memory associated with continued progression of diabetic retinopathy.

PURPOSE: Diabetic retinopathy fails to halt after cessation of hyperglycemic insult, and a vicious cycle of mitochondria damage continues. The aim of our study was to investigate the effect of termination of hyperglycemia on retinal mtDNA replication, and elucidate the mechanism responsible for the continued mtDNA damage. METHODS: Polymerase gamma 1 (POLG1), the catalytic subunit of the mitochondrial DNA replication enzyme, and the damage to the displacement loop region of mtDNA (D-loop) were analyzed in the retina from streptozotocin-diabetic rats maintained in poor glycemic control (PC, glycated hemoglobin ∼11%) or in good glycemic control (GC, glycated hemoglobin ∼6%) for 6 months, or in PC for three months followed by GC for three months (Rev). To understand the mechanism DNA methylation status of POLG1 promoter was investigated by methylation-specific PCR. The key parameters were confirmed in the isolated retinal endothelial cells exposed to high glucose, followed by normal glucose. RESULTS: POLG1 continued to be down-regulated, the D-loop region damaged, and the CpG islands at the regulatory region of POLG hyper-methylated even after three months of GC that had followed three months of PC (Rev group). Similar results were observed in the retinal endothelial cells exposed to normal glucose after being exposed to high glucose. CONCLUSIONS: Continued hypermethylation of the CpG sites at the regulatory region of POLG affects its binding to the mtDNA, compromising the transcriptional activity. Modulation of DNA methylation using pharmaceutic or molecular means could help maintain mitochondria homeostasis, and prevent further progression of diabetic retinopathy.