Effects of curcumin on retinal oxidative stress and inflammation in diabetes

Abstract Background Oxidative stress and inflammation are implicated in the pathogenesis of retinopathy in diabetes. The aim of this study is to examine the effect of curcumin, a polyphenol with antioxidant and anti-inflammatory properties, on diabetes-induced oxidative stress and inflammation in the retina of rats. Methods A group of streptozotocin-induced diabetic rats received powdered diet supplemented with 0.05% curcumin (w/w), and another group received diet without curcumin. The diets were initiated soon after induction of diabetes, and the rats were sacrificed 6 weeks after induction of diabetes. The retina was used to quantify oxidative stress and pro-inflammatory markers. Results Antioxidant capacity and the levels of intracellular antioxidant, GSH (reduced form of glutathione) levels were decreased by about 30–35%, and oxidatively modified DNA (8-OHdG) and nitrotyrosine were increased by 60–70% in the retina of diabetic rats. The levels of interleukin-1β (IL-1β) and vascular endothelial growth factor (VEGF) were elevated by 30% and 110% respectively, and the nuclear transcription factor (NF-kB) was activated by 2 fold. Curcumin administration prevented diabetes-induced decrease in the antioxidant capacity, and increase in 8-OHdG and nitrotyrosine; however, it had only partial beneficial effect on retinal GSH. Curcumin also inhibited diabetes-induced elevation in the levels of IL-1β, VEGF and NF-kB. The effects of curcumin were achieved without amelioration of the severity of hyperglycemia. Conclusion Thus, the beneficial effects of curcumin on the metabolic abnormalities postulated to be important in the development of diabetic retinopathy suggest that curcumin could have potential benefits in inhibiting the development of retinopathy in diabetic patients

Oxidative Stress and Diabetic Retinopathy

Oxygen metabolism is essential for sustaining aerobic life, and normal cellular homeostasis works on a fine balance between the formation and elimination of reactive oxygen species (ROS). Oxidative stress, a cytopathic consequence of excessive production of ROS and the suppression of ROS removal by antioxidant defense system, is implicated in the development of many diseases, including Alzheimer's disease, and diabetes and its complications. Retinopathy, a debilitating microvascular complication of diabetes, is the leading cause of acquired blindness in developed countries. Many diabetes-induced metabolic abnormalities are implicated in its development, and appear to be influenced by elevated oxidative stress; however the exact mechanism of its development remains elusive. Increased superoxide concentration is considered as a causal link between elevated glucose and the other metabolic abnormalities important in the pathogenesis of diabetic complications. Animal studies have shown that antioxidants have beneficial effects on the development of retinopathy, but the results from very limited clinical trials are somewhat ambiguous. Although antioxidants are being used for other chronic diseases, controlled clinical trials are warranted to investigate potential beneficial effects of antioxidants in the development of retinopathy in diabetic patients

Mixed-lineage kinase-3 (MLK3) plays a negative modulatory role in insulin secretion from the pancreatic β-cell

Glucose-stimulated insulin secretion (GSIS) from the pancreatic β-cell in response to elevated levels of glucose is controlled by a variety of signals including intracellular calcium and nucleotides such as cAMP, ATP and GTP. These cellular signals are responsible for activation of specific kinases that mediate phosphorylation of key exocytotic proteins that lead to GSIS. In the context of protein kinases, mixed-lineage kinases (MLKs) have been implicated in an assortment of cellular functions, including cell proliferation and apoptosis. However, very little is known on potential regulatory roles of MLKs in islet β-cell function, including GSIS. The goal of this study is to determine the roles of MLK3 in GSIS. Initial data indicated that MLK3 is expressed in clonal β-cells (INS-1 832/13) and human islets. INS-1 832/13 cell exposure to stimulatory glucose (20mM) resulted in a time-dependent increase in phosphorylation of MLK3 at T277 and S281 with peak phosphorylation within 20 minutes. URMC-099, a known inhibitor of MLK3, markedly suppressed glucose-induced MLK3 phosphorylation under stimulatory glucose conditions. Moreover, URMC-099 significantly increased insulin secretion under basal (2.5mM) and stimulatory glucose exposure conditions, suggesting a suppressive role for MLK3 in insulin secretion. Together, these studies provide evidence that MLK3 is in fact expressed in clonal and primary β-cells and inhibition of glucose-induced phosphorylation of MLK3 results in potentiation of GSIS. Studies are underway to further confirm our pharmacological findings using molecular biological (siRNA-MLK3) approaches

Metabolic Memory Phenomenon and Accumulation of Peroxynitrite in Retinal Capillaries

Aim. Diabetic retinopathy resists reversal after good glycemic control (GC) is reinitiated, and preexisting damage at the time of intervention is considered as the major factor in determining the outcome of the GC. This study is to investigate the role of peroxynitrite accumulation in the retinal capillaries in the failure of retinopathy to reverse after reestablishment of GC, and to determine the effect of this reversal on the activity of the enzyme responsible for scavenging mitochondrial superoxide, MnSOD. Methods. In streptozotocin-diabetic rats, 6 months of poor glycemic control (PC, glycated hemoglobin, GHb > 12.0%) was followed by 6 additional months of GC (GHb about 6%). The trypsin-digested retinal microvessels were prepared for immunostaining of nitrotyrosine (a measure of peroxynitrite) and for counting the number of acellular capillaries (a measure of histopathology). The retina from the other eye was used to quantify nitrotyrosine concentration, MnSOD activity and the total antioxidant capacity. Results. Reversal of hyperglycemia after 6 months of PC had no significant effect on nitrotyrosine concentration in the retina, on the nitrotyrosine-positive retinal capillary cells and on the number of acellular capillaries; the values were similar in PC-GC and PC groups. In the same rats retinal MnSOD activity remained inhibited and the total antioxidant capacity was subnormal 6 months after cessation of PC. Conclusions. Peroxynitrite accumulation in the retinal microvasculature, the site of histopathology, fails to normalize after reversal of hyperglycemia, and superoxide remains inadequately scavenged. This failure of reversal of peroxynitrite accumulation could be, in part, responsible for the resistance of diabetic retinopathy to reverse after termination of PC

Activation of Acetyl-CoA Carboxylase by a Glutamate- and Magnesium-Sensitive Protein Phosphatase in the Islet β-Cell

Acetyl-CoA carboxylase (ACC) catalyzes the formation of malonyl-CoA, a precursor in the biosynthesis of long-chain fatty acids, which have been implicated in physiological insulin secretion. The catalytic function of ACC is regulated by phosphorylation (inactive)−dephosphorylation (active). In this study we investigated whether similar regulatory mechanisms exist for ACC in the pancreatic islet β-cell. ACC was quantitated in normal rat islets, human islets, and clonal β-cells (HIT-15 or INS-1) using a [ 14 C]bicarbonate fixation assay. In the β-cell lysates, ACC was stimulated by magnesium in a concentration-dependent manner. Of all the dicarboxylic acids tested, only glutamate, albeit ineffective by itself, significantly potentiated magnesium-activated ACC in a concentration-dependent manner. ACC stimulation by glutamate and magnesium was maximally demonstrable in the cytosolic fraction; it was markedly reduced by okadaic acid (OKA) in concentrations

Sirt1, a Negative Regulator of Matrix Metalloproteinase-9 in Diabetic Retinopathy

Citation: Kowluru RA, Santos JM, Zhong Q. Sirt1, a negative regulator of matrix metalloproteinase-9 in diabetic retinopathy. Invest Ophthalmol Vis Sci. 2014;55:5653-5660. DOI: 10.1167/iovs.14-14383 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-jB), and the activation of NF-jB 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 H 2 O 2 . 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 H 2 O 2 , 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. Keywords: diabetic retinopathy, MMP-9, NF-kB, posttranslational modification, Sirt1 R etinopathy remains one of the most feared complications of diabetes. Clinical and experimental studies have shown a strong relationship between chronic hyperglycemia and the development of diabetic retinopathy. 1,2 Many metabolic abnormalities that are triggered in hyperglycemic milieu, including polyol pathway, protein kinase C activation, and oxidative stress 3,4 -and a number of genes associated with these pathways 5 -have been implicated in the development of diabetic retinopathy, but the underlying mechanism of how hyperglycemia causes its development remains elusive. Diabetic environment also stimulates secretion of matrix metalloproteinases (MMPs), a class of approximately 25 zincdependent proteinases important in degrading at least one component of the extracellular matrix, and increased levels of MMP-2 and MMP-9 are observed in the vitreous, retina, and retinal capillary cells of diabetic patients and rodents. The goal of this study is to investigate the role of Sirt1 in the activation of MMP-9 in the development of diabetic retinopathy. Using retinal endothelial cells, we investigated the effect of high glucose on Sirt1 activity. The role of Sirt1 in the regulation of MMP-9 was evaluated by using Sirt1 activator, resveratrol, o