Muthuswamy Balasubramanyam

Curcumin-induced inhibition of cellular reactive oxygen species generation: novel therapeutic implications.

There is evidence for increased levels of circulating reactive oxygen species (ROS) in diabetics, as indirectly inferred by the findings of increased lipid peroxidation and decreased antioxidant status. Direct measurements of intracellular generation of ROS using fluorescent dyes also demonstrate an association of oxidative stress with diabetes. Although phenolic compounds attenuate oxidative stress-related tissue damage, there are concerns over toxicity of synthetic phenolic antioxidants and this has considerably stimulated interest in investigating the role of natural phenolics in medicinal applications. Curcumin (the primary active principle in turmeric,Curcuma longa Linn.) has been claimed to represent a potential antioxidant and antiinflammatory agent with phytonutrient and bioprotective properties. However there are lack of molecular studies to demonstrate its cellular action and potential molecular targets. In this study the antioxidant effect of curcumin as a function of changes in cellular ROS generation was tested. Our results clearly demonstrate that curcumin abolished both phorbol-12 myristate-13 acetate (PMA) and thapsigargin-induced ROS generation in cells from control and diabetic subjects. The pattern of these ROS inhibitory effects as a function of dose-dependency suggests that curcumin mechanistically interferes with protein kinase C (PKC) and calcium regulation. Simultaneous measurements of ROS and Ca2+ influx suggest that a rise in cytosolic Ca2+ may be a trigger for increased ROS generation. We suggest that the antioxidant and antiangeogenic actions of curcumin, as a mechanism of inhibition of Ca2+ entry and PKC activity, should be further exploited to develop suitable and novel drugs for the treatment of diabetic retinopathy and other diabetic complications.

Telomere shortening occurs in Asian Indian Type 2 diabetic patients

Aim  Telomere shortening has been reported in several diseases including atherosclerosis and Type 1 diabetes. Asian Indians have an increased predilection for Type 2 diabetes and premature coronary artery disease. The aim of this study was to determine whether telomeric shortening occurs in Asian Indian Type 2 diabetic patients.

Oxidative stress is independently associated with non-alcoholic fatty liver disease (NAFLD) in subjects with and without type 2 diabetes.

OBJECTIVE Our work is aimed at exploring the interrelationship of oxidative stress and insulin resistance in NAFLD subjects with and without type 2 diabetes in a population-based study. METHODS Subjects [n=200] were recruited from the Chennai Urban Rural Epidemiology Study. 1: Normal glucose tolerance (NGT) subjects without NAFLD; 2: NGT with NAFLD; 3: type 2 diabetic subjects [T2DM] without NAFLD and 4: T2DM with NAFLD. Thiobarbituric acid reactive substances (TBARS), protein carbonyl (PCC) and glutathione levels were measured by standard methods. Ultrasound of the liver was used to diagnose NAFLD. RESULTS TBARS and PCC levels were significantly elevated and GSH/GSSG ratio was significantly decreased in diabetic subjects with NAFLD compared to all other groups (p trend <0.001). Oxidative stress markers significantly associated with NAFLD even after adjusting for age, gender, BMI and glycemic status. CONCLUSIONS Increased oxidative stress is independently associated with NAFLD in Asian Indians without and with T2DM.

Differential gene expression of NADPH oxidase (p22phox) and hemoxygenase-1 in patients with Type 2 diabetes and microangiopathy.

Aims  While the downstream effects of increased reactive oxygen species (ROS) in the pathogenesis of diabetes were well studied, only a few studies have explored the cellular sources of ROS. We examined whether protection against oxidative stress is altered in patients with diabetes and microangiopathy by examining changes in NADPH oxidase (p22phox) and hemoxygenase‐1 (HO‐1) levels.

Transcriptional regulation of cytokines and oxidative stress by gallic acid in human THP-1 monocytes.

Increased inflammation/prooxidation has been linked not only to Type 2 diabetes but also in prediabetes state. In this study we investigated hyperglycemia-mediated proinflammatory/prooxidant effects in THP-1 monocytes and tested whether gallic acid could attenuate changes in gene expression induced by high-glucose. Cells were treated either with 5.5mM glucose or 25mM glucose in the absence and presence of gallic acid. While oxidative DNA damage was assessed by COMET assay, GSH and GSSG levels were estimated fluorimetrically. Gene expression patterns were determined by RT-PCR. Cells treated with high-glucose showed increased DNA damage and glutathione depletion and this was attenuated in the presence of gallic acid. High-glucose treated cells exhibited increased mRNA expression of TNF-alpha, IL-6, NADPH oxidase and TXNIP and gallic acid attenuated these proinflammatory and prooxidant effects. Cells treated with high-glucose revealed a deficiency in mounting SOCS-3 expression and gallic acid upregulates this feedback regulatory signal. Gallic acid attenuates DNA damage, maintains glutathione turnover, and suppresses hyperglycemia-induced activation of proinflammatory and prooxidant gene expression. Gallic acid beneficially modulate transcription of functionally diverse groups of genes and its regulation of SOCS-3 and TXNIP signals is a newly identified mechanism that has therapeutic implications.

Gallic acid protects RINm5F β‐cells from glucolipotoxicity by its antiapoptotic and insulin‐secretagogue actions

Gallic acid is claimed to possess antioxidant, antiinflammatory and cytoprotective effects. Since pancreatic islets from Type 2 diabetic patients have functional defects, it was hypothesized that glucolipotoxicity might induce apoptosis in β‐cells and gallic acid could offer protection. To test this, RINm5F β‐cells were exposed to high glucose (25 μM) or palmitate (500 μM) or a combination of both for 24 h in the presence and absence of gallic acid. Cells subjected to glucolipotoxicity in the absence and presence of gallic acid were assessed for DNA damage by comet assay. Apoptosis was inferred by caspase‐3 protein expression and caspase‐3 activity and changes in Bcl‐2 mRNA. RT‐PCR was used to analyse PDX‐1, insulin and UCP‐2 mRNA expression in RINm5F β‐cells and insulin levels were quantified from the cell culture supernatant. NFκB signal was studied by EMSA, immunofluorescence and Western blot analysis. While RINm5F β‐cells subjected to glucolipotoxicity exhibited increased DNA damage, apoptotic markers and NFκB signals, all these apoptotic perturbations were resisted by gallic acid. Gallic acid dose‐dependently increased insulin secretion in RINm5F β‐cells and upregulated mRNA of PDX‐1 and insulin. It is suggested that the insulin‐secretagogue and transcriptional regulatory action of gallic acid is a newly identified mechanism in our study. Copyright

Curcumin Modulates SDF-1α/CXCR4–Induced Migration of Human Retinal Endothelial Cells (HRECs)

PURPOSE The stromal-derived factor (SDF)-1alpha and the CXC receptor (CXCR)-4 jointly regulate the trafficking of various cell types and play a pivotal role in cell migration, proliferation, and survival. The purpose of this study was to assess whether curcumin inhibits human retinal endothelial cell (HREC) migration by interfering with SDF-1alpha/CXCR4 signaling. METHODS Primary HREC culture was established and maintained in endothelial growth medium. The viability and proliferation of HRECs were assessed by MTT and thymidine uptake assays, respectively. The effect of SDF-1alpha-induced HREC migration (chemotaxis) in the presence and absence of curcumin was determined using the Boyden chamber migration assay. Intracellular Ca(2+) concentration was measured by fluorometric analysis. Immunofluorescence and Western blot analyses were performed to quantify CXCR4, phosphorylated AKT, and PI3-kinase expression levels. RESULTS HREC migration increased in a dose-dependent manner (1, 10, 50, and 100 ng/mL; P < 0.001) in SDF-1alpha-treated cells. In contrast, AMD3100, an inhibitor of CXCR4 effectively inhibited HREC migration dose dependently. HREC migration was decreased when the cells were exposed to EGTA, a chelator of Ca(2+). Curcumin also blocked Ca(2+) influx, an important signal for HREC migration. In addition, curcumin significantly (P < 0.001) decreased SDF-1alpha-induced HRECs migration and downregulated SDF-1alpha-induced expression of CXCR4, phospho-AKT, phospho-phosphatidylinositol-3-kinase (PI3-K), and eNOS. CONCLUSIONS This study indicates that curcumin has an inhibitory effect on SDF-1alpha-induced HREC migration. The plausible mechanism of action could be upstream blockage of Ca(2+) influx and the downstream reduction of PI3-K/AKT signals.

Is insulin signaling molecules misguided in diabetes for ubiquitin-proteasome mediated degradation?

Recent mining of the human and mouse genomes, use of yeast genetics, and detailed analyses of several biochemical pathways, have resulted in the identification of many new roles for ubiquitin–proteasome mediated degradation of proteins. In the context of last year’s award of Noble Prize (Chemistry) work, the ubiquitin and ubiquitin-like modifications are increasingly recognized as key regulatory events in health and disease. Although the ATP-dependent ubiquitin–proteasome system has evolved as premier cellular proteolytic machinery, dysregulation of this system by several different mechanisms leads to inappropriate degradation of specific proteins and pathological consequences. While aberrations in the ubiquitin–proteasome pathway have been implicated in certain malignancies and neurodegenerative disorders, recent studies indicate a role for this system in the pathogenesis of diabetes and its complications. Inappropriate degradation of insulin signaling molecules such as insulin receptor substrates (IRS-1 and IRS-2) has been demonstrated in experimental diabetes, mediated in part through the up-regulation of suppressors of cytokine signaling (SOCS). It appears that altered ubiquitin–proteasome system might be one of the molecular mechanisms of insulin resistance in many pathological situations. Drugs that modulate the SOCS action and/or proteasomal degradation of proteins could become novel agents for the treatment of insulin resistance and Type 2 diabetes.

Evidence for mechanistic alterations of Ca2+ homeostasis in Type 2 diabetes mellitus.

Altered cytosolic Ca2+ is implicated in the aetiology of many diseases including diabetes but there are few studies on the mechanism(s) of the altered Ca2+ regulation. Using human lymphocytes, we studied cytosolic calcium (Cai) and various Ca2+ transport mechanisms in subjects with Type 2 diabetes mellitus and control subjects. Ca2+-specific fluorescent probes (Fura-2 and Fluo-3) were used to monitor the Ca2+ signals. Thapsigargin, a potent and specific inhibitor of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), was used to study Ca2+- store dependent Ca2+ fluxes. Significant (P < 0.05) elevation of basal Cai levels was observed in lymphocytes from diabetic subjects. Cai levels were positively correlated with fasting, plasma glucose and HbAlc. There was also a significant (P < 0.05) reduction in plasma membrane calcium (PMCA) ATPase activity in diabetic subjects compared to controls. Cells from Type 2 diabetics exhibited an increased Ca2+ influx (as measured both by Fluo-3 fliorescence and C45a assays) as a consequence of of thapsigargin-mediated Ca2+ store depletion. Upon addition of Mn2+ (a surrogate of Ca2+), the fura-2 fluorescence decayed in an exponential fashion and the rate and extent of this decline was steeper and greater in cells from type 2 diabetic patients. There was also a significant (P < 0.05) difference in the Na+/Ca2+ exchange activity in Type 2 diabetic patients, both under resting conditions and after challenging the cells with thapsigargin, when the internal store Ca2+ sequestration was circumvented. Pharmacological activation of protein kinase C (PKC) in cells from patients resulted in only partial inhibition of Ca2+ entry. We conclude that cellular Ca2+ accumulation in cells from Type 2 diabetes results from (a) reduction in PMCA ATPase activity, (b) modulation of Na+/Ca2+ exchange and (3) increased Ca2+ influx across the plasma membrane.

Development of Diagnostic Fragment Ion Library for Glycated Peptides of Human Serum Albumin: Targeted Quantification in Prediabetic, Diabetic, and Microalbuminuria Plasma by Parallel Reaction Monitoring, SWATH, and MSE

Human serum albumin is one of the most abundant plasma proteins that readily undergoes glycation, thus glycated albumin has been suggested as an additional marker for monitoring glycemic status. Hitherto, only Amadori-modified peptides of albumin were quantified. In this study, we report the construction of fragment ion library for Amadori-modified lysine (AML), N(ε)-(carboxymethyl)lysine (CML)-, and N(ε)-(carboxyethyl)lysine (CEL)-modified peptides of the corresponding synthetically modified albumin using high resolution accurate mass spectrometry (HR/AM). The glycated peptides were manually inspected and validated for their modification. Further, the fragment ion library was used for quantification of glycated peptides of albumin in the context of diabetes. Targeted Sequential Window Acquisition of all THeoretical Mass Spectra (SWATH) analysis in pooled plasma samples of control, prediabetes, diabetes, and microalbuminuria, has led to identification and quantification of 13 glycated peptides comprised of four AML, seven CML, and two CEL modifications, representing nine lysine sites of albumin. Five lysine sites namely K549, K438, K490, K88, and K375, were observed to be highly sensitive for glycation modification as their respective m/z showed maximum fold change and had both AML and CML modifications. Thus, peptides involving these lysine sites could be potential novel markers to assess the degree of glycation in diabetes.