Amelioration of Glucolipotoxicity-Induced Endoplasmic Reticulum Stress by a “Chemical Chaperone” in Human THP-1 Monocytes

Chronic ER stress is emerging as a trigger that imbalances a number of systemic and arterial-wall factors and promote atherosclerosis. Macrophage apoptosis within advanced atherosclerotic lesions is also known to increase the risk of atherothrombotic disease. We hypothesize that glucolipotoxicity might mediate monocyte activation and apoptosis through ER stress. Therefore, the aims of this study are (a) to investigate whether glucolipotoxicity could impose ER stress and apoptosis in THP-1 human monocytes and (b) to investigate whether 4-Phenyl butyric acid (PBA), a chemical chaperone could resist the glucolipotoxicity-induced ER stress and apoptosis. Cells subjected to either glucolipotoxicity or tunicamycin exhibited increased ROS generation, gene and protein (PERK, GRP-78, IRE1α, and CHOP) expression of ER stress markers. In addition, these cells showed increased TRPC-6 channel expression and apoptosis as revealed by DNA damage and increased caspase-3 activity. While glucolipotoxicity/tunicamycin increased oxidative stress, ER stress, mRNA expression of TRPC-6, and programmed the THP-1 monocytes towards apoptosis, all these molecular perturbations were resisted by PBA. Since ER stress is one of the underlying causes of monocyte dysfunction in diabetes and atherosclerosis, our study emphasize that chemical chaperones such as PBA could alleviate ER stress and have potential to become novel therapeutics

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

Increased glutathionylated hemoglobin (HbSSG) in type 2 diabetes subjects with microangiopathy

Objective: Protein glutathionylation is considered an important post-translational modification in the pathogenesis of complex diseases. The aim of this study was to examine whether hemoglobin (Hb) is modified by reduced glutathione (GSH) via oxidation of the thiol groups present in diabetes and its associated microangiopathy and to determine whether oxidative imbalance has any correlation with glutathionylated Hb (HbSSG) levels. Methods: The study group consisted of a total of 130 subjects which included non-diabetic healthy control subjects (n = 30) and type 2 diabetic patients with (n = 53) and without (n = 47) microangiopathy. All subjects were assessed for glycemic and lipidemic status, while diabetic subjects were also assessed for the diagnosis of retinopathy and nephropathy. RBC lysates from all the subjects were analyzed by liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS) for HbSSG β-globin chains. Levels of GSH and thiobarbituric acid substances (TBARS) levels were measured by spectrophotometric and fluorimetric methods, respectively. Results: The positivity for HbSSG in diabetic subjects with microangiopathy was significantly higher (69%) compared to diabetics without microangiopathy (22%) and control subjects (14%). In univariate regression analysis, HbSSG levels were significantly associated with the duration of diabetes, HbA1c, and TBARS levels. GSH levels were negatively correlated (r = -0.57, P &lt; 0.001) with HbSSG in diabetic subjects. A significant inverse correlation (r = -0.42, P &lt; 0.001) between the GSH levels and HbA1c levels was also seen in diabetic subjects. Conclusions: This is perhaps the largest LC-MS-based study to demonstrate that HbSSG levels are markedly increased in diabetic subjects with microangiopathy. Since diabetic subjects also exhibited increased lipid peroxidation and decreased GSH levels, it appears that enhanced oxidative stress may account for the increased HbSSG concentrations and altered reduction-oxidation (redox) signaling

Research and Policy to Achieve Healthy Aging in Asia: Recommendations from an Expert Workshop

Open Longevity Science71-1

Increased glutathionylated hemoglobin (HbSSG) in type 2 diabetes subjects with microangiopathy

Abstract Objective: Protein glutathionylation is considered an important post-translational modification in the pathogenesis of complex diseases. The aim of this study was to examine whether hemoglobin (Hb) is modified by reduced glutathione (GSH) via oxidation of the thiol groups present in diabetes and its associated microangiopathy and to determine whether oxidative imbalance has any correlation with glutathionylated Hb (HbSSG) levels. Methods: The study group consisted of a total of 130 subjects which included non-diabetic healthy control subjects (n = 30) and type 2 diabetic patients with (n = 53) and without (n = 47) microangiopathy. All subjects were assessed for glycemic and lipidemic status, while diabetic subjects were also assessed for the diagnosis of retinopathy and nephropathy. RBC lysates from all the subjects were analyzed by liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS) for HbSSG h-globin chains. Levels of GSH and thiobarbituric acid substances (TBARS) levels were measured by spectrophotometric and fluorimetric methods, respectively. Results: The positivity for HbSSG in diabetic subjects with microangiopathy was significantly higher (69%) compared to diabetics without microangiopathy (22%) and control subjects (14%). In univariate regression analysis, HbSSG levels were significantly associated with the duration of diabetes, HbA1c, and TBARS levels. GSH levels were negatively correlated (r = À0.57, P &lt; 0.001) with HbSSG in diabetic subjects. A significant inverse correlation (r = À0.42, P &lt; 0.001) between the GSH levels and HbA1c levels was also seen in diabetic subjects. Conclusions: This is perhaps the largest LC-MS-based study to demonstrate that HbSSG levels are markedly increased in diabetic subjects with microangiopathy. Since diabetic subjects also exhibited increased lipid peroxidation and decreased GSH levels, it appears that enhanced oxidative stress may account for the increased HbSSG concentrations and altered reduction -oxidation (redox) signaling

Convergence of innate immunity and insulin resistance as evidenced by increased nucleotide oligomerization domain (NOD) expression and signaling in monocytes from patients with type 2 diabetes

Despite the well known role of nucleotide oligomerization domain (NOD) receptor proteins in innate immunity, their association with diabetes is less explored. Here we report the transcriptional level of NODs and their downstream molecular signatures in CD14(+) monocytes from subjects with different grades of glucose tolerance. NOD1 and NOD2 mRNA expression were significantly up-regulated in monocytes from patients with type 2 diabetes (T2DM) and positively correlated with HOMA-IR and poor glycemic control. Patients with T2DM also exhibited increased monocyte activation markers (CD11b and CD36) and proinflammatory signals downstream of NOD (RIPK2 and NFκB) along with the increased circulatory levels of TNF-α and IL-6. In vitro stimulation of monocytes with NOD specific ligands-i-EDAP and MDP significantly up regulated the mRNA expression of NOD1 and NOD2 respectively in T2DM. Our study exposes up regulation of NODs in monocytes as an important component of inflammation and insulin resistance in patients with T2DM

Linking a role of lncRNAs (long non-coding RNAs) with insulin resistance, accelerated senescence, and inflammation in patients with type 2 diabetes

Abstract Background Studying epigenetics is expected to provide precious information on how environmental factors contribute to type 2 diabetes mellitus (T2DM) at the genomic level. With the progress of the whole-genome resequencing efforts, it is now known that 75–90% of the human genome was transcribed to generate a series of long non-coding RNAs (lncRNAs). While lncRNAs are gaining widespread attention as potential and robust biomarkers in the genesis as well as progression of several disease states, their clinical relevance and regulatory mechanisms are yet to be explored in the field of metabolic disorders including diabetes. Despite the fact that Asian Indians are highly insulin resistant and more prone to develop T2DM and associated vascular complications, there is virtually lack of data on the role of lncRNAs in the clinical diabetes setting. Therefore, we sought to evaluate a panel of lncRNAs and senescence-inflammation signatures in peripheral blood mononuclear cells (PBMCs) from patients with type 2 diabetes (T2DM; n = 30) compared to individuals with normal glucose tolerance (NGT; n = 32). Results Compared to control subjects, expression levels of lncRNAs in PBMCs from type 2 diabetes patients showed significantly (p < 0.05) increased levels of HOTAIR, MEG3, LET, MALAT1, MIAT, CDKN2BAS1/ANRIL, XIST, PANDA, GAS5, Linc-p21, ENST00000550337.1, PLUTO, and NBR2. In contrast, lncRNA expression patterns of THRIL and SALRNA1 were significantly (p < 0.05) decreased in patients with T2DM compared to control subjects. At the transcriptional level, senescence markers (p53, p21, p16, and β-galactosidase), proinflammatory markers (TNF-α, IL6, MCP1, and IL1-β), and epigenetic signature of histone deacetylase-3 (HDAC3) were significantly (p < 0.05) elevated in patients with type 2 diabetes compared to control subjects. Interestingly, mRNA expression of Sirt1 and telomere length were significantly (p < 0.05) decreased in patients with type 2 diabetes compared to control subjects. Majority of the altered lncRNAs were positively correlated with poor glycemic control, insulin resistance, transcriptional markers of senescence, inflammation, and HDAC3 and negatively correlated with telomere length. Logistic regression analysis revealed a significant association of altered lncRNA signatures with T2DM, but this association was lost after adjusting for insulin resistance (HOMA-IR) and senescence markers. Conclusion Our study provides a clinically relevant evidence for the association of altered lncRNAs with poor glycemic control, insulin resistance, accelerated cellular senescence, and inflammation

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 5.5 mM glucose or 25 mM 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-α, 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

Association of hypoadiponectinemia with hypoglutathionemia in NAFLD subjects with and without type 2 diabetes

Objective: The aim of this study is to measure the extent of oxidative stress and to see whether it has any correlation to changes in adiponectin levels in NAFLD subjects with and without Type 2 diabetes. Methods: Subjects recruited from the Chennai Urban Rural Epidemiology Study comprise of 1: Normal Glucose Tolerance (NGT) subjects without NAFLD; 2: NGT with NAFLD; 3: Type 2 Diabetic patients [T2DM] without NAFLD and 4: T2DM with NAFLD. Thiobarbituric acid reactive substances (TBARS), protein carbonyl (PCO), glutathione and adiponectin levels were measured by standard methods. Ultrasound of the liver was used to diagnose NAFLD. Results: T2DM subjects with NAFLD had significantly (p&lt; 0.001) higher levels of thiobarbituric acid reactive substances (TBARS) and protein carbonyls (PCO) but lower (p&lt; 0.001) GSH/GSSG ratio and adiponectin levels compared to other three groups. The association of hypoadiponectinemia with NAFLD/Type 2 diabetes was significant even after adjusting for age, gender and BMI, but lost when adjusted for parameters of oxidative stress. While palmitate significantly reduced GSH/GSSG ratio in hepatocytes, addition of exogenous recombinant adiponectin restored the GSH/GSSG ratio comparable to those of untreated cells. Conclusion: There exists an association of hypoglutathionemia and hypoadiponectinemia in subjects with NAFLD and/or T2DM. In addition to the known beneficial effects, out study also exposes the antioxidant nature of adiponectin