Umesh C. S. Yadav

Oxidative stress and metabolic disorders: Pathogenesis and therapeutic strategies.

Increased body weight and metabolic disorder including insulin resistance, type 2 diabetes and cardiovascular complications together constitute metabolic syndrome. The pathogenesis of metabolic syndrome involves multitude of factors. A number of studies however indicate, with some conformity, that oxidative stress along with chronic inflammatory condition pave the way for the development of metabolic diseases. Oxidative stress, a state of lost balance between the oxidative and anti-oxidative systems of the cells and tissues, results in the over production of oxidative free radicals and reactive oxygen species (ROS). Excessive ROS generated could attack the cellular proteins, lipids and nucleic acids leading to cellular dysfunction including loss of energy metabolism, altered cell signalling and cell cycle control, genetic mutations, altered cellular transport mechanisms and overall decreased biological activity, immune activation and inflammation. In addition, nutritional stress such as that caused by high fat high carbohydrate diet also promotes oxidative stress as evident by increased lipid peroxidation products, protein carbonylation, and decreased antioxidant system and reduced glutathione (GSH) levels. These changes lead to initiation of pathogenic milieu and development of several chronic diseases. Studies suggest that in obese person oxidative stress and chronic inflammation are the important underlying factors that lead to development of pathologies such as carcinogenesis, obesity, diabetes, and cardiovascular diseases through altered cellular and nuclear mechanisms, including impaired DNA damage repair and cell cycle regulation. Here we discuss the aspects of metabolic disorders-induced oxidative stress in major pathological conditions and strategies for their prevention and therapy.

Mitogenic responses of vascular smooth muscle cells to lipid peroxidation-derived aldehyde 4-hydroxy-trans-2-nonenal (HNE): Role of aldose reductase-catalyzed reduction of the HNE-glutathione conjugates in regulating cell growth

Products of lipid peroxidation such as 4-hydroxy-trans-2-nonenal (HNE) trigger multiple signaling cascades that variably affect cell growth, differentiation, and apoptosis. Because glutathiolation is a significant metabolic fate of these aldehydes, we tested the possibility that the bioactivity of HNE depends upon its conjugation with glutathione. Addition of HNE or the cell-permeable esters of glutathionyl-4-hydroxynonenal (GS-HNE) or glutathionyl-1,4-dihydroxynonene (GS-DHN) to cultures of rat aortic smooth muscle cells stimulated protein kinase C, NF-κB, and AP-1, and increased cell growth. The mitogenic effects of HNE, but not GS-HNE or GS-DHN, were abolished by glutathione depletion. Pharmacological inhibition or antisense ablation of aldose reductase (which catalyzes the reduction of GS-HNE to GS-DHN) prevented protein kinase C, NF-κB, and AP-1 stimulation and the increase in cell growth caused by HNE and GS-HNE, but not GS-DHN. The growth stimulating effect of GS-DHN was enhanced in cells treated with antibodies directed against the glutathione conjugate transporters RLIP76 (Ral-binding protein) or the multidrug resistance protein-2. Overexpression of RLIP76 abolished the mitogenic effects of HNE and its glutathione conjugates, whereas ablation of RLIP76 using RNA interference promoted the mitogenic effects. Collectively, our findings suggest that the mitogenic effects of HNE are mediated by its glutathione conjugate, which has to be reduced by aldose reductase to stimulate cell growth. These results raise the possibility that the glutathione conjugates of lipid peroxidation products are novel mediators of cell signaling and growth.

Aldose reductase inhibition suppresses oxidative stress-induced inflammatory disorders.

Oxidative stress-induced inflammation is a major contributor to several disease conditions including sepsis, carcinogenesis and metastasis, diabetic complications, allergic asthma, uveitis and after cataract surgery posterior capsular opacification. Since reactive oxygen species (ROS)-mediated activation of redox-sensitive transcription factors and subsequent expression of inflammatory cytokines, chemokines and growth factors are characteristics of inflammatory disorders, we envisioned that by blocking the molecular signals of ROS that activate redox-sensitive transcription factors, various inflammatory diseases could be ameliorated. We have indeed demonstrated that ROS-induced lipid peroxidation-derived lipid aldehydes such as 4-hydroxy-trans-2-nonenal (HNE) and their glutathione-conjugates (e.g. GS-HNE) are efficiently reduced by aldose reductase to corresponding alcohols which mediate the inflammatory signals. Our results showed that inhibition of aldose reductase (AKR1B1) significantly prevented the inflammatory signals induced by cytokines, growth factors, endotoxins, high glucose, allergens and auto-immune reactions in cellular as well as animal models. We have demonstrated that AKR1B1 inhibitor, fidarestat, significantly prevents tumor necrosis factor-alpha (TNF-α)-, growth factors-, lipopolysachharide (LPS)-, and environmental allergens-induced inflammatory signals that cause various inflammatory diseases. In animal models of inflammatory diseases such as diabetes, cardiovascular, uveitis, asthma, and cancer (colon, breast, prostate and lung) and metastasis, inhibition of AKR1B1 significantly ameliorated the disease. Our results from various cellular and animal models representing a number of inflammatory conditions suggest that ROS-induced inflammatory response could be reduced by inhibition of AKR1B1, thereby decreasing the progression of the disease and if the therapy is initiated early, the disease could be eliminated. Since fidarestat has already undergone phase III clinical trial for diabetic neuropathy and found to be safe, though clinically not very effective, our results indicate that it can be developed for the therapy of a number of inflammation-related diseases. Our results thus offer a novel therapeutic approach to treat a wide array of inflammatory diseases.

Combined treatment of sodium orthovanadate and Momordica charantia fruit extract prevents alterations in lipid profile and lipogenic enzymes in alloxan diabetic rats

Momordica charantia Linn., commonly called bitter gourd, is a medicinal plant used in the Ayurvedic system of medicine for treating various diseases including diabetes mellitus. Sodium orthovanadate (SOV) is also well-known insulin mimetic and an antidiabetic compound. Our laboratory has been using reduced doses of SOV along with administration of herbal extracts to alloxan diabetic rats and has established this combination as a good antihyperglycemic agent. The present study was undertaken to investigate the effects of treatment of Momordica fruit extract (MFE) and sodium orthovanadate, separately and in combination, on serum and tissue lipid profile and on the activities of lipogenic enzymes in alloxan induced diabetic rats. The results show that there was a significant (p < 0.01) increase in serum total lipids, triglycerides and total cholesterol levels after 21 days of alloxan diabetes. In the liver and kidney of diabetic rats the levels of total lipids and triglycerides also increased significantly (p < 0.01) while levels of total cholesterol decreased significantly (p < 0.01 and p < 0.05, respectively). The lipogenic enzymes showed decreased activity in the diabetic liver, while in kidney they showed an increased activity. When compared with the controls these changes were significant. The treatment of alloxan diabetic rats with MFE and SOV prevented these alterations and maintained all parameters near control values. Most effective prevention was however observed in a combined treatment of Momordica with a reduced dose of SOV (0.2%). The results suggest that Momordica fruit extract and SOV exhibit hypolipidemic as well as hypoglycemic effect in diabetic rats and their effect is pronounced when administered in combination. (Mol Cell Biochem 268: 111–120, 2005)

Regulation of NF-κB-induced inflammatory signaling by lipid peroxidation-derived aldehydes.

Oxidative stress plays a critical role in the pathophysiology of a wide range of diseases including cancer. This view has broadened significantly with the recent discoveries that reactive oxygen species initiated lipid peroxidation leads to the formation of potentially toxic lipid aldehyde species such as 4-hydroxy-trans-2-nonenal (HNE), acrolein, and malondialdehyde which activate various signaling intermediates that regulate cellular activity and dysfunction via a process called redox signaling. The lipid aldehyde species formed during synchronized enzymatic pathways result in the posttranslational modification of proteins and DNA leading to cytotoxicity and genotoxicty. Among the lipid aldehyde species, HNE has been widely accepted as a most toxic and abundant lipid aldehyde generated during lipid peroxidation. HNE and its glutathione conjugates have been shown to regulate redox-sensitive transcription factors such as NF-κB and AP-1 via signaling through various protein kinase cascades. Activation of redox-sensitive transcription factors and their nuclear localization leads to transcriptional induction of several genes responsible for cell survival, differentiation, and death. In this review, we describe the mechanisms by which the lipid aldehydes transduce activation of NF-κB signaling pathways that may help to develop therapeutic strategies for the prevention of a number of inflammatory diseases.

Effects of sodium-orthovanadate andTrigonella foenum-graecum seeds on hepatic and renal lipogenic enzymes and lipid profile during alloxan diabetes

Sodium-orthovanadate (SOV) and seed powder ofTrigonella foenum graecum Linn. (common name: fenugreek, family: Fabaceae) (TSP) besides being potential hypoglycemic agents have also been shown to ameliorate altered lipid metabolism during diabetes. This study evaluates the short-term effect of oral administration of SOV and TSP separately and in concert (for 21 days) on total lipid profile and lipogenic enzymes in tissues of alloxan diabetic rats. Diabetic rats showed 4-fold increase in blood glucose. The level of total lipids, triglycerides and total cholesterol in blood serum increased significantly during diabetes. During diabetes the level of total lipids increased significantly (P < 0001) in liver and in kidney by 48% and 55%, respectively, compared to control. Triglycerides level increased by 32% (P < 001) in liver and by 51% (P < 0005) in kidney, respectively, compared to control. Total cholesterol level also increased significantly in both liver and kidney (P < 0.01 andP < 0001, respectively). The activities of NADP-linked enzymes; namely glucose-6-phosphate dehydrogenase (G6PDH), malic enzyme (ME), isocitrate dehydrogenase (ICDH), and the activities of lipogenic enzymes namely ATP-citrate lyase ΜTP-CL) and fatty acid synthase (FAS) were decreased significantly in liver and increased in kidney during diabetes as compared to control. SOV and TSP administration to diabetic animals prevented the development of hyperglycemia and alteration in lipid profile in plasma and tissues and maintained it near normal. Maximum prevention was observed in the combined treatment with lower dose of SOV (0.2%) after 21 days. We are presenting for the first time effectiveness of combined treatment of SOV and TSP in amelioration of altered lipid metabolism during experimental type-I diabetes

Pharmacological effects of Trigonella foenum-graecum L. in health and disease

Abstract Context: The health benefits and medicinal properties of herbal food products are known since antiquity. Fenugreek [Trigonella foenum-graecum Linn. (Fabaceae)], a seed spice used to enhance flavor, color and texture of food, is employed for medicinal purposes in many traditional systems. A number of epidemiological studies and laboratory research have unraveled the biological actions of fenugreek. Objective: Research on fenugreek in recent years has identified a number of health benefits and physiological attributes in both experimental animals as well as clinical trials in humans. In this study we have reviewed the available scientific literature on fenugreek. Methods: This review article summarizes and reviews published experimental studies and scientific literature from the databases including PubMed, Google and local library searches. Results: The information available in the literature on the health benefits and pharmaceutical effects of Trigonella accounts for its known medicinal properties and adds new therapeutic effects in newer indications. Besides its known medicinal properties such as carminative, gastric stimulant, antidiabetic and galactogogue (lactation-inducer) effects, newer research has identified hypocholesterolemic, antilipidemia, antioxidant, hepatoprotective, anti-inflammatory, antibacterial, antifungal, antiulcer, antilithigenic, anticarcinogenic and other miscellaneous medicinal effects of fenugreek. Although most of these studies have used whole seed powder or different forms of extracts, some have identified active constituents from seeds and attributed them medicinal values for different indications. Conclusion: The resarch on Trigonella exhibits its health benefits and potential medicinal properties in various indications and has little or no side effects, suggesting its pharmaceutical, therapeutic and nutritional potential.

Cell‐death assessment by fluorescent and nonfluorescent cytosolic and nuclear staining techniques

Apoptosis, a genetically programmed cellular event leads to biochemical and morphological changes in cells. Alterations in DNA caused by several factors affect nucleus and ultimately the entire cell leading to compromised function of the organ and organism. DNA, a master regulator of the cellular events, is an important biomolecule with regards to cell growth, cell death, cell migration and cell differentiation. It is therefore imperative to develop the staining techniques that may lead to visualize the changes in nucleus where DNA is housed, to comprehend the cellular pathophysiology. Over the years a number of nuclear staining techniques such as propidium iodide, Hoechst‐33342, 4’, 6‐diamidino‐2‐phenylindole (DAPI), Acridine orange–Ethidium bromide staining, among others have been developed to assess the changes in DNA. Some nonnuclear staining techniques such as Annexin‐V staining, which although does not stain DNA, but helps to identify the events that result from DNA alteration and leads to initiation of apoptotic cell death. In this review, we have briefly discussed some of the most commonly used fluorescent and nonfluorescent staining techniques that identify apoptotic changes in cell, DNA and the nucleus. These techniques help in differentiating several cellular and nuclear phenotypes that result from DNA damage and have been identified as specific to necrosis or early and late apoptosis as well as scores of other nuclear deformities occurring inside the cells.

Protective effects of magnesium lithospermate B against diabetic atherosclerosis via Nrf2-ARE-NQO1 transcriptional pathway

Hyperglycemia-induced oxidative stress is known to play an important role in the development of several diabetic complications, including atherosclerosis. Although a number of antioxidants are available, none have been found to be suitable for regulating the oxidative stress response and enhancing antioxidative defense mechanisms. In this study, we evaluated the effects of magnesium lithospermate B (LAB) against oxidative stress. We also endeavored to identify the target molecule of LAB in vascular smooth muscle cells (VSMCs) and the underlying biochemical pathways related to diabetic atherosclerosis. Modified MTT and transwell assays showed that the increased proliferation and migration of rat aortic VSMCs in culture with high glucose was significantly inhibited by LAB. LAB also attenuated neointimal hyperplasia after balloon catheter injury in diabetic rat carotid arteries. To determine molecular targets of LAB, we studied the effects of LAB on aldose reductase (AR) activity, O-GlcNAcylation, and protein kinase C (PKC) activity in VSMCs under normoglycemic or hyperglycemic conditions and showed the improvement of major biochemical pathways by LAB. Potential involvement of the nuclear factor erythroid 2-related factor-2 (Nrf2)--antioxidant responsive element (ARE)-NAD(P)H: quinone oxidoreductase-1 (NQO1) pathway was assessed using siRNA methods. We found that LAB activates the NQO1 via the Nrf2-ARE pathway, which plays an important role in inhibition of the major molecular mechanisms that lead to vascular damage and the proliferation and migration of VSMCs. Together, these findings demonstrate that the induction of the Nrf2-ARE-NQO1 pathway by LAB could be a new therapeutic strategy to prevent diabetic atherosclerosis.

Prevention of Endotoxin-Induced Uveitis in Rats by Benfotiamine, a Lipophilic Analogue of Vitamin B1

PURPOSE To study the amelioration of ocular inflammation in endotoxin-induced uveitis (EIU) in rats by benfotiamine, a lipid-soluble analogue of thiamine. METHODS EIU in Lewis rats was induced by subcutaneous injection of lipopolysaccharide (LPS) followed by treatment with benfotiamine. The rats were killed 3 or 24 hours after LPS injection, eyes were enucleated, aqueous humor (AqH) was collected, and the number of infiltrating cells, protein concentration, and inflammatory marker levels were determined. Immunohistochemical analysis of eye sections was performed to determine the expression of inducible-nitric oxide synthase (iNOS), cyclooxygenase (Cox)-2, protein kinase C (PKC), and transcription factor NF-kappaB. RESULTS Infiltrating leukocytes, protein concentrations, and inflammatory cytokines and chemokines were significantly elevated in the AqH of EIU rats compared with control rats, and benfotiamine treatment suppressed these increases. Similarly increased expression of inflammatory markers iNOS and Cox-2 in ciliary body and retinal wall was also significantly inhibited by benfotiamine. The increased phosphorylation of PKC and the activation of NF-kappaB in the ciliary body and in the retinal wall of EIU rat eyes were suppressed by benfotiamine. CONCLUSIONS These results suggest that benfotiamine suppresses oxidative stress-induced NF-kappaB-dependent inflammatory signaling leading to uveitis. Therefore, benfotiamine could be used as a novel therapeutic agent for the treatment of ocular inflammation, especially uveitis.