Maleppillil Vavachan Vijayakumar

The hypoglycaemic activity of fenugreek seed extract is mediated through the stimulation of an insulin signalling pathway

The in vivo hypoglycaemic activity of a dialysed fenugreek seed extract (FSE) was studied in alloxan (AXN)‐induced diabetic mice and found to be comparable to that of insulin (1.5 U kg−1). FSE also improved intraperitoneal glucose tolerance in normal mice. The mechanism by which FSE attenuated hyperglycaemia was investigated in vitro. FSE stimulated glucose uptake in CHO‐HIRc‐mycGLUT4eGFP cells in a dose‐dependent manner. This effect was shown to be mediated by the translocation of glucose transporter 4 (GLUT4) from the intracellular space to the plasma membrane. These effects of FSE on GLUT4 translocation and glucose uptake were inhibited by wortmannin, a phosphatidylinositol 3‐kinase (PI3‐K) inhibitor, and bisindolylmaleimide 1, a protein kinase C (PKC)‐specific inhibitor. In vitro phosphorylation analysis revealed that, like insulin, FSE also induces tyrosine phosphorylation of a number of proteins including the insulin receptor, insulin receptor substrate 1 and p85 subunit of PI3‐K, in both 3T3‐L1 adipocytes and human hepatoma cells, HepG2. However, unlike insulin, FSE had no effect on protein kinase B (Akt) activation. These results suggest that in vivo the hypoglycaemic effect of FSE is mediated, at least in part, by the activation of an insulin signalling pathway in adipocytes and liver cells.

Hypolipidemic Effect of Fenugreek Seeds Is Mediated Through Inhibition of Fat Accumulation and Upregulation of LDL Receptor

Fenugreek (Trigonella foenum‐graecum) seeds, used as a condiment, are documented for health benefits including amelioration of abnormalities in lipid homeostasis due to its hypolipidemic properties. However, molecular mechanisms underlying the hypolipidemic effect of fenugreek seeds remain obscure. In this study, hypolipidemic effect of a novel thermostable extract of fenugreek seeds (TEFS) was evaluated in vitro by employing differentiating and differentiated 3T3‐L1 cells, and HepG2 cells cultured in normal or sterol‐enriched conditions. Hypolipidemic effect was studied by quantifying decrease in accumulation of fat or by western blot analysis of adipogenic and lipogenic factors. At molecular level, TEFS inhibited accumulation of fat in differentiating and differentiated 3T3‐L1 cells via decreased expression of adipogenic factors such as peroxisome proliferators activated‐receptor‐γ (PPAR‐γ), sterol regulatory element‐binding protein‐1 (SREBP‐1), and CAAT element‐binding proteins‐α (c/EBP‐α). We also show that following TEFS treatment, cellular triglycerides (TGs), and cholesterol concentrations decreased significantly (P < 0.05) in HepG2 cells via reduced expression of SREBP‐1, at mRNA as well as protein level. Under sterol enriched condition, TEFS upregulated low‐density lipoprotein receptor (LDLR) expression resulting in enhanced LDL uptake. Treating fat supplement fed C57BL6/J mice with TEFS for 15 days resulted in decrease of serum TG, LDL‐cholesterol (LDLc), and body weight in a dose‐ and time‐dependent manner (P < 0.05). Results indicate that hypolipidemic effect of TEFS is due to inhibition of fat accumulation and upregulation of LDLR. Taken together, the study suggests that TEFS may have potential application in the management of dyslipidemia and its associated metabolic disorders.

Diet-induced obesity increases melanoma progression: Involvement of Cav-1 and FASN

Recent population‐based epidemiological studies strongly hint towards a link between obesity and its occurrence as well as progression of several cancers including melanoma. Although effects of obesity on breast, colon and liver cancers have been extensively investigated, the links between obesity and melanoma remain largely unexplored. Present study aimed to understand the effect of high fat diet‐induced weight gain on susceptibility of C57BL/6J mice to melanoma. For this, mice routinely were fed on high fat diet for 6 months (HFD mice). Subsequently, mouse melanoma cells were injected subcutaneously in control as well as HFD mice and followed for tumor initiation and progression. We provide strong evidence that diet‐induced obesity leads to increased melanoma progression in male C57BL/6J mice. We observed that increased melanoma progression is associated with enhanced Cav‐1 and FASN expression in tumors from HFD mice. Cav‐1 and FASN are co‐ordinately regulated and Cav‐1 interacts with FASN in melanoma cells. Enhanced levels of Cav‐1, FASN and pAkt control melanoma cell proliferation. Our study establishes a causative relationship between diet‐induced obesity and melanoma progression as well as demonstrates that obesity affects important tumorigenic pathways in melanoma.

Cdk5 phosphorylates non-genotoxically overexpressed p53 following inhibition of PP2A to induce cell cycle arrest/apoptosis and inhibits tumor progression

Backgroundp53 is the most studied tumor suppressor and its overexpression may or may not cause cell death depending upon the genetic background of the cells. p53 is degraded by human papillomavirus (HPV) E6 protein in cervical carcinoma. Several stress activated kinases are known to phosphorylate p53 and, among them cyclin dependent kinase 5 (Cdk5) is one of the kinase studied in neuronal cell system. Recently, the involvement of Cdk5 in phosphorylating p53 has been shown in certain cancer types. Phosphorylation at specific serine residues in p53 is essential for it to cause cell growth inhibition. Activation of p53 under non stress conditions is poorly understood. Therefore, the activation of p53 and detection of upstream kinases that phosphorylate non-genotoxically overexpressed p53 will be of therapeutic importance for cancer treatment.ResultsTo determine the non-genotoxic effect of p53; Tet-On system was utilized and p53 inducible HPV-positive HeLa cells were developed. p53 overexpression in HPV-positive cells did not induce cell cycle arrest or apoptosis. However, we demonstrate that overexpressed p53 can be activated to upregulate p21 and Bax which causes G2 arrest and apoptosis, by inhibiting protein phosphatase 2A. Additionally, we report that the upstream kinase cyclin dependent kinase 5 interacts with p53 to phosphorylate it at Serine20 and Serine46 residues thereby promoting its recruitment on p21 and bax promoters. Upregulation and translocation of Bax causes apoptosis through intrinsic mitochondrial pathway. Interestingly, overexpressed activated p53 specifically inhibits cell-growth and causes regression in vivo tumor growth as well.ConclusionPresent study details the mechanism of activation of p53 and puts forth the possibility of p53 gene therapy to work in HPV positive cervical carcinoma.

Hypoglycemic Effect of a Novel Dialysed Fenugreek Seeds Extract is Sustainable and is Mediated, in Part, by the Activation of Hepatic Enzymes

A novel preparation of a dialysed aqueous extract of fenugreek seeds (FSE) that stimulates the insulin signalling pathway was reported previously (Vijayakumar et al., 2005). The present study was designed to investigate the long‐term effects (multiple dose effect) of this FSE preparation on the blood glucose level and body weight, and a short‐term effect (single dose effect) on serum insulin and hepatic enzymes, in experimentally induced diabetic conditions. The multiple dose effect of FSE on the glucose level and body weight was studied in alloxan (AXN)‐diabetic mice in comparison with the vehicle treated control diabetic mice. Intraperitoneal (i.p.) administration of FSE (15 mg/kg body weight (BW)) for 5 consecutive days reduced hyperglycemia in AXN‐diabetic mice on day 5 and this effect was further sustained for 10 days. The FSE‐induced hypoglycemic effect was accompanied without any reduction in the body weight compared with the diabetic mice in which the body weight was reduced significantly. A single dose effect of FSE on hepatic glucokinase (GK) and hexokinase (HK) enzymes was studied in streptozotocin (STZ)‐diabetic mice. Intraperitoneal administration of FSE (15 mg/kg BW) by 90 min decreased the blood glucose levels significantly (p < 0.01) in STZ‐diabetic mice and the effect was comparable to that achieved by insulin (1.5 U/kg BW) injection. This effect was associated with a significant enhancement in the liver GK and HK activities on a par with that of insulin. In normal glucose loaded mice, FSE improved the intraperitoneal glucose tolerance accompanied by a reduction in serum insulin concentration. These results are indicative of an extra‐pancreatic mode of action of FSE. The present study concludes that this novel FSE preparation corrects metabolic alterations associated with diabetes by exhibiting insulin‐like properties and has a potential for clinical applications. Copyright

Obesity induced rapid melanoma progression is reversed by orlistat treatment and dietary intervention: Role of adipokines

Obesity, owing to adiposity, is associated with increased risk and development of various cancers, and linked to their rapid growth as well as progression. Although a few studies have attempted to understand the relationship between obesity and melanoma, the consequences of controlling body weight by reducing adiposity on cancer progression is not well understood. By employing animal models of obesity, we report that controlling obesity either by orlistat treatment or by restricting caloric intake significantly slows down melanoma progression. The diminished tumor progression was correlated with decreased fat mass (adiposity) in obese mice. Obesity associated factors contributing to tumor progression were decreased in the experimental groups compared to respective controls. In tumors, protein levels of fatty acid synthase (FASN), caveolin (Cav)‐1 and pAkt, which are tumor promoting molecules implicated in melanoma growth under obese state, were decreased. In addition, increased necrosis and reduction in angiogenesis as well as proliferative markers PCNA and cyclin D1 were observed in tumors of the orlistat treated and/or calorically restricted obese mice. We observed that growth of melanoma cells cultured in conditioned medium (CM) from orlistat‐treated adipocytes was reduced. Adipokines (leptin and resistin), via activating Akt and modulation of FASN as well as Cav‐1 respectively, enhanced melanoma cell growth and proliferation. Together, we demonstrate that controlling body weight reduces adipose mass thereby diminishing melanoma progression. Therefore, strategic means of controlling obesity by reduced caloric diet or with antiobesity drugs treatment may render obesity‐promoted tumor progression in check and prolong survival of patients.

Demonstration of a visual cell-based assay for screening glucose transporter 4 translocation modulators in real time

Insulin-stimulated translocation of glucose transporter 4 (GLUT4) to cell membrane leading to glucose uptake is the rate-limiting step in diabetes. It is also a defined target of antidiabetic drug research. Existing GLUT4 translocation assays are based on time-consuming immunoassays and are hampered by assay variability and low sensitivity. We describe a real-time, visual, cell-based qualitative GLUT4 translocation assay using CHO-HIRc-myc-GLUT4eGFP cells that stably express myc- and eGFP-tagged GLUT4 in addition to human insulin receptor (HIRc). GLUT4 translocation is visualized by live cell imaging based on GFP fluorescence by employing a cooled charge-coupled device camera attached to a fluorescent microscope. This video imaging method and further quantitative analysis of GLUT4 on the cell membrane provide rapid and foolproof visual evidence that this method is suitable for screening GLUT4 translocation modulators.

Real time qualitative and quantitative GLUT4 translocation assay.

Insulin-stimulated glucose transporter 4 (GLUT4) translocation promoting glucose uptake is vital to glucose homeostasis and is a defined target of antidiabetic drug research. Existing functional assays to detect the process of GLUT4 translocation are hampered due to assay variability and low sensitivity, thus slowing down the progress towards the development of preferred alternative to insulin. This chapter describes a real time, visual, cell-based qualitative GLUT4 translocation assay suitable for screening insulin mimetics. The basic strategy consists of establishment of insulin-sensitive CHO-HIRc-myc-GLUT4eGFP cells those stably express myc and eGFP-tagged GLUT4 in addition to human insulin receptor (HIRc). GLUT4 translocation is visualized by tracking the movement of GLUT4 associated GFP fluorescence from perinuclear space to the plasma membrane by employing cooled charge-coupled device (CCD) camera attached to a simple fluorescent microscope. This video imaging method and further quantitative analysis of GLUT4 on the cell membrane provides rapid and fool-proof visual evidence suitable for screening GLUT4 translocation modulators. This assay is further validated by complementary assays.

Elevated circulatory levels of leptin and resistin impair therapeutic efficacy of dacarbazine in melanoma under obese state

BackgroundObesity is associated with increased risk, poor prognosis and outcome of therapy, in various cancers. Obesity-associated factors or adipokines, especially leptin and resistin, are purported to promote growth, survival, proliferation, and invasiveness of cancer cells. However, the mechanistic link between these adipokines and therapeutic response in malignancies is not clearly understood.Methodsob/ob and db/db mouse models were used in this study to evaluate the role of leptin and resistin towards the outcome of dacarbazine (DTIC) therapy in melanoma. Unique in vitro approaches were employed to complement in vivo findings by culturing melanoma cells in the serum collected from the experimental mice.ResultsHere, we have shown the role of important adipokines leptin and resistin in growth and the outcome of DTIC therapy in melanoma. Both leptin and resistin not only enhance proliferation of melanoma cells but also are involved in impairing the therapeutic efficacy of DTIC. Leptin and resistin treatment caused an increase in the protein levels of fatty acid synthase (FASN) and caveolin 1 (Cav-1) respectively, through their stabilization in A375 cells. Further, it was observed that leptin and resistin impaired the response of melanoma cells to DTIC via upregulation of heat shock protein 90 (Hsp90) and P-glycoprotein (P-gp) respectively.ConclusionThese findings unraveled the involvement of adipokines (leptin and resistin) in melanoma progression, and more importantly, in the outcome of DTIC therapy.