Cardiovascular disease and PPARdelta: Targeting the risk factors

Metabolism, in part, is regulated by the peroxisome proliferator-activated receptors (PPARs). The PPARs act as nutritional lipid sensors and three mammalian PPAR subtypes designated PPARalpha (NR1C1), PPARgamma (NR1C3) and PPARdelta (NR1C2) have been identified. This subgroup of nuclear hormone receptors binds DNA and controls gene expression at the nexus of pathways that regulate lipid and glucose homeostasis, energy storage and expenditure in an organ-specific manner. Recent evidence has demonstrated activation of PPARdelta in the major mass peripheral tissue (ie, adipose and skeletal muscle). It enhances glucose tolerance, insulin-stimulated glucose disposal, lipid catabolism, energy expenditure, cholesterol efflux and oxygen consumption. These effects positively influence the blood-lipid profile. Furthermore, PPARdelta activation produces a predominant type I/slow twitch/oxidative muscle fiber phenotype that leads to increased endurance, insulin sensitivity and resistance to obesity. PPARdelta has rapidly emerged as a potential target in the battle against dyslipidemia, insulin insensitivity, type II diabetes and obesity, with therapeutic efficacy in the treatment of cardiovascular disease risk factors. GW-501516 is currently undergoing phase II safety and efficacy trials in human volunteers for the treatment of dyslipidemia. The outcome of these clinical trials are eagerly awaited against a background of conflicting reports about cancer risks in genetically predisposed animal models. This review focuses on the potential pharmacological utility of selective PPARdelta agonists in the context of risk factors associated with metabolic and cardiovascular disease.

Treating type 2 diabetes through insulin resistance

Type 2 diabetes is an insidious disorder, with micro and/or macrovascular and nervous damage occurring in many patients before diagnosis. This damage is caused by hyperglycaemia and the diverse effects of insulin resistance. Obesity, in particular central obesity, is a strong pre-disposing factor for type 2 diabetes. Skeletal muscle is the main site of insulin-stimulated glucose disposal and appears to be the first organ that becomes insulin resistant in the diabetic state, with later involvement of adipose tissue and the liver. This study has investigated the use of novel agents to ameliorate insulin-resistance in skeletal muscle as a means of identifying intervention sites against insulin resistance and of improving glucose uptake and metabolism by skeletal muscle. Glucose uptake was measured in vitro by cultured L6 myocytes and isolated muscles from normal and obese diabetic ob/ob mice, using either the tritiated non-metabolised glucose analogue 2-deoxy-D-glucose or by glucose disposal. Agents studied included lipoic acid, isoferulic acid, bradykinin, lipid mobilising factor (provisionally synonymous with Zinca2 glycoprotein) and the trace elements lithium, selenium and chromium. The putative role of TNFa in insulin resistance was also investigated. Lipoic acid improved insulin-stimulated glucose uptake in normal and insulin resistance murine muscles, as well as cultured myocytes. Isoferulic acid, bradykinin and LMF also produced a transient increase in glucose uptake in cultured myocytes. Physiological concentrations of TNFa were found to cause insulin resistance in cultured, but no in excised murine muscles. The effect of the M2 metabolite of the satiety-inducing agent sibutramine on lipolysis in excised murine and human adipocytes was also investigated. M2 increased lipolysis from normal lean and obese ob/ob mouse adipocytes. Arguably the most important observation was that M2 also increased the lipolytic rate in adipocytes from catecholamine resistant obese subjects. The studies reported in this thesis indicate that a diversity of agents can improve glucose uptake and ameliorate insulin resistance. It is likely that these agents are acting via different pathways. This thesis has also shown that M2 can induce lipolysis in both rodent and human adipocytes. M2 hence has potential to directly reduce adiposity, in addition to well documented effects via the central nervous system.

Palmitate induces insulin resistance and a pro-atherogenic phenotype in monocytes

The present study investigated the effect of the two most abundant FFA in plasma – palmitate and oleate – on insulin sensitivity and vascular function (monocyte phenotype and adhesion to endothelium) using in vitro cell culture models and Wistar rats. Palmitate at 300µM for 6h induced insulin resistance in THP-1 monocytes and L6 monocytes. The ceramide synthesis pathway partly accounted for the palmitate-induced insulin resistance in THP-1 monocytes but not for L6 myotubes. Oleate treatment did not induce insulin resistance in either cell type and co-incubation with oleate protected cells from palmitate-induced insulin resistance. Palmitate at 300µN for 24h significantly increased cell surface CD11b and CD36 expression in U937 monocytes. The increase in CD11b and CD36 expression was effectively inhibited by Fumonisin B1, an inhibitor of ceramide synthesis. Oleate treatment did not show any effect on CD11b and CD36 expression and co-incubation with oleate antagonised the effect of palmitate on CD11b and CD36 expression in U937 monocytes. The increase in CD11b expression did not affect U937 monocyte adhesion to ICAM-1. Treating Wistar rats with palmitate for 6h caused a transient delay in glucose disposal and an increase in adhesion of U937 monocytes to the aortic endothelium, particularly at bifurcations. In conclusion, the present study demonstrates that the saturated free fatty acid palmitate induces insulin resistance and a pro-atherogenic phenotype for monocytes, whereas the unsaturated free fatty acid oleate does not. In vivo studies also confirmed that palmitate induces insulin resistance and an increase in monocyte adhesion to aorta.

Role of β-adrenergic receptors in the oral activity of zinc-α2-glycoprotein (ZAG)

Zinc-a2-glycoprotein (ZAG) is an adipokine with the potential as a therapeutic agent in the treatment of obesity and type 2 diabetes. In this study we show that human ZAG, which is a 41-kDa protein, when administered to ob/ob mice at 50 µg/d-1 orally in the drinking water produced a progressive loss of body weight (5 g after 8 d treatment), together with a 0.5 C increase in rectal temperature and a 40% reduction in urinary excretion of glucose. There was also a 33% reduction in the area under the curve during an oral glucose tolerance test and an increased sensitivity to insulin. These results were similar to those after iv administration of ZAG. However, tryptic digestion was shown to inactivate ZAG. There was no evidence of human ZAG in the serum but a 2-fold elevation of murine ZAG, which was also observed in target tissues such as white adipose tissue. To determine whether the effect was due to interaction of the human ZAG with the ß-adrenergic (ß-AR) in the gastrointestinal tract before digestion, ZAG was coadministered to ob/ob mice together with propanolol (40 mg/kg-1), a nonspecific ß-AR antagonist. The effect of ZAG on body weight, rectal temperature, urinary glucose excretion, improvement in glucose disposal, and increased insulin sensitivity were attenuated by propanolol, as was the increase in murine ZAG in the serum. These results suggest that oral administration of ZAG increases serum levels through interaction with a ß-AR in the upper gastrointestinal tract, and gene expression studies showed this to be in the esophagus.

Metformin

Metformin is the only biguanide antihyperglycemic agent used in the treatment of type 2 (non-insulin dependent) diabetes mellitus. It counters insulin resistance partly by increased insulin action (so-called insulin sensitizing effects) and partly via actions that are not directly insulin dependent. Metformin reduces hepatic glucose output by suppression of gluconeogenesis and glycogenolysis. In skeletal muscle, metformin increases insulin-mediated glucose uptake and glycogen storage. Other actions of metformin that contribute to its blood glucose-lowering effect are reduced fatty acid oxidation and increased glucose turnover, the latter occurring particularly in the splanchnic bed .... © 2007 Copyright © 2007 Elsevier Inc.

Nouveaux indices de suppression de la lipolyse par l'insuline déterminés lors de l'hyperglycémie provoquée par voie orale : comparaisons avec le clamp euglycémique-hyperinsulinémique et les paramètres métaboliques chez les femmes

Résumé : Une dysrégulation de la lipolyse des tissus adipeux peut conduire à une surexposition des tissus non-adipeux aux acides gras non-estérifiés (AGNE), qui peut mener à un certain degré de lipotoxicité dans ces tissus. La lipotoxicité constitue, par ailleurs, l’une des causes majeures du développement de la résistance à l’insuline et du diabète de type 2. En plus de ses fonctions glucorégulatrices, l’insuline a pour fonction d’inhiber la lipolyse et donc de diminuer les niveaux d’AGNE en circulation, prévenant ainsi la lipotoxicité. Il n’y a pas d’étalon d’or pour mesurer la sensibilité de la lipolyse à l’insuline. Le clamp euglycémique hyperinsulinémique constitue la méthode étalon d’or pour évaluer la sensibilité du glucose à l’insuline mais il est aussi utilisé pour mesurer la suppression de la lipolyse par l’insuline. Par contre, cette méthode est couteuse et laborieuse, et ne peut pas s’appliquer à de grandes populations. Il existe aussi des indices pour estimer la fonction antilipolytique de l’insuline dérivés de l’hyperglycémie provoquée par voie orale (HGPO), un test moins dispendieux et plus simple à effectuer à grande échelle. Cette étude vise donc à : 1) Étudier la relation entre les indices de suppressibilité des AGNE par l’insuline dérivés du clamp et ceux dérivés de l’HGPO; et 2) Déterminer laquelle de ces mesures corrèle le mieux avec les facteurs connus comme étant reliés à la dysfonction adipeuse : paramètres anthropométriques et indices de dysfonction métabolique. Les résultats montrent que dans le groupe de sujets étudiés (n=29 femmes, 15 témoins saines et 14 femmes avec résistance à l’insuline car atteintes du syndrome des ovaires polykystiques), certains indices de sensibilité à l’insuline pour la lipolyse dérivés de l’HGPO corrèlent bien avec ceux dérivés du clamp euglycémique hyperinsulinémique. Parmi ces indices, celui qui corrèle le mieux avec les indices du clamp et les paramètres anthropométriques et de dysfonction adipeuse est le T50[indice inférieur AGNE] (temps nécessaire pour diminuer de 50% le taux de base – à jeun – des AGNE). Nos résultats suggèrent donc que l’HGPO, facile à réaliser, peut être utilisée pour évaluer la sensibilité de la lipolyse à l’insuline. Nous pensons que la lipo-résistance à l’insuline peut être facilement quantifiée en clinique humaine.

Surgical infection and malnutrition

Background: Malnutrition in surgical patients is associated with delayed recovery, higher rates of morbidity and mortality, prolonged hospital stay, increased healthcare costs and a higher early re-admission rate. Methods: Data synthesis after review of pertinent literature. Results: The aetiology of malnutrition is multifactorial. In cancer patients, there is an abnormal peripheral glucose disposal, gluconeogenesis, and whole-body glucose turnover. Malnourished cancer patients undergoing major operations are at significant risk from perioperative complications such as infectious complications. Surgical aggression generates an inflammatory response which worsens intermediary metabolism. Conclusions: Nutritional evaluation and nutritional support must be performed in all surgical patients, in order to minimize infectious complications. Enteral nutrition early in the postoperative period is effective and well tolerated reducing infectious complications, improving wound healing and reducing length of hospital stay. Pharmaconutrition is indicated in those patients, who benefit from enteral administration of arginine, omega 3 and RNA, as well as parenteral glutamine supplementation. When proximal sutures are used, tubes allowing early jejunal feeding should be used.

Insulin sensitivity and secretion in obese Type 2 diabetic women after various bariatric operations

Objective: To compare the effects of biliopancreatic diversion (BPD) and laparoscopic gastric banding (LAGB) on insulin sensitivity and secretion with the effects of laparoscopic gastric plication (P). Methods: A total of 52 obese women (age 30-66 years) suffering from type 2 diabetes mellitus (T2DM) were prospectively recruited into three study groups: 16 BPD; 16 LAGB, and 20 P. Euglycemic clamps and mixed meal tolerance tests were performed before, at 1 month and at 6 months after bariatric surgery. Beta cell function derived from the meal test parameters was evaluated using mathematical modeling. Results: Glucose disposal per kilogram of fat free mass (a marker of peripheral insulin sensitivity) increased significantly in all groups, especially after 1 month. Basal insulin secretion decreased significantly after all three types of operations, with the most marked decrease after BPD compared with P and LAGB. Total insulin secretion decreased significantly only following the BPD. Beta cell glucose sensitivity did not change significantly post-surgery in any of the study groups. Conclusion: We documented similar improvement in insulin sensitivity in obese T2DM women after all three study operations during the 6-month postoperative follow-up. Notably, only BPD led to decreased demand on beta cells (decreased integrated insulin secretion), but without increasing the beta cell glucose sensitivity.

High Glucose-Mediated Oxidative Stress Impairs Cell Migration

Deficient wound healing in diabetic patients is very frequent, but the cellular and molecular causes are poorly defined. In this study, we evaluate the hypothesis that high glucose concentrations inhibit cell migration. Using CHO.K1 cells, NIH-3T3 fibroblasts, mouse embryonic fibroblasts and primary skin fibroblasts from control and diabetic rats cultured in 5 mM D-glucose (low glucose, LG), 25 mM D-glucose (high glucose, HG) or 25 mM L-glucose medium (osmotic control - OC), we analyzed the migration speed, protrusion stability, cell polarity, adhesion maturation and the activity of the small Rho GTPase Rac1. We also analyzed the effects of reactive oxygen species by incubating cells with the antioxidant N-Acetyl-Cysteine (NAC). We observed that HG conditions inhibited cell migration when compared to LG or OC. This inhibition resulted from impaired cell polarity, protrusion destabilization and inhibition of adhesion maturation. Conversely, Rac1 activity, which promotes protrusion and blocks adhesion maturation, was increased in HG conditions, thus providing a mechanistic basis for the HG phenotype. Most of the HG effects were partially or completely rescued by treatment with NAC. These findings demonstrate that HG impairs cell migration due to an increase in oxidative stress that causes polarity loss, deficient adhesion and protrusion. These alterations arise, in large part, from increased Rac1 activity and may contribute to the poor wound healing observed in diabetic patients.

cAMP prevents the glucose-mediated stimulation of GLUT2 gene transcription in hepatocytes.

Glucose homoeostasis necessitates the presence in the liver of the high Km glucose transporter GLUT2. In hepatocytes, we and others have demonstrated that glucose stimulates GLUT2 gene expression in vivo and in vitro. This effect is transcriptionally regulated and requires glucose metabolism within the hepatocytes. In this report, we further characterized the cis-elements of the murine GLUT2 promoter, which confers glucose responsiveness on a reporter gene coding the chloramphenicol acetyl transferase (CAT) gene. 5'-Deletions of the murine GLUT2 promoter linked to the CAT reporter gene were transfected into a GLUT2 expressing hepatoma cell line (mhAT3F) and into primary cultured rat hepatocytes, and subsequently incubated at low and high glucose concentrations. Glucose stimulates gene transcription in a manner similar to that observed for the endogenous GLUT2 mRNA in both cell types; the -1308 to -212 bp region of the promoter contains the glucose-responsive elements. Furthermore, the -1308 to -338 bp region of the promoter contains repressor elements when tested in an heterologous thymidine kinase promoter. The glucose-induced GLUT2 mRNA accumulation was decreased by dibutyryl-cAMP both in mhAT3F cells and in primary hepatocytes. A putative cAMP-responsive element (CRE) is localized at the -1074/-1068 bp region of the promoter. The inhibitory effect of cAMP on GLUT2 gene expression was observed in hepatocytes transfected with constructs containing this CRE (-1308/+49 bp fragment), as well as with constructs not containing the consensus CRE (-312/+49 bp fragment). This suggests that the inhibitory effect of cAMP is not mediated by the putative binding site located in the repressor fragment of the GLUT2 promoter. Taken together, these data demonstrate that the elements conferring glucose and cAMP responsiveness on the GLUT2 gene are located within the -312/+49 region of the promoter.

Assay for high glucose-mediated islet cell sensitization to apoptosis induced by streptozotocin and cytokines

Pancreatic beta-cell apoptosis is known to participate in the beta-cell destruction process that occurs in diabetes. It has been described that high glucose level induces a hyperfunctional status which could provoke apoptosis. This phenomenon is known as glucotoxicity and has been proposed that it can play a role in type 1 diabetes mellitus pathogenesis. In this study we develop an experimental design to sensitize pancreatic islet cells by high glucose to streptozotocin (STZ) and proinflammatory cytokines [interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma]-induced apoptosis. This method is appropriate for subsequent quantification of apoptotic islet cells stained with Tdt-mediated dUTP Nick-End Labeling (TUNEL) and protein expression assays by Western Blotting (WB).

Oxygen/Glucose deprivation induces a reduction in synaptic AMPA receptors on hippocampal CA3 neurons mediated by mGluR1 and adenosine A3 receptors

Hippocampal CA1 pyramidal neurons are highly sensitive to ischemic damage, whereas neighboring CA3 pyramidal neurons are less susceptible. It is proposed that switching of AMPA receptor (AMPAR) subunits on CA1 neurons during an in vitro model of ischemia, oxygen/glucose deprivation (OGD), leads to an enhanced permeability of AMPARs to Ca2+, resulting in delayed cell death. However, it is unclear whether the same mechanisms exist in CA3 neurons and whether this underlies the differential sensitivity to ischemia. Here, we investigated the consequences of OGD for AMPAR function in CA3 neurons using electrophysiological recordings in rat hippocampal slices. Following a 15 min OGD protocol, a substantial depression of AMPAR-mediated synaptic transmission was observed at CA3 associational/commissural and mossy fiber synapses but not CA1 Schaffer collateral synapses. The depression of synaptic transmission following OGD was prevented by metabotropic glutamate receptor 1 (mGluR1) or A3 receptor antagonists, indicating a role for both glutamate and adenosine release. Inhibition of PLC, PKC, or chelation of intracellular Ca2+ also prevented the depression of synaptic transmission. Inclusion of peptides to interrupt the interaction between GluA2 and PICK1 or dynamin and amphiphysin prevented the depression of transmission, suggesting a dynamin and PICK1-dependent internalization of AMPARs after OGD. We also show that a reduction in surface and total AMPAR protein levels after OGD was prevented by mGluR1 or A3 receptor antagonists, indicating that AMPARs are degraded following internalization. Thus, we describe a novel mechanism for the removal of AMPARs in CA3 pyramidal neurons following OGD that has the potential to reduce excitotoxicity and promote neuroprotection

Triphenylmethane dyes, an alternative for mediated electronic transfer systems in glucose oxidase biofuel cells

The bioelectrochemical behavior of three triphenylmethane (TPM) dyes commonly used as pH indicators, and their application in mediated electron transfer systems for glucose oxidase bioanodes in biofuel cells was investigated. Bromophenol Blue, Bromothymol Blue, Bromocresol Green were compared bio-electrochemically against two widely used mediators, benzoquinone and ferrocene carboxy aldehyde. Biochemical studies were performed in terms of enzymatic oxidation, enzyme affinity, catalytic efficiency and co-factor regeneration. The different features of the TPM dyes as mediators are determined by the characteristics in the oxidation/reduction processes studied electrochemically. The reversibility of the oxidation/reduction processes was also established through the dependence of the voltammetric peaks with the sweep rates. All three dyes showed good performances compared to the FA and BQ when evaluated in a half enzymatic fuel cell. Potentiodynamic and power response experiments showed maxima power densities of 32.8 mu W cm(-2) for ferrocene carboxy aldehyde followed by similar values obtained for TPM dyes around 30 mu W cm(-2) using glucose and mediator concentrations of 10 mmol L(-1) and 1.0 mmol L(-1), respectively. Since no mediator consumption was observed during the bioelectrochemical process, and also good redox re-cycled processes were achieved, the use of triphenylmethane dyes is considered to be promising compared to other mediated systems used with glucose oxiclase bioanodes and/or biofuel cells. (C) 2011 Elsevier Inc. All rights reserved.

Ethylene-Mediated Phospholipid Catabolic Pathway in Glucose-Starved Carrot Suspension Cells1

Glucose (Glc) starvation of suspension-cultured carrot (Daucus carota L.) cells resulted in sequential activation of phospholipid catabolic enzymes. Among the assayed enzymes involved in the degradation, phospholipase D (PLD) and lipolytic acyl hydrolase were activated at the early part of starvation, and these activities were followed by β-oxidation and the glyoxylate cycle enzymes in order. The activity of PLD and lipolytic acyl hydrolase was further confirmed by in vivo-labeling experiments. It was demonstrated that Glc added to a medium containing starving cells inhibited the phospholipid catabolic activities, indicating that phospholipid catabolism is negatively regulated by Glc. There was a burst of ethylene production 6 h after starvation. Ethylene added exogeneously to a Glc-sufficient medium activated PLD, indicating that ethylene acts as an element in the signal transduction pathway leading from Glc depletion to PLD activation. Activation of lipid peroxidation, suggestive of cell death, occurred immediately after the decrease of the phospholipid degradation, suggesting that the observed phospholipid catabolic pathway is part of the metabolic strategies by which cells effectively survive under Glc starvation.