Ezetimibe (EZE) and glucuronidated EZE (EZE-Glu) differentially target Niemann-Pick C1-like 1 (NPC1L1) and CD13 (aminopeptidase-N) to inhibit intestinal cholesterol absorption and cholesterol processing in other cells, although the precise molecular mechanisms are not fully elucidated. Cellular effects of EZE, EZE-Glu, and the low-absorbable EZE-analogue S6130 were investigated on human monocyte-derived macrophages upon loading with atherogenic lipoproteins. EZE and S6130, but not EZE-Glu disturbed the colocalization of CD13 and its coreceptor CD64 (Fcγ receptor I) in membrane microdomains, and decreased the presence of both receptors in detergent-resistant membrane fractions. Biotinylated cholesterol absorption inhibitor C-5 (i.e., derivative of EZE) was rapidly internalized to perinuclear tubular structures of cells, resembling endoplasmic reticulum (ER), but CD13 was detected on extracellular sites of the plasma membrane and endolysosomal vesicles. Administration of EZE, but not of EZE-Glu or S6130, was associated with decreased cellular cholesteryl ester content, indicating the sterol-O acyltransferase 1 (SOAT1)-inhibition by EZE. Furthermore, EZE decreased the expression of molecules involved in cholesterol uptake and synthesis, in parallel with increased apolipoprotein A-I-mediated cholesterol efflux and upregulation of efflux-effectors. However, NPC1L1 the other claimed molecular target of EZE, was not detected in macrophages, thereby excluding this protein as target for EZE in macrophages. Thus, EZE is very likely a CD13-linked microdomain-disruptor and SOAT1-inhibitor in macrophages leading to in vitro anti-atherosclerotic effects through a decrease of net cellular cholesterol content. © 2019 International Society for Advancement of Cytometry.

The sodium-dependent organic anion transporter SOAT specifically transports sulfated steroid hormones and is supposed to play a role in testicular steroid regulation and male fertility. The present study aimed to identify novel specific SOAT inhibitors for further in vitro and in vivo studies on SOAT function. More than 100 compounds of different molecular structures were screened for inhibition of the SOAT-mediated transport of dehydroepiandrosterone sulfate in stably transfected SOAT-HEK293 cells. Twenty-five of these with IC50 values covering four orders of magnitude were selected as training set for 3D pharmacophore modelling. The SOAT pharmacophore features were calculated by CATALYST and consist of three hydrophobic sites and two hydrogen bond acceptors. By substrate database screening, compound T 0511-1698 was predicted as a novel SOAT inhibitor with an IC50 of 15 μM. This value was confirmed by cell-based transport assays. Therefore, the developed SOAT pharmacophore model demonstrated its suitability in predicting novel SOAT inhibitors.

Membrane transporters are major determinants for the pharmacokinetic, safety and efficacy behavior of drugs. Available technologies to study function and structure of transport proteins has strongly stimulated research in transporter biology and uncovered their importance for the drug discovery and development process, especially for drug absorption and disposition. Physiological transport systems are investigated as potential ferries to improve drug absorption and membrane permeation and to achieve organ-specific drug action. In particular, the bile acid transport systems in the liver and the small intestine and the oligopeptide transporters are of significant importance for molecular drug delivery.

Male obese Zucker Diabetic Fatty (ZDF) rats develop type 2 diabetes around eight weeks of age, and are widely used as a model for human diabetes and its complications. The objective of the study was to test whether the complications manifested in the kidney and nerves of ZDF rats really correspond to human diabetic complications in their being related to the hyperglycaemic state. Four groups of ZDF rats were used. One lean (Fa/?) and one obese (fa/fa) untreated group served as non-diabetic and diabetic controls. In two further groups of obese (fa/fa) rats, diabetes was prevented by pioglitazone or delayed by food restriction. All rats were monitored up to 35 weeks of age with respect to their blood glucose, HbA1c and insulin levels, their kidney function (urinary glucose excretion, renal glucose filtration, glomerular filtration rate, albumin/creatinine ratio), and their nerve function (tactile and thermal sensory threshold and nerve conduction velocity). Pioglitazone prevented the development of diabetes, while food restriction delayed its onset for 8–10 weeks. Accordingly, kidney function parameters were similar to lean non-diabetic rats in pioglitazone-treated rats and significantly improved in food-restricted rats compared with obese controls. Kidney histology paralleled the functional results. By contrast, nerve functional evaluations did not mirror the differing blood glucose levels. We conclude that the ZDF rat is a good model for diabetic nephropathy, while alterations in nerve functions were not diabetes-related.

This is the first study to examine the effect of subchronic olanzapine (OLZ) on energy homeostasis in rats, covering all aspects of energy balance, including energy intake as metabolizable energy, storage, and expenditure. We further analyzed whether, and by which mechanism, the CB1‐antagonist AVE1625 might attenuate OLZ‐induced body weight gain. For this purpose, we selected juvenile female Hanover Wistar rats that robustly and reproducibly demonstrated weight gain on OLZ treatment, accepting limitations to model the aberrations on lipid and carbohydrate metabolism. Rats received 2 mg/kg OLZ orally twice daily for 12 days. Body weight and body composition were analyzed. Moreover daily food intake, energy expenditure, and substrate oxidation were determined in parallel to motility and body core temperature. OLZ treatment resulted in substantial body weight gain, in which lean and fat mass increased significantly. OLZ‐treated rats showed hyperphagia that manifested in increased carbohydrate oxidation and lowered fat oxidation (FO). Energy expenditure was increased, motility decreased, but there was no indication for hypothermia in OLZ‐treated rats. Coadministration of OLZ and AVE1625 (10 mg/kg orally once daily) attenuated body weight gain, diminishing the enhanced food intake while maintaining increased energy expenditure and decreased motility. Our data reveal that energy expenditure was enhanced in OLZ‐treated rats, an effect not critically influenced by motility. Energy uptake, however, exceeded energy expenditure and led to a positive energy balance, confirming hyperphagia as the major driving factor for OLZ‐induced weight gain. Combination of OLZ treatment with the CB1‐antagonist AVE1625 attenuated body weight gain in rats.

The glucagon-like peptide-1 (GLP-1) receptor represents an established therapeutic target in type 2 diabetes mellitus (T2DM). Agents that activate this receptor improve glucose tolerance alongside a low risk of hypoglycaemia, and have the potential to modify disease progression. Lixisenatide is a new potent and selective GLP-1 receptor agonist currently in development. The preclinical pharmacological profile of Lixisenatide suggests actions that are highly relevant to the long-term maintenance of glucose homeostasis. Lixisenatide protected Ins-1 cells (a rat-derived β-cell line) from both lipid- and cytokine-induced apoptosis. More importantly, Lixisenatide also prevented lipotoxicity-induced insulin depletion in human islets and preserved insulin production, storage and pancreatic β-cell function in vitro. Enhancement of insulin biosynthesis and pancreatic β-cell volume could also be demonstrated in animal models of type 2 diabetes. The improvement of glucose-stimulated insulin secretion provided by Lixisenatide occurred in a strictly glucose-dependent manner. In animal models of diabetes, Lixisenatide improved basal blood glucose and HbA1c with a rapid onset and sustained duration of action, and prevented the deterioration of pancreatic responsiveness and glucose homeostasis. Lixisenatide also delayed gastric emptying and reduced food intake. The efficacy/safety profile of Lixisenatide is currently being studied further in an extensive ongoing Phase III clinical study programme. This article reviews the preclinical pharmacological profile of Lixisenatide.

Synthesis and degradation of lipids in mammalian adipocytes are tightly and coordinatedly regulated by insulin, fatty acids, reactive oxygen species and drugs. Conversely, the lipogenic or lipolytic state of adipocytes is communicated to other tissues by the secretion of soluble adipocytokines. Here we report that insulin, palmitate, H(2)O(2) and the antidiabetic sulfonylurea drug glimepiride induce the release of the typical lipid droplet (LD) protein, perilipin-A, as well as typical plasma membrane microdomain (DIGs) proteins, such as caveolin-1 and the glycosylphosphatidylinositol (GPI)-anchored proteins, Gce1 and CD73 from rat adipocytes. According to biochemical and morphological criteria these LD and GPI-proteins are embedded within two different types of phospholipid-containing membrane vesicles, collectively called adiposomes. Adiposome release was not found to be causally related to cell lysis or apoptosis. The interaction of Gce1 and CD73 with the adiposomes apparently depends on their intact GPI anchor. Pull-down of caveolin-1, perilipin-A and CD73 together with phospholipids (via binding to annexin-V) as well as mutually of caveolin-1 with CD73 or perilipin-A (via coimmunoprecipitation) argues for their colocalization within the same adiposome vesicle. Taken together, certain lipogenic and anti-lipolytic agents induce the specific release of a subset of LD and DIGs proteins, including certain GPI-proteins, in adiposomes from primary rat adipocytes. Given the (c)AMP-degrading activities of Gce1 and CD73 and LD-forming function of perilipin-A and caveolin-1, the physiological relevance of the release of adiposomes from adipocytes may rely on the intercellular transfer of lipogenic and anti-lipolytic information.

Aim: To investigate the efficacy of the peroxisome proliferator-activated receptor-α (PPARα) agonist, AVE8134, in cellular and experimental models of cardiac dysfunction and heart failure. Methods: In Sprague Dawley rats with permanent ligation of the left coronary artery (post-MI), AVE8134 was compared to the PPARγ agonist rosiglitazone and in a second study to the ACE inhibitor ramipril. In DOCA-salt sensitive rats, efficacy of AVE8134 on cardiac hypertrophy and fibrosis was investigated. Finally, AVE8134 was administered to old spontaneously hypertensive rats (SHR) at a non-blood pressure lowering dose with survival as endpoint. In cellular models, we studied AVE8134 on hypertrophy in rat cardiomyocytes, nitric oxide signaling in human endothelial cells (HUVEC) and LDL-uptake in human MonoMac-6 cells. Results: In post-MI rats, AVE8134 dose-dependently improved cardiac output, myocardial contractility and relaxation and reduced lung and left ventricular weight and fibrosis. In contrast, rosiglitazone exacerbated cardiac dysfunction. Treatment at AVE8134 decreased plasma proBNP and arginine and increased plasma citrulline and urinary NOx/creatinine ratio. In DOCA rats, AVE8134 prevented development of high blood pressure, myocardial hypertrophy and cardiac fibrosis, and ameliorated endothelial dysfunction. Compound treatment increased cardiac protein expression and phosphorylation of eNOS. In old SHR, treatment with a low dose of AVE8134 improved cardiac and vascular function and increased life expectancy without lowering blood pressure. AVE8134 reduced phenylephrine-induced hypertrophy in adult rat cardiomyocytes. In HUVEC, Ser-1177-eNOS phosphorylation but not eNOS expression was increased. In monocytes, AVE8134 increased the expression of CD36 and the macrophage scavenger receptor 1, resulting in enhanced uptake of oxidized LDL. Conclusion: The PPARα agonist AVE8134 prevents post-MI myocardial hypertrophy, fibrosis and cardiac dysfunction. AVE8134 has beneficial effects against hypertension-induced organ damages, resulting in decreased mortality. The compound exerts its protective properties by a direct effect on cardiomyocyte hypertrophy, but also indirectly via monocyte signaling and increased endothelial NO production.

The CB1 receptor antagonist, rimonabant, affects the endocannabinoid system and causes a sustained reduction in body weight (BW) despite the transient nature of the reduction in food intake. Therefore, in a multiple-dose study, female candy-fed Wistar rats were treated with rimonabant (10 mg/kg) and matched with pair-fed rats to distinguish between hypophagic action and hypothesized effects on energy expenditure. Within the first week of treatment, rimonabant reduced BW nearly to levels of standard rat chow-fed rats. Evaluation of energy balance (energy expenditure measured by indirect calorimetry in relation to metabolizable energy intake calculated by bomb calorimetry) revealed that increased energy expenditure based on increased fat oxidation contributed more to sustained BW reduction than reduced food intake. A mere food reduction through pair feeding did not result in comparable effects because animals reduced their energy expenditure to save energy stores. Because fat oxidation measured by indirect calorimetry increased immediately after dosing in the postprandial state, the acute effect of rimonabant on lipolysis was investigated in postprandial male rats. Rimonabant elevated free fatty acids postprandially, demonstrating an inherent pharmacological activity of rimonabant to induce lipolysis and not secondarily postabsorptively due to reduced food intake. We conclude that the weight-reducing effect of rimonabant was due to continuously elevated energy expenditure based on increased fat oxidation driven by lipolysis from fat tissue as long as fat stores were elevated. When the amount of endogenous fat stores declined, rimonabant-induced increased energy expenditure was maintained by a re-increase in food intake.

The mitochondrial pyruvate dehydrogenase complex (PDC) is inactivated in many tissues during starvation and diabetes. We investigated carbohydrate oxidation (CHO) and the regulation of the PDC in lean and obese Zucker diabetic fatty (ZDF) rats during fed and starved conditions as well as during an oral glucose load without and with pharmacologically reduced levels of free fatty acids (FFA) to estimate the relative contribution of FFA on glucose tolerance, CHO, and PDC activity. The increase in total PDC activity (20-45%) was paralleled by increased protein levels ( approximately 2-fold) of PDC subunits in liver and muscle of obese ZDF rats. Pyruvate dehydrogenase kinase-4 (PDK4) protein levels were higher in obese rats, and consequently PDC activity was reduced. Although PDK4 protein levels were rapidly downregulated (57-62%) in both lean and obese animals within 2 h after glucose challenge, CHO over 3 h as well as the peak of PDC activity (1 h after glucose load) in liver and muscle were significantly lower in obese rats compared with lean rats. Similar differences were obtained with pharmacologically suppressed FFA by nicotinic acid, but with significantly improved glucose tolerance in obese rats, as well as increased CHO and delta increases in PDC activity (0-60 min) both in muscle and liver. These results demonstrated the suppressive role of FFA acids on the measured parameters. Furthermore, the results clearly demonstrate a rapid reactivation of PDC in liver and muscle of lean and obese rats after a glucose load and show that PDC activity is significantly lower in obese ZDF rats.

Objective: The severity of obesity is often more determined by the distribution of fat depots rather than by body weight itself. Therefore, the effect of rimonabant on fat distribution pattern was investigated in female candy-fed Wistar rats. Design: Female Wistar rats were fed a high fat, high carbohydrate (candy-) diet for 12 weeks. During the last 6 weeks rats were treated with rimonabant. Food intake and body weight development were investigated, as well as effects on total body fat, especially visceral fat and ectopic lipid accumulation in skeletal muscle and liver, determined by in vivo magnetic resonance imaging/magnetic resonance spectroscopy. Results: Candy-diet increased body weight, which was predominantly due to the increased total fat mass with predominance of visceral fat accumulation. Treatment with rimonabant fully reversed the weight gain and fat deposition in the visceral cavity and skeletal muscle, in contrast to pair feeding. In spite of an only transient reduction of food intake, body weight reduction, as well as normalized body fat, reduced visceral fat and intramyocellular lipids were maintained over the treatment period. Conclusions: We conclude that additional factors other than reduced caloric intake must be responsible for the improvements in these lipid parameters. The complete cluster of results is consistent with increased lipid oxidation caused by rimonabant.

AVE2268, a substituted glycopyranoside, is an orally active and selective inhibitor of sodium-dependent glucose transporter 2 (SGLT2; IC50=13 nmol/L). Investigation of the pharmacological profile of AVE2268 on urinary glucose excretion (UGE) and blood glucose after glucose challenge (po or intraperitoneal) was performed in mice and rats. AVE2268 caused a dose-dependent increase of UGE in mice (ID30 = 79 +/- 8.1 mg/kg p.o.) and rats (ID30 = 39.8 +/- 4.0 mg/kg p.o.). AVE2268 in mice was more potent to decrease blood glucose ascent when glucose was given intraperitoneally (ID50 = 13.2 +/- 3.9 mg/kg), compared to orally administered glucose (ID50 = 26.1 +/- 3.9 mg/kg), showing that AVE2268 has no effects on SGLT I in the gut in vivo, which is in accordance with ist very low affinity to the SGLT I in vitro (IC50 > 10,000 mnol/L). During an oral glucose tolerance test, AVE2268 dose-dependently increased UGE, with subsequent decreases of AUC and blood glucose. A highly significant inverse correlation between AUC and UGE was found (p<0.001). The increase in UGE is linked to the inhibition of SGLT2 only. This profile renders AVE2268 as a new antidiabetic drug for the treatment of type 2 diabetes.