Impact of oral lipid and glucose tolerance tests on the postprandial concentrations of angiopoietin-like proteins (Angptl) 3 and 4.

BACKGROUND: The postprandial regulation of angiopoietin-like proteins (Angptls) and their expression in adipocytes is poorly characterized. OBJECTIVE: Circulating Angptl3 and 4 were analyzed in healthy individuals undergoing either an oral lipid tolerance test (OLTT; n = 98) or an oral glucose tolerance test (OGTT; n = 99). Venous blood was drawn after 0, 2, 4, and 6 h during OLTT and after 0, 1, and 2 h during OGTT. Anthropometric and laboratory parameters were assessed and concentrations of Angptls were quantified by enzyme-linked immunosorbent assay. Angptl gene expression in 3T3-L1 adipocytes and in murine adipose tissues and cellular fractions was analyzed by quantitative real-time PCR. RESULTS: Angptl3 concentrations significantly decreased while Angptl4 levels continuously increased during OLTT. Both proteins remained unaffected during OGTT. Angptl3 and Angptl4 were expressed in murine subcutaneous and visceral AT with higher mRNA levels in mature adipocytes when compared to the stroma-vascular cell fraction. Both proteins were strongly induced during 3T3-L1 adipocyte differentiation and they were unresponsive to glucose in mature fat cells. Adipocyte Angptl3 (but not Angptl4) mRNA expression was inhibited by the polyunsaturated fatty acids arachidonic acid and docosahexaenoic acid, whereas nine types of dietary fatty acids remained without any effect. CONCLUSIONS: There is evidence of short-time regulation of Angptl3/4 levels upon metabolic stress. Angptl4 expression is high and Angptl3 expression is low in AT and restricted mainly to mature adipocytes without any differences concerning fat compartments. Whereas dietary fatty acids and glucose are without any effect, omega-3/-6-polyunsaturated fatty acids inhibited Anptl3 expression in adipocytes.

Accelerating the discovery of DGAT1 inhibitors through the application of parallel medicinal chemistry (PMC).

The parallel medicinal chemistry (PMC) was effectively applied to accelerate the optimization of diacylglycerol O-acyltransferase I (DGAT-1) inhibitors. Through a highly collaborative and iterative library design, synthesis and testing, a benzimidazole lead was rapidly and systematically advanced to a highly potent, selective and bioavailable DGAT1 inhibitor with the potential for further development.

CTRP-3 is permeable to the blood-brain barrier and is not regulated by glucose or lipids in vivo.

BACKGROUND: C1q/TNF-related protein-3 (CTRP-3) represents a novel anti-inflammatory and antidiabetic adipokine secreted by adipose tissue. The physiological and postprandial regulation of CTRP-3 remains obscure and it is not known whether CTRP-3 is permeable to the brain. The postprandial regulation of CTRP-3 during an oral glucose tolerance test (n = 100) and an oral lipid tolerance test (n = 100) in humans was investigated. Moreover, CTRP-3 concentrations were measured in paired serum and cerebrospinal fluid (CSF) samples of patients (n = 270) undergoing neurological evaluation. The expression of CTRP-3 mRNA was investigated in adipocytes upon stimulation with glucose, sex hormones and a broad panel of fatty acids. MATERIALS AND METHODS: Serum and CSF CTRP-3 concentrations were measured by ELISA. 3T3-L1 adipocytes were used for stimulation experiments. CTRP-3 mRNA expression was quantified by using real-time PCR analysis. RESULTS: CTRP-3 is present in human cerebrospinal fluid with a mean CSF/serum ratio of 110 ± 64 × 10-3 . CTRP-3 is not regulated postprandially by carbohydrates or lipids in the healthy state. Females have slightly higher levels of CTRP-3 when compared to males. A significant and positive correlation of CTRP-3 to LDL cholesterol serum levels is reproducible in several cohorts and deserves further mechanistic investigation. Whereas glucose concentrations do not influence CTRP-3 mRNA expression in 3T3-L1 adipocytes in vitro, fatty acids differentially modulate CTRP-3 expression. CONCLUSIONS: The novel adipokine CTRP-3 is detectable in human cerebrospinal fluid (proof of principle). Due to its blood-brain barrier permeability, CTRP-3 represents a novel putative candidate for a physiological regulator molecule affecting central nervous functions.

Pro-inflammatory chemokines CCL2, chemerin, IP-10 and RANTES in human serum during an oral lipid tolerance test.

BACKGROUND: There is a strong coincidence of obesity and a chronic state of modest inflammation. Secretion of pro-inflammatory cytokines from adipocytes and immune cells represents a key mechanism in this process and is affected by fatty acids. MATERIAL AND METHODS: A study cohort of 100 overnight fasted healthy volunteers underwent an oral lipid tolerance test (OLTT) by ingestion of 160ml of a protein- and sugar-free lipid emulsion of defined composition. Venal blood was drawn at 0h (fasting) and at 2, 4, and 6h after lipid ingestion. Subjects were characterized by anthropometric and standard laboratory parameters. Serum concentrations of CCL2, IP-10, chemerin, and RANTES were measured by enzyme-linked immunosorbent assay (ELISA). Murine 3T3-L1 adipocytes were stimulated with free fatty acids (FA) and with sex steroids and concentrations of CCL2 and chemerin in cell culture supernatants were measured by ELISA. RESULTS: A significant reduction of circulating CCL2, IP-10, and chemerin concentrations was observed as a consequence of triglyceride ingestion whereas RANTES levels were increased. CCL2 serum concentrations were positively correlated with resistin and visfatin levels and with LDL/HDL ratio and negatively with adiponectin. There were significant differences in chemerin and RANTES serum concentrations in female and male subjects. CCL2 secretion from 3T3-L1 adipocytes was inhibited by treatment with linoleic (LA) and oleic acid (OA) whereas chemerin secretion was induced. Chemerin release from 3T3-L1 adipocytes was inhibited by testosterone. CONCLUSIONS: Oral lipid loading is linked to reduced circulating pro-inflammatory chemokines CCL2, IP-10, and chemerin and to increased RANTES levels, suggesting that dietary lipids affect immune function.

Identification of 2-[2-(4-tert-butylphenyl)ethyl]-N-(4-fluorophenyl)-1,2,3,4-tetrahydroisoquinoline-6-sulfonamide (29) as an orally available MGAT2 inhibitor.

MGAT2 (monoacylglycerol acyltransferase 2) is expected to be an attractive target for the drug treatment of obesity, diabetes, and other disease. We describe our exploration and structure-activity relationship (SAR) study of 2,3-dihydro-1H-isoindole-5-sulfonamide derivatives. In this study, we identified 29 as an orally available inhibitor of MGAT2 through optimization especially in terms of solubility. This compound exhibited moderate potency in the enzyme inhibitory assay (IC50 = 1522 nM) and significant suppression of fat absorption (57% inhibition) in mice oral lipid tolerance test.

The HuMet Repository: Watching human metabolism at work.

Metabolism oscillates between catabolic and anabolic states depending on food intake, exercise, or stresses that change a multitude of metabolic pathways simultaneously. We present the HuMet Repository for exploring dynamic metabolic responses to oral glucose/lipid loads, mixed meals, 36-h fasting, exercise, and cold stress in healthy subjects. Metabolomics data from blood, urine, and breath of 15 young, healthy men at up to 56 time points are integrated and embedded within an interactive web application, enabling researchers with and without computational expertise to search, visualize, analyze, and contextualize the dynamic metabolite profiles of 2,656 metabolites acquired on multiple platforms. With examples, we demonstrate the utility of the resource for research into the dynamics of human metabolism, highlighting differences and similarities in systemic metabolic responses across challenges and the complementarity of metabolomics platforms. The repository, providing a reference for healthy metabolite changes to six standardized physiological challenges, is freely accessible through a web portal.

Investigating the Postprandial Metabolome after Challenge Tests to Assess Metabolic Flexibility and Dysregulations Associated with Cardiometabolic Diseases.

This review focuses on the added value provided by a research strategy applying metabolomics analyses to assess phenotypic flexibility in response to different nutritional challenge tests in the framework of metabolic clinical studies. We discuss findings related to the Oral Glucose Tolerance Test (OGTT) and to mixed meals with varying fat contents and food matrix complexities. Overall, the use of challenge tests combined with metabolomics revealed subtle metabolic dysregulations exacerbated during the postprandial period when comparing healthy and at cardiometabolic risk subjects. In healthy subjects, consistent postprandial metabolic shifts driven by insulin action were reported (e.g., a switch from lipid to glucose oxidation for energy fueling) with similarities between OGTT and mixed meals, especially during the first hours following meal ingestion while differences appeared in a wider timeframe. In populations with expected reduced phenotypic flexibility, often associated with increased cardiometabolic risk, a blunted response on most key postprandial pathways was reported. We also discuss the most suitable statistical tools to analyze the dynamic alterations of the postprandial metabolome while accounting for complexity in study designs and data structure. Overall, the in-depth characterization of the postprandial metabolism and associated phenotypic flexibility appears highly promising for a better understanding of the onset of cardiometabolic diseases.

Evolving data analysis of an Oral Lipid Tolerance Test toward the standard for the Oral Glucose Tolerance Test: Cross species modeling effects of AZD7687 on plasma triacylglycerol.

We have developed a novel mechanistic pharmacokinetic-pharmacodynamic (PK/PD) model to describe the time course of plasma triglyceride (TAG) after Oral Lipid Tolerance Test (OLTT) and the effects of AZD7687, an inhibitor of diacylglycerol acyltransferase 1 (DGAT1), in humans, rats, and mice. Pharmacokinetic and plasma TAG data were obtained both in animals and in two phase I OLTT studies. In the PK/PD model, the introduction of exogenous TAG is represented by a first order process. The endogenous production and removal of TAG from plasma are described with a turnover model. AZD7687 inhibits the contribution of exogenous TAG into circulation. One or two compartment models with first order absorption was used to describe the PK of AZD7687 for the different species. Nonlinear mixed effect modeling was used to fit the model to the data. The effects of AZD7687 on the plasma TAG time course during an OLTT as well as interindividual variability were well described by the model in all three species. Meal fat content or data from single vs repeated dosing did not affect model parameter estimates. Body mass index was found to be a significant covariate on the plasma TAG baseline. The system parameters of the model will facilitate analysis for other compounds and provide tools to bring the standard of OLTT data analysis closer to the analyses of Oral Glucose Tolerance Test data maximizing knowledge gain.

Regulation of natriuretic peptides postprandially in vivo and of their receptors in adipocytes by fatty acids in vitro.

BACKGROUND AND AIM: Natriuretic peptides (NPs) and their receptors gain attention regarding adipocyte function. It was the aim to investigate the expression of natriuretic peptide receptors NPR-A, NPR-B and NPR-C during adipocyte differentiation (AD), upon stimulation with fatty acids (FA), and in murine and human adipose tissue depots (AT) of patients undergoing bariatric surgery (n = 44). PATIENTS, MATERIAL AND METHODS: The postprandial regulation of NT-proANP and NT-proBNP levels was measured by ELISA and was studied in two cohorts of healthy individuals undergoing an oral glucose tolerance test (OGTT) (n = 100) and an oral lipid tolerance test (OLTT) (n = 100). Adipocyte mRNA expression was investigated by quantitative real-time PCR. RESULTS: During AD, an early expression pattern could be described for NPR-C, a bimodal expression for NPR-B and a late expression pattern for NPR-A. NPR-A and NPR-B expression was high in epididymal and subcutaneous AT but low in peri-renal AT of mice. NPR-C showed a differential expression profile. FA stimulation caused a significant and differential regulation of NPRs in adipocytes. Serum NT-proANP and NT-proBNP concentrations did not change during OGTT, whereas NT-proANP significantly declined during OLTT. Basal NT-proANP and NT-proBNP concentrations were positively correlated with each other and with FGF-19 and FGF-21 levels. CONCLUSION: Adipocytes and AT show a characteristic expression of NPRs. FA are able to regulate NPR expression differentially. There is a postprandial and negative regulation of serum NT-proANP concentrations after OLTT and of NPR-A after FA stimulation. Both effects could represent a novel hypothetical negative feedback mechanism on adipocyte lipolysis.

Effect of DDT exposure on lipids and energy balance in obese Sprague-Dawley rats before and after weight loss.

Dichlorodiphenyltrichloroethane (DDT) and its metabolites accumulate in adipose tissue through dietary exposure, and have been proposed to contribute to the development of abdominal obesity, insulin resistance and dyslipidemia. Toxicity may also result when DDT and its metabolites are released from adipose tissue into the bloodstream as a result of rapid weight loss. We hypothesized that DDT-exposed rats fed a high fat diet (HFD) followed by 60% calorie restriction would have an adverse metabolic response to rapid weight loss. To test this, we exposed obese Sprague-Dawley (SD) rats to DDT and a HFD over one month followed by 60% calorie restricted diet for two weeks, and examined metabolic parameters throughout the study. During the HFD feeding period, DDT-exposed rats had significantly elevated postprandial non-esterified fatty acids (NEFAs) and decreased body temperature compared with control rats. During calorie restriction, DDT-exposed rats had lowered food efficiency (weight gained/calories consumed), body temperature, and circulating TSH. Our findings suggest that exposure to DDT may impairs metabolic substrate utilization in rats during dynamic periods of weight gain and weight loss.

Discovery of a Potent and Selective DGAT1 Inhibitor with a Piperidinyl-oxy-cyclohexanecarboxylic Acid Moiety.

We report the discovery of a novel series of DGAT1 inhibitors in the benzimidazole class with a piperdinyl-oxy-cyclohexanecarboxylic acid moiety. This novel series possesses significantly improved selectivity against the A2A receptor, no ACAT1 off-target activity at 10 µM, and higher aqueous solubility and free fraction in plasma as compared to the previously reported pyridyl-oxy-cyclohexanecarboxylic acid series. In particular, 5B was shown to possess an excellent selectivity profile by screening it against a panel of more than 100 biological targets. Compound 5B significantly reduces lipid excursion in LTT in mouse and rat, demonstrates DGAT1 mediated reduction of food intake and body weight in mice, is negative in a 3-strain Ames test, and appears to distribute preferentially in the liver and the intestine in mice. We believe this lead series possesses significant potential to identify optimized compounds for clinical development.

Potent DGAT1 Inhibitors in the Benzimidazole Class with a Pyridyl-oxy-cyclohexanecarboxylic Acid Moiety.

We report the design and synthesis of a series of novel DGAT1 inhibitors in the benzimidazole class with a pyridyl-oxy-cyclohexanecarboxylic acid moiety. In particular, compound 11A is a potent DGAT1 inhibitor with excellent selectivity against ACAT1. Compound 11A significantly reduces triglyceride excursion in lipid tolerance tests (LTT) in both mice and dogs at low plasma exposure. An in vivo study in mice with des-fluoro analogue 10A indicates that this series of compounds appears to distribute in intestine preferentially over plasma. The propensity to target intestine over plasma could be advantageous in reducing potential side effects since lower circulating levels of drug are required for efficacy. However, in the preclinical species, compound 11A undergoes cis/trans epimerization in vivo, which could complicate further development due to the presence of an active metabolite.