Analysis of the transcriptome of differentiating and non-differentiating preadipocytes from rats and humans by next generation sequencing.

Alongside cell lines such as 3T3-L1 cells, primary cell culture models of adipogenesis have helped in developing an understanding of the process of adipocyte recruitment and maintenance, which may lead to therapeutic advances to treat the growing epidemic of obesity. Recently, it has been demonstrated that fat cell progenitors (DFAT) established through ceiling culture of adipocytes retain an enhanced ability to undergo adipocyte differentiation compared to preadipocytes isolated from the stromal vascular fraction of adipose tissue. Clonal expansion of rat DFAT cells identified differentiation capable and incapable cell strains. To understand the mechanisms underlying these differences, comparison of their transcriptomes by next generation sequencing was performed. Two hundred seventy-eight genes with a significant fold change of 1.4 were detected as being consistently deregulated between differentiating and non-differentiating strains. Bioinformatic network analyses identified components of the extra-cellular matrix and PPARγ as important genes in this process, suggesting crosstalk between ECM and transcription factors influences differentiation. Analyses of the transcriptomes of human DFAT cells in early and late passage (non-differentiating) confirmed the importance of these pathways in maintaining an adipogenic potential.

Challenges in tackling energy expenditure as obesity therapy: From preclinical models to clinical application.

BACKGROUND: A chronic imbalance of energy intake and energy expenditure results in excess fat storage. The obesity often caused by this overweight is detrimental to the health of millions of people. Understanding both sides of the energy balance equation and their counter-regulatory mechanisms is critical to the development of effective therapies to treat this epidemic. SCOPE OF REVIEW: Behaviors surrounding ingestion have been reviewed extensively. This review focuses more specifically on energy expenditure regarding bodyweight control, with a particular emphasis on the organs and attractive metabolic processes known to reduce bodyweight. Moreover, previous and current attempts at anti-obesity strategies focusing on energy expenditure are highlighted. Precise measurements of energy expenditure, which consist of cellular, animal, and human models, as well as measurements of their translatability, are required to provide the most effective therapies. MAJOR CONCLUSIONS: A precise understanding of the components surrounding energy expenditure, including tailored approaches based on genetic, biomarker, or physical characteristics, must be integrated into future anti-obesity treatments. Further comprehensive investigations are required to define suitable treatments, especially because the complex nature of the human perspective remains poorly understood.

Deletion of the diabetes candidate gene Slc16a13 in mice attenuates diet-induced ectopic lipid accumulation and insulin resistance.

Genome-wide association studies have identified SLC16A13 as a novel susceptibility gene for type 2 diabetes. The SLC16A13 gene encodes SLC16A13/MCT13, a member of the solute carrier 16 family of monocarboxylate transporters. Despite its potential importance to diabetes development, the physiological function of SLC16A13 is unknown. Here, we validate Slc16a13 as a lactate transporter expressed at the plasma membrane and report on the effect of Slc16a13 deletion in a mouse model. We show that Slc16a13 increases mitochondrial respiration in the liver, leading to reduced hepatic lipid accumulation and increased hepatic insulin sensitivity in high-fat diet fed Slc16a13 knockout mice. We propose a mechanism for improved hepatic insulin sensitivity in the context of Slc16a13 deficiency in which reduced intrahepatocellular lactate availability drives increased AMPK activation and increased mitochondrial respiration, while reducing hepatic lipid content. Slc16a13 deficiency thereby attenuates hepatic diacylglycerol-PKCε mediated insulin resistance in obese mice. Together, these data suggest that SLC16A13 is a potential target for the treatment of type 2 diabetes and non-alcoholic fatty liver disease.

EGFL6 is increasingly expressed in human obesity and promotes proliferation of adipose tissue-derived stromal vascular cells.

With increasing rates of obesity driving the incidence of type 2 diabetes and cardiovascular diseases to epidemic levels, understanding of the biology of adipose tissue expansion is a focus of current research. Identification and characterization of secreted proteins of the adipose tissue could provide further insights into the function of adipose tissue and might help to therapeutically influence the development of obesity and associated metabolic disorders. In the present study, we identified human epidermal growth factor-like domain multiple-6 (EGFL6) as an adipose tissue-secreted protein. EGFL6 expression in human subcutaneous adipose tissue significantly increased with obesity and decreased after weight loss. Further, expression and secretion of EGFL6 increased with in vitro differentiation of human preadipocytes, suggesting that mature adipocytes are the main source of EGFL6. Containing epidermal growth factor (EGF)-like repeats, an Arg-Gly-Asp (RGD) integrin binding motif and a mephrin, A5 protein and receptor protein-tyrosine phosphatase mu (MAM) domain, EGFL6 was suggested to be an extra-cellular matrix protein. Recombinant human EGFL6 protein mediated cell adhesion of human adipose tissue-derived stromal vascular cells (AD-SVC) in an RGD-dependent manner. FACS analyses revealed specific binding of the protein to the cell surface of AD-SVC with the binding being predominantly mediated by the EGF-like repeats. Recombinant EGFL6 enhanced proliferation of human AD-SVC as measured by MTS assay and [(14)C]-thymidine incorporation. These results indicate that human EGFL6 is a paracrine/autocrine growth factor of adipose tissue up-regulated in obesity and potentially involved in the process of adipose tissue expansion and the development of obesity.

Identification of potent agonists acting at an endogenous atypical beta3-adrenoceptor state that modulate lipolysis in rodent fat cells.

Small molecules interacting with aminergic G-protein coupled receptors represent a number of very successful drugs. G-protein coupled receptors continue to be a significant group of targets for pharmaceutical intervention, and modifying their activity through small molecules is a major focus of drug development. Previously, these small molecules could be easily fit in models, as agonists, partial agonists or antagonists. More recently, however, these lines have been blurred as it is increasingly recognized that ligands can interact with receptors in various ways. Analysis of beta-adrenoceptors has revealed that several sites or states exist for the individual receptors. The putative atypical beta(4)-adrenoceptor identified on heart and adipose tissue is now recognized as a unique beta(1)-adrenoceptor state. Similarly, a unique beta(3)-adrenoceptor state has been identified using the aryloxypropanolamine CGP-12,177 and cloned receptor systems. Here we expand upon these observations, by describing an atypical state of the beta(3)-adrenoceptor that exists endogenously in adipose tissue. Furthermore, we describe novel arylethanolamine ligands that interact with this atypical state of the beta(3)-adrenoceptor with high affinity and provide additional tools to investigate the atypical beta(3)-adrenoceptor state to determine whether it can be influenced for therapeutic purposes.

SGLT6 - A pharmacological target for the treatment of obesity?

Despite increased knowledge of nutrient intake regulation and energy homeostasis, treatment options for obesity remain limited. Food reward consists of two branches: gustatory and post-ingestive nutritive information. Drosophila lacking dSLC5A11 (sodium/glucose cotransporter 6-SGLT6) prefer L-glucose over D-glucose independently of their state of satiety. Human SGLT6 is an active transporter of myo-inositol and D-glucose. We investigated expression of SGLT6 in human tissue and found a significant expression in the small intestine and brain. The preference between a metabolizable and a non-metabolizable sugar was tested in 3 mouse models with a selective and potent SGLT6 inhibitor. No influence on sugar preference was seen with SGLT6 inhibition. These studies suggest that SGLT6 does not play a significant role in nutrient sensing in mammals.

Influence of sub-chronic selective 11ß-hydroxysteroid dehydrogenase 1 inhibition on the hypothalamic-pituitary-adrenal axis in female cynomolgus monkeys.

Inhibition of local cortisol regeneration from circulating cortisone by blocking 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) has been shown to ameliorate the risk factors associated with the metabolic syndrome. Chronic modulation of glucocorticoid homeostasis may result in hypothalamic-pituitary-adrenal (HPA) axis stimulation. HPA axis over-activation leading androgen excess would be undesirable in a therapeutic intervention designed to treat a chronic condition such as the metabolic syndrome. To address whether 11ß-HSD1 inhibition would lead to excess androgens, we treated female cynomolgus monkeys with a selective inhibitor, BI 135558, for 4 weeks. Continual action of the compound over the dosing period was confirmed by constant plasma exposure, and a maintained change in urinary glucocorticoid metabolites consistent with 11ß-HSD1 inhibition. No significant changes in adrenal function, as evidenced by an adrenocorticotropic hormone (ATCH) challenge, were observed. An examination of androgenic hormones revealed a slight increase in dehydroepiandrosterone sulfate (DHEA-S), while other hormones such as testosterone remained within reference values. Overall, treatment with BI 135558 in monkeys did not result in obvious over-activation of the HPA axis.

Pharmacological characterization of the selective 11ß-hydroxysteroid dehydrogenase 1 inhibitor, BI 135585, a clinical candidate for the treatment of type 2 diabetes.

To combat the increased morbidity and mortality associated with the developing diabetes epidemic new therapeutic interventions are desirable. Inhibition of intracellular cortisol generation from cortisone by blocking 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) has been shown to ameliorate the risk factors associated with the metabolic syndrome. A challenge in developing 11ß-HSD1 inhibitors has been the species selectivity of small molecules, as many compounds are primate specific. Here we describe our strategy to identify potent selective 11ß-HSD1 inhibitors while ensuring target engagement in key metabolic tissues, liver and fat. This strategy enabled the identification of the clinical candidate, BI 135585.

Characterization of the NPGP receptor and identification of a novel short mRNA isoform in human hypothalamus.

Recently, an orphan G protein coupled receptor (GPCR) termed NPGPR was described. A shorter variant of this receptor lacking exon 1 was shown to have subnanomolar affinity for neuropeptide FF (NPFF), a pain modulatory peptide, and therefore was named NPFF(2) receptor. Here, we characterize the full-length cloned NPGPR and identify a novel short form lacking exon 2 with a differential pattern of mRNA abundance in several tissues and organs. The NPGPR is most similar to the recently cloned neuropeptide FF (NPFF) receptor which lacks exon 1, but also shows high homology to the orexin and neuropeptide Y (NPY) receptor families, two neuropeptides involved in food intake regulation. Therefore, we used binding studies to examine the interaction of NPFF, orexin and NPY with the NPGPR. [125I] NPFF was displaced by NPFF with an IC(50) of 14.7 +/- 8.8 nM, whereas [125I] Orexin B was displaced by Orexin B with an IC(50) of 415 +/- 195 nM. We conclude that orexins interact with the NPGPR and that the affinity of NPFF for NPGPR is approximately 100-fold lower than for the NPFF2 receptor. We postulate that NPGPR is a splice variant of the family of NPFF receptors and displays a binding profile different from the other members of the NPFF receptor family due to the presence of exon 1. In order to evaluate whether NPGPR levels are affected by the feeding status, we examined the mRNA level using real-time PCR in two feeding models, i.e. before and after diet-induced body weight increase as well as after chronic food restriction in rats. However, hypothalamic NPGPR mRNA was unchanged in both models. Therefore, our evidence does not support the hypothesis that NPGPR is involved in feeding regulation.

Inhibition of fatty acid synthase prevents preadipocyte differentiation.

Inhibition of fatty acid synthase (FAS) reduces food intake in rodents. As adipose tissue expresses FAS, we sought to investigate the effect of reduced FAS activity on adipocyte differentiation. FAS activity was suppressed either pharmacologically or by siRNA during differentiation of 3T3-L1 cells. Cerulenin (10 microM), triclosan (50 microM), and C75 (50 microM) reduced dramatically visible lipid droplet accumulation, while incorporation of [1-(14C)]acetate into lipids was reduced by 75%, 70%, and 90%, respectively. Additionally, the substances reduced FAS, CEBPalpha, and PPARgamma mRNA by up to 85% compared to that of control differentiated cells. Transient transfection with FAS siRNA suppressed FAS mRNA and FAS activity, and this was accompanied by reduction of CEBPalpha and PPARgamma mRNA levels, and complete prevention of lipid accumulation. CD36, a late marker of differentiation, was also reduced. Together, these results suggest that FAS generated signals may be essential to support preadipocyte differentiation.

Chronic application of MTII in a rat model of obesity results in sustained weight loss.

OBJECTIVE: To examine the effects of a cafeteria diet and a chronic treatment with melanocortin agonist (MTII) on mature weight-stable female rats. RESEARCH METHODS AND PROCEDURES: Ex-breeder Chbb:Thom rats (350 to 400 g) were divided into two groups: highly palatable food (HPF) and normal rat chow (RC). Both groups had ab libitum access to rat chow. The HPF group had access to chocolate bars, cookies, cheese, and nuts (approximately 20 g/d). After 21 days, the rats in each group were then divided into control and treated groups. Mini-pumps delivering saline or MTII (1 mg/kg per day) for minimally 28 days were implanted. Oxygen consumption was measured for 17 days in a second group of rats implanted with mini-pumps containing MTII (1 mg/kg per day) or saline. RESULTS: HPF rats ate less (<50%) rat chow than RC rats. After 20 days, the HPF group had reached a plateau and weighed significantly more (p < 0.005) than the RC group (411.7 +/- 9.3 g; n = 17 vs. 365.1 +/- 9.4 g; n = 16). HPF rats and RC rats receiving MTII reduced their pellet intake and body weight in the initial 2 weeks of treatment (day 14, RC-saline: -1.6 +/- 1.8 g; RC-MTII, -22.5 +/- 3.7 g; HPF-saline, -7.1 +/- 1.7 g; HPF-MTII, -30.7 +/- 4.8 g). Subsequently, pellet intake returned to pre-implantation values, although body weights remained reduced in both HPF and RC groups. Oxygen consumption was increased in rats treated with MTII. DISCUSSION: This suggests that MTII initially reduced body weight by limiting food intake; however, maintenance of weight is most likely due to increased energy expenditure under conditions of normal and highly palatable diets in mature animals.