RESUMO
Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) is one of two known DGAT enzymes that catalyze the final step in triglyceride synthesis. Findings from genetically modified mice as well as pharmacological studies suggest that inhibition of DGAT1 is a promising strategy for the treatment of obesity and type 2 diabetes. Here we characterize a tool DGAT1 inhibitor compound, T863. We found that T863 is a potent inhibitor for both human and mouse DGAT1 in vitro, which acts on the acyl-CoA binding site of DGAT1 and inhibits DGAT1-mediated triacylglycerol formation in cells. In an acute lipid challenge model, oral administration of T863 significantly delayed fat absorption and resulted in lipid accumulation in the distal small intestine of mice, mimicking the effects of genetic ablation of DGAT1. In diet-induced obese mice, oral administration of T863 for 2 weeks caused weight loss, reduction in serum and liver triglycerides, and improved insulin sensitivity. In addition to the expected triglyceride-lowering activity, T863 also lowered serum cholesterol. Hepatic IRS2 protein was dramatically up-regulated in mice treated with T863, possibly contributing to improved insulin sensitivity. In differentiated 3T3-L1 adipocytes, T863 enhanced insulin-stimulated glucose uptake, suggesting a possible role for adipocytes to improve insulin sensitivity upon DGAT1 inhibition. These results reveal novel mechanistic insights into the insulin-sensitizing effects of DGAT1 inhibition in mouse models. Taken together, our study provides a comprehensive evaluation of a small molecule inhibitor for DGAT1 and suggests that pharmacological inhibition of DGAT1 holds promise in treating diverse metabolic disorders.
Assuntos
Diacilglicerol O-Aciltransferase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Resistência à Insulina , Fígado/enzimologia , Redução de Peso/efeitos dos fármacos , Células 3T3-L1 , Administração Oral , Animais , Sítios de Ligação , Diacilglicerol O-Aciltransferase/genética , Diacilglicerol O-Aciltransferase/metabolismo , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/farmacocinética , Humanos , Camundongos , Camundongos Obesos , Triglicerídeos/sangueRESUMO
Strong genetic data link the Tyrosine kinase receptor B (TrkB) and its major endogenous ligand brain-derived neurotrophic factor (BDNF) to the regulation of energy homeostasis, with loss-of-function mutations in either gene causing severe obesity in both mice and humans. It has previously been reported that peripheral administration of the endogenous TrkB agonist ligand neurotrophin-4 (NT-4) profoundly decreases food intake and body weight in rodents, while paradoxically increasing these same parameters in monkeys. We generated a humanized TrkB agonist antibody, TAM-163, and characterized its therapeutic potential in several models of type 2 diabetes and obesity. In vitro, TAM-163 bound to human and rodent TrkB with high affinity, activated all aspects of the TrkB signaling cascade and induced TrkB internalization and degradation in a manner similar to BDNF. In vivo, peripheral administration of TAM-163 decreased food intake and/or body weight in mice, rats, hamsters, and dogs, but increased food intake and body weight in monkeys. The magnitude of weight change was similar in rodents and non-human primates, occurred at doses where there was no appreciable penetration into deep structures of the brain, and could not be explained by differences in exposures between species. Rather, peripherally administered TAM-163 localized to areas in the hypothalamus and the brain stem located outside the blood-brain barrier in a similar manner between rodents and non-human primates, suggesting differences in neuroanatomy across species. Our data demonstrate that a TrkB agonist antibody, administered peripherally, causes species-dependent effects on body weight similar to the endogenous TrkB ligand NT-4. The possible clinical utility of TrkB agonism in treating weight regulatory disorder, such as obesity or cachexia, will require evaluation in man.
Assuntos
Anticorpos Monoclonais/farmacologia , Depressores do Apetite/farmacologia , Peso Corporal/efeitos dos fármacos , Receptor trkB/agonistas , Administração Intravenosa , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacocinética , Depressores do Apetite/administração & dosagem , Depressores do Apetite/farmacocinética , Cricetinae , Dieta Hiperlipídica/efeitos adversos , Cães , Avaliação Pré-Clínica de Medicamentos , Ingestão de Energia/efeitos dos fármacos , Feminino , Células HEK293 , Humanos , Hipotálamo/metabolismo , Macaca fascicularis , Macaca mulatta , Masculino , Mesocricetus , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/etiologia , Obesidade/patologia , Ratos , Ratos Sprague-Dawley , Ratos Zucker , Receptor trkB/metabolismo , Distribuição TecidualRESUMO
Coiled-coil domain containing 80 (Ccdc80) is a secreted protein highly enriched in mouse and human white adipose tissue (WAT) that plays an important role during adipocyte differentiation in vitro. To investigate the physiological function of Ccdc80 in energy and glucose homeostasis, we generated mice in which the gene encoding Ccdc80 was disrupted. Mice lacking Ccdc80 showed increased sensitivity to diet-induced hyperglycemia and glucose intolerance while displaying reduced glucose-stimulated insulin secretion in vivo. Gene expression analysis by microarray revealed that only 10 transcripts were simultaneously altered in pancreas, skeletal muscle, and WAT from Ccdc80(-/-) mice, including some components of the circadian clock. Expression of the core clock member Arntl/Bmal1 was reduced whereas that of the oscillating transcription factors Dbp and Tef was increased in all tissues examined. Furthermore, knockdown of Ccdc80 in 3T3-L1 cells led to an increase of Dbp mRNA levels during adipocyte differentiation, suggesting that Ccdc80 might be involved in the regulation of this gene in a cell-autonomous manner. Importantly, transcriptional alterations in Ccdc80(-/-) mice were associated with changes in feeding behavior, increased caloric intake, decreased energy expenditure, and obesity. Taken together, our results suggest that Ccdc80 is a novel modulator of glucose and energy homeostasis during diet-induced obesity.
Assuntos
Glucose/metabolismo , Glicoproteínas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Obesidade/metabolismo , Células 3T3-L1 , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Tecido Adiposo Branco/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas da Matriz Extracelular , Glicoproteínas/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , Camundongos Obesos , Músculo Esquelético/metabolismo , Obesidade/genética , Pâncreas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Development of novel therapies for polycystic kidney disease (PKD) requires assays that adequately reflect disease biology and are adaptable to high-throughput screening. Here we describe an embryonic cystic kidney organ culture model and demonstrate that a new mutant allele of the Pkd1 gene (Pkd1(tm1Bdgz)) modulates cystogenesis in this model. Cyst formation induced by cAMP is influenced by the dosage of the mutant allele: Pkd1(tm1Bdgz) -/- cultures develop a larger cystic area compared with +/+ counterparts, while Pkd1(tm1Bdgz) +/- cultures show an intermediate phenotype. A similar relationship between the degree of cystogenesis and mutant gene dosage is seen in cystic kidney organ cultures derived from mice with a mutated Nek8 gene (Nek8(jck)). Both Pkd1- and Nek8- cultures display altered cell-cell junctions, with reduced E-cadherin expression and altered desmosomal protein expression, similar to ADPKD epithelia. Additionally, characteristic ciliary abnormalities are identified in cystic kidney cultures, with elevated ciliary polycystin 1 expression in Nek8 homozygous cultures and elevated ciliary Nek8 protein expression in Pkd1 homozygotes. These data suggest that the Nek8 and Pkd1 genes function in a common pathway to regulate cystogenesis. Moreover, compound Pkd1 and Nek8 heterozygous adult mice develop a more aggressive cystic disease than animals with a mutation in either gene alone. Finally, we validate the kidney organ culture cystogenesis assay as a therapeutic testing platform using the CDK inhibitor roscovitine. Therefore, embryonic kidney organ culture represents a relevant model for studying molecular cystogenesis and a rapid tool for the screening for therapies that block cystic growth.