RESUMEN
Secretory proteins are an essential component of interorgan communication networks that regulate animal physiology. Current approaches for identifying secretory proteins from specific cell and tissue types are largely limited to in vitro or ex vivo models which often fail to recapitulate in vivo biology. As such, there is mounting interest in developing in vivo analytical tools that can provide accurate information on the origin, identity, and spatiotemporal dynamics of secretory proteins. Here, we describe iSLET (in situ Secretory protein Labeling via ER-anchored TurboID) which selectively labels proteins that transit through the classical secretory pathway via catalytic actions of Sec61b-TurboID, a proximity labeling enzyme anchored in the ER lumen. To validate iSLET in a whole-body system, we express iSLET in the mouse liver and demonstrate efficient labeling of liver secretory proteins which could be tracked and identified within circulating blood plasma. Furthermore, proteomic analysis of the labeled liver secretome enriched from liver iSLET mouse plasma is highly consistent with previous reports of liver secretory protein profiles. Taken together, iSLET is a versatile and powerful tool for studying spatiotemporal dynamics of secretory proteins, a valuable class of biomarkers and therapeutic targets.
Asunto(s)
Retículo Endoplásmico/metabolismo , Canales de Translocación SEC/metabolismo , Vías Secretoras/fisiología , Animales , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Hígado/metabolismo , Hígado/patología , Ratones , Ratones Endogámicos C57BL , Células 3T3 NIH , Proteoma/metabolismo , ProteómicaRESUMEN
Insulin resistance is a cornerstone of obesity-related complications such as type 2 diabetes, metabolic syndrome, and nonalcoholic fatty liver disease. A high rate of lipolysis is known to be associated with insulin resistance, and inhibiting adipose tissue lipolysis improves obesity-related insulin resistance. Here, we demonstrate that inhibition of serotonin (5-hydroxytryptamine [5-HT]) signaling through serotonin receptor 2B (HTR2B) in adipose tissues ameliorates insulin resistance by reducing lipolysis in visceral adipocytes. Chronic high-fat diet (HFD) feeding increased Htr2b expression in epididymal white adipose tissue, resulting in increased HTR2B signaling in visceral white adipose tissue. Moreover, HTR2B expression in white adipose tissue was increased in obese humans and positively correlated with metabolic parameters. We further found that adipocyte-specific Htr2b-knockout mice are resistant to HFD-induced insulin resistance, visceral adipose tissue inflammation, and hepatic steatosis. Enhanced 5-HT signaling through HTR2B directly activated lipolysis through phosphorylation of hormone-sensitive lipase in visceral adipocytes. Moreover, treatment with a selective HTR2B antagonist attenuated HFD-induced insulin resistance, visceral adipose tissue inflammation, and hepatic steatosis. Thus, adipose HTR2B signaling could be a potential therapeutic target for treatment of obesity-related insulin resistance.
Asunto(s)
Resistencia a la Insulina , Grasa Intraabdominal/metabolismo , Obesidad/metabolismo , Receptor de Serotonina 5-HT2B/metabolismo , Serotonina/metabolismo , Adipocitos/citología , Adipocitos Blancos , Tejido Adiposo , Tejido Adiposo Blanco/metabolismo , Adulto , Animales , Dieta Alta en Grasa , Epidídimo , Femenino , Glicerol/metabolismo , Humanos , Inflamación , Insulina/metabolismo , Lipólisis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Fosforilación , Transducción de Señal , Adulto JovenRESUMEN
Tryptophan hydroxylase 1 (TPH1) has been recently suggested as a promising therapeutic target for treating obesity and fatty liver disease. A new series of 1,2,4-oxadiazolylphenyl alanine derivatives were identified as TPH1 inhibitors. Among them, compound 23a was the most active in vitro, with an IC50 (half-maximal inhibitory concentration) value of 42 nM, showed good liver microsomal stability, and showed no significant inhibition of CYP and hERG. Compound 23a inhibited TPH1 in the peripheral tissue with limited BBB penetration. In high-fat diet-fed mice, 23a reduced body weight gain, body fat, and hepatic lipid accumulation. Also, 23a improved glucose intolerance and energy expenditure. Taken together, compound 23a shows promise as a therapeutic agent for the treatment of obesity and fatty liver diseases.
Asunto(s)
Alanina/síntesis química , Alanina/farmacología , Fármacos Antiobesidad/síntesis química , Fármacos Antiobesidad/farmacología , Hígado Graso/tratamiento farmacológico , Triptófano Hidroxilasa/antagonistas & inhibidores , Adiposidad/efectos de los fármacos , Alanina/análogos & derivados , Animales , Fármacos Antiobesidad/uso terapéutico , Dieta Alta en Grasa , Metabolismo Energético/efectos de los fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Intolerancia a la Glucosa/tratamiento farmacológico , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Aumento de Peso/efectos de los fármacosRESUMEN
Nonalcoholic fatty liver disease (NAFLD) is increasingly prevalent worldwide, causing serious liver complications, including nonalcoholic steatohepatitis. Recent findings suggest that peripheral serotonin (5-hydroxytryptamine, 5HT) regulates energy homeostasis, including hepatic lipid metabolism. More specifically, liver-specific 5HT2A knockout mice exhibit alleviated hepatic lipid accumulation and hepatic steatosis. Here, structural modifications of pimavanserin (CNS drug), a 5HT2A antagonist approved for Parkinson's disease, led us to synthesize new peripherally acting 5HT2A antagonists. Among the synthesized compounds, compound 14a showed good in vitro activity, good liver microsomal stability, 5HT subtype selectivity, and no significant inhibition of CYP and hERG. The in vitro and in vivo blood-brain barrier permeability study proved that 14a acts peripherally. Compound 14a decreased the liver weight and hepatic lipid accumulation in high-fat-diet-induced obesity mice. Our study suggests new therapeutic possibilities for peripheral 5HT2A antagonists in NAFLD.