Characterizing the obesogenic and fatty liver-inducing effects of Acetyl tributyl citrate (ATBC) plasticizer using both in vivo and in vitro models.

The global incidence of obesity and non-alcoholic fatty liver disease (NAFLD) is rising rapidly in recent years. Environmental factors including usage of plastics and exposure to chemicals have been proposed as important contributors to the obesity pandemic. Acetyl tributyl citrate (ATBC) is a non-phthalate plasticizer widely used in food packaging, personal care products, medical devices and children's toys etc. Due to its high leakage rate from plastics, exposure risk of ATBC keeps increasing. Although there are some studies investigating the safety of ATBC on human health, these studies mainly focused on high dosages and information regarding ATBC safety at environmental-relevant low levels is still limited. In this study, we aimed to evaluate the safety of subchronic exposure to environmentally-relevant concentrations of ATBC. C57BL/6J mice were orally exposed to ATBC for 6 or 14 weeks. Results indicated that ATBC exposure increased the body weight gain, the body fat content and the size of adipocytes, induced liver steatosis in mice. Consistent with in vivo effects, ATBC treatment increased the intracellular lipid accumulation in vitro hepatocytes. Transcriptome sequencing, qRT-PCR analysis and western blotting revealed that ATBC exposure affected the expression of genes involved in de novo lipogenesis and lipid uptake. Therefore, based on our subchronic and in vitro results, it suggested that ATBC might be a potential environmental obesogen with metabolism-disturbing and fatty liver-inducing risk, and its application in many consumer products should be carefully re-evaluated.

RIP1 kinase activity promotes steatohepatitis through mediating cell death and inflammation in macrophages.

Hepatocyte cell death and liver inflammation have been well recognized as central characteristics of nonalcoholic steatohepatitis (NASH), however, the underlying molecular basis remains elusive. The kinase receptor-interacting protein 1 (RIP1) is a multitasking molecule with distinct functions in regulating apoptosis, necroptosis, and inflammation. Dissecting the role of RIP1 distinct functions in different pathophysiology has absorbed huge research enthusiasm. Wild-type and RIP1 kinase-dead (Rip1K45A/K45A) mice were fed with high-fat diet (HFD) to investigate the role of RIP1 kinase activity in the pathogenesis of NASH. Rip1K45A/K45A mice exhibited significantly alleviated NASH phenotype of hepatic steatosis, liver damage, fibrosis as well as reduced hepatic cell death and inflammation compared to WT mice. Our results also indicated that both in vivo lipotoxicity and in vitro saturated fatty acids (palmitic acid) treatment were able to induce the kinase activation of RIP1 in liver macrophages. RIP1 kinase was required for mediating inflammasome activation, apoptotic and necrotic cell death induced by palmitic acid in both bone marrow-derived macrophage and mouse primary Kupffer cells. Results from chimeric mice established through lethal irradiation and bone marrow transplantation further confirmed that the RIP1 kinase in hematopoietic-derived macrophages contributed mostly to the disease progression in NASH. Consistent with murine models, we also found that RIP1 kinase was markedly activated in human NASH, and the kinase activation mainly occurred in liver macrophages as indicated by immunofluorescence double staining. In summary, our study indicated that RIP1 kinase was phosphorylated and activated mainly in liver macrophages in both experimental and clinical NASH. We provided direct genetic evidence that the kinase activity of RIP1 especially in hematopoietic-derived macrophages contributes to the pathogenesis of NASH, through mediating inflammasome activation and cell death induction. Macrophage RIP1 kinase represents a specific and potential therapeutic target for NASH.

Tributyltin triggers lipogenesis in macrophages via modifying PPARγ pathway.

Tributyltin (TBT), a bioaccumulative and persistent environmental pollutant, has been proposed as a metabolism disruptor and obesogen through targeting peroxisome proliferator-activated receptor gamma (PPARγ) receptor pathway. However, it remains unknown whether this biological effect occurs in macrophage, a cell type which cooperates closely with hepatocytes and adipocytes to regulate lipid metabolism. This study for the first time investigated the effect of TBT on PPARγ pathway in macrophages. Our results indicated that nanomolar levels of TBT was able to strongly activate PPARγ in human macrophages. TBT treatment also markedly increased the intracellular lipid accumulation, and enhanced the expression of lipid metabolism-related genes in macrophages, while these effects were all significantly down-regulated in PPARγ-deficient macrophages, confirming the involvement of PPARγ in TBT-induced lipogenesis. Next, a mouse model that C57BL/6 mice were orally exposed to TBT with the doses (250 and 500 µg/kg body weight) lower than NOAEL (no observed adverse effect level) was used to further investigate the in vivo mechanisms. And the in vivo results were consistent with cellular assays, confirming the induction of PPARγ and the increased expression of lipogenesis-regulating and lipid metabolism-related genes by TBT in vivo. In conclusion, this study not only provided the first evidence that TBT stimulated lipogenesis, activated PPARγ and related genes in human macrophages, but also provided insight into the mechanism of TBT-induced metabolism disturbance and obesity through targeting PPARγ via both in vitro cellular assays and in vivo animal models.