The liver plays a central role in whole-body lipid metabolism by regulating the uptake, synthesis, oxidation and export of fatty acids (FAs) and lipids. Dysfunction of lipid metabolism in liver underlies the development of obesity, diabetes, nonalcoholic fatty liver disease (NAFLD) and cardiovascular disease. Although the mechanisms that regulate the hepatic uptake, activation, and metabolism of fatty acids (FAs) are not fully understood, nearly all pathways of FA metabolism require conversion of FAs to acyl-CoAs by acyl-CoA synthetases. Long chain acyl-CoA synthetase (ACSL) is a family of five enzymes (ACSL1, 3, 4, 5 and 6) that catalyze the formation of fatty acyl-CoAs from ATP, CoA, and long chain fatty acids. Within ACSL family members, ACSL4 has unique substrate specificity for arachidonic acid (AA) and hepatic ACSL4 has been demonstrated being abnormally expressed in pathological conditions including hepatocarcinoma and NAFLD. However, to date, no literature reports have clearly defined the specific roles played by ACSL4 in liver lipid metabolism under normal and disease state such as the benign form of NAFLD (steatosis) and the nonalcoholic steatohepatitis (NASH). During the last funding period, we have obtained two major findings related to the regulation of hepatic ACSL4. First, we demonstrated that expression and activity of hepatic ACSL4 are upregulated by activators of peroxisome proliferator-activated receptor ? (PPAR?) in vivo and in vitro. Through conducting lipidomic studies we further demonstrated that depletion of ACSL4 in hepatic cells selectively reduced cellular contents of several PC species including PC(18:0/18:1), a critical lipid mediator in PPAR? signaling pathway. Second, we uncovered a novel substrate-induced posttranslational regulatory mechanism by which AA specifically downregulates ACSL4 protein abundance in hepatic cells by promoting its ubiquitination and proteasomal degradation. Since AA-derived eicosanoids have been implicated in the pathogenesis of NAFLD, the AA-induced ACSL4 degradation could be one contributing factor to the progression of hepatic steatosis to NASH with increased hepatic inflammation caused by imbalance between AA-CoA and unesterified AA that leads to increased production of AA-derived eicosanoids. Recently, through proteomic studies, we have identified the intracellular vesicular transport factor p115 as a major ACSL4 interacting protein and demonstrated that p115 interaction with ACSL4 is markedly enhanced by exposure of HepG2 cells to AA but not to other FAs. Since AA specifically induces ACSL4 degradation, our observations suggest that p115 might be a key mediator in the process of ACSL4 degradation through ubiquitin-proteasomal pathway (UPP). The overall goals of this revised Merit Review Renewal are to fully characterize two new regulatory mechanisms that uniquely impact the expression and function of ACSL4 in liver tissue to reach a better understanding of its specific roles in hepatic lipid metabolism under healthy and pathophysiological conditions including hyperlipidemia and NAFLD.
The specific aims of this Merit Review renewal application are to: 1) comprehensively define the metabolic functions of hepatic ACSL4 and its critical roles in PPAR? regulated lipid metabolism under normal and hyperlipidemic conditions in two complementary animal models with acute deletion and chronic deletion of ACSL4 in liver tissues and 2) utilize various molecular and biochemical approaches to characterize the ubiquitination motifs of ACSL4 protein and to define the functional roles of the newly identified ACSL4-interacting proteins including p115 and other three ACSL4 binding proteins in the degradation process of ACSL4 via UPP in response to treatment with AA in vitro and in vivo. The successful completion of our proposed studies is likely to identify novel targets that can be exploited in the development of new therapies to treat hyperlipidemia and steatosis in metabolic diseases such as obesity, NAFLD and cardiovascular disease.
Our proposed work in this renewal application is highly relevant to Veteran?s health. Many Veterans suffer from dyslipidemia-induced cardiovascular disease, diabetes, obesity and NAFLD. Reduction of elevated cholesterol, triglycerides and fatty acid levels in blood circulation and in the liver are beneficial to these diseases and may have direct therapeutic relevance. We will use state-of-the-art molecular, cellular, proteomic, lipidomic and gene deletion and knockdown technologies to fully elucidate two novel cellular mechanisms that regulate the expression and functions of long chain acyl-CoA synthetase 4 (ACSL4) that has critical roles in lipid metabolism and are implicated in human metabolic diseases such as NAFLD. Thus, the information obtained from our proposed studies will provide valuable insight for developing new therapeutic interventions based upon PPAR biology to treat hyperlipidemia and its associated diseases, which would contribute directly to better health of Veterans and also lower the cost of health care.
