The liver plays a central role in whole-body lipid metabolism by regulating the uptake, synthesis, oxidation and export of lipids. Dysfunction of lipid metabolism in liver underlies the development of obesity, diabetes, and hepatic steatosis. 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-CoAs are formed by a family of five acyl-CoA synthetases (ACSL1, ACSL3, ACSL4, ACSL5, and ACSL-6). Gain-of-function as well as loss-of-function studies suggest that each of ACSL isoform has a distinct function in directing acyl-CoAs to one or more specific downstream pathways. Thus, the level of expression of individual ACSL isozymes could directly influence the FA metabolic fates in liver tissue. Currently, the cellular mechanisms that regulate the hepatic expression of ACSLs under either physiological or pathological conditions remain largely unexplored. Previously, our laboratory demonstrated that the isozyme ACSL3 was transcriptionally upregulated by the cytokine oncostatin M (OM) in HepG2 cells accompanied by reduced cellular triglyceride content and enhanced FA 2-oxidation. During the last funding period we investigated the molecular mechanism underlying the OM-induced activation of ACSL3 gene transcription and identified the peroxisome proliferator-activated receptor 4 (PPAR4) as the critical trans-activator that mediated the OM induction of ACSL3 gene transcription. Our work, for the first time, revealed an important functional link between ACSL family and PPAR4, a key transcription factor for modulating a cascade of gene expressions in lipid metabolism. Interestingly, we have also observed an increase in mRNA levels of two other members of the ACSL family (ACSL5 and ACSL4) along with ACSL3 in HepG2 cells that were treated with the PPAR4 agonist L165041. This observation suggested that PPAR4 might regulate the transcription of multiple ACSL isozymes in liver cells. The expression level of PPAR4 directly correlates with its transactivating capacity in modulation of gene expression. Thus, it has become important to understand how PPAR4 expression is regulated in liver. However, currently, little is known about the cellular mechanisms governing hepatic PPAR4 transcription. Furthermore, the in vivo role of PPAR4 in regulating ACSL isozymes under normolipidemic and hyperlipidemic conditions has not been examined. Therefore, the overall objectives of this renewal application are to comprehensively define all the molecular determinants that control the basal and OM-induced gene transcription of PPAR4 in hepatic cells and to use hamsters as our in vivo model to thoroughly examine the roles of PPAR4 in gene expression of the ACSL family and in lipid catabolic process of the liver tissue.
The specific aims of this Merit Review renewal application are to: 1) functionally characterize PPAR4 promoter to identify critical regulatory sequences for the basal transcriptional activity of PPAR4 in liver cells and to precisely map the OM-responsive element;2) comprehensively identify the trans-activator that mediates OM stimulatory effect on PPAR4 transcription as well as those transcriptional factors that are critical for PPAR4 gene transcription in responding to hepatic metabolic demands;and 3) thoroughly investigate the in vivo regulatory role of PPAR4 in the hepatic expression of ACSL isozymes under normolipidemic and hyperlipidemic conditions in two hamster models that either increase or knockdown expression levels of PPAR? in liver tissue through applying adenoviral-mediated gene delivery approaches. Metabolic disorders such as obesity and type II diabetes exhibit altered FA metabolism, to which dysregulated expression of ACSL isozymes could contribute to the disease development. Through these proposed studies we hope to reach a better understanding of the interplay between the ACSL enzyme family and PPAR4 under normolipidemic and hyperlipidemic conditions to gain insights for identifying new therapeutic targets to treat hyperlipidemia.
Our proposed work is highly relevant to Veteran's health. Many Veterans suffer from dyslipidemia-induced cardiovascular disease, hypertension, diabetes, obesity, and hepatic steatosis. Reduction of elevated triglycerides and free fatty acid levels in blood circulation and in liver are beneficial to these diseases and may have direct therapeutic relevance. We will use state-of-the-art molecular, cellular, proteomic and viral gene delivery approaches to fully characterize a new cellular mechanism utilized by cytokine oncostatin M to reduce plasma and hepatic accumulations of triglycerides and fatty acids through upregulation of the critical transcription activator PPAR4 and its target genes of acyl-CoA synthetase family enzymes, which play important roles in lipid and fatty acid metabolism. The information obtained from our studies will provide insight for developing new therapeutic interventions based upon PPAR biology to treat hyperlipidemia, which would contribute directly to better health of Veterans and also lower the cost of health care.