Bioactive Lipids and Metabolic Syndrome

Non-alcoholic fatty liver disease is strongly associated with hepatic insulin resistance; however, the key lipid species and molecular mechanisms linking these conditions are widely debated. We developed a subcellular fractionation method combined with liquid chromatography-tandem mass spectrometry to quantify diacylglycerol (DAG) stereoisomers and ceramides in the endoplasmic reticulum, mitochondria, lipid droplets, cytosol and plasma membrane. Acute knockdown (KD) of diacylglycerol acyltransferase-2 in liver induced hepatic insulin resistance in rats. This could be attributed to plasma membrane sn-1,2-DAG accumulation, which promoted Protein Kinase C-epsilon activation, and insulin receptor kinase (IRK)-T1160 phosphorylation resulting in decreased IRK-T1162 phosphorylation. Plasma membrane sn-1,2-DAG content and IRK-T1160 phosphorylation in the liver were also higher in humans with hepatic insulin resistance. In rats, liver-specific PKC-epsilon KD ameliorated high-fat diet-induced hepatic insulin resistance by lowering IRK-T1160 phosphorylation, while liver-specific overexpression of constitutively active PKC-epsilon-induced HIR by promoting IRK-T1160 phosphorylation. In contrast there was no consistent association between hepatic ceramide content and hepatic insulin resistance. These studies identify sn-1,2-DAGs in the plasma membrane as the key bioactive lipid and intracellular compartment that are responsible for mediating lipid-induced hepatic insulin resistance and that hepatic PKC-epsilon is both necessary and sufficient in mediating HIR [1]. We also show that increases in plasma membrane sn-1,2-DAGs leading to activation of novel PKCs are also responsible for lipid-induced insulin resistance in skeletal muscle [2] and white adipose tissue [3] and that this hypothesis explains the dissociation between increases in ectopic lipid content in liver and skeletal muscle and insulin resistance in these organs under certain conditions.

Non-resolving inflammation is the underpinning of several prevalent diseases including atherosclerosis. Understanding new mechanisms to promote the resolution of inflammation in atherosclerosis are of interest. Resolution is mediated in part by specialized pro-resolving mediators, including resolvins such as Resolvin D1 (RvD1). We recently showed that RvD1 prevents lesional necrosis in Ldlr^-/- mice. However, the mechanisms underlying RvD1’s protective actions remain unknown. In this regard, the accumulation of senescent cells has recently emerged as a driver of plaque necrosis. Senescent cells are particularly harmful because they possess a highly pro-inflammatory and proteolytic phenotype. Because RvD1 decreased lesional necrosis, we questioned whether RvD1’s actions were through limiting the senescent cells in plaques. We administered RvD1 to Ldlr^-/- mice during advanced atherosclerosis and observed a significant decrease in senescent cells and overall lesion necrosis compared with vehicle controls. To explore mechanism, we developed a new macrophage senescence model and found that senescent cells have defective clearance mechanisms that can be rescued by RvD1. This talk will focus on mechanisms underlying delayed resolution programs by senescent cells and how RvD1 rescues this impairment.

Prostaglandin E2 (PGE2) signaling at its receptors is terminated when PGE2 is taken up across the plasma membrane by the carrier PGT. By controlling cell surface [PGE2], PGT controls PGE2 signaling. In liver, fasting reduces PGT expression, suggesting that PGT/PGE2 may control hepatic metabolism. We used single cell RNA-Seq (scRNASeq) and single-molecule fluorescence in situ hybridization to confirm reports of others that PGT expression predominates in hepatocytes at the portal end of the hepatic lobule. PGT in this location would effectively clear PGE2 entering via the portal vein, allowing little to reach the central vein end of the lobule. In accord with this idea, control mouse liver in vivo extracted portal PGE2, whereas liver of mice lacking PGT (PGT-KO) passed portal PGE2 on to the hepatic vein. PGT-KO liver analyzed by scRNASeq exhibited suppressed gene expression in mid-lobule and central vein hepatocytes. In alignment with these data, Q-PCR showed that PGT KO liver has reduced expression of lipogenesis genes (fatty acid synthase, acetyl-CoA carboxylase-1, steroyl-CoA desaturase-1, peroxisome proliferator activated receptor gamma). PGT-KO mice had markedly reduced hepatic steatosis on high-fat or high-fat-fructose diets. These effects were mimicked by a systemically administered PGT inhibitor. We conclude that fasting or PGT inhibition reduces portal zone PGT activity, which causes PGE2 to be passed axially down the hepatic lobule, thereby suppressing lipogenesis.

The number of individuals with non-alcoholic fatty liver disease (NAFLD) is at epidemic levels worldwide. Although thought of as benign, NAFLD can progress to more severe forms of the disease that include non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and hepatocellular carcinoma. Presently no therapies exist that can treat NAFLD and/or NASH even though this area of drug discovery has been highly active. Intestinal monoacylglycerol acyltransferase 2 (MGAT2) catalyzes the resynthesis of triglycerides from dietary triglyceride-derived monoacylglycerol and fatty acids. mMgat2-/- mice are resistant to hepatic steatosis, suggesting that targeting MGAT2 may be a viable option to treat NAFLD. Here, we generated transgenic hMgat2+/+ mice and characterized their responses to being fed different metabolic diets. hMgat2+/+ mice fed a steatotic diet acquired NAFLD and had elevated levels of several inflammatory cytokines in the liver, indicating the presence of ongoing NASH. Hydroxyproline levels were elevated and correlated with the degree of fibrosis seen by histology. These deleterious effects were attenuated by treatment with the PPARα/δ agonist, elafibranor. hMgat2+/+ mice fed a high fat diet became obese, were glucose intolerant, and acquired insulin resistance, suggesting the onset of diabetes.. hMgat2+/+ mice may be an excellent “humanized” model for studying how hMGAT2 affects overall triglyceride metabolism and the progression of NAFLD.

Lysophosphatidic acid (LPA) is an essential bioactive lysolipid mediator that regulate a range of developmental and physiological processes by acting on cell surface receptors. In the cardiovascular system, LPA is poised to serve as a mediator of vascular inflammation, particularly in the setting of hyperlipidemia where it is found in lipoprotein particles and accumulates in atherosclerotic plaque. Extracellular levels of LPA are largely controlled by the enzyme autotaxin (ATX), and the lipid is degraded to a receptor-inactive form by lipid phosphate phosphatase 3 (LPP3). The signaling nexus regulates vascular inflammation, including IL-6 levels, smooth muscle cell migration and differentiation, fibrosis, and endothelial barrier function. Growing evidence has linked the ATX/LPA/LPP3 signaling nexus to experimental atherosclerosis, including our work demonstrating that genetic deficiency of LPA receptors attenuates lesion formation. Genome-wide association studies of coronary artery disease identified a striking association between the PLPP3 locus (encoding LPP3) and myocardial infarction. We established that the risk variant disrupts an intronic enhancer to increases transcription of the gene and LPP3 expression. We have functionally validated these observations by demonstrating that genetic loss of LPP3 in mice increases in plaque LPA and accelerates disease progression.

The bioactive sphingolipid metabolites ceramide and sphingosine-1-phosphate (S1P) are a relatively new addition to the lipids accumulated in obesity and have emerged as important molecular players in metabolic diseases. In this lecture, I will summarize evidence that dysregulation of sphingolipid metabolism correlates with pathogenesis of metabolic diseases in mice and humans. I will also discuss the current understanding of how ceramide regulates signaling and metabolic pathways to exacerbate metabolic diseases and the Janus faces for its further metabolite S1P, the kinases that produce it, and the multifaceted and at times opposing actions of S1P receptors in various tissues. Gaps and limitations in current knowledge will be highlighted together with the need to further decipher the full array of their actions in tissue dysfunction underlying metabolic pathologies, pointing out prospects to move this young field of research toward the development of effective therapeutics.