Hepatic steatosis, defi ned as excessive lipid accumulation in the liver, is observed in >40% of patients with type 2 diabetes ( 1, 2 ). Although a causal relationship between hepatic steatosis and insulin resistance has been diffi cult to defi ne ( 1 ), infl ammation has been implicated as a contributing factor to dysregulated hepatic insulin signaling ( 3 ). As a consequence, hyperinsulinemia-mediated lipogenesis ensues, which along with suppressed FA oxidation contributes to ectopic lipid deposition ( 4 ). Prolonged Abstract PPAR ␦ regulates systemic lipid homeostasis and infl ammation, but its role in hepatic lipid metabolism remains unclear. Here, we examine whether intervening with a selective PPAR ␦ agonist corrects hepatic steatosis induced by a high-fat, cholesterol-containing (HFHC) diet. Ldlr ؊ / ؊ mice were fed a chow or HFHC diet (42% fat, 0.2% cholesterol) for 4 weeks. For an additional 8 weeks, the HFHC group was fed HFHC or HFHC plus GW1516 (3 mg/kg/ day). GW1516-intervention significantly attenuated liver TG accumulation by induction of FA ␤ -oxidation and attenuation of FA synthesis. In primary mouse hepatocytes, GW1516 treatment stimulated AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) phosphorylation in WT hepatocytes, but not AMPK ␤ 1 ؊ / ؊ hepatocytes. However, FA oxidation was only partially reduced in AMPK ␤ 1 ؊ / ؊ hepatocytes, suggesting an AMPK-independent contribution to the GW1516 effect. Similarly, PPAR ␦ -mediated attenuation of FA synthesis was partially due to AMPK activation, as GW1516 reduced lipogenesis in WT hepatocytes but not AMPK ␤ 1 ؊ / ؊ hepatocytes. HFHC-fed animals were hyperinsulinemic and exhibited selective hepatic insulin resistance, which contributed to elevated fasting FA synthesis and hyperglycemia. GW1516 intervention normalized fasting hyperinsulinemia and selective hepatic insulin resistance and attenuated fasting FA synthesis and hyperglycemia. The HFHC diet polarized the liver toward a proinfl ammatory M1 state, which was reversed by GW1516 intervention. Thus, PPAR ␦ agonist treatment inhibits the progression of preestablished hepatic steatosis. -Bojic, L.

Objective— The peroxisome proliferator–activated receptor (PPAR) δ regulates systemic lipid homeostasis and inflammation. However, the ability of PPARδ agonists to improve the pathology of pre-established lesions and whether PPARδ activation is atheroprotective in the setting of insulin resistance have not been reported. Here, we examine whether intervention with a selective PPARδ agonist corrects metabolic dysregulation and attenuates aortic inflammation and atherosclerosis. Approach and Results— Low-density lipoprotein receptor knockout mice were fed a chow or a high-fat, high-cholesterol (HFHC) diet (42% fat, 0.2% cholesterol) for 4 weeks. For a further 8 weeks, the HFHC group was fed either HFHC or HFHC plus GW1516 (3 mg/kg per day). GW1516 significantly attenuated pre-established fasting hyperlipidemia, hyperglycemia, and hyperinsulinemia, as well as glucose and insulin intolerance. GW1516 intervention markedly reduced aortic sinus lesions and lesion macrophages, whereas smooth muscle α-actin was unchanged and collagen deposition enhanced. In aortae, GW1516 increased the expression of the PPARδ-specific gene Adfp but not PPARα- or γ-specific genes. GW1516 intervention decreased the expression of aortic proinflammatory M1 cytokines, increased the expression of the anti-inflammatory M2 cytokine Arg1 , and attenuated the iNos / Arg1 ratio. Enhanced mitogen-activated protein kinase signaling, known to induce inflammatory cytokine expression in vitro, was enhanced in aortae of HFHC-fed mice. Furthermore, the HFHC diet impaired aortic insulin signaling through Akt and forkhead box O1, which was associated with elevated endoplasmic reticulum stress markers CCAAT-enhancer-binding protein homologous protein and 78kDa glucose regulated protein. GW1516 intervention normalized mitogen-activated protein kinase activation, insulin signaling, and endoplasmic reticulum stress. Conclusions— Intervention with a PPARδ agonist inhibits aortic inflammation and attenuates the progression of pre-established atherosclerosis.

Objective-Hypertriglyceridemia is an important risk factor for cardiovascular disease. Elevated plasma very low-density lipoprotein (VLDL) puts insulin-resistant patients at risk for atherosclerosis. VLDL readily induces macrophage lipid accumulation and inflammatory responses, for which targeted therapeutic strategies remain elusive. We examined the ability of VLDL to induce macrophage foam cells and the inflammatory response and sought to define the cell signaling cascades involved. We further examined the potential of peroxisome proliferator-activated receptor (PPAR) δ activation to attenuate both VLDL-stimulated lipid accumulation and cytokine expression. Methods and Results-THP-1 macrophages exposed to VLDL displayed significant triglyceride accumulation, which was attenuated by PPARδ activation. PPARδ agonists stimulated a transcriptional program resulting in inhibition of lipoprotein lipase activity, activation of fatty acid uptake, and enhanced β-oxidation. VLDL-treated macrophages significantly increased the expression of activator protein 1 associated cytokines interleukin-1β, macrophage inflammatory protein 1α, and intercellular adhesion molecule-1. VLDL treatment significantly increased the phosphorylation of both extracellular signal-related kinase 1 and 2 and p38. VLDL reduced AKT phosphorylation as well as its downstream effector forkhead box protein O1, concomitant with increased nuclear forkhead box protein O1. Cells treated with PPARδ agonists were completely resistant to VLDL-induced expression of inflammatory cytokines, mediated by normalization of mitogenactivated protein kinase (MAPK) erk and AKT/forkhead box protein O1 signaling. Conclusion-The

Recent advances in the understanding of PPARδ reveal that activation of this receptor represents a multifaceted therapeutic strategy for the prevention and treatment of insulin-resistant syndromes and atherosclerosis.

Oxylipins are oxidation products of polyunsaturated fatty acids (PUFAs) that affect a broad range of physiological processes, including cell proliferation, inflammation, inflammation resolution, and vascular function. Moreover, oxylipins are readily detectable in plasma, and certain subsets of oxylipins have been detected in human atherosclerotic lesions. Taken together, we set out to produce a detailed quantitative assessment of plasma and plaque oxylipins in a widely used model of atherosclerosis, to identify potential biomarkers of disease progression. We administered regular chow or regular chow supplemented with 0.5% cholesterol (HC) to male New Zealand white rabbits for 12 weeks to induce hypercholesterolemia and atherosclerosis. Our targeted lipidomic analyses of oxylipins on plaques isolated from rabbits fed the HC diet detected 34 oxylipins, 28 of which were in compliance with our previously established quality control acceptance criteria. The arachidonic acid (AA) metabolite derived from the COX pathway, 6-keto-PGF1α was the most abundant plaque oxylipin, followed by the linoleic acid (LA) metabolites 9-HODE, 13-HODE and 9,12,13-TriHOME and the arachidonic acid (AA)-derivatives 11-HETE and 12-HETE. We additionally found that the most abundant oxylipins in plasma were three of the five most abundant oxylipins in plaque, namely 11-HETE, 13-HODE, and 9-HODE. The studies reported here make the first step towards a comprehensive characterization of oxylipins as potentially translatable biomarkers of atherosclerosis.

The cellular, macromolecular and neutral lipid composition of the atherosclerotic plaque has been extensively characterized. However, a comprehensive lipidomic analysis of the major lipid classes within atherosclerotic lesions has not been reported. The objective of this study was to produce a detailed framework of the lipids that comprise the atherosclerotic lesion of a widely used pre-clinical model of plaque progression. Male New Zealand White rabbits were administered regular chow supplemented with 0.5% cholesterol (HC) for 12 weeks to induce hypercholesterolemia and atherosclerosis. Our lipidomic analyses of plaques isolated from rabbits fed the HC diet, using ultra-performance liquid chromatography (UPLC) and high-resolution mass spectrometry, detected most of the major lipid classes including: Cholesteryl esters, triacylglycerols, phosphatidylcholines, sphingomyelins, diacylglycerols, fatty acids, phosphatidylserines, lysophosphatidylcholines, ceramides, phosphatidylglycerols, phosphatidylinositols and phosphatidylethanolamines. Given that cholesteryl esters, triacylglycerols and phosphatidylcholines comprise greater than 75% of total plasma lipids, we directed particular attention towards the qualitative and quantitative assessment of the fatty acid composition of these lipids. We additionally found that sphingomyelins were relatively abundant lipid class within lesions, and compared the abundance of sphingomyelins to their precursor phosphatidylcholines. The studies presented here are the first approach to a comprehensive characterization of the atherosclerotic plaque lipidome.

Objective: The peroxisome proliferator-activated receptor (PPAR) delta has been implicated in systemic lipid homeostasis and inflammation. However, the role of PPARdelta agonists as anti-atherogenic agents remains unclear. In the present study, we used low-density lipoprotein receptor-null mice (Ldlr-/-) fed a high fat (HF) diet to test the hypothesis that a selective PPARdelta agonist corrects metabolic dysregulation and attenuates inflammation associated with atherosclerosis. Methods and Results: Ldlr -/- mice were fed chow or HF (42% fat, 0.2% cholesterol) for 4 weeks. Subsequently, the HF group was fed either HF or HF plus GW1516 (3mg/kg/d) for a further 8 weeks. Fasting plasma triglyceride, total cholesterol and free fatty acids were significantly decreased (-50%) by intervention with GW1516. In addition, GW1516 normalized fasting blood glucose and improved glucose and insulin tolerance. GW1516 also enhanced total energy expenditure compared to HF-fed mice. In the aorta, ER-stress markers CHOP and GRP78 were significantly elevated in HF-fed mice, which were markedly attenuated by GW1516-intervention. Aortae of HF-fed mice also showed marked elevations in the expression of proinflammatory cytokines including Ccl3, Il1beta, Icam1, Tnf, Il6 and Ccl2. Furthermore, HF-aortae, compared to chow, displayed reduced expression of the M2 macrophage marker arginase-1(Arg1). Intervention with GW1516 significantly attenuated aortic expression of all examined proinflammatory cytokines, and restored Arg1 expression. Enhanced MAPKerk signalling and decreased AKT/FoxO1 signalling are known to induce inflammatory cytokine expression in vitro. HF-feeding induced phosphorylation (p) of the MAP kinases ERK1/2 and p38 and dampened levels of pAKT and pFoxO1 in the aorta. In contrast, aortae of GW1516-treated animals displayed normalized levels of pERK1/2, p-p38, pAKT and pFoxO1. Conclusions: These studies demonstrate that PPARdelta activation ameliorates dyslipidemia and insulin resistance in HF-fed Ldlr -/- mice. Furthermore, PPARdelta activation inhibits aortic ER-stress as well as dysregulation of MAPK and AKT/FoxO1 signalling induced by HF-feeding, resulting in inhibition of the inflammatory response within the aorta.

Dyslipidemia associated with insulin resistance and obesity are core features of the metabolic syndrome and type 2 diabetes, which contribute significantly to atherosclerosis. In mouse models of diet-induced metabolic dysregulation, the citrus flavonoids naringenin and nobiletin prevent obesity, hepatic steatosis, apoB100 overproduction, dyslipidemia, insulin resistance and atherosclerosis. To elucidate the mechanism of action in liver we assessed flavonoid-induced activation of AMP-activated protein kinase (AMPK), the major regulator of cellular energy homeostasis, in primary mouse hepatocytes. Stimulated AMPK activity promotes catabolic, ATP-generating processes such as fatty acid (FA) oxidation while inhibiting anabolic processes such as FA synthesis. In primary C57BL/6 (WT) hepatocytes, naringenin and nobiletin increased phosphorylation (P) of AMPK and its downstream target acetyl-CoA carboxylase (ACC) in a time- and dose-dependent manner. This was associated with decreased apoB100 secretion. Phosphorylation of ACC by AMPK inhibits the formation of malonyl-CoA reducing substrate for FA synthesis in the cytosol while relieving inhibition of mitochondrial FA oxidation by malonyl-CoA. Under insulin resistant conditions stimulated by high glucose media, reduced pAMPK and pACC were reversed by flavonoid treatment in WT hepatocytes, whereas these effects were lost in Ampkβ1-/- hepatocytes. Sterol receptor element binding protein-1c, which stimulates lipogenesis, was also phosphorylated (inhibited) by flavonoid-induced AMPK activation. BAPTA, a calcium chelator or STO609, an inhibitor of Ca2+/calmodulin-dependent protein kinase kinase-beta (CaMKKβ), did not block flavonoid-induced pACC, suggesting that CaMKKβ is not required for AMPK activation by flavonoids. In chow-fed Ldlr-/- mice, acute i.p. injection of nobiletin following a fasting-refeeding protocol, depressed the respiratory exchange ratio indicative of a switch to FA oxidation. Freeze-clamped liver samples from these mice sacrificed 90 min. post injection showed marked induction of pAMPK and pACC. These results suggest that naringenin and nobiletin attenuate hepatic steatosis and metabolic dysregulation, in part, through activation of hepatic AMPK.