FARE2017 WINNERS Sorted By Study Section National Cancer Institute - Center for Cancer Research (NCI-CCR) Chad Brocker Research FellowResearch Fellow Biochemistry - General and Lipids Liver-specific peroxisome proliferator-activated receptor alpha (PPARA) knockout reveals a role for extrahepatic PPARA in glucose homeostasis Peroxisome proliferator-activated receptor alpha (PPARA) is a ligand-activated transcription factor and a key mediator of systemic lipid homeostasis. PPARA is activated in response to fasting and promotes the uptake, utilization, and catabolism of fatty acids by regulating fatty acid transport and peroxisome and mitochondrial beta-oxidation. Moreover, constitutive agonist-induced PPARA activation causes hepatocellular carcinoma in mice. PPARA has profound effects on the fasting response, and PPARA activation protects against fasting-induced oxidative stress. After fasting, full-body PPARA knockout (KO) mice exhibit hepatic lipid accumulation leading to steatosis and elevated oxidative stress, lipid peroxidation and lipotoxicity. In order to elucidate the relationships between PPARA, fasting-induced hepatosteatosis, and oxidative stress, a liver-specific PPARA knockout (LKO) mouse was generated. Serum chemistry panels revealed markedly elevated serum phospholipids, cholesterol and triglycerides in fasted LKO mice when compared to KO. These endpoints were confirmed by LC/MS-based lipidomics, which showed significantly higher levels of circulating medium- and long-chain fatty acids. H&E and Oil Red O staining indicated significantly less hepatic lipid accumulation in LKO when compared to KO. These data were supported by TBARS assays revealing that lipid peroxidation was significantly lower in LKO mouse livers. Unlike KO mice, LKO mice do not display hypoglycemia in response to fasting. Furthermore, only KO mice exhibited insulin resistance as revealed by glucose tolerance tests. Alterations in glucose metabolism were supported by PAS staining which showed that impaired glycogen mobilization in KO was dramatically reduced in LKO mice. Differential glycogen metabolism was further supported by analysis of glycogenolytic gene expression that indicated slight but significant changes in expression of rate-limiting enzymes. Taken together, these studies provide strong evidence that extrahepatic PPARA expression plays a major role in regulating lipid and glucose homeostasis during the acute fasting response. Moreover, these studies serve as the foundation for future work utilizing cell- type specific knockout mice to expand our current understanding of PPARA’s role during physiological response to fasting and agonist-induced carcinogenesis. National Cancer Institute - Center for Cancer Research (NCI-CCR) Cen Xie Visiting FellowVisiting Fellow Biochemistry - General and Lipids Intestinal farnesoid X receptor signaling modulates hepatic gluconeogenesis Enhanced hepatic gluconeogenesis is a major contributor to hyperglycemia in type 2 diabetes. To date, the underlying molecular mechanism of increased hepatic gluconeogenesis in type 2 diabetes remains largely unknown. Farnesoid X receptor (FXR) belongs to the nuclear receptor superfamily and is a crucial modulator that senses and regulates hepatic glucose levels and lipid metabolism. Recent studies revealed that gut microbiota influences metabolic disease by metabolizing bile acids into metabolites that inhibit intestinal FXR signaling. Manipulation of the gut microbiota-FXR signaling axis profoundly impacts glucose intolerance, but the molecular mechanism for this effect was poorly understood. Here, a combination of lipidomics, biochemical assays and use of intestine epithelial cell-specific FXR knockout (FXR-dIE) mice were employed to dissect the precise mechanism by which inhibition of intestinal FXR signaling improves glucose homeostasis. Absence of intestinal FXR in FXR-dIE mice was correlated with lowered intestinal-derived serum ceramides due to the direct control by FXR of genes involved in ceramide synthesis in the intestine. Lower serum ceramides resulted in decreased hepatic mitochondrial acetyl-CoA levels and pyruvate carboxylase activities, which attenuated hepatic gluconeogenesis. These processes were found to be independent of body weight change and hepatic insulin signaling. Further, ceramide administration to the FXR-dIE mice and primary hepatocytes substantially attenuated hepatic mitochondrial citrate synthase activities, primarily through the induction of endoplasmic reticulum (ER) stress, ER-mitochondria tethering, and calcium influx, which triggers increased hepatic mitochondrial acetyl-CoA levels and pyruvate carboxylase (PC) activities. Collectively, this study revealed a novel mechanism by which intestinal FXR regulates hepatic gluconeogenesis through a novel intestinal FXR- ceramide pathway, leading to decreased hepatic mitochondrial acetyl-CoA levels and PC activities, without affecting hepatic insulin signaling. Similar results as were obtained with FXR-dIE mice, could be achieved by the direct inhibition of intestinal FXR using an FXR antagonist, thus demonstrating that FXR is a potential target for the treatment of metabolic diseases such as type 2 diabetes. National Institute on Alcohol Abuse and Alcoholism (NIAAA) Janos Paloczi Visiting FellowVisiting Fellow Biochemistry - General and Lipids Novel clinically relevant model of alcohol-induced cardiomyopathy associated with impaired cardiovascular function and cardiometabolic dysregulation Chronic alcoholism ranks among the top five risk factors for disease and premature death. Binge drinking is the most common pattern of excessive alcohol use in the United States, particularly among young adults. Long-term, heavy alcohol consumption impairs vascular function and leads to development of cardiomyopathy and heart failure. However, reproducible and clinically relevant models of alcohol-induced cardiomyopathy in mice are lacking. Here we describe new mouse models of alcoholic cardiomyopathies induced by chronic and binge ethanol (EtOH)-feeding and characterize detailed hemodynamic and metabolic alterations, mitochondrial dysfunction, and impaired redox signaling. Mice were fed with 5% EtOH for 10, 20, 40 days (d) combined with single/multiple EtOH- binges. Isocalorically pair-fed mice served as controls. Left ventricular (LV) function was assessed by sophisticated pressure-volume approach (allowing characterization of multiple load- and heart rate- independent indices of left ventricular performance) and by echocardiography. Mitochondrial complex (I, II, IV) activities, mitochondrial biogenesis, oxidative stress markers and histopathology were also investigated. Chronic and binge EtOH-feeding was characterized by contractile dysfunction (decreased slope of end-systolic pressure-volume relationship and preload recruitable stroke work), impaired relaxation (decreased time constant of LV pressure decay and dP/dtmin) and vascular dysfunction (impaired arterial elastance and lower total peripheral resistance). This was accompanied by enhanced myocardial oxidative/nitrative stress and deterioration of mitochondrial complex I, II, IV activities and mitochondrial biogenesis, myocardial hypertrophy, and excessive cardiac steatosis. The above described deleterious effects of alcohol consumption were dramatically more pronounced in groups exposed to chronic drinking combined with alcohol binges. Our results demonstrate that chronic plus binge EtOH- feeding in mice leads to alcohol-induced cardiomyopathy and vascular dysfunction characterized by increased myocardial oxidative/nitrative stress, impaired mitochondrial function and biogenesis, enhanced cardiac steatosis and hypertrophy. These results also suggest that combination of regular drinking with binges even within a short period of time may profoundly affect cardiac and vascular function and metabolism. National Institute of Environmental Health Sciences (NIEHS) Erin Romes Postdoctoral FellowPostdoctoral Fellow Biochemistry - Proteins The Crystal Structure of the Ubiquitin-like Domain of Ribosome Assembly Factor WDR12 and Characterization of Its Interaction with the AAA-ATPase Midasin The synthesis of eukaryotic ribosomes is a complex, energetically demanding process requiring the aid of numerous non-ribosomal assembly factors such as the PeBoW complex, Midasin, and Nle1. Ribosomal RNA (rRNA) transcription accounts for 60 percent of total cellular RNA transcription in growing cells and can lead to aberrant cell growth when improperly regulated. Although the structure and mechanics of the fully assembled ribosome are well understood, much less is known about the process of assembly. The mammalian PeBoW complex, comprised of Pes1, Bop1, and WDR12, is essential for processing the 32S pre-rRNA. Without removal of the PeBoW complex from pre-60S particles by the large dynein-like AAA-ATPase, Midasin, ribosome maturation is stalled, which activates the p53 response and ultimately leads to cell death. Our objective is to determine how Midasin recognizes its substrates; WDR12 and Nle1, and removes them from pre-60S particles. We solved the crystal structure of the ubiquitin-like (UBL) domain of the WDR12 homolog from S. cerevisiae at 1.7 Angstrom resolution, which revealed that the domain contains a beta-grasp fold and a well-conserved hydrophobic core. Through pull-down assays in HEK293 cells, we were able to demonstrate that Midasin contains a C-terminal metal ion-dependent adhesion site (MIDAS) domain that specifically interacts with the N-terminal