Non-bile acid cholephils such as bilirubin and sulfobromophthalein (BSP), free fatty acids (FFA), and bile acids represent 3 distinct classes of organic anions efficiently taken up by the liver despite tight albumin binding. Organic anion uptake is, in fact, a major hepatic function, which may be impaired in disease. This proposal seeks to clarify the role in carrier mediated organic anion uptake of BSP/bilirubin binding protein (BSP/BR-BP) and hepatic plasma membrane fatty acid binding protein (h- FABP/pm), two proteins in the basolateral plasma membrane of the hepatocyte with high affinities for BSP/bilirubin and FFA, respectively, identified and isolated during years 1-11 of this project. Specifically, we will complete the physicochemical characterization of the proteins including aminoacid sequencing; confirm their transport function in liposome reconstitution studies; prepare polyclonal and monoclonal antibodies against each; complete ongoing development of immunoassays to quantitate cell surface expression of both proteins; and correlate changes in the uptake velocities of radiolabeled BSP/bilirubin and FFA produced by phenobarbital or clofibrate administration, or by fibroblast to adipocyte differentiation in 3T3-L1 cells, with changes in the cell surface expression of the corresponding proteins. Additionally, we will use the antibodies to screen a rat liver cDNA library in bacteriophage lambda-gt11; clone the cDNA inserts from positive isolates into an appropriate plasmid and use them screen for full length cDNAs; restriction map and sequence the inserts/cDNAs and use these data to search for homologies between these proteins and with other known proteins; radiolabel the inserts with [32P] and determine the tissue distribution and response to pharmacologic manipulation of the relevant mRNA's by slot blot, and the size and number of the corresponding messages in the liver by Northern hybridization; and estimate the sizes of the relevant genes by Southern hybridization. We will further confirm protein function by using the cloned cDNAs in genetic reconstitution studies in, e.g., Xenopus oocytes, and COS cells. Finally, the inserts will be used to screen genomic libraries. A long term goal is to study the promoter/enhancer regions of the genes for phenobarbital responsive and clofibrate responsive (FFA binding) hepatic proteins, to determine whether the apparently coordinated response to each agent is mediated by common regulatory sequences within each of the responsive genes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK026438-13
Application #
3227890
Study Section
General Medicine A Subcommittee 2 (GMA)
Project Start
1979-12-01
Project End
1995-11-30
Budget Start
1992-12-01
Budget End
1993-11-30
Support Year
13
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Mount Sinai School of Medicine
Department
Type
Schools of Medicine
DUNS #
City
New York
State
NY
Country
United States
Zip Code
10029
Bradbury, Michael W; Stump, Decherd; Guarnieri, Frank et al. (2011) Molecular modeling and functional confirmation of a predicted fatty acid binding site of mitochondrial aspartate aminotransferase. J Mol Biol 412:412-22
Berk, Paul D (2008) Regulatable fatty acid transport mechanisms are central to the pathophysiology of obesity, fatty liver, and metabolic syndrome. Hepatology 48:1362-76
Petrescu, O; Fan, X; Gentileschi, P et al. (2005) Long-chain fatty acid uptake is upregulated in omental adipocytes from patients undergoing bariatric surgery for obesity. Int J Obes (Lond) 29:196-203
Berk, Paul D; Zhou, Shengli; Bradbury, Michael W (2005) Increased hepatocellular uptake of long chain fatty acids occurs by different mechanisms in fatty livers due to obesity or excess ethanol use, contributing to development of steatohepatitis in both settings. Trans Am Clin Climatol Assoc 116:335-44; discussion 345
Grodstein, Francine; Fretts, Ruth; Lifford, Karen et al. (2003) Association of age, race, and obstetric history with urinary symptoms among women in the Nurses' Health Study. Am J Obstet Gynecol 189:428-34
Fan, Xinqing; Bradbury, Michael W; Berk, Paul D (2003) Leptin and insulin modulate nutrient partitioning and weight loss in ob/ob mice through regulation of long-chain fatty acid uptake by adipocytes. J Nutr 133:2707-15
Cechetto, J D; Sadacharan, S K; Berk, P D et al. (2002) Immunogold localization of mitochondrial aspartate aminotransferase in mitochondria and on the cell surface in normal rat tissues. Histol Histopathol 17:353-64
Stump, D D; Fan, X; Berk, P D (2001) Oleic acid uptake and binding by rat adipocytes define dual pathways for cellular fatty acid uptake. J Lipid Res 42:509-20
Bradbury, M W; Berk, P D (2000) Mitochondrial aspartate aminotransferase: direction of a single protein with two distinct functions to two subcellular sites does not require alternative splicing of the mRNA. Biochem J 345 Pt 3:423-7
Berk, P D; Zhou, S; Kiang, C et al. (1999) Selective up-regulation of fatty acid uptake by adipocytes characterizes both genetic and diet-induced obesity in rodents. J Biol Chem 274:28626-31

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