The goal is to identify site of regulation of hepatic production of apo B-lipoproteins. Emphasis is placed on the role of insulin in models of altered insulin states including hyperinsulinemia produced by insulin pumps, streptozotocin-induced hypoinsulinemic diabetes, and fasting- hypoinsulinemia in rats. Insulin and the opposing effects of counterregulatory hormones will be examined in vitro in primary hepatocyte cultures in established animal models of altered insulin action. Levels of hepatic regulation of apo B production will be identified by measurement of apo B MRNA (edited and unedited), apo B synthesis including apo Bh (B100) and apo BL (B48), apo B degradation, apo B pool size, apo B secretion and apolipoprotein and lipid composition of secretory lipoproteins. Studies will identify transcriptional, translational, and pre-secretory events. Evaluation of apo E will be compared with apo B.
Specific Aim 1 : Studies will investigate transcriptional regulation of apo B and apo E in altered insulin states. Studies include measurement of hepatic apo B MRNA (total, GLN, UAA) with comparison to MRNA of other proteins including apo E. Preliminary evidence indicates that increased apo B MRNA are present with insulin treatment of hypoinsulinemic diabetes with preferential expression of apo B MRNA UAA (apo BL). Studies will explore whether insulin in other states induces apo B MRNA expression. Whole liver analysis will be compared to hepatocytes and in vitro effects of insulin and counterreulatory hormones will be assessed in cultures extending to 72h. Apo BH, apo BL and apo E synthesis will be studied in hepatocytes using steady-state labeling and pulse-chase labeling experiments to evaluate secretory pathways. Translation rates for proteins will be studied using dual label techniques combined with immunoprecipitation of subcellular fractions.
Specific Aim 2 : Studies will investigate intracellular degradation of apo B in altered insulin states in vitro. Intracellular degradation is evaluated using pulse-chase protocols with amino acid label. Studies will include use of protease inhibitors to distinguish lysosomal and non-lysosomal pathways. Effects of perturbations will be evaluated with respect to the apo B intracellular pool. Subcellular fractionation studies will determine the kinetics of intracellular apo B movement.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL029837-08
Application #
2216514
Study Section
Metabolism Study Section (MET)
Project Start
1983-04-01
Project End
1997-12-31
Budget Start
1994-01-01
Budget End
1994-12-31
Support Year
8
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of Rochester
Department
Pathology
Type
Schools of Dentistry
DUNS #
208469486
City
Rochester
State
NY
Country
United States
Zip Code
14627
Sowden, M P; Collins, H L; Smith, H C et al. (1999) Apolipoprotein B mRNA and lipoprotein secretion are increased in McArdle RH-7777 cells by expression of betaine-homocysteine S-methyltransferase. Biochem J 341 ( Pt 3):639-45
Sparks, J D; Phung, T L; Bolognino, M et al. (1998) Lipoprotein alterations in 10- and 20-week-old Zucker diabetic fatty rats: hyperinsulinemic versus insulinopenic hyperglycemia. Metabolism 47:1315-24
Van Mater, D; Sowden, M P; Cianci, J et al. (1998) Ethanol increases apolipoprotein B mRNA editing in rat primary hepatocytes and McArdle cells. Biochem Biophys Res Commun 252:334-9
Sparks, J D; Collins, H L; Sabio, I et al. (1997) Effects of fatty acids on apolipoprotein B secretion by McArdle RH-7777 rat hepatoma cells. Biochim Biophys Acta 1347:51-61
Phung, T L; Mooney, R A; Kulas, D T et al. (1997) Suppression of the protein tyrosine phosphatase LAR reduces apolipoprotein B secretion by McA-RH7777 rat hepatoma cells. Biochem Biophys Res Commun 237:367-71
Phung, T L; Roncone, A; Jensen, K L et al. (1997) Phosphoinositide 3-kinase activity is necessary for insulin-dependent inhibition of apolipoprotein B secretion by rat hepatocytes and localizes to the endoplasmic reticulum. J Biol Chem 272:30693-702
Phung, T L; Sowden, M P; Sparks, J D et al. (1996) Regulation of hepatic apolipoprotein B RNA editing in the genetically obese Zucker rat. Metabolism 45:1056-8
Sparks, J D; Sparks, C E (1996) Chromatographic method for isolation and quantification of apolipoproteins B-100 and B-48. Methods Enzymol 263:104-20
Schock, D; Kuo, S R; Steinburg, M F et al. (1996) An auxiliary factor containing a 240-kDa protein complex is involved in apolipoprotein B RNA editing. Proc Natl Acad Sci U S A 93:1097-102
Sparks, J D; Phung, T L; Bolognino, M et al. (1996) Insulin-mediated inhibition of apolipoprotein B secretion requires an intracellular trafficking event and phosphatidylinositol 3-kinase activation: studies with brefeldin A and wortmannin in primary cultures of rat hepatocytes. Biochem J 313 ( Pt 2):567-74

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