Abstract

We have found that the urinary sediment lactosylceramide (LacCer) levels are elevated 15 fold in homozygous familial hpypercholesterolemic (FH) human subjects compared to normal. LacCer was localized in the cytoplasmic vesicles of FH urinary proximal tubular cells. Plasma exchange treatment and portacaval shunt operation decreased the urinary excretion of LacCer and emptied the cytoplasmic vesicles in the proximal tubular (PT) cells. Our overall objectives are: 1) to characterize proximal tubular cells maintained in culture; 2) to determine the biochemical mechanism for the elevated urinary levels of LacCer in FH homozygotes; 3) to isolate and characterize the cytoplasmic vesicles in PT cells. The uptake of 14C glucosylceramide on LDL and [3H] galactose (galactose oxidase [3H] KBH4 reduction) labeled LacCer on LDL by cells will be studied in order to assess whether or not the exogenously derived LacCer on LDL contributes directly to the increased cellular levels of LacCer or indirectly following the uptake of glucosylceramide and its conversion to LacCer in the urinary cells. The capacity of cells to incorporate [14C] labeled galactose, glucose, serine and palmitate into LacCer and the UDP-galactose: GlcCer, galactosyltransferase activity will be measured in order to assess the endogenous synthesis of LacCer in these cells. The proximal tubular cell cytoplasmic vesicles will be isolated by homogenization, centrifugation, and sucrose density gradient centrifugation. The cytoplasmic vesicles will be characterized employing marker enzyme assays, immunochemical techniques and electron microscopy. Whether or not the cytoplasmic vesicles are capable of synthesizing LacCer or acquiring LacCer directly from LDL will be assessed. Major hypothesis. Increased urinary cell LacCer levels in homozygous FH may be due to a non-receptor mediated uptake of exogenous LacCer on LDL and increased endogenous synthesis of LacCer or both. These studies will provide new insights into glycosphingolipid and lipoprotein metabolism in proximal tubular cells from normal and FH subjects and the pathophysiology of LacCer storage in FH.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK031722-03
Application #
3230291
Study Section
Pathobiochemistry Study Section (PBC)
Project Start
1983-12-01
Project End
1987-06-30
Budget Start
1985-12-01
Budget End
1987-06-30
Support Year
3
Fiscal Year
1986
Total Cost
Indirect Cost

  Institution

Name
Johns Hopkins University
Department
Type
Schools of Medicine
DUNS #
045911138
City
Baltimore
State
MD
Country
United States
Zip Code
21218

  Publications

Chatterjee, Subroto; Alsaeedi, Nezar (2012) Lactosylceramide synthase as a therapeutic target to mitigate multiple human diseases in animal models. Adv Exp Med Biol 749:153-69
Chatterjee, Subroto; Pandey, Ambarish (2008) The Yin and Yang of lactosylceramide metabolism: implications in cell function. Biochim Biophys Acta 1780:370-82
Chatterjee, Subroto; Neill, Roger; Shupp, Jeffrey W et al. (2007) Identification of staphylococcal enterotoxin B domains involved in binding to cultured human kidney proximal tubular cells: imparting proliferation and death. Exp Biol Med (Maywood) 232:1142-51
Martin, Sergio F; Williams, Niesha; Chatterjee, Subroto (2006) Lactosylceramide is required in apoptosis induced by N-Smase. Glycoconj J 23:147-57
Chatterjee, Subroto; Berliner, Judith A; Subbanagounder, Ganesamoorthy G et al. (2004) Identification of a biologically active component in minimally oxidized low density lipoprotein (MM-LDL) responsible for aortic smooth muscle cell proliferation. Glycoconj J 20:331-8
Yeh, L H; Kinsey, A M; Chatterjee, S et al. (2001) Lactosylceramide mediates shear-induced endothelial superoxide production and intercellular adhesion molecule-1 expression. J Vasc Res 38:551-9
Jan, J T; Chatterjee, S; Griffin, D E (2000) Sindbis virus entry into cells triggers apoptosis by activating sphingomyelinase, leading to the release of ceramide. J Virol 74:6425-32
Chatterjee, S (2000) Assay of lactosylceramide synthase and comments on its potential role in signal transduction. Methods Enzymol 311:73-81
Chatterjee, S; Han, H; Rollins, S et al. (1999) Molecular cloning, characterization, and expression of a novel human neutral sphingomyelinase. J Biol Chem 274:37407-12
Chatterjee, S (1999) Neutral sphingomyelinase: past, present and future. Chem Phys Lipids 102:79-96

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