Expression of liver-specific genes, especially their mRNA synthesis, was found dependent upon cooperative effects of peptide hormones and heparins (HPs). Comparison of more than 40+ species of intact, native glycosaminoglycans (GAGs) and PGs indicates that only HPs or PGS with HP- like GAG chains, can cooperate with peptide hormones to regulate mRNA synthesis of tissue-specific genes. By contrast, heparin sulfates (HSs), at all concentrations and from all sources, proved inactive or even inhibitory. Posttranscriptional effects were observed with all tested PGs, and of the GAGs, with all species of HPs and weakly with dermantan sulfates (DSs). The rank order of potency of the PGs with respect to posttranscriptional effects was HP-PGs>>HS-PGs>>>DS-PGs>>chondroitin sulfate-PG (CS-PG) and of the active GAGs, HPs>>>>DSs. We propose to complete a structure-function analysis, ongoing, to define the chemistry of active PGs and GAGs enabling them to regulate gene expression. Our completed studies allow us to focus on HPs, the biologically active components of HP-PGs, the most active species of PG able to regulate gene expression in liver cells. HSs are closely related chemically, are inactive, and will be used as negative controls. The biological assays, all documented to be HP sensitive, will be: [1) morphological assays to detect nuclear shape changes;] 2) molecular hybridization assays for mRNA synthesis and abundance of insulin-responsive genes; and 3) electrophysiological assays for gap junctions. Cells will be treated with a test HP or HP fraction when in serum-free medium supplemented only with completely defined and purified hormones or growth factors, the composition and concentrations tailored for each gene. The format of the analysis is to: 1) complete the screens of HPs and of polyanions with HP-like activity (e.g. suramin) allowing us to identify especially active species; 2) dissect the most active ones for contributions of molecular weight (chain length), charge density, degree and form of sulfation, and extent of anticoagulant activity; [3) chemically and structurally analyze the most active fractions of the most active HPs and polyanions; 4) deduce from the chemical analysis what the active fractions have in common and prepare modified versions accordingly; 5) assess the biological activity of the modified versions and 6) repeat 3-5 until the active saccharide sequences (s) are identified.] The most active HP saccharide(s) identified will be used to initiate studies on the signal transduction mechanism(s) involved in HP/insulin's regulation of gene expression. We will focus initially on studies of calcium and intracellular pH, since our prior studies indicated that membrane-permanent analogues of one known second messenger, cAMP, were unable to synergize with HPs in the regulation of transcription rates for any liver-specific gene assayed. [We will also assess whether HPs modify insulin receptor levels or the binding affinity of insulin for its own receptor or for the receptors for IGF I or II.]

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
7R01DK044266-03
Application #
2143662
Study Section
Pathobiochemistry Study Section (PBC)
Project Start
1992-05-01
Project End
1997-04-30
Budget Start
1995-05-01
Budget End
1996-04-30
Support Year
3
Fiscal Year
1995
Total Cost
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Physiology
Type
Schools of Medicine
DUNS #
078861598
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Brill, S; Zvibel, I; Reid, L M (1999) Expansion conditions for early hepatic progenitor cells from embryonal and neonatal rat livers. Dig Dis Sci 44:364-71
Fiorino, A S; Diehl, A M; Lin, H Z et al. (1998) Maturation-dependent gene expression in a conditionally transformed liver progenitor cell line. In Vitro Cell Dev Biol Anim 34:247-58
Zvibel, I; Fiorino, A S; Brill, S et al. (1998) Phenotypic characterization of rat hepatoma cell lines and lineage-specific regulation of gene expression by differentiation agents. Differentiation 63:215-23
Agelli, M; Dello Sbarba, P; Halay, E D et al. (1997) Putative liver progenitor cells: conditions for long-term survival in culture. Histochem J 29:205-17
Reid, L M; Luntz, T L (1997) Ex vivo maintenance of differentiated mammalian cells. Methods Mol Biol 75:31-57
Reid, L M (1996) Stem cell-fed maturational lineages and gradients in signals: relevance to differentiation of epithelia. Mol Biol Rep 23:21-33
Fiorino, A S; Zvibel, I (1996) Disruption of cell-cell adhesion in the presence of sodium butyrate activates expression of the 92 kDa type IV collagenase in MDCK cells. Cell Biol Int 20:489-99
Zvibel, I; Brill, S; Reid, L M (1995) Insulin-like growth factor II regulation of gene expression in rat and human hepatomas. J Cell Physiol 162:36-43
Brill, S; Zvibel, I; Reid, L M (1995) Maturation-dependent changes in the regulation of liver-specific gene expression in embryonal versus adult primary liver cultures. Differentiation 59:95-102
Agelli, M; Halay, E D (1995) Flow cytometry and monoclonal antibodies identify normal liver cell populations antigenically related to oval cells. Eur J Histochem 39:175-82

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