The synthesis and degradation of metallothionein (MT) was studied in streptozotocin-induced diabetic rats and monolayer cultures of adult rat hepatocytes. Elevated levels of MT-I and MT-II were identified in the liver and kidney of untreated diabetic rats. The relative rates of hepatic and renal MT synthesis were significantly higher in STZ-diabetic rats than in controls. The changes in the relative rate of MT synthesis were maximal by 4 and 10 days in liver and kidney, respective, after administration of streptozotocin. The relative rate of cytoplasmic MT turnover was also increased in liver, but largely unaffected in the kidney, of diabetic rats. The altered metabolism of hepatic MT in diabetic rats was attributed primarily to chronic changes in the levels of pancreatic and adrenal hormones in plasma. In contrast, increased synthesis of renal MT in the diabetic rat was due largely to accumulation of excessive dietary Cu in the kidney.
Critical analysis of in vivo studies with diabetic rats and other literature revealed that cytoplasmic turnover of MT may not reflect actual degradation of this protein. Therefore, the characteristics of MT degradation in primary cultures of hepatocytes were investigated in subsequent studies. Hepatocytes were incubated in medium containing ³⁵S-cysteine and 100 uM zn overnight to induce MT synthesis. The level of ³⁵S-MT was quantified in heat stable extracts of cell homogenates by Fast Protein Liquid Chromatography (FPIC). When Zn was removed from medium, the rate of ³⁵S-MT turnover (t<sub>1/2</sub>= 7 hours) was four times faster than general ³H-protein (t<sub>1/2</sub>= 29 hours). This decrease in cellular MT level reflected degradation since less than 1% of cellular MT was secreted. The rate of MT degradation was inversely proportional to cellular Zn status. Cycloheximide, chloroquine and tosyl-lysine chloromethyl ketone (TLCK) inhibited ³⁵S-MT degradation by 33, 65 and 50%, respectively, without affecting cellular Zn status. Degradation of ³H-protein was inhibited by 41, 41 and 16% in the presence of cycloheximide, chloroquine and TI.CK, respectively. Removal of insulin increased ³H-protein degradation by 30%, but did not alter ³⁵S-MT degradation. Together, these data suggest that hepatic MT degradation (a) is primarily regulated by cellular Zn status and (b) occurs in both lysosomal and cytoplasmic compartments. / Ph. D.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/54319 |
| Date | January 1988 |
| Creators | Chen, Ming Long |
| Contributors | Biochemistry and Nutrition, Failla, Mark L., Keenan, Thomas W., Gregory, Eugene "Mick", Sitz, Thomas O., Webb, Ryland E. |
| Publisher | Virginia Polytechnic Institute and State University |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | en_US |
| Detected Language | English |
| Type | Dissertation, Text |
| Format | xi, 142 leaves, application/pdf, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
| Relation | OCLC# 17863633 |
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