Glycerol metabolism in smelt (Osmerus mordax)

Ditlecadet, Delphine (2012) Glycerol metabolism in smelt (Osmerus mordax). Doctoral (PhD) thesis, Memorial University of Newfoundland.

[English] PDF (Migrated (PDF/A Conversion) from original format: (application/pdf)) - Accepted Version Available under License - The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. Download (42MB) [English] PDF - Accepted Version Available under License - The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. (Original Version)

Abstract

Rainbow smelt (Osmerus mordax) is a small fish that accumulates high amounts of glycerol in winter as a cryoprotectant. The principal objective of my thesis was to investigate the metabolic control of the synthesis and clearance (loss/catabolism) of glycerol in this species. As rainbow smelt lose 4-13% of their glycerol content daily, they have to vigorously synthesize this metabolite at temperatures below 0ﾟC to reach the exceptional levels reported (> 200 mM). In this context, the two main hypotheses of my thesis were: 1) glycerol loss/catabolism might be turned-off during the accumulation period and be turned on during the decrease period; and 2) the last step of glycerol synthesis, leading from glycerol-3-phosphate (G3P) to glycerol might be direct, involving a single reaction catalyzed by glycerol 3-phosphatase (G3Pase), an enzyme not yet convincingly described in any animal species. -- Activity and mRNA levels of glycerol kinase (GK), the first enzyme involved in glycerol breakdown, were measured in liver, the tissue displaying the highest activity in vertebrates. mRNA levels did not change over the cycle but GK activity did, suggesting a regulation at a post transcriptional level. Overall, GK activity was so low in liver, and in all other tissues assayed, that it likely has a minor quantitative role in glycerol clearance. Daily loss toward water was also determined over the cycle but was not lower over the accumulation period, ruling out the presence of any mechanisms to retain glycerol during this period. Taken together, these results suggest that glycerol levels are dictated primarily by the rate of glycerol synthesis. -- To produce glycerol, G3P can be directly dephosphorylated by a G3Pase, as in yeast (Saccharomyces cerevisiae) or can enter the triacylglycerol pool and then be released as glycerol via reactions well recognized in mammals. Lipid content and activities of G3Pase and key enzymes involved in lipid metabolism were measured over the glycerol cycle in liver, the site of glycerol synthesis. Lipid content did not change over the cycle and was too low to account for a significant amount of glycerol, but activities of G3Pase and of enzymes involved in lipid degradation were up-regulated early in the cycle, at the onset of glycerol accumulation. A second set of experiments was performed using isolated smelt hepatic cells incubated in optimal conditions to produce glycerol over 72h in the presence or absence of a potent inhibitor of lipid synthesis. It was shown that neither on-board lipids nor newly synthesized lipids could be the source of the glycerol produced, pointing out for the first time a direct dephosphorylation by G3Pase as the main source of glycerol in an animal species. -- The last objective of my thesis was to partially purify and characterize G3Pase from smelt liver. The enzyme is most likely a cytosolic, acidic, low molecular weight phosphatase, in contrast to other species where the enzyme was previously purified and that has an optimal activity at neutral pH. -- These results are crucial in terms of understanding glycerol metabolism in rainbow smelt but also for the elucidation of pathways involved in glycerol synthesis in vertebrates as they clearly showed that glycerol is not necessarily only a by-product of lipid degradation.

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