Date of Award

6-2002

Degree Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

Supervisor

Professor Chris M. Wood

Abstract

A biochemical approach was employed to examine the oxidative utilization of lipid and carbohydrate in red and white muscle of rainbow trout (Oncorhynchus mykiss) during high- and low- intensity exercise and during recovery from high-intensity exercise. Measurements of the activation state of the rate-limiting enzymes, glycogen phosphorylase (Phos) and pyruvate dehydrogenase (PDH), and their allosteric regulation of pathways involved in lipid and carbohydrate oxidation in fish muscle.

High-intensity exercise in trout is powered predominately by white muscle. During the first 10 s of exercise, high ATP turnover rates (3.7 μmol·g-1 wet tissue·s-1) are achieved by rapid creatine phosphate (CrP) hydrolysis and glycolysis. Activation of glycolysis is achieved by a large transformation of Phos into its active form. Exercise performed from 10 s to exhaustion (52 s) occurs at a lower ATP turnover rate (0.5 to 1.2 μmol·g-1 wet tissue·s-1) and is primarily supported by glycosis. A gradual transformation of PDH into its active form points to a minor contribution of oxidative phosphorylation of carbohydrate to support ATP turnover. The gradual activation of PDH and an increasing mitchondria redox ([NAD+]/[NAHD]) in white muscle during exercise suggests that O2 is not limiting during high-intensity exercise and thus anaerobiosis may not be responsible for lactate production in trout white muscle.

During recovery from high-intensity exercise, there is a rapid activation of pathways involved in lipid oxidation to provide ATP for CrP, ATP, and glycogen synthesis. A rapid transformation of PDH into its inactive form spares lactate from an oxidative fate, likely to serve as the substrate for in situ glycogen re-synthesis. Decreases in free carnitine and increases in long-chain fatty acyl (LCFA) carnitine, acetyl-CoA, and acetyl canitine indicate an activation of pathways involved in lipid oxidation. Increases in white muscle malonyl-CoA during recovery suggest that malonyl-CoA may not regulate carnitine palimtoyltransferase-1 in trout white muscle.

Sustainable, "aerobic" exercise at 30 and 60% Ucrit is characterized by an inital (2 min) oxidative utilization of carbohydrate for ATP production. Withink 15 min swimming at 30 and 60% Ucrit, PDH activation returns to resting values, and at 240 min increases in LCFA-carnitine and decreases in malonyl-CoA indicate an overall enhancement of lipid oxidation. Non-sustainable swimming at 90% Ucrit is characterized by a sustained activation of red muscle PDH. Overall, sustained swimming at 30 and 60% Ucrit is supported by approximately equal contributions of carbohydrate (~45%) and lipid (~35%) oxidation, whereas non-sustainable swimming is supported primarily by carbohydrate oxidation.

Palmitate uptake by red and white muscle membrane vesicles is facilitated by a saturable transport mechanism which is sensitive to phloretin, 4,4'diisothiocyanatostilbene-2,2'-disulfonic acid, (DIDS), and mercury. Red and white muscle transporters have similar affinities for palmitate (Km=26±6 and 33±8 nM free palmitate, respectively), but red muscle exhibits a significantly higher maximal uptake rate compared to white muscle (Vmax = 476±41 and 227±23 pmol·mg-1 protein·s-1, respectively). Prolonged swimming and chronic cortisol elevatins do not stimulate palmitate uptake by muscle, indicating that the mechanisms involved in LCFA uptake by muscle cells may not limit lipid oxidation in trout muscle.

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