Introduction
Energy expenditure represents the conversion of food or stored forms of energy to carbon dioxide, water, heat and work on the environment (such as muscle contraction, protein synthesis and ion pumping). The expression and activity of key proteins directly involved in energy expenditure are tightly regulated by nutritional state (type of diet or starvation) and level of physical exercise. The aim of this study was to quantitate the specific responses of the Na,K-ATPase a- and b-isoforms, the mitochondrial uncoupling protein-3 (UCP-3) and the glucose transporter-4 (GLUT4) to in response to high fat feeding and exercise training in rat skeletal muscle.
Methods
Female Wistar rats receive a either standard rat chow or a high fat diet (55% of calories). After three weeks chow-fed and fat-fed rats were randomly assigned to two subgroups: 5-day exercised-trained or sedentary control. Exercise-trained rats performed two three-hour swimming exercise bouts, separated by one 45-min rest period. Approximately 16 h after the last exercise bout, rats were anesthetized and muscles were removed. The Animal Ethical Committee of the Karolinska Institute approved all protocols. Mitochondria-enriched and total membrane fractions were obtained from homogenates of gastrocnemius muscles by differential centrifugation. Aliquots of membrane fractions (10 µg) were separated by SDS-PAGE and expression of proteins of interest were analyzed by Western blot with appropriate antibody
Results
After 4 weeks of high fat feeding, Na,K-ATPase a1-subunit protein levels, as measured by immunoblotting, were increased 1.6 fold (p<0.05), whereas a2-subunit protein levels were decreased 2-fold (p<0.01). Exercise training completely restored a-subunit expression to control levels. Na,K-ATPase b1-subunit protein level was decreased 2.2 fold (p<0.05) in muscle from fat-fed rats. Exercise training restored protein expression of the Na,K-ATPase b1-subunit in fat-fed rats. Na,K-ATPase b2-subunit protein level was not altered by high fat diet. b2-subunit protein level were decreased 2-fold (p<0.05) by exercise-training in chow and high fat fed rats.
High fat diet increased expression of UCP-3 in mitochondria 1.7 fold (p<0.01) and this was further increased 1.5 -fold after exercise-training (p<0.05). Exercise training along has no effect on UCP-3 expression in skeletal muscle.
Exercise training led to 1.8-fold (p<0.05) increase in GLUT4 protein expression and 2.1 fold (p<0.05) increase in insulin-regulated aminopeptidase protein (IRAP) content. High fat feeding did not alter expression of either GLUT4 or IRAP
Conclusions
High fat diet alters expression of proteins directly involved in energy expenditure. For Na,K-ATPase a- and b-subunits, the effect is isoform-specific. Expression of mitochondrial uncoupling protein-3 was elevated by high fat diet. In contrast to the changes in Na, K-ATPase a1-, a2- and b1 subunits expression, elevated level of UCP-3 is likely to occur in direct response to fat feeding rather than peripheral insulin resistance.
References