CALORIC RESTRICTION AND ITS INFLUENCE ON GLYCOLYSIS
K. Hagopian* and R. Weindruch
University of Wisconsin Medical School, Dept. of Medicine, and
VA Hospital, GRECC-D5, 2500 Overlook Terrace, Madison, WI 53705
Caloric restriction (CR) is an intervention that offsets and delays age-associated changes and extends maximum life span in a variety of species. Studies in laboratory rodents have shown that a decrease of 30-60% in food intake resulted in a corresponding increase in the average and maximum life span. The purpose of this work was to study the effects of CR on glycolytic enzymes and metabolites and to understand the role of CR in metabolism.
Young (3 months) and old (30 months) male C57BL/6J mice were used. Mice on CR diet received 25% fewer calories than the controls. The livers were freeze-clamped in situ and removed, as rapidly as possible, and powdered under liquid nitrogen. Extracts prepared from livers were assayed for glucokinase, phosphoglucose isomerase, phosphofructokinase-1, aldolase, triosphosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase and pyruvate kinase activities. For the assay of glycolytic metabolites, perchloric acid extracts were prepared and the levels of Glc, G6P, F6P, F1,6BP, GAP, DHAP, 3PG, 2PG, PEP, Pyr and Lac were determined. All enzyme activities and metabolite levels were determined spectrophotometrically.
Results showed that CR decreased glycolytic activity. Statistically significant decreases in all enzyme activities in old CR mice were observed when compared with old controls, except for aldolase, triosephosphate isomerase and phosphoglycerate mutase. Statistically significant decreases in all enzyme activities in the young CR mice were also observed when compared with young controls, except for aldolase, triosephosphate isomerase and enolase. In the case of hepatic metabolites, statistically significant decreases in the levels of Glc, G6P, F6P, Pyr and Lac and increases in the levels of F1,6BP, GAP, DHAP, 3PG and PEP were observed in old CR mice when compared with old controls, while 2PG showed no significant changes. On the other hand, hepatic metabolites Glc, G6P, F6P and Pyr showed statistically significant decrease while PEP levels increased significantly in young CR mice when compared with young controls. Cross-over analysis has shown two regulatory points at phosphofructokinase-1 and pyruvate kinase. Moreover, hepatic ketone body concentration also increased significantly in old CR mice, while the levels were unchanged in the young.
Our results indicate that decreased hepatic glucose concentration in CR is accompanied by a decreased glycolytic activity. Moreover, this decrease in energy source must be compensated and the result has been an increase in ketone body production, as an alternative source of fuel. The increase in the levels of F1,6BP, GAP and DHAP indicate that other sources of energy are also being utilized as fuel and since these metabolites are shared by glycolysis and gluconeogenesis, it indicates that gluconeogenesis is in progress. These also are the points of entry of other sources into the glycolytic/gluconeogenic pathway.
Key words:
Caloric restriction, Glycolysis, Metabolism, Mice, Liver
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