Abstract
The suckling-weaning transition naturally begins at around 16 days of age in rats and continues to 25-30 days when the pups consume the solid, maternal diet. Throughout this transition, the dietary content changes from dominantly fats in the milk to carbohydrates in the adult diet. This marked change in diet is accompanied by dramatic changes in the metabolic priorities and the carbon flux. Although the role of pyruvate dehydrogenase in controlling the carbon flux during other metabolic adaptations, such as the starved-refed transition and diabetes, has already been shown to be essential, this enzyme has not been investigated in detail during the suckling-weaning transition. This has been addressed in this thesis. Pyruvate dehydrogenase activity was measured spectro-photometrically in isolated liver and heart of rats between 10 and 30 days old, and compared to citrate synthase activity. Initially, the rat pups were left to wean themselves naturally (spontaneously). Hepatic pyruvate dehydrogenase activity was found to be low during suckling (<7. 3mU/U) and to increase gradually throughout the suckling-weaning transition, reaching a maximum approximately 4-times suckling levels, at 25 days. Cardiac pyruvate dehydrogenase activity increased more rapidly and doubled between suckling (< 17. 0mU/U) and weaning, peaking at 22 days. Weaning could be condensed and an accurate starting point for the post-weaning period established, by imposing weaning by removing the mother and forcing the pups to adapt to the new diet immediately. Weaning was imposed at 20 days, and pyruvate dehydrogenase activity measured. Hepatic pyruvate dehydrogenase activity rose 10-fold between 20 day and 22 days, faster and to a higher peak (52. 4mU/U) than seen during spontaneous weaning. Cardiac activity, however, was largely unaffected by the altered weaning conditions and rose as in the spontaneously weaned animals. Weaning was also imposed at 18 days in order to minimise the carbohydrate intake by the pups before weaning. Premature weaning in this way advanced the activation of both hepatic and cardiac pyruvate dehydrogenase, by two days and one day respectively. In both cases the post-weaning peak reached was higher than after weaning at 20 days (94. 4 and 38. 5mU/U respectively). Total pyruvate dehydrogenase was measured spectro-photometrically after activation of the inactive enzyme with dichloroacetic acid, during suckling and weaning at 20 days. Total pyruvate dehydrogenase was found to be 3- to 8- fold higher than the active form during suckling and weaning in the liver and heart, indicating that the pyruvate dehydrogenase complex was present in high levels even at 10 days of age, but remained largely inactive until weaning. The increase in PDH activity at weaning is therefore due to activation of enzyme already present and not due to synthesis of enzyme. In order to distinguish the effects of the increased carbohydrate content of the diet from the effects of the weaning process itself, weaning to a synthetic high-fat/low-carbohydrate diet was imposed at 20 days. Pyruvate dehydrogenase activity did not increase after weaning in either the liver or the heart. Total activity increased as with carbohydrate weaning suggesting that the dietary content and not the weaning process itself was responsible for activation of pyruvate dehydrogenase. Hepatic glycogen content was measured during both spontaneous and imposed weaning to high-carbohydrate and high-fat diets. The glycogen content of the liver increased 2-fold after weaning to a high-carbohydrate diet but not a high-fat diet. The increase after imposed weaning to a high-carbohydrate diet, occurred 24hrs in advance of the activation of hepatic pyruvate dehydrogenase. The expression of hepatic pyruvate dehydrogenase kinase mRNA levels throughout the suckling-weaning transition were investigated by Northern Blotting. However, due to the apparent lack of specificity of the cDNA probe used, no indication of the developmental profile of this enzyme was obtained. The immediate synthesis of glycogen and delay in pyruvate dehydrogenase activation in the liver, after weaning to a carbohydrate-rich diet suggests a regulatory role for pyruvate dehydrogenase in promoting glycogen synthesis before allowing flow of carbohydrate metabolites into the citric acid cycle or lipogenesis. Hepatic pyruvate dehydrogenase is therefore likely to be an important enzyme regulating the carbon flux during the suckling-weaning transition. Cardiac pyruvate dehydrogenase activation, however, is not delayed after weaning, possibly due to the low glycogen content of the heart and the difference in the tissue function, and thus may not be instrumental in controlling carbon flux during the suckling-weaning transition.