Carbohydrate and lipid metabolism in insulin resistant states and the development of in vivo magnetic resonance spectroscopic techniques for the analysis of gluconeogenesis and glycogen synthesis

Abstract

This thesis will provide new information on carbohydrate and lipid metabolic control in various related physiological states both in vitro and in vivo in the rat model. Pregnancy is one such state which elicits many complex changes both physiologically and metabolically, one major consequence of which is the development of insulin resistance/glucose intolerance by the mother. The aim was to evaluate the mother's capacity to utilise glucose during the various stages of pregnancy. For the first time, an unrestrained, unanaesthetised rat model was used to produce indices of glucose utilisation (GUI) in a wide range of tissue types ranging from non-working postural muscles to continually contracting muscles like the heart. In the various fed conditions investigated, significant reductions in the GUI of up to 80% were seen during late pregnancy in some tissues. A method of feeding was also applied to the pregnant model producing results contrary to normal pregnant glucose utilisation values ie. increasing the GUI up to 90% of normal pregnancy values in some tissues. Lipogenic rates in five major tissue sites were concurrently investigated which showed increases of between 2.2 and 4 fold during late pregnancy compared with virgin controls. Analysis of glycogen levels and an attempt to probe the pyruvate dehydrogenase activities of the heart and diaphragm in the various physiological states are also discussed. To obtain further insight into glycogen synthesis/non-oxidated disposal of glucose in the liver, a less invasive approach was then followed to explore, by non-invasive techniques, the extent to which insulin resistant states, like pregnancy, can be explained by in vivo hepatic glucose metabolic studies. For this, carbon-13 magnetic resonance spectroscopy (MRS) was employed to determine the fate of 1-13C glucose in the liver of rats in the same physiological states as those studied in the above methodology. Bolus infusions of 13C-glucose were administered into the hepatic portal vein of the rats under investigation. The incorporation or degradation of the glucose was followed in real time by observing rapid proton-decoupled carbon spectra. From this methodology, continuous real time glucose utilisation rates together with glycogen synthesis rates were measured. 13C incorporation into the glycogen macromolecule was seen to be negligible in 20 day pregnant ad libitum fed rats however in routine meal fed (RMF - allowed free access to food for 2h per 24h day) 20 day pregnant rats, incorporation reached a level 2.3 fold higher than the former. Maximal increases of 13C incorporation were shown by the 10 day pregnant RMF rats which attained levels 2.4 fold higher than the 20 day pregnant RMF group. Results indicate an enhancement of the direct route for glycogen deposition during RMF regimes in pregnancy. A consequence to the above MRS study led an investigation into gluconeogenic control in the rat model. Gluconeogenesis is a major metabolic pathway which may be perturbed in insulin resistant states like non-insulin dependent diabetes mellitus. In this study 13P MRS was employed as a tool to look at the relative changes in vivo of the hepatic phosphorus spectrum. This study involved external manipulation of hepatic gluconeogenesis by the infusion of alanine. Large increases in the phosphomonoester region of the phosphorous spectrum were seen both in vivo and in vitro which was secondary to increases in 3-phosphoglycerate. The inorganic phosphates also increased dramatically together with concomitant declines in ATP both of which recovered to pre-infusion values. This study shows many promising features which could be applied to clinical studies in humans.