The proposal consists of two projects: (A) The Precursors of Glycogen Several investigators including myself have presented evidence that the major precursors of hepatic glycogen are 3-carbon precursors, rather than glucose. There is some contrary evidence and the issue is controversial. A novel approach, using 3HOH incorporation, to resolve the problem is proposed. 3HOH will be administered in vivo and the distribution of tritium in glucose will be determined. 3HOH yield in C-2 should represent glycogen synthesis from all precursors, including glucose, whereas that on C-6, synthesis from pyruvate. The tritium pattern in circulating glucose, liver and muscle glycogen and lactate will be determined. The effects of diet, diabetes and hormones on the labelling pattern will be determined. (B) Determination of Gluconeogenesis in vivo Oxalacetate is a shared intermediate for synthesis of phosphoenolpyruvate and the tricarboxylic acid cycle. Therefore, in the conversion of pyruvate to PEP, the carbon of PEP is diluted by carbon from acetyl CoA and CO2. To calculate the contribution of alanine and lactate to gluconeogenesis, it is essential to evaluate this dilution. A theory to obtain this dilution is presented. Two labelled species, such as 1-13C and 2-14C alanine or lactate, are required to calculate the true rate of gluconeogenesis. Experiments with the administration of two tracers (13C and 14C) and methods to calculate the true contribution of alanine and lactate to glucose production are planned. Methods to quantitate gluconeogenesis from the incorporation of 14CO2 and 14C acetate will be examined. Fed, starved, diabetic and hormone-treated animals will be used.