The relation between cellular uracil nucleotides and ability to synthesize glycogen was studied in rat diaphragm incubated in vitro. In the absence of exogenous uridine the tissue content of UTP and rate of glycogen synthesis decreased with time. Uridine added to the medium increased cellular UTP and UDPG and stimulated glycogen synthesis. Insulin significantly increased the synthesis of UTP from extracellular uridine. This action of insulin appeared to be due to a stimulation of phosphorylation of the nucleoside and not to an effect on transport at the concentrations of uridine studied. However, an effect of insulin on transport of uridine at low concentrations cannot be excluded.Studies of the regulation of glycogen synthesis in muscle have been concerned primarily with the properties of glycogen synthase (EC 2.4.1.11; UDP glucose:glycogen 4-a-glucosyltransferase), the rate-limiting enzyme that catalyzes the reaction: UDPG + (glucose),, = UDP + (glucose),,+,. This enzyme exists in two or more forms (1, 2), the best characterized being designated as the independent (I or a) and dependent (D or b) forms differing in their Ka values for activation by glucose-6-P and in their apparent Km values for UDPG. Glycogen synthase b is probably inactive physiologically since it is strongly inhibited by tissue metabolites and its Km for UDPG is in the order of 0.2 mM, whereas glycogen synthase a is less sensitive to inhibition and has a Km of about 0.06 mM (2). Since the actual concentration of UDPG in muscle is approximately 0.03 mM (3), it is clear that any changes in the cellular concentration of this substrate would markedly alter the rate of glycogen synthesis.The formation of UDPG occurs by the reaction: UTP + glucose-1-P = UDPG + P-P and is catalyzed by UDPG pyrophosphorylase (EC 2.7.7.9; UTP:a-D-glucose-l-phosphate uridylyltransferase), an enzyme that has been found not to be rate limiting in the synthesis of glycogen in muscle (4). However, it seemed possible to us that the availability of UTP for the synthesis of UDPG could be a limiting factor in glycogen formation and hence the rate of synthesis of this polysaccharide could be influenced by the intracellular concentration of UTP. If this is true, additional supply of UDPG from exogenous uridine by the pyrimidine nucleotide salvage pathway should lead to an increase in the rate of formation of glycogen. The experiments reported here were designed to study this problem. MATERIALS AND METHODSMaterials. [U-14C]Glucose and [U-14C]uridine were obtained from New England Nuclear, and uridine was from Calbiochem. The insulin preparation used was crystalline porcine insulin (Eli Lilly and Co.; lot 615-DG3-10).Tissue Incubation. Male Wistar rats (125-150 g) fed freely were killed by decapitation and the diaphragms were removed. and collected in chilled 0.15 M NaCl. Wet weights were determined on a torsion balance after gentle blotting on filter paper. The results of metabolic measurements are expressed in units/g of wet weight. The tissues were incubated at 370 for...