Joseph Larner

Insulin treatment and increased UDPG-glycogen transglucosylase activity in muscle.

Abstract Phosphoglucomutase, UDP-glucose pyrophosphorylase, phosphorylase, and UDP-glucose glycogen transglucosylase activities were compared in extracts prepared from diaphragms incubated without and with insulin. No differences in the activities of the first three enzymes were found, but UDP-glucose glycogen transglucosylase activity was found to be increased in extracts of diaphragms incubated with insulin when assayed without addition of glucose-6-P. When glucose-6-P was added in the assay, the activities of extracts prepared from control and insulin-treated diaphragms were both increased and were the same. The increase in enzyme activity by insulin was not duplicated by incubation of diaphragms in an increased concentration of glucose. Further, it was not obtained after incubation of diaphragms with insulin at 0 °C. Enzyme activity was increased, however, after incubation of diaphragms with insulin without glucose in the incubation medium. The increased enzyme activity was retained after dialysis or ammonium sulfate precipitation. The increase in transglucosylase activity is in keeping with increased glycogen content after insulin treatment and supports the role of UDP-glucose glycogen transglucosylase in glycogen 1,4-bond synthesis. An explanation of the partial increase of enzyme activity after insulin treatment based on the presence of two activity forms of the enzyme is presented. These studies demonstrate a new mode of hormonal control of glycogen metabolism.

Levels of activity of the enzymes of the glycogen cycle in rat tissues.

Abstract Levels of phosphorylase, UDPG pyrophosphorylase, and phosphoglucomutase activities have been studied in rat tissues. Glycogen content of the same tissues has been determined in fed and fasted rats. The results obtained appear to be compatible with a cyclic mechanism of glycogen synthesis and degradation.

The influence of insulin and epinephrine on adenosine 3′,5′-phosphate and glycogen transferase in muscle

Abstract 1. 1. The role of adenosine 3′,5′-phosphate (cyclic AMP) in the mechanism of action of insulin on the enzyme glycogen transferase (UDP glucose: α-1,4-glucan α-4-glucosyltransferase, EC 2.4.1.11) was investigated in rat diaphragm muscle. 2. 2. Incubation of intact rat diaphragms with insulin alone did not alter the cyclic AMP content of the muscle. When the diaphragms were exposed to both insulin and epinephrine simultaneously, the increase in the cyclic AMP concentration was the same as with epinephrine alone. Under these conditions, insulin produced the previously observed increase in the proportion of glycogen transferase in the glucose-6-phosphate-independent ( I ) form, but had no significant effect on glycogen phosphorylase (α-1,4-glucan: orthophosphate glucosyltransferase, EC 2.4.1.1). 3. 3. When diaphragms were pe-incubated with insulin, there was a diminished rise in cyclic AMP levels in the first few minutes after the addition of epinephrine. An increase in activity of the I form of glycogen transferase was observed, but even under these conditions, phosphorylase activity was not altered significantly by insulin. 4. 4. These results suggest that the effect of insulin on glycogen transferase activity in skeletal muscle does not depend upon changes in the tissue concentration of cyclic AMP.

INSULIN‐STIMULATED GLYCOGEN FORMATION IN RAT DIAPHRAGM*

Insulin-stimulated glucose uptake and glycogen synthesis in isolated rat diaphragm was originally demonst rated by Gemmill and Hamman. Brown et ul.? have reported that, following a 30-min. incubation period, about 75 per cent of the increased glucose uptake in the presence of insulin could be accounted for as tissue glycogen. In the present report it is shown that, after a 10-min. incubation period at 38C., more than 90 per cent of the increased glucose uptake in the presence of insulin could be accounted for as tissue glycogen, the major increase being in a perchloric acid-soluble glycogen fraction. Analyses of tissue acid-soluble phosphate intermediates, including inorganic phosphate, creatine phosphate, adenosine diphosphate (ADP), and adenosine triphosphate (ATP) showed no difference between control and insulin-treated diaphragms. A small but statistically significant increase in hexose-6-phosphate content of the insulin-stimulated diaphragms was observed at 2 medium glucose levels. (ilycogen synthesis occurred with ratios of inorganic phosphate to glucose-lphosphate that would markedly favor glycogen degradation by phosphorylase as studied in soluble systems. Enzyme studies in broken-cell preparations indicate the presence in skeletal muscle of 2 enzymes, uridine diphosphate glucose (UDPG) pyropho~phorylase,~ and UDPG glycogen transgluc~sylase,~ constituting a reaction sequence (independent of inorganic phosphate) converting glucose-1-phosphate to glycogen. These enzymes are of sufficient activity to account for the insulin-stimulated glycogen formation observed.6

Uridinediphosphate glucose pyrophosphorylase from skeletal muscle

Abstract Uridinediphosphate glucose pyrophosphorylase has been purified from skeletal muscle over 1300-fold. It appears to be present with an activity of 30 mmoles/100 g. muscle/hr. at 30 °C. The enzyme seems to be specific for glucose 1-phosphate. The K m for UDPG is 4.5 × 10 −5 M ; the calculated K m for both Mg ++ and pyrophosphate is 1 + 10 −4 M . The pH optimum of stability is 9.8, and the pH optimum of activity is 7.5.

Insulin-stimulated glycogen formation in rat diaphragm. Levels of tissue intermediates in short-time experiments.

Abstract Studies carried out with rat hemidiaphragms indicated that, under the experimental conditions outlined, over 90% of the increased uptake of glucose in the presence of insulin, on a balance basis, could be accounted for as tissue glycogen. Hexose 6-phosphate content was increased in diaphragms incubated in the presence of insulin, in agreement with an increased conversion of extracellular glucose to intracellular hexose 6-phosphate. Glucose 1-phosphate contents were unchanged, suggesting a rapid removal of glucose 1-phosphate. Glycogen formation in the presence of insulin took place at ratios of inorganic phosphate to glucose 1-phosphate of 305. ADP, ATP, inorganic phosphate, and creatine phosphate were not changed, a finding compatible with the association of the increased glycogen formation to phosphorylative reactions.