T. W. Rall

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.

Conditions for the formation, partial purification and assay of an inhibitor adenosine 3′,5′-monophosphate

Abstract Particle preparations from dog and rat heart and dog liver were used to catalyze the formation of 3′,5′-AMP from ATP in the presence of Mg 2+ . These incubation mixtures also formed a maternal which noncompetitively inhibited the 3′, 5′-AMP-stimulated activation of phosphorylase in extracts of liver and heart. Inhibitory activity was quantified using an assay based on this effect. Conditions which increased the accumulation of the inhibitor were very similar to those that enhanced the accumulation of 3′,5′-AMP. These included the presence of a low-speed particle fraction, ATP, Mg 2+ , l -epinephrine, F − and caffeine. The β-adrenergic-blocking agent [I-(3,4-dichlorophenyl)-2-isopropylaminoethanol·HCl] prevented the stimulating effect of epinephrine on accumulation of the inhibitor and 3′,5′-AMP. With heart preparations, carbachol decreased the accumulation of both compounds. With liver preparations, glucagon increased the formation of both. Added 3′,5′-AMP also increased the formation of the inhibitor, whereas N 6 -monobutyryl 3′,5′-AMP prevented its formation. Both the inhibitor and 3′,5′-AMP were inactivated by treatment with 3′,5′-nucleotide phosphodiesterase, but were unaffected by several phosphataes. The inhibitor was not separated from 3′,5′-AMP by a number of chromatographic procedures. Both materials were, however, separated from other compounds that interfered with the effect of 3′,5′-AMP in the assay. The inhibitor was found in acidic extracts from brain, heart, liver, lung, adipose tissue and skeletal muscle, and the levels paralleled the 3′,5′-AMP concentration.

The interconversion of phosphorylase a and phosphorylase b from dog heart muscle

Abstract 1. 1. The purification from dog heart of phosphorylase a, a phosphorylase phosphatase and a dephosphophosphorylase kinase has been described. 2. 2. Inorganic phosphate was formed during the conversion of heart phosphorylase a to b by the phosphorylase phosphatases from heart and liver. No inorganic phosphate formation was detected when this conversion was catalyzed by trypsin. 3. 3. Heart phosphorylase b, formed by the action of the phosphorylase phosphatases, could be converted to the a form in the presence of ATP, Mg++ and the dephosphophosphorylase kinases from either heart or liver. Heart phosphorylase b, formed by the action of trypsin, was not converted to the a form.

Hormonal Regulation of Incorporation of Alanine-U-14C into Glucose in Human Fetal Liver Explants: Effect of Dibutyryl Cyclic AMP, Glucagon, Insulin, and Triamcinolone

Incorporation of alanine-U-14C into glucose and liver glycogen increased linearly over sixty-five hours in culture of human fetal liver explants. This rate of incorporation was stimulated two- to tenfold by incubation with N6,2′O-dibutyry I adenosine 3′-5′:cyclic monophosphate (dibutyryl cyclic AMP) (0.1 mM) plus theophylline (0.5 mM) or glucagon (7.5 μg./ml.) plus theophylline. No apparent lag period was detected, and the hormonal effect continued throughout the observation period. Insulin (1 U./ml.) significantly decreased both the basal rate of incorporation and the stimulated rate resulting from dibutyryl cyclic AMP or glucagon incubation. These effects were observed at both high (10 mM) and low (2.8 mM) media glucose and from both 2.3 μM and 5 mM alanine-U-14C. Triamcinolone (20 μg./ml.) alone stimulated the rate of alanine-U-14C incorporation into glucose, whereas triamcinolone in the presence of dibutyryl cyclic AMP produced an increase in incorporation greater than the sum of the individual effects. The basal incorporation of alanine-U-14C into glucose by these human fetal liver explants provide a rate of approximately 4 nmoles glucose/gm. min, which is discussed in relation to the physiologic needs of the fetus and newborn.

Hormonal Regulation of Glycogen Metabolism in Human Fetal Liver: I. Normal Development and Effects of Dibutyryl Cyclic AMP, Glucagon, and Insulin in Liver Explants

Glycogen accumulates in human fetal liver beginning at the eighth week of gestation. A parallel increase in total glycogen synthase activity is found, although the I-form activity remains low and constant throughout the first two thirds of gestation. Total phosphorylase activity increases slightly during this period, with the proportion in the active form amounting to about one half of the total throughout. After an initial rapid decline, the glycogen concentration in explants of human fetal liver remained constant for twenty to forty hours at about 20 per cent of the in vivo level. Incubation with glucagon, cyclic AMP (adenosine 3ʹ,5ʹ-monophosphate) or its dibutyryl derivative markedly reduced tissue glycogen concentrations while insulin brought about a small increase. The effect of maximal doses of dibutyryl cyclic AMP and glucagon were the same, and the combination of agents produced no further effect. The response to dibutyryl cyclic AMP was apparent by one hour and maximal by three to six hours, whereas the response to insulin required about six hours to be detected, and it continued for at least eighteen hours. Insulin antagonized the glycogenolytic effect of low doses of glucagon or theophylline but was without significant effect in the presence of high glucagon concentrations. Glucagon stimulated cyclic AMP output from explants, and this effect was further augmented by theophylline. Insultin had no consistent effect on cyclic AMP output in either the presence or the absence of glucagon or theophylline. Incubation with dibutyryl cyclic AMP resulted in a decrease of glycogen synthase I-form activity, while insulin tended to increase this enzyme activity. In neither circumstance was the proportion of active phosphorylase altered. These results suggest that the regulation of glycogen levels in human fetal liver by cyclic AMP, glucagon, and insulin may entail alterations in the activity of glycogen synthase activity without necessitating alterations in phosphorylase activity. Cyclic AMP or glucagon was capable of depleting tissue glycogen stores in tissue from fetuses of six weeks’ gestation. Insulin increased tissue glycogen concentrations in tissue from fetuses of seven or more weeks.

Hormonal Regulation of Glycogen Metabolism in Human Fetal Liver: II. Regulation of Glycogen Synthase Activity

During the first two thirds of gestation, the concentrations of UDPG, ATP, ADP, and Mg++ in human fetal liver remain constant, whereas the concentration of Pi decreases twofold and the G-6-P and AMP concentrations increase. Incubation of human fetal liver explants with glucagon or insulin did not alter the concentrations of any of these intermediates. ATP, ADP, and Pi are inhibitors of human fetal liver glycogen synthase D-form activity, while G-6-P and AMP and Mg++ are stimulators. Ca++ at concentrations of < 0.1 mM was found to stimulate glycogen synthase D activity. This effect of Ca++ was also observed in “physiologic” mixtures containing UDPG, G-6-P, ATP, ADP, AMP, Pi, and Mg++ at concentrations found either in liver in utero or in explants. 45Ca++ efflux from perifused (rat) fetal liver explants was stimulated by glucagon. These data provide a picture of the metabolite regulation of human fetal liver glycogen synthase activity in which the D-form may largely control glycogen synthesis in utero and hormonal effects on glycogen synthase may be induced by effects of Ca++ on the D-form.

Effect of dibutyryl cyclic amp on glucose-6-phosphatase activity in human fetal liver explants.

Abstract Glucose-6-phosphatase (EC 3.1.3.9) activity in human fetal liver remains constant at 8–28 nmoles/min per mg protein from the 8th week of gestation to at least week 28 and this value is approximately 25–35% of that found in the adult. This enzyme activity was well maintained for 2–3 days in organ culture of fetal liver explants. Incubation with dibutyryl cyclic AMP (0.1 mM) and theophylline (0.5 mM) increased glucose-6-phosphatase activity 4–8-fold within 24 h. Theophylline alone was ineffective, but markedly potentiated the effects of dibutyryl cyclic AMP. This increase in enzyme activity was completely abolished by simultaneous incubation with cycloheximide or actinomycin D. Insulin clearly decreased glucose-6-phosphatase activity in control tissues after 24 h incubation and tended to diminish the elevated glucose-6-phosphatase activity which resulted from pre-incubation with dibutyryl cyclic AMP. The smallest specimen obtained (36 mm crown-rump length = 6 weeks gestation) was capable of elevating glucose-6-phosphatase activity more than 3-fold in response to dibutyryl cyclic AMP incubation, suggesting that the human fetal liver has the competence to respond to hormonal agents at a very early stage of development.

Regulation of glucose-6-phosphatase activity in human fetal liver in vitro

G-6-Pase activity in human fetal liver remains constant at 8-28nmoles/min/mg protein from the 8th week of gestation to week 28, this value being appr. 25-35% of that found in the adult. This enzyme activity is well maintained for 2-3 days in organ culture of fetal liver explants. Incubation with dibutyryl cyclic AMP (0.1mM) and theophylline (0.5mM) increased G-6-Pase activity 4-8-fold within 24h. Theophylline alone was ineffective, but markedly potentiated the effects of dBcAMP. The increase in enzyme activity was completely abolished by simultaneous incubation with cycloheximide or actinomycin D. Insulin significantly decreased G-6-Pase activity to 50% of the control value after 24h incubation. In addition, insulin tended to diminish the elevated G-6-Pase activity which resulted from preincubation with dBcAMP. The smallest fetus obtained (36mm crown-rump length) was capable of elevating liver G-6-Pase activity more than 3-fold in response to dBcAMP, suggesting that the human fetal liver has the competence to respond to hormonal agents at a very early stage.