Frank Q. Nuttall

Radioactive method for the assay of glycogen phosphorylases

Abstract A new assay for glycogen phosphorylase (EC 2.4.1.1) is presented. This assay employs a filter paper technique in which newly formed glycogen-14C is precipitated on paper squares using 66% ethanol while other radioactivity is removed by washing. It is rapid and reproducible and may be employed with crude as well as purified enzyme preparations. An additional advantage of the method is the potential for increased sensitivity. It should find utility for assay of extremely small tissue samples and should be useful in kinetic studies.

Udpglucose: α-1,4-glucan α-4-glucosyltransferase in heart regulation of the activity of the transferase in vivo and in vitro in rat. A dissociation in the action of isulin on transport and on transferase conversion

Abstract In the isolated perfused rat heart the nonhormonal control of transferase by glycogen was readily demonstrated. The transferase phosphate converting the D to I form was shown to be inhibited by glycogen in vitro. No hormonal effect of epinephrine, insulin or caffeine on transferase was demonstrated in this preparation although the usual effect of insulin to increase glucoge uptake was presen. In vivo the effect of insulin to increase transferase I activity in heart was readily demonstrated. These findings suggest a ‘dissociation’ in the action of insulin on the prefused rat heart in terms of the control of transport and the control of transferase conversion. This dissociation is discussed in terms of other dissociations of these two parameters noted in the literature, and it is concluded that there is no required temporal sequencing in the action of insulin on glucose transport and glycogen synthesis

Evidence for the non-identity of proteins having synthase phosphatase, phosphorylase phosphatase and histone phosphatase activity in rat liver

Synthase phosphatase, phosphorylase phosphatase and histone phosphatase in rat liver were measured using as substrates purified liver synthase D, phosphorylase alpha and 32P-labelled phosphorylated f1 histone, respectively. The three phosphatase enzymes had different sedimentation characteristics. Both synthase phosphatase and phosphorylase phosphatase were found to sediment with the microsomal fraction under our experimental conditions. Only 10% of histone phosphatase was in this fraction; the majority was in the cytosol. No change in histone phosphatase was observed in the adrenalectomized fasted rat whereas synthase phosphatase and phosphorylase phosphatase activities were decreased 5-10 fold. Fractionation of liver extract with ethanol produced a dissociation of the three phosphatase activities. When a partially purified fraction was put on a DEAE-cellulose column, synthase phosphatase and phosphorylase phosphatase both exhibited broad elution profiles but their activity peaks did not coincide. Histone phosphatase eluted as a single discrete peak. When the supernatant of CaCl2-treated microsomal fraction was put on a Sepharose 4B column, the majority of synthase phosphatase was found to elute with the larger molecular weight proteins whereas the majority of phosphorylase phosphatase eluted with the smaller species. Histone phosphatase migrated as a single peak and was of intermediate size. Synthase phosphorylase phosphatase by synthase D (Ki approximately 2 units/ml). The inhibition of synthase phosphatase by phosphorylase alpha was kinetically non-competitive with substrate. Histone phosphatase activity was not inhibited by synthase D or by phosphorylase alpha. The above results suggest that different proteins are involved in the dephosphorylation of synthase D, phosphorylase alpha and histone in the cell.

Stimulation of liver glycogen particle synthase D phosphatase activity by caffeine, AMP, and glucose 6-phosphate

Abstract Caffeine stimulates synthase and phosphorylase phosphatase activities by independent mechanisms. Both effects of caffeine are concentration-dependent with different apparent A 0.5 for each reaction. Stimulation of the synthase phosphatase reaction was independent of the initial phosphorylase a concentration, was immediate, and did not follow in sequence the depletion of phosphorylase a . Glucose 6-phosphate also was stimulatory to the synthase phosphatase reaction ( A 0.5 = 0.14 mM) with little effect on phosphorylase phosphatase. In combination glucose 6-phosphate and caffeine effects were additive suggesting the existence of separate binding sites. The synthase phosphatase reaction also was stimulated by AMP (binding affinity 2.3 m m ) but with no effect on phosphorylase phosphatase activity. Together caffeine and AMP effects were not additive suggesting that they share a common binding site or closely interrelated sites. The location of the AMP and caffeine site(s) has not yet been determined.

The role of ATP and glucose 6-phosphate in the regulation of glycogen synthetase D phosphatase

Abstract Glycogen synthetase D phosphatase catalyzes the conversion of synthetase D to synthetase I. The phosphatase reaction has been found to be inhibited by ATP, ADP and UDP; however, only ATP inhibited at a physiological concentration. ATP inhibition was enhanced by glycogen. Glucose 6-phosphate (G 6-P) stimulated the phosphatase reaction and at least partially relieved the ATP inhibition. A possible physiological role for ATP, G 6-P and glycogen in the regulation of the synthetase D phosphatase reaction has been proposed.

The regulation of liver glycogen synthetase D phosphatase by ATP and glucose.

Abstract Synthetase D phosphatase activity in a liver glycogen pellet preparation is inhibited by ATP (physiological concentration). The inhibition can be reversed by glucose concentrations within the usual physiological range. Phosphophosphorylase activity decreases concomitantly with increasing synthetase I activity during the phosphatase incubation but the decrease is modest even in the presence of glucose. The glucose reversal of ATP inhibition is not the result of ATPase or glucokinase activities, which would reduce the ATP concentration. ATP, glucose and glucose 6-phosphate concentrations remain stable during the phosphatase assay.

Effect of 24 hours of starvation on plasma glucose and insulin concentrations in subjects with untreated non-insulin-dependent diabetes mellitus.

Adherence to a low-calorie diet often results in a decrease in blood glucose concentration in persons with non-insulin-dependent diabetes mellitus (NIDDM). Whether this is due to the resultant weight loss or to a decrease in caloric intake has been uncertain. We have obtained data previously that indicated a very short-term reduction in caloric intake (5 hours) resulted in a significant decrease in plasma glucose concentration in subjects with NIDDM. The purpose of the present study was to determine if a further decrease in glucose would occur if the fast was extended from 5 to 24 hours. Seven male subjects with untreated NIDDM were studied after an 11-hour overnight fast. For the subsequent 24-hour period, subjects were given only water. Blood was obtained for glucose, insulin, C-peptide, triglycerides, nonesterified fatty acids (NEFA) alpha-amino acid nitrogen, urea nitrogen, and glucagon at hourly intervals for 24 hours beginning at 8 AM. The amount of glycogen degraded was calculated based on the potassium balance. Plasma glucose decreased from 158 mg/dL at 8 AM to a nadir of 104 mg/dL at 7 PM. It then increased by 30 mg/dL. Corresponding changes occurred in insulin and C-peptide. Serum glucagon remained unchanged. Serum alpha-amino acid nitrogen and urea nitrogen decreased. Triglycerides and NEFA increased. The calculated glycogen utilized over this period was approximately 167 g. This would provide approximately 700 kcal energy. The elevated blood glucose concentration in mild to moderately severe untreated NIDDM subjects was normalized following short-term fasting. Plasma insulin concentrations also decreased to within normal limits. These decreases were highly significant. Glycogenolysis is an important source of fuel during this period.

Direct glucose stimulation of glycogen synthase phosphatase activity in a liver glycogen particle preparation

In glycogen particle suspensions prepared from fed rats given either glucagon or glucose in order to increase or decrease the phosphorylase a concentration, respectively, glucose stimulation of synthase phosphatase activity was observed. In preparations from glucagon-treated rats, addition of glucose stimulated synthase and phosphorylase phosphatase simultaneously and not sequentially. Synthase phosphatase stimulation was glucose concentration dependent even when phosphorylase a had been rapidly reduced to a low level. The estimated A0.5 for glucose stimulation of synthase phosphatase activity was 27 mM. An A0.5 for glucose stimulation of phosphorylase phosphatase activity could not be estimated since activity was still increasing with concentrations of glucose as high as 200 mM. In preparations from glucose-treated rats which contain virtually no phosphorylase a, glucose stimulation was still apparent but the A0.5 was increased modestly (36 mM). Stimulation of synthase phosphatase activity was specific for glucose. Several other monosaccharides and the polyhydric alcohol sorbitol were ineffective.

Influence of fructose on the glycogen synthase and phosphorylase systems in rat liver

Fructose and glucose, when administered as a single, large intravenous dose (500 mg/kg) produced opposite effects on key regulatory enzymes of glycogen metabolism in intact normal fed animals. Glucose rapidly stimulated glycogen synthase phosphatase activity and increased the proportion of glycogen synthase in the active (I) form as expected; fructose reduced synthase phosphatase activity and the proportion of synthase in the I form. Glucose also stimulated a reduction in the proportion of phosphorylase in the active (a) form, whereas fructose stimulated an increase in the proportion of phosphorylase in thea form. The effect of fructose was not mediated by an increase in cyclic adenylate (cAMP) concentration nor by a conversion of phosphorylase kinase b to phosphorylase kinase a. As expected, the concentration of ATP decreased significantly. The increase in proportion of phosphorylase in the a form may be due to stimulation of phosphorylase kinase b activity by a decrease in the intracellular ATP:Mg++ ratio or by increase in intracellular Ca++ concentration. The mechanism of the fructose-induced change in synthase phosphatase activity and in synthase I activity is unknown.

Effects of graded intravenous doses of fructose on glycogen synthase in the liver of fasted rats

We have examined in fasted rats the effects of graded doses of intravenous fructose (50 to 500 mg/kg) in order to determine potential mechanisms by which different concentrations of fructose reaching the liver may modify the activity of glycogen synthase (and phosphorylase). With increasing fructose doses the % synthase I increased threefold to a maximum at a dose of 125 mg/kg and then decreased progressively after higher fructose doses were given. The % phosphorylase a decreased by 30% to a minimum at a dose of 125 mg/kg but increased with higher doses to 370% of the control values. Both the % synthase I and the % phosphorylase a were elevated above the control values at fructose doses of 175 to 225 mg/kg. The increase in % synthase I after low doses of fructose occurred with a significant increase in glucose-6-P but no significant change in hepatic fructose, glucose, UDPglucose, ATP/Mg++, Pi, cAMP, plasma insulin, or glucagon concentrations. The reciprocal decrease in % synthase I and increase in % phosphorylase a occurred despite increases in glucose and glucose-6-P, at fructose doses resulting in no change in ATP/Mg++, Pi or cAMP, and only a small increase (0.39 mmol/L) in the fructose-1-P concentration. We propose that activation of synthase phosphatase by a rise in the glucose-6-P concentration is responsible for the increase in % synthase I after low doses of fructose. The mechanism by which higher fructose doses overcome the expected activation of synthase phosphatase by glucose and glucose-6-P and a decreased ATP/Mg++ ratio is uncertain.(ABSTRACT TRUNCATED AT 250 WORDS)