Effect of orally administered phenylalanine with and without glucose on insulin, glucagon and glucose concentrations.

OBJECTIVE: As part of our studies of the metabolic effects of ingested proteins, we are currently investigating the effects of ingestion of individual amino acids. The objective of the present study was to determine whether ingested phenylalanine stimulates insulin and/or glucagon secretion, and if phenylalanine ingested with glucose modifies the insulin, glucagon or glucose response to the ingested glucose. DESIGN: Six healthy subjects were tested on 4 separate occasions. Plasma phenylalanine, glucose, insulin, glucagon, and total alpha amino nitrogen (AAN) (i.e., total amino acids) concentrations were measured at various times during a 2.5 h period after ingestion of 1 mmol phenylalanine/kg lean body mass, 25 g glucose, 1 mmol phenylalanine/kg lean body mass+25 g glucose, or water only, given in random order. RESULTS: Following phenylalanine ingestion, the circulating phenylalanine concentration increased approximately 14 fold and remained elevated for the duration of the experiment. Glucagon and AAN increased, insulin increased modestly, and glucose was unchanged when compared to water ingestion. When glucose was ingested with phenylalanine, the circulating phenylalanine, glucagon, AAN, and insulin area responses were approximately the sum of the responses to phenylalanine alone and glucose alone. However, the plasma glucose area response was decreased 66% when phenylalanine was co-ingested with glucose. CONCLUSION: In summary, phenylalanine in an amount moderately greater than that in a large protein meal stimulates an increase in insulin and glucagon concentration. It markedly attenuates the glucose-induced rise in plasma glucose when ingested with glucose.

Effect of protein ingestion on the glucose appearance rate in people with type 2 diabetes.

Amino acids derived from ingested protein are potential substrates for gluconeogenesis. However, several laboratories have reported that protein ingestion does not result in an increase in the circulating glucose concentration in people with or without type 2 diabetes. The reason for this has remained unclear. In people without diabetes it seems to be due to less glucose being produced and entering the circulation than the calculated theoretical amount. Therefore, we were interested in determining whether this also was the case in people with type 2 diabetes. Ten male subjects with untreated type 2 diabetes were given, in random sequence, 50 g protein in the form of very lean beef or only water at 0800 h and studied over the subsequent 8 h. Protein ingestion resulted in an increase in circulating insulin, C-peptide, glucagon, alpha amino and urea nitrogen, and triglycerides; a decrease in nonesterified fatty acids; and a modest increase in respiratory quotient. The total amount of protein deaminated and the amino groups incorporated into urea was calculated to be approximately 20-23 g. The net amount of glucose estimated to be produced, based on the quantity of amino acids deaminated, was approximately 11-13 g. However, the amount of glucose appearing in the circulation was only approximately 2 g. The peripheral plasma glucose concentration decreased by approximately 1 mM after ingestion of either protein or water, confirming that ingested protein does not result in a net increase in glucose concentration, and results in only a modest increase in the rate of glucose disappearance.

Glucose appearance rate after the ingestion of galactose.

Galactose is one of the monosaccharides of importance in human nutrition. It is converted to glucose-1-phosphate in the liver and subsequently stored as glycogen, or is converted to glucose and released into the circulation. The increase in plasma glucose is known to be modest following galactose ingestion. Whether this is due to a small increase in hepatic glucose output, or to a relatively large increase in hepatic glucose output but a concomitant increase in glucose disposal, is not known in humans. Therefore, the rates of glucose appearance (Ra) and disappearance (Rd) were determined over an 8-hour period in normal subjects using an isotope dilution technique. The subjects ingested 50 g galactose or water alone in random order at 8 AM on separate occasions. Plasma glucose, glucagon, lactate, urea nitrogen, total amino acids, and uric acid and serum insulin and triglycerides also were determined. Following galactose ingestion, there was a modest transient increase in peripheral glucose and insulin concentrations. This was associated with a modest increase in the glucose Ra. The calculated amount of glucose appearing in the circulation as a result of galactose ingestion was 9.8 g, while the amount of glucose disappearing over the 8 hours was 9.9 g. Thus, following ingestion of 50 g galactose by overnight-fasted men, approximately 20% appears as additional glucose in the circulation. Data obtained in animals suggest that a large amount of the galactose is stored as glucose in glycogen. Nevertheless, the conversion of galactose to glucose in the liver may have been greater than suggested by the increase in glucose appearance in the circulation due to substitution for other gluconeogenic substrates.

Uric acid inhibits liver phosphorylase a activity under simulated in vivo conditions.

We have reported that glycogen synthesis and degradation can occur in vivo without a significant change in the amount of phosphorylase a present. These data suggest the presence of a regulatable mechanism for inhibiting phosphorylase a activity in vivo. Several effectors have been described. AMP stimulates, whereas ADP, ATP, and glucose inhibit activity. Of these effectors, only the glucose concentration changes under normal conditions; thus it could regulate phosphorylase a activity in vivo. We previously have reported that, when all of these effectors were present at physiological concentrations, the net effect was no change in phosphorylase a activity. Addition of caffeine, an independent inhibitor of activity, to the above effectors not only resulted in inhibition but also restored a glucose concentration-dependent inhibition. Because uric acid is an endogenous xanthine derivative, we decided to determine whether it had an effect on phosphorylase a activity. Independently, uric acid did not affect activity; however, when added at a presumed physiological concentration in combination with AMP, ADP, ATP, and glucose, it inhibited activity. A modest but not statistically significant glucose concentration-dependent inhibition was also present. Thus uric acid may play an important role in regulating phosphorylase a activity in vivo.

Peripheral glucose appearance rate following fructose ingestion in normal subjects.

Ingested fructose is rapidly utilized by the liver and is either stored as glycogen, converted to glucose, or oxidized to CO2 for energy. The glycemic response to fructose is known to be modest. However, the relative importance of these pathways in humans is unclear. In the present study, a tritiated glucose tracer dilution technique was used to determine the effect of fructose ingestion on the glucose appearance rate (Ra) in the peripheral circulation over an 8-hour period beginning at 8:00 AM. Six normal healthy males ingested 50 g fructose with 500 mL water. On a separate occasion, the same subjects received 500 mL water without fructose as a control. Serum insulin, triglycerides, plasma glucagon, glucose, lactate, alanine, urea nitrogen, and total amino acids also were determined. The plasma glucose concentration was not significantly different following ingestion of fructose or water, other than a transient increase beginning at 8:30 AM of 0.8 mmol/L in response to ingested fructose. Glucose appearing in the peripheral circulation as a result of ingestion of 50 g fructose was calculated to be 9.8 +/- 2.4 g. Following the ingestion of fructose, there was a small increase in glucagon but a 2-fold increase in insulin concentration. There was a large transient increase in lactate and alanine concentrations. The total amino acid concentration remained unchanged, as did the urea production rate. In summary, in men fasted overnight, ingestion of 50 g fructose resulted in a modest increase in the circulating glucose concentration. However, it is likely that a larger proportion of the ingested fructose was converted to glucose in the liver and stored as glycogen and that fructose substituted, at least in part, for lactate and alanine as a gluconeogenic substrate. The increase in glucose production occurred even in the presence of an increase in the insulin concentration and an unchanged glucagon concentration. The metabolic fate of the remaining fructose is yet to be determined.

Acute metabolic response to high-carbohydrate, high-starch meals compared with moderate-carbohydrate, low-starch meals in subjects with type 2 diabetes.

OBJECTIVE: The monosaccharides resulting from the digestion of ingested carbohydrates are glucose, fructose, and galactose. Of these three monosaccharides, only ingested glucose resulted in a large increase in the plasma glucose concentration. Fructose (Metabolism 41:510-517, 1992) and galactose (Metabolism 42:1560-1567, 1993) had only a minor effect. Therefore, we were interested in determining whether we could design a mixed meal, using foods of known monosaccharide, disaccharide, and starch composition, the ingestion of which would result in only a small rise in plasma glucose concentration. RESEARCH DESIGN AND METHODS: The experimental meal was composed of very little readily digestible starch but rather large amounts of fruits and vegetables. It contained 43% carbohydrate, 22% protein, and 34% fat. The results were compared with a second type of meal that contained 55% carbohydrate, 15% protein, and 30% fat, with an emphasis on complex carbohydrates (starch). It also was compared with a third meal that contained 40% carbohydrate, 20% protein, and 40% fat, typical of that consumed by the average American. The test meals were ingested in random order by people with type 2 diabetes who were not treated with oral hypoglycemic agents or insulin. Each subject ingested each type of meal. The same identical meal was ingested at 0800, 1200, and 1700. RESULTS: The integrated 24-h plasma glucose area response was statistically significantly smaller (P < 0.05) after ingestion of the low-starch meals compared with the high-starch, high-carbohydrate meals or the typical American meals. The 24-h integrated serum insulin area response also was statistically significantly less (P < 0.05) after ingestion of the low-starch meals compared with the high-starch meals or the typical American meals. The serum triglyceride area response was similar after ingestion of all three test diets. CONCLUSIONS: A diet in which fruits, nonstarch vegetables, and dairy products are emphasized may be useful for people with type 2 diabetes.

Liver glycogenin activity in diabetic and adrenalectomized rats.

The glycogen concentration in liver is altered in various pathophysiologic states. In fasted rats, it is higher in diabetic, and lower in adrenalectomized rats compared to control animals. In fed rats, it is lower in diabetic, and little changed in adrenalectomized animals compared to controls. We were interested in determining whether the activity of glycogenin, a self-glycosylating protein that initiates the synthesis of new glycogen molecules, could explain these differences in liver glycogen concentration. Glycogenin activity was measured by the incorporation of 14C-glucose from UDP-U-14C-glucose into an acid-precipitable product before and after amylase treatment of liver extracts. The glycogenin activity was similar in normal, diabetic and adrenalectomized fasted animals, regardless of the hepatic glycogen concentration. In fasted rats, glycogenin was present predominantly as the free-form of the enzyme, i.e., not attached to an amylase-digestible glycan, presumably glycogen. In contrast, in fed rats, the majority, if not all of the glycogenin was incorporated into a glycogen-like (proteoglycan) molecule. Proteoglycan synthase activity, previously identified in normal fed rats, also was present in diabetic and adrenalectomized fed rats, and the activity was similar. Thus, the altered ability to store hepatic glycogen in diabetic fed and fasted and adrenalectomized fasted rats cannot be explained by decreases in glycogenin or proteoglycan synthase activities, at least as measured using the present assays.

State-space models of insulin and glucose responses to diets of varying nutrient content in men and women.

Discrete-time state-space models were developed to describe contemporaneous responses of plasma insulin and glucose of normal human subjects. Male and female subjects ingested three consecutive identical meals from isocaloric diets classified as high-carbohydrate, high-fat, high-protein, or standard. Distinctly different glucose and insulin responses were measured in men and women. A seven-state system of linear equations, three in insulin and four in glucose, was identified and estimated to describe responses in men. A six-state system, three in insulin and three in glucose, describes responses in women. Model simulations at 15-min intervals closely match measured concentrations over a 12-h period. Effects of diet content and meal timing on insulin and glucose concentrations were quantified. Dynamic insulin and glucose responses to isocaloric meals of pure carbohydrate, fat, and protein diets were projected on the basis of models developed from mixed diets. The symmetry of the projections indicates that positive excursions in glucose concentrations associated with carbohydrate intake may be matched with negative excursions associated with fat and protein intake to help manage postmeal glucose excursions.

Mutations in the liver glycogen synthase gene in children with hypoglycemia due to glycogen storage disease type 0.

Glycogen storage disease type 0 (GSD-0) is a rare form of fasting hypoglycemia presenting in infancy or early childhood and accompanied by high blood ketones and low alanine and lactate concentrations. Although feeding relieves symptoms, it often results in postprandial hyperglycemia and hyperlactatemia. The glycogen synthase (GS) activity has been low or immeasurable in liver biopsies, whereas the liver glycogen content has been only moderately decreased. To investigate whether mutations in the liver GS gene (GYS2) on chromosome 12p12.2 were involved in GSD-0, we determined the exon-intron structure of the GYS2 gene and examined nine affected children from five families for linkage of GSD-0 to the GYS2 gene. Mutation screening of the 16 GYS2 exons was done by single-strand conformational polymorphism (SSCP) and direct sequencing. Liver GS deficiency was diagnosed from liver biopsies (GS activity and glycogen content). GS activity in the liver of the affected children was extremely low or nil, resulting in subnormal glycogen content. After suggestive linkage to the GYS2 gene had been established (LOD score = 2.9; P < 0.01), mutation screening revealed several different mutations in these families, including a premature stop codon in exon 5 (Arg246X), a 5'-donor splice site mutation in intron 6 (G+1T--> CT), and missense mutations Asn39Ser, Ala339Pro, His446Asp, Pro479Gln, Ser483Pro, and Met491Arg. Seven of the affected children carried mutations on both alleles. The mutations could not be found in 200 healthy persons. Expression of the mutated enzymes in COS7 cells indicated severely impaired GS activity. In conclusion, the results demonstrate that GSD-0 is caused by different mutations in the GYS2 gene.

Stimulation of insulin secretion by fructose ingested with protein in people with untreated type 2 diabetes.

OBJECTIVE: Ingested protein provides substrate for gluconeogenesis and strongly stimulates insulin and glucagon secretion, but it has little effect on the glucose concentration in people with type 2 diabetes. Ingested fructose also is a substrate for gluconeogenesis, modestly stimulates insulin and glucagon secretion, and has little effect on the plasma glucose. Therefore we were interested in determining if ingestion of fructose along with protein would result in an additive, greater than additive, or less than additive effect on circulating insulin, glucagon, and glucose concentrations. RESEARCH DESIGN AND METHODS: Seven male subjects with untreated type 2 diabetes were fasted overnight and then were given either 25 g fructose, 25 g protein, 25 g fructose plus 25 g protein, or water only at 0800. Subjects also ingested 50 g glucose on two separate occasions. Plasma glucose, insulin, C-peptide, glucagon, alpha-amino nitrogen, urea nitrogen, nonesterified fatty acids, and triglyceride concentrations were determined over the subsequent 5 h. RESULTS: The glucose concentration was only modestly increased and the area responses were similar when protein, fructose, or the combination was ingested. Thus, the glucose response to the combination was less than additive. The insulin area response to protein was 2.5-fold greater than to fructose, and the response to the two nutrients was additive and quantitatively similar to the response to 50 g glucose. The glucagon area response was less than additive, i.e., there was an interaction between the protein and fructose that resulted in a smaller than expected response. CONCLUSIONS: When protein and fructose were ingested together, the insulin response was similar to that following ingestion of 50 g glucose. It also was as expected based on the response to the individual nutrients. In contrast, the glucose and glucagon responses were significantly less than expected. These data may be useful in dietary planning for subjects with type 2 diabetes.

Effect of feeding, fasting, and diabetes on liver glycogen synthase activity, protein, and mRNA in rats.

Hepatic glycogen synthase activity is increased in diabetic animals. However, the relationship between enzymic activity, enzyme protein mass, and mRNA abundance has not been well characterized. In the present study, these relationships were determined in 3- and 8-day diabetic, fed and fasted rats. The results were compared to data obtained in normal fed and fasted animals. In normal rats, total synthase specific activity and protein mass were similar in the fed and fasted state. However, in fed animals, the synthase mRNA abundance was increased 1.7-fold. In 3-day diabetic rats, total synthase specific activity was increased approximately 29% compared to normal controls. It was unaffected by feeding and fasting and was associated with an approximate 15% increase in enzyme mass. Synthase mRNA was increased 1.8 and 2.6-fold in fasted and fed animals, respectively. In 8-day diabetic rats, total synthase specific activity was increased more than 2-fold compared to controls. However, the enzyme protein mass was decreased by approximately 20%. The mRNA abundance in 8-day diabetic fasted rats was only 30% of controls, while in fed rats it was increased by 40%. These data indicate that feeding and fasting have a major effect on synthase mRNA abundance which is independent of synthase activity, or protein mass, or both, in normal and diabetic animals. Total synthase specific activity increased with duration of diabetes. This was associated with only a modest change in protein mass. Thus, diabetes induces an increase in synthase catalytic efficacy. The specific activity of phosphorylase is decreased in diabetic rats.

Allosteric regulation of liver phosphorylase a: revisited under approximated physiological conditions.

Phosphorylase removes glucosyl units from the terminal branches of glycogen through phosphorolysis, forming glucose-1-P. It is present in two interconvertible forms, phosphorylase a and b. The a form is the active form and is rate limiting in glycogen degradation. The activities of phosphorylase a and of total phosphorylase as conventionally measured exceed the activities of glycogen synthase R (active form) and of total synthase by approximately 10- and 20-fold. Thus, unless phosphorylase a is inhibited or compartmentalized or its substrates are exceedingly low in vivo, net glycogen synthesis could not occur. In addition, following an administered dose of glucose, phosphorylase a activity changes little when glycogen is being synthesized, is stable, or is being degraded, suggesting an important role for allosteric effectors in regulation. Therefore, we have determined the effect of potential modifiers of enzyme activity at estimated intracellular concentrations. Purified liver phosphorylase a was used. Activity was measured in the direction of glycogenolysis, at 37 degrees C, pH 7.0, and under initial rate conditions. Both a Km and a near-saturating concentration of inorganic phosphate (substrate) were used in the assays. A physiological concentration of AMP was saturating. It decreased the Km for Pi by approximately 50% and stimulated activity. ADP, ATP, and glucose inhibited activity. Fructose-1-P inhibited activity only at a high and nonphysiological concentration. Glucose-6-P and UDP-glucose were not significant inhibitors. Inhibition of activity by ADP was little affected by the addition of AMP. However, AMP partially abolished the inhibitory effect of ATP and completely abolished the inhibitory effect of glucose. When AMP, ADP, ATP, glucose-6-P, UDP-glucose, glucose, and fructose-1-P were added together, the net effect was no change in phosphorylase a activity compared to the activity without any effectors. In addition, changes in glucose concentration did not affect activity. K glutamine modestly stimulated activity. Numerous other metabolites were tested and were without effect. The present data indicate that the known endogenous allosteric effectors cannot explain the smaller than expected in vivo phosphorylase a activity or the regulation of phosphorylase a activity.

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.

Effects of glucagon with or without insulin administration on liver glycogen metabolism.

Rats fed ad libitum were given insulin alone (4 U/kg), glucagon alone (25 micrograms/kg), or insulin and glucagon sequentially. Phosphorylase a and synthase R activities, hepatic glycogen, uridine diphosphoglucose, inorganic phosphate (Pi), and plasma glucose, lactate, glucagon, and insulin concentrations were determined over the subsequent 40 min. In separate animals, muscle extraction of 2-deoxy-D-[3H]glucose also was determined. After glucagon administration, glycogen phosphorylase a and plasma glucose were increased within 5 min. However, the glycogen concentration did not decrease for 20 min. Glucagon administration to rats pretreated with insulin stimulated a similar increase in phosphorylase a activity. Again, glycogen was not degraded for 20 min. After insulin only, glycogen concentration remained unchanged. Plasma glucose decreased as expected. In each group, muscle extraction of 2-deoxy-D-[3H]glucose increased compared with the controls (P < 0.05). In summary, glucagon and/or insulin administration did not stimulate significant glycogen degradation for 20 min, even though phosphorylase was activated. The mechanism remains to be determined.

Physiological doses of oral casein affect hepatic glycogen metabolism in normal food-deprived rats.

In a previous study, administration of casein hydrolysate to food-deprived rats at a dose of 4 g/kg body wt resulted in an increase in portal plasma glucagon concentration. This was associated with an activation of phosphorylase a and a decrease in hepatic glycogen concentration. The present study was undertaken to determine whether similar results would be obtained with smaller doses. Doses of 1 and 2 g/kg body wt were administered to food-deprived rats. At a dose of 2 g/kg, portal plasma glucagon concentration was significantly elevated. This was associated with a slight increase in phosphorylase a activity (P < 0.05) and a 50% decrease in hepatic glycogen concentration (P < 0.01). At a dose of 1 g casein hydrolysate/kg body wt, changes in portal plasma glucagon concentration, phosphorylase a activity and hepatic glycogen concentration generally were not observed. Hepatic glucose, uridine diphosphoglucose, ATP and glucose-6-phosphate concentrations were unaffected by either dose of casein hydrolysate. The data indicate a dose-response relationship between casein hydrolysate administration and effects on glycogen metabolism in the liver. Protein-induced glycogenolysis is likely to occur when rats ingest a moderate amount of a pure protein meal.

Effect of added fat on the plasma glucose and insulin response to ingested potato given in various combinations as two meals in normal individuals.

OBJECTIVE: In normal subjects, ingestion of fat with potato in a morning meal resulted in a decrease in the glucose response. Therefore, we wished to determine whether a fat-induced decrease in blood glucose also would be observed after a second identical meal. In addition, we were interested in determining if fat ingestion with a morning meal would have an effect on the blood glucose and insulin responses to a second meal not containing fat. RESEARCH DESIGN AND METHODS: Nine healthy male subjects ingested two meals consisting of an amount of potato containing 50 g carbohydrate, either alone or with 50 g fat as butter. The meals were served in four combinations as follows: 1) potato for the first meal, potato for the second meal; 2) potato for the first meal, potato with fat for the second meal; 3) potato with fat for the first meal, potato for the second meal; and 4) potato with fat for the first meal, potato with fat for the second meal. Meals were ingested at 8:00 A.M. and noon. Plasma glucose and C-peptide, serum insulin, triglyceride, and free fatty acid (FFA) concentrations were determined over an 8-h period. The integrated area responses to the meals were quantified over the subsequent 4-h period using the fasting value or the noon value as baseline for the first and second meals, respectively. RESULTS: When the first meal contained potato only, the glucose area response to the second meal was significantly less when the second meal contained fat. However, fat ingestion had no effect on the glucose area response to the second meal when fat was present in the first meal. The insulin area responses to the first and second meals were similar after ingestion of potato or potato with fat. However, the insulin response to the second meal always was less than that to the first meal. The C-peptide area responses after ingestion of the second meal also were all higher than those after the first meal. The triglyceride area responses were slightly negative after ingestion of potato alone in the first meal. When fat was ingested, they were positive. When the first meal contained fat but the second meal did not, there was a rise in triglyceride concentration after the second meal as well as after the first meal. That is, a rise occurred without ingestion of fat with the second meal. If fat was present in the second meal the rise was even greater. The FFA area responses were similar to the triglyceride area responses. CONCLUSIONS: When fat was ingested with carbohydrate in either the first or second meal, the glucose area response was decreased. However, when both meals contained fat, a decrease in the glucose area response did not occur with the second meal. The glucose area responses all were greater after the second meal compared with those after the first meal, i.e., the opposite of a Staub-Traugott effect was observed. The insulin area responses to the first and second meals were similar whether fat was ingested or not.

Liver glycogen synthase, phosphorylase, and the glycogen concentration in rats given a glucose load orally: a 24-hour study.

Fasted rats were given 4 g/kg glucose orally. Synthase R (active forms), total synthase, and phosphorylase alpha activities, and hepatic glycogen, glucose 6-phosphate (glucose-6P), uridine diphosphoglucose (UDP-glucose), glucose, and plasma glucose concentrations were determined over the subsequent 24 h. The resulting glycogen concentration changes could be divided into three distinct phases. A glycogen synthetic phase (between 0 and 4 h), a stability phase (between 4 and 12 h), and a degradation phase (between 12 and 24 h). Synthase R activity increased rapidly and reached a maximum at 20 min. With the onset of glycogen synthesis it gradually decreased below the control values, reaching a nadir by 4 h. During the glycogen stability phase it gradually increased again up to the control value. It then remained stable during the subsequent glycogen degradation phase. Phosphorylase a activity did not change throughout the entire 24-h period. Glucose-6-P concentration increased almost twofold at 20 min. It then decreased but was above the control values at the 24th h. The plasma and hepatic glucose concentrations increased as expected after the glucose load. They then decreased but remained above the control value at all subsequent time points. In summary, the synthase R, phosphorylase a activities, or changes in the known allosteric modifiers of these enzymes could not explain the changes in glycogen concentration. The reasons for these discrepancies remain to be determined.

Incorporation of glycogenin into a hepatic proteoglycogen after oral glucose administration.

Glycogenin is a 37-kDa protein upon which new glycogen molecules are considered to be constructed. Therefore, we were interested in determining its role in liver glycogen synthesis following glucose administration. Twenty-four-hour fasted rats were given 4 g/kg glucose orally. Glycogenin and synthase R activities and glycogen were determined over the subsequent 24 h. In fasted rats given just water, glycogenin activity was present and did not change over the subsequent 24 h. Following glucose, glycogenin activity also was not different for 1 h, i.e. the glycogenin was not incorporated into glycogen even though the glycogen concentration had increased. Subsequently, the glycogenin activity became unmeasurable. Presumably, the glycogenin was incorporated into a proteoglycan product since after amylase treatment, glycogenin activity was again present and was quantitatively unchanged. Free glycogenin activity remained unmeasurable until after 12 h. At this time, glycogen began to decrease, and, by 15 h, free glycogenin activity again appeared. The results indicate that in fasted rats, essentially all of the glycogenin was free. Following administration of oral glucose, glycogenin was incorporated into a proteoglycan product but only 60 min after glycogen synthesis had begun. Free glycogenin did not reappear until the 15th h after glucose was given and after the glycogen concentration had decreased by approximately 60%.

Primary structure of human liver glycogen synthase deduced by cDNA cloning.

The cDNA for human liver glycogen synthase was isolated by screening a human liver cDNA library constructed in lambda gt11. The full cDNA was 2912 bp in length. It coded for a protein of 703 amino acid residues with a molecular mass of 80.9 kDa. The number of amino acids was identical to and the deduced amino acid sequence homology was 92% that of the rat liver enzyme. The human and rat liver glycogen synthases are truncated by 34 amino acids compared to the human muscle enzyme, and by 32 amino acids compared to the rabbit muscle enzyme. The amino acid similarity between human liver and human muscle glycogen synthase was only 69%. It was least similar in the N and C terminal regions of the molecule. Two highly conserved regions are present in all published amino acid sequences for glycogen synthase, including those of the two yeast enzymes. These regions include the amino acid sequences from 201 to 400 and 501 to 600. This high conservation suggests that the catalytic site and the glucose-6-P and nucleotide allosteric sites are included in these regions.