The effect of streptozotocin-induced diabetes on glycogen metabolism in rat kidney and its relationship to the liver system.

The effects in kidney of streptozotocin-induced diabetes and of insulin supplementation to diabetic animals on glycogen-metabolizing enzymes were determined. Kidney glycogen levels were approximately 30-fold higher in diabetic animals than in control or insulintreated diabetic animals. The activities of glycogenolytic enzymes i.e., phosphorylase (both a and b), phosphorylase kinase, and protein kinase were not significantly altered in the diabetic animals. Glycogen synthase (I form) activity decreased in the diabetic animals whereas total glycogen synthase (I + D) activity significantly increased in these animals. The activities were restored to control values after insulin therapy. Diabetic animals also showed a 3-fold increase in glucose 6-phosphate levels. These data suggest that higher accumulation of glycogen in kidneys of diabetic animals is due to increased amounts of total glycogen synthase and its activator glucose 6-phosphate.     

Fetal lung development in streptozotocin-induced experimental diabetes: cytidylyl transferase activity, disaturated phosphatidyl choline and glycogen levels

The influence of streptozotocin-induced maternal diabetes on choline phosphate cytidylyltransferase activity (EC.2.7.7.15) glycogen content and disaturated phosphatidyl choline in fetal lung was studied between 19 and 21 days of gestation. In this experimental model, induction of maternal diabetes two days after mating, resulted in fetal hyperglycemia and hyperinsulinemia; the fetuses were neither macrosomic nor showed any evidence of fetal growth retardation. The glycogen content of lungs on days 19 and 20, but not on day 21 of gestation was significantly higher in fetuses of diabetic rats than in controls. The pulmonary cytosol cytidylyltransferase activity was similar in the two groups of fetuses on days 19 and 20. On day 21 of gestation the enzyme activity was significantly lower in fetuses of diabetic rats than in those of controls. On day 21 of gestation and in newborns of diabetic mothers, although there was no difference in the total pulmonary phospholipids, the levels of disaturated phosphatidyl cholines were significantly lower than in controls.     

Regulation of rabbit fetal glycogen: effect of in utero fetal decapitation on the metabolism of glycogen in fetal heart, lung, and liver

To understand the control mechanisms involved in the regulation of fetal glycogen, we have studied the effect of in utero fetal decapitations on glycogen metabolism in rabbit fetal heart, lung, and liver. In utero fetal decapitations were performed between days 18 and 21 of gestation. Two to four fetuses on one side of the horn were decapitated. Fetuses were delivered between days 23 and 26 or between days 28 and 30 of gestation. Fetal heart, lungs, and liver were analyzed for DNA, protein, glycogen, glycogen synthase (I and D forms), glycogen phosphorylase (a and b forms), phosphofructokinase, pyruvate kinase, and lactic dehydrogenase. In fetal heart and lung, no difference was observed in any of the above measurements in the intact and decapitated fetuses. In contrast, fetal liver does not appear to develop the glycogen system as indicated by the very low levels of glycogen (0.02 mg/mg DNA) in decapitated fetuses as compared with intact fetuses (0.4 mg/mg DNA). Similarly the levels of glycogen synthase and phosphorylase were two to three times lower in livers from decapitated fetuses as compared with the livers from intact fetuses. The three enzymes phosphofructokinase, pyruvate kinase, and lactic dehydrogenase were not affected by fetal decapitation in all three tissues. These results indicate that the fetal hypothalamic-pituitary-adrenal (thyroid) axis is not required at least after day 18 of gestation for the normal accumulation and subsequent utilization of glycogen in fetal heart and lungs, while it is an absolute requirement for the development of the fetal liver glycogen system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Control of glycogen synthase activity in developing rat liver.

Fasting newborn and growing young rats, though capable of synthesizing liver glycogen when fed, are, unlike adult fasted animals, insensitive to glucocorticoid stimulation of the rate of glucose and lactate incorporation into glycogen. Hormone resistance parallels a decreased liver capability for the synthase b to a conversion reaction up to 2 days after birth, after which the b to a transformation becomes adult type in nature. A comparison of the level of glucose 6-phosphate in liver to the effect of the activator on the synthase activity from newborn rat shows that the enzyme has a greater affinity toward the activator than comparable enzyme from the adult, suggesting the presence of an intermediate metabolite-regulated form of synthase in neonatal liver.     

Carbohydrate metabolism in the fetal pig during late gestation.

In acute experiments on pregnant sows under sodium pentobarbitone anaesthesia, acid base balance, oxygenation and plasma metabolite concentrations were well maintained in the dam and all fetuses which remained undisturbed in utero, irrespective of the duration of the experiment. Fetal liver glycogen concentrations were also unaffected by the time of removal of the fetus. By contrast, intravascular catheterization and withdrawal of blood led to fetal hyperglycaemia and depletion of hepatic glycogen although blood gas and pH values were not changed by these procedures. In the 1 1/2--2 h sampling period following catheterization the normal positive umbilical venous-arterial differences in plasma glucose and lactate generally became reversed. These changes were prevented by the administration of hexamethonium (10--15 mg . kg-1 i.v.) but the drug did not block the fall in hepatic glycogen in catheterized fetuses. Both adrenaline and noradrenaline, which were each infused intravenously at 2.7--3.9 or 0.6--0.9 microgram . kg-1 . min-1, resulted in fetal hyperglycaemia and lacticacidemia together with a fall in arterial blood pH; hepatic glycogen concentrations in these fetuses were also reduced. The apparent sensitivity of the glycogenolytic mechanism to surgical trauma and haemorrhage in the fetal piglet is discussed in relation to findings in other species.     

Effects of glucose infusion on surfactant and glycogen regulation in fetal lamb lung

To investigate the increased incidence of respiratory distress syndrome (RDS) that occurs in infants of diabetic mothers (IDM) with poor maternal glucose homeostasis, we infused glucose intravenously at a rate of 14 +/- 2 (SD) mg.kg-1.min-1 into eight twin and four singleton chronically catheterized fetal lambs from 112 days (0.77) gestation onward. Twelve catheterized and seven uncatheterized fetuses served as controls, including the eight twins of the glucose-treated fetuses. Glucose infusion resulted in a twofold elevation in fetal serum glucose levels and a 2.2-fold elevation in fetal serum insulin levels. Before 113 days (0.9) gestation, pulmonary disaturated phosphatidylcholine (DSPC) content was 1.5-fold higher in the glucose-infused fetuses than in the controls. However, after 0.9 gestation, pulmonary DSPC content increased 2.2-fold in the controls but did not increase significantly in the glucose-infused fetuses. In addition, the DSPC content of lung lavage was 5.0-fold higher in the controls and lung stability to air inflation was 2.0-fold greater and to deflation was 2.2-fold greater than in the glucose-infused fetuses. Pulmonary adenosine 3',5'-cyclic monophosphate-dependent protein kinase activity was also 1.5-fold higher, and pulmonary protein kinase C activity was 1.3-fold higher in the controls than in the glucose-infused fetuses. In contrast, glucose infusion was associated with a 1.8-fold increase in pulmonary glycogen content and with increased activities of glycogen phosphorylase kinase and glycogen phosphorylase. We conclude that the effects of chronic glucose infusion on fetal lamb lung DSPC and lung stability are compatible with a predisposition of the fetus to develop RDS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Control mechanisms in the acceleration of hepatic glycogen degradation during hypoxia

Hepatic glycogen metabolism in aerobic and hypoxic conditions has been assessed with respect to glycogenolysis, phosphorylase a activity and nucleotide content. Insulin did not inhibit glycogen breakdown nor stimulate lipogenesis in the aerobic perfused liver.Partial ischaemia induced glycogen breakdown, release of glucose and changes in nucleotide content in the perfused liver. Phosphorylase a content increased within 2 min in response to total ischaemia, in vivo and in the perfused liver. This change was paralleled by an increase in hepatic AMP. Glycogen synthase a activity decreased, as did the hepatic content of both cyclic AMP and cyclic GMP.     

Development of glycogen and phospholipid metabolism in fetal and newborn rat lung.

Glucose, a major metabolic substrate for the mammalian fetus, probably makes significant contributions to surface active phospholipid synthesis in adult lung. We examined the developmental patterns of glycogen content, glycogen synthase activity, glycogen phosphorylase activity and glucose oxidation in fetal and newborn rat lung. These patterns were correlated with the development of phosphatidylcholine synthesis, content and the activities of enzymes involved in phosphatidylcholine synthesis. Fetal lung glycogen concentration increased until day 20 of gestation (term is 22 days) after which it declined to low levels. Activity of both glycogen synthase I and total glycogen synthase (I + D) in fetal lung increased late in gestation. Increased lung glycogen concentration preceded changes in enzyme activity. Glycogen phosphorylase a and total glycogen phosphorylase (a + b) activity in fetal lung increased during the period of prenatal glycogen depletion. The activity of the pentose phosphate pathway, as measured by the ratio of CO2 derived from oxidation of C1 and C6 of glucose, declined after birth. Fetal lung total phospholipid, phosphatidycholine and disaturated phosphatidylcholine content increased by 60, 90 and 180%, respectively, between day 19 of gestation and the first postnatal day. Incorporation of choline into phosphatidylcholine and disaturated phosphatidylcholine increased 10-fold during this time. No changes in phosphatidylcholine enzyme activities were noted during gestation, but both choline phosphate cytidylyltransferase and phosphatidate phosphatase activity increased after birth. The possible contributions of carbohydrate derived from fetal lung glycogen to phospholipid synthesis are discussed.     

Some effects of glucose concentration and anoxia on glycolysis and metabolite concentrations in the perfused liver of fetal guinea pig.

Effects of glucose concentration and anoxia upon the metabolite concentrations and rates of glycolysis and respiration have been investigated in the perfused liver of the fetal guinea pig. In most cases the metabolite concentrations in the perfused liver were similar to those observed in vivo. Between 50 days and term there was a fall in the respiratory rate and in the concentration of ATP and fructose 1,6-diphosphate and an increase in the concentration of glutamate, glycogen and glucose. Reducing the medium glucose concentration from 10 mM to 1 mM or 0.1 mM depressed lactate production and the concentration of most of the phosphorylated intermediates (except 6-phosphogluconate) in the liver of the 50-day fetus. This indicates a fall in glycolytic rate which is not in accord with the known kinetic properties of hexokinase in the fetal liver. Anoxia increased lactate production by, and the concentrations of, the hexose phosphates ADP and AMP in the 50-day to term fetal liver, while the concentration of ribulose 5-phosphate, ATP and some triose phosphates fell. These results are consistent with an activation of glycolysis, particularly at phosphofructokinase and of a reduction in pentose phosphate pathway activity, particularly at 6-phosphogluconate dehydrogenase. The calculated cytosolic NAD+/NADH ratio for the perfused liver was similar to that measured in vivo and evidence is presented to suggest that the dihydroxyacetone phosphate/glycerol 3-phosphate ratio gives a better indication of cytosolic redox than the lactate/pyruvate ratio. The present observations indicate that phosphofructokinase hexokinase and possibly pyruvate kinase control the glycolytic rate and that glyceraldehyde-3-phosphate dehydrogenase is at equilibrium in the perfused liver of the fetal guinea pig.     

Effects of diabetes and glycogen on the distribution between soluble and particulate fractions of activities of enzymes involved in hepatic glycogen metabolism.

The large decreases in hepatic glycogen associated with alloxan diabetes in fed rats were accompanied by apparent decreases in total activities of glycogen synthase, phosphorylase, protein kinase and synthase phosphatase determined on 8000 × g supernatants of liver homogenates. Inclusion of 4% glycogen in the extraction buffer normalized total soluble activities of synthase in the diabetic. Whereas inclusion of 4% glycogen in the extraction buffer doubled total soluble phosphorylase, total activity remained lower in the diabetic than in the normal. Extraction and assay of soluble protein kinase were unaffected by added glycogen. When activities were determined on whole homogenates, total glycogen synthase activities were the same in normal and diabetic liver. Although the decreases in total activities of phosphorylase, kinase and phosphatase were less when determined on whole homogenates of livers from diabetic rats, the diabetes-related decreases in total activities remained significant. Therefore, it appears that while alloxan diabetes results in absolute decreases in total hepatic activities of phosphorylase, kinase and phosphatase, it may also result in redistribution of hepatic synthase and phosphorylase between soluble and particulate fractions, a phenomenon possibly related to tissue glycogen concentrations. Such a redistribution might be involved in the lack of control of hepatic glycogenesis observed in alloxan diabetic rats.     

AUTOPHAGIC DEGRADATION OF GLYCOGEN IN SKELETAL MUSCLES OF THE NEWBORN RAT

Large amounts of glycogen accumulate in rat skeletal muscle fibers during the late fetal stages and are mobilized in the first postnatal days. This glycogen depletion is relatively slow in the immature leg muscles, in which extensive deposits are still found 24 hr after birth and, to some extent, persist until the 3rd day. In the more differentiated psoas muscle and especially in the diaphragm, the glycogen stores are completely mobilized already during the early hours. Section of the sciatic nerve 3 days before birth or within the first 2 hr after delivery does not affect glycogen depletion in the leg muscles. Neonatal glycogenolysis in rat muscle fibers takes place largely by segregation and digestion of glycogen particles in autophagic vacuoles. These vacuoles: (a) are not seen in fetal muscle fibers or at later postnatal stages, but appear concomitantly with the process of glycogen depletion and disappear shortly afterwards; (b) are prematurely formed in skeletal muscles of fetuses at term treated with glucagon; (c) contain almost exclusively glycogen particles and no other recognizable cell constituents; (d) have a double or, more often, single limiting membrane and originate apparently from flattened sacs sequestering glycogen masses; (e) are generally found to contain reaction product in preparations incubated from demonstration of acid phosphatase activity. The findings emphasize the role of the lysosomal system in the physiological process of postnatal glycogen mobilization and appear relevant in the interpretation of type II glycogen storage disease.     

Isolation and Identification of l-Deoxy-l-(L-asparagino)-D-fructose Formed in the Autoclaved Culture Medium

To elucidate the effect of nutrition during induction on peripheral muscle responsiveness to insulin, the incorporation of radiolabeled glucose to glycogen and the uptake of radiolabeled deoxyglucose were studied in isolated diaphragms from the fetuses of normal and diabetic pregnant rats in vitro. Basal- and insulin-stimulated incorporation of [1-¹⁴C]glucose into diaphragm glycogen were greater in the fetuses of diabetic mothers (IDM) than in normal fetuses, but there was no difference in the degree of stimulation by insulin of labeled glucose into glycogen between normal fetuses and IDM. Diaphragms from normal fetuses and IDM had the same basal uptake of 2-deoxy-[1-³H]glucose as well as insulin-stimulated uptake. Consequently the sensitivity of glucose uptake to insulin was similar both in normal fetuses and IDM. These data indicate that glucose utilization (incorporation of labeled glucose into glycogen) was increased in IDM, but that the response of glucose uptake and glycogenesis to insulin was not altered.     

Regulation of glycogen metabolism in primary cultures of rat hepatocytes. Restoration of acute effects of insulin and glucose in cells from diabetic rats

Defects in the deposition of glycogen and the regulation of glycogen synthesis in the livers of severely insulin-deficient rats can be reversed, in vivo, within hours of insulin administration. Using primary cultures of hepatocytes isolated from normal and diabetic rats in a serum-free chemically defined medium, the present study addresses the chronic action of insulin to facilitate the direct effects of insulin and glucose on the short term regulation of the enzymes controlling glycogen metabolism. Primary cultures were maintained in the presence of insulin, triiodothyronine, and cortisol for 1-3 days. On day 1 in alloxan diabetic cultures, 10(-7) M insulin did not acutely activate glycogen synthase over a period of 15 min or 1 h, whereas insulin acutely activated synthase in cultures of normal hepatocytes. By day 3 in hepatocytes isolated from alloxan diabetic rats, insulin effected an approximate 30% increase in per cent synthase I within 15 min as was also the case for normal cells. The acute effect of insulin on synthase activation was independent of changes in phosphorylase alpha. Whereas glycogen synthase phosphatase activity could not be shown to be acutely affected by insulin, the total activity in diabetic cells was restored to normal control values over the 3-day culture period. The acute effect of 30 mM glucose to activate glycogen synthase in cultured hepatocytes from normal rats after 1 day of culture was missing in hepatocytes isolated from either alloxan or spontaneously diabetic (BB/W) rats. After 3 days in culture, glucose produced a 50% increase in glycogen synthase activity during a 10-min period under the same conditions. These studies clearly demonstrate that insulin acts in a chronic manner in concert with thyroid hormones and steroids to facilitate acute regulation of hepatic glycogen synthesis by both insulin and glucose.     

Combined action of Escherichia coli glycogen synthase and branching enzyme in the so-called "unprimed" polyglucoside synthesis.

Mutants of Escherichia coli which are unable to synthesize glycogen were used to study the so-called “unprimed” synthesis of glycogen. The glycogen synthase has been partially purified from these mutants. During the purification, attempts were made to separate the activity which requires the addition of an exogenous primer (primed activity) from the activity which does not require a primer but is highly dependent on the presence of some salts such as citrate and EDTA (unprimed activity). No separation between these two activities could be achieved but the results obtained by chromatography on DEAE-Sephadex indicate that there is a single form of glycogen synthase which is responsible for both unprimed and primed activity. The evidence that a single protein was necessary to catalyze these two reactions was given by the findings that mutants defective in glycogen synthase activity were unable to catalyze glucosyl transfer without added primer. At low concentration, the glycogen synthase purified from a branching enzyme negative mutant catalyzed the unprimed reaction at a slow rate even in presence of salts. A protein activator of this reaction was found in mutants lacking glycogen synthase but not in mutants lacking branching enzyme. The hypothesis that this activator is the branching enzyme itself was supported by the observation that it co-purified with the branching enzyme from a E. coli strain defective in glycogen synthase activity. EDTA or Triton X-100 increased the stimulation of the unprimed synthesis by the branching enzyme. The apparent affinity of the glycogen synthase for glycogen was increased twofold in the presence of EDTA but the branching enzyme further increased the effect of EDTA. The combined action of the glycogen synthase and the branching enzyme on the endogenous glucan associated with the synthase may account for the unprimed activity observed in vitro.     

Rat liver glycogen metabolism in the perinatal period

The correlation between blood glucose levels, the concentration of glycogen, the activities of glycogen sythase and phosphorylase and their respective kinases and phosphatases was examined in liver of rat fetuses between day 18 of gestation and one day after birth. Between day 18 and 21 there is a rapid increase in the concentration of glycogen and in the activity of synthase a and a much slower increase in the activity of phosphorylase a. The activity of the respective kinases increased rapidly during this period and reached maximun on day 21. The activity of synthase phosphatase and phosphorylase phosphatase increased after day 18, to reach a maximum on day 19 and 20, respectively, but decreased again towards day 21. The possibility that the changes in glycogen concentration and enzyme activities were related to an effect of glucose of AMP on the respective phosphatases was considered. It was found that the Km of phosphatase for glucose in the prenatal period was 5–7 mM, as in the adult. Since the level of blood glucose during this period was constant (2.8 mM), an effect of glucose on phosphatase activity seems unlikely. AMP concentration increased between day 18 and 21 from 6–15 nmol/g. In view of the low level of phosphorylase a activity during this period, the increase in AMP concentration is not considered to be important in the regulation of glycogen breakdown at this time.Immediately after birth blood glucose levels dropped to 5 mg/dl. This was accompanied by a rapid decrease in glycogen concentration and in the activity of glycogen synthase and a rise in phosphorylase activity. Blood glucose levels returned to the initial level within 1 h after birth, whereas the changes in glycogen concentration and enzyme activities continued for at least 3 h after birth. On day 22 all parameters examined had reached the level found in adult rat liver.It is suggested that the rapid changes observed immediately after birth are due to an effect of hypoglycemia mediated by hormones and cannot be ascribed to direct effects of metabolites on the enzyme systems involved.     

Some effects of glucose concentration and anoxia on glycolysis and metabolite concentrations in the perfused liver of fetal guinea pig

Effects of glucose concentration and anoxia upon the metabolite concentrations and rates of glycolysis and respiration have been investigated in the perfused liver of the fetal guinea pig. In most cases the metabolite concentrations in the perfused liver were similar to those observed in vivo. Between 50 days and term there was a fall in the respiratory rate and in the concentration of ATP and fructose 1,6-diphosphate and an increase in the concentration of glutamate, glycogen and glucose. Reducing the medium glucose concentration from 10 mM to 1 mM or 0.1 mM depressed lactate production and the concentration of most of the phosphorylated intermediates (except 6-phosphogluconate) in the liver of the 50-day fetus. This indicates a fall in glycolytic rate which is not in accord with the known kinetic properties of hexokinase in the fetal liver. Anoxia increased lactate production by, and the concentrations of, the hexose phosphates ADP and AMP in the 50-day to term fetal liver, while the concentration of ribulose 5-phosphate, ATP and some triose phosphates fell. These results are consistent with an activation of glycolysis, particularly at phosphofructokinase and of a reduction in pentose phosphate pathway activity, particularly at 6-phosphogluconate dehydrogenase.The calculated cytosolic NAD⁺/NADH ratio for the perfused liver was similar to that measured in vivo and evidence is presented to suggest that the dihydroxyacetone phosphate/glycerol 3-phosphate ratio gives a better indication of cytosolic redox than the lactate/pyruvate ratio. The present observations indicate that phosphofructokinase and hexokinase and possibly pyruvate kinase control the glycolytic rate and that glyceraldehyde-3-phosphate dehydrogenase is at equilibrium in the perfused liver of the fetal guinea pig.     

Insulin action on protein phosphatase-1 activation is enhanced by the antidiabetic agent pioglitazone in cultured diabetic hepatocytes

Effect of the antidiabetic agent pioglitazone on the insulin-mediated activation of protein phosphatase-1 was examined in diabetic hepatocytes. Streptozotocin-induced diabetes in Sprague Dawley rats caused a significant decrease in the activation of glycogen synthase in hepatocytes isolated from these animals. There was an inverse correlation between the in vivo hyperglycemic condition and the in vitro activation of glycogen synthase in liver cells (r = 0.93, p > 0.001). Long term incubation of diabetic hepatocytes with insulin and dexamethasone caused significant (p > 0.001) improvement in the activation of glycogen synthase activation. When incubated along with hormones, pioglitazone enhanced their action (p > 0.05-0.01). Diabetic hepatocytes were also characterized by 50% decrease in the activity of protein phosphatase-1, the enzyme which dephosphorylates and activates glycogen synthase. Pioglitazone potentiated the acute stimulatory effect of insulin on protein phosphatase-1 in normal hepatocytes but not in diabetic hepatocytes. Long term incubation of diabetic hepatocytes with insulin ameliorated the decrease in the protein phosphatase -1 activity in these cells. This stimulatory long-term effect of insulin was significantly (p > 0.05) enhanced by the antidiabetic agent pioglitazone.     

Environmental stress alters the developmental pattern of delta 5-3 beta-hydroxysteroid dehydrogenase activity in Leydig cells of fetal rats: a quantitative cytochemical study

Quantitative cytochemistry was used to determine the effect of subjecting pregnant rats to environmental stress on the activity of delta 5-3 beta hydroxysteroid dehydrogenase (3 beta-HSD) in Leydig cells of their fetuses. Enzyme activity was measured by microspectrophotometry in individual Leydig cells in cryostat sections of fetal testes on Days 16-21 postconception. Fetuses of stressed mothers lacked the peak of enzyme activity on Days 18 and 19 of gestation that is characteristic of Leydig cells of normal fetuses at this time. In addition, both before and after these 2 days, 3 beta-HSD activity in Leydig cells of stressed fetuses was significantly higher than normal. The altered developmental pattern of 3 beta-HSD activity in the stressed fetuses largely corresponds to the changes in plasma testosterone found previously in male fetuses of mothers exposed to the same regimen of stress. Thus, in the fetal Leydig cell, the activity of 3 beta-HSD, a key steroidogenic enzyme, can be modified by environmental stress, and provides an index of steroidogenic activity of the fetal testes and of the titers of circulating testosterone.     

Appearance of a nonfunctional isozyme of hepatic glycogen synthase in late gestation

Glycogen levels, glycogen synthase activities, and glycogen synthase protein levels were determined in liver tissues obtained from 14- to 19-day-old fetal mice, newborn mice, and adult mice. The results of these experiments demonstrate a significant increase in the quantity of hepatic glycogen synthase beginning at Day 17 of gestation and reaching adult levels at birth. However, during the same time period, there is a dramatic decrease in total glycogen synthase activity suggesting that the accumulating glycogen synthase molecules are unable to transfer UDP-glucose to glycogen. These inversely coordinated changes in the quantity and activity of glycogen synthase are consistent with the suggestion that glycogen synthesis in the near-term fetal mouse is being maintained by preexisting enzyme, while accumulating enzyme molecules may represent a quiescent isozyme.