The influence of diurnal rhythms of carbohydrate metabolism in adult rat liver on the metabolic characteristics of isolated liver parenchymal cells.

Rats trained to the "8 + 16" controlled feeding cycle where food is only available for the first 8 h of the 12 h dark period exhibit a pronounced diurnal rhythm of hepatic glycogen metabolism. Glycogen is stored within the liver parenchymal cells during the dark period and subsequently mobilized for energy production during the light period. Hepatocytes, isolated by collagenase perfusion, from livers of such animals have differing capacities for glycogen synthesis when incubated with glucose. Cells prepared at the end of the 16 h period without food have very little capacity for synthesis compared with much higher rates obtained in cells obtained during the feeding period. Cells obtained from liver containing a large glycogen concentration produce a net breakdown of glycogen during incubations with glucose, however experiments using radioactively labelled glucose indicate that synthesis does occur in these cells. The changes in the capacity of the cells for glycogen synthesis appear to be due, in part, to changes in the percentage of the cell population involved in synthesis and in the activity of glycogen synthetase a. Attempts of influence the rate of glycogen synthesis at any time of day with insulin or dexamethasone were unsuccessful.     

The influence of diurnal rhythms of carbohydrate metabolism in adult rat liver on the metabolic characteristics of isolated liver parenchymal cells

Rats trained to the “8 + 16” controlled feeding cycle where food is only available for the first 8 h of the 12 h dark period exhibit a pronounced diurnal rhythm of hepatic glycogen metabolism. Glycogen is stored within the liver parenchymal cells during the dark period and subsequently mobilized for energy production during the light period. Hepatocytes, isolated by collagenase perfusion, from livers of such animals have differing capacities for glycogen synthesis when incubated with glucose. Cells prepared at the end of the 16 h period without food have very little capacity for synthesis compared with much higher rates obtained in cells obtained during the feeding period. Cells obtained from livers containing a large glycogen concentration produce a net breakdown of glycogen during incubations with glucose, however experiments using radioactively labelled glucose indicate that synthesis does occur in these cells. The changes in the capacity of the cells for glycogen synthesis appear to be due, in part, to changes in the percentage of the cell population involved in synthesis and in the activity of glycogen synthetase $\text{a}$. Attempts to influence the rate of glycogen synthesis at any time of day with insulin or dexamethasone were unsuccessful.     

Effects of hormones and serum on glycogen metabolism in adult rat liver parenchymal cell primary cultures.

Parenchymal cells from adult rat liver, isolated by a collagenase perfusion technique, have been maintained in primary culture and a detailed study on carbohydrate metabolism carried out over the initial 48-hour culture period. The glucose concentration of the medium exerts a major influence on glycogen accumulation by the cells. Insulin, particularly at high glucose concentrations, stimulates glycogen biosynthesis, whereas glucagon prevents glycogen accumulation. Dexamethasone was without effect on glycogen metabolism. Glucose appears to stimulate glycogen accumulation by activation of glycogen synthetase enzyme. However, there is a gradual loss of synthetase activity throughout the culture period. Similar decreases in activity were noted for pyruvate kinase, aldolase and hexokinase. Glucose, insulin and dexamethasone were unable to prevent these decreases in enzyme activity. Foetal bovine serum contains fructose and this hexose appears to be the factor in serum which is responsible for the activation of glycogen accumulation in the presence of physiological glucose concentrations. The lactic acid content of the serum may also stimulate glycogen accumulation. In general, there is a gradual loss of the pattern of carbohydrate metabolism typical of differentiated hepatocytes during the culture period.     

Effects of Feeding a Histidine-excess Diet and Subsequent Starvation on Liver and Muscle Glycogen,and on Serum Glucose

The effects of feeding with a histidine-excess diet and subsequent starvation on liver and muscle glycogen, and on serum glucose were investigated in young and adult rats.

Feeding with a histidine-excess diet resulted in the accumulation of liver glycogen in both young and adult rats. The hepatic glycogen continued to decrease during starvation, and the liver became almost totally depleted of glycogen after starvation for 48 hr. Glycogen in the liver of young rats starved for 24 hr after previous feeding with a histidine-excess diet was significantly higher than that of young rats starved for 24 hr after previous feeding with a basal diet.

Muscle glycogen after feeding and subsequent starvation was not affected by the types of diets fed previously, muscle glycogen during starvation showing a slight decrease in young rats and a slight increase in adult rats.

Feeding with a histidine-excess diet caused a significant decrease of serum glucose in young rats, but not in adult rats. Serum glucose in young rats was markedly reduced by starvation after previous feeding with a basal diet, but not after previous feeding with a histidine-excess diet. In adult rats, there were no changes in serum glucose between rats starved after feeding with either a basal diet or a histidine-excess diet, and serum glucose was decreased slightly by starvation after feeding with the test diets.

The overall results indicate that the maintenance of serum glucose in young rate even during starvation after previous feeding with a histidine-excess diet might be partially concerned with the export of glucose from the accumulated glycogen in the liver due to the diet.     

Adenovirus-mediated transfer of the muscle glycogen phosphorylase gene into hepatocytes confers altered regulation of glycogen metabolism.

The muscle isozyme of glycogen phosphorylase is potently activated by the allosteric ligand AMP, whereas the liver isozyme is not. In this study we have investigated the metabolic impact of expression of muscle phosphorylase in liver cells. To this end, we constructed a replication-defective, recombinant adenovirus containing the muscle glycogen phosphorylase cDNA (termed AdCMV-MGP) and used this system to infect hepatocytes in culture. AMP-activatable glycogen phosphorylase activity was increased 46-fold 6 days after infection of primary liver cells with AdCMV-MGP. Despite large increases in phosphorylase activity, glycogen levels were only slightly reduced in AdCMV-MGP-infected liver cells compared to uninfected cells or cells infected with wild-type adenovirus. The lack of correlation of phosphorylase activity and glycogen content suggests that the liver cell environment can inhibit the muscle phosphorylase isozyme. This inhibition can be overcome, however, by addition of carbonyl cyanide m-chlorophenylhydrazone (CCCP), which increases AMP levels by 30-fold and causes a much larger decrease in glycogen levels in AdCMV-MGP-infected cells than in uninfected or wild-type adenovirus-infected controls. CCCP treatment also caused a preferential decrease in glycogen content relative to glucagon treatment in AdCMV-MGP-infected hepatocytes (74% versus 11%, respectively), even though the two drugs caused equal increases in phosphorylase a activity. Introduction of muscle phosphorylase into hepatocytes therefore confers a capacity for glycogenolytic response to effectors that is not provided by the endogenous liver phosphorylase isozyme. The remarkable efficiency of adenovirus-mediated gene transfer into primary hepatocytes and the demonstration of altered regulation of glycogen metabolism as a consequence of expression of a non-cognate phosphorylase isozyme may have implications for gene therapy of glycogen storage diseases.     

Glycogen degradation during migration of presumptive cell types in Dictyostelium discoideum.

During the time course of differentiation in Dictyostelium discoideum, glycogen was found to accumulate from the amoebae stage to the culmination stage of development. Upon sorocarp formation (23 h), glycogen was rapidly degraded. Ultramicrotechniques, utilizing amplification of glycogen by enzymatic cycling, were used to follow glycogen metabolism in pre-stalk and prespore cells during the differentiation cycle. Both cell types accumulated glycogen at nearly the same rate. By the pseudoplasmodium stage of development glycogen had accumulated to 50% of its maximum value, and no differences were found between pre-stalk and pre-spore cells. Glycogen was degraded as pre-stalk cells migrated into the position for stalk construction. At the culmination stage of development stalk cells near the base were devoid of glycogen while pre-stalk cells near the apex of the stalk showed no loss of glycogen. The complete loss of glycogen from stalk cells occurred over a distance occupied by approximately 100 cells, and over a time period of approx. 1 h. Pre-spore cells at the culmination stage showed no loss of glycogen even though separated from stalk cells by only a thin cellulose sheath. The degradation of prespore cell glycogen did not commence until stalk construction was completed and the pre-spore mass had reached the apex of the stalk. Pre-spore cells at the culmination stage contained high levels of glycogen while only 2 h later, total degradation had occurred.     

Effect of insulin on glycogen metabolism in isolated catfish hepatocytes

Insulin effect on carbohydrate metabolism in catfish hepatocytes consisted of a significant decrease of cell glycogen concentration both in the absence and in the presence of glucose in the medium. The hormone did not influence either the output of glucose from the cell or the intracellular glucose level. Experiments with radioactive glucose showed a very low uptake of the sugar by the hepatocytes; correspondingly the incorporation of radioactivity into glycogen was very low and not influenced by insulin. The glycogen content in catfish liver cells was influenced by the hormone in the opposite way to rat liver cells.     

Carbohydrate metabolism in Manduca sexta during late larval development

The carbohydrate metabolism in Manduca sexta underwent significant changes during late larval development. Approximately 10% of fat body glycogen phosphorylase was active during the feeding period of the 5th instar, pharate-pupal development and after the pupal moult; it is concluded that glycogen synthesis prevailed. During the last larval and the pupal moult, as well as the wandering stage the percentage of active phosphorylase was significantly increased indicating that fat body glycogen stores were broken down to supply substrates to meet the demands of carbohydrate metabolism. In the course of the last larval moult and the wandering stage the fat body glycogen content decreased significantly from about 300 to about 200 μg mg⁻¹ dry mass substantiating that carbohydrates were released from the fat body. Prior to phosphorylase activation, the concentrations of total haemolymph sugars decreased significantly from about 12 to about 6 mg trehalose equivalents ml⁻¹ (last larval moult) and from about 18 to about 12 mg ml⁻¹ (wandering stage), and increased again slightly when phosphorylase was activated. The haemolymph glucose concentration decreased significantly from about 1.1 to 0.3 mg ml⁻¹ (last larval moult) and in the course of the 5th-instar feeding period from about 1.1 to 0.2 mg ml⁻¹, and remained at this level until the beginning of adult development. The amount of chitosan present in the cuticle increased steadily during the feeding period of the 5th instar from about 10 to 110 mg. It appears that fat body glycogen might be broken down during the last larval moult and the wandering period to provide substrates for chitin synthesis. A dramatic decrease in the amount of chitosan was observed prior to the pupal moult.     

Cytophotometric analysis of glycogen, protein and DNA of a glycogen-storing rat hepatoma (N13) cell line.

This study examines the behavior of glycogen-storing rat hepatoma (N13) in vitro using cytophotometric techniques. A significant increase in glycogen is observed in these cells after 30 min incubation in a buffered solution containing 0.1 mM glucose, that is 80 times lower than the physiological glucose concentration in rat blood. N13 hepatoma cells grow exponentially in culture using RPMI 1640 tissue culture medium supplemented with 10% fetal bovine serum. During the first day in culture these cells store a large amount of glycogen and this increase is also observed in serum-free cultures. In more prolonged cultures the amount of glycogen per cell gradually becomes lower, although the culturing conditions are maintained. Similar variations of protein are also observed during the initial period of culture. DNA distribution does not show significant changes, although in serum-free cultures an increase in the proportion of cells in S and G2/M phases is observed. The addition of glucagon, epinephrine and cyclic AMP derivatives to serum-free cultures does not impede the storage of glycogen. Nevertheless, addition of either 2 mM N6,O2'-dibutyryl cyclic AMP or 0.1 mM 8-(4-chlorophenylthio)-cyclic AMP blocks the cell cycle at G0/G1 and glycogen content does not decrease after the first day in culture. We believe that this cell line offers an appropriated model to study glycogen metabolism and its involvement in the neoplastic process.     

Glucose 6-phosphate plays a central role in the regulation of glycogen synthesis in a glycogen-storing liver cell line

Two substrains of the epithelial liver cell line C1I, one storing large amounts of glycogen, the other one being very poor in glycogen were used as a model for studying glycogen synthesis. The glycogen content of glycogen-rich cells doubled during the proliferative phase and remained high in plateau phase although glycogen synthase I activity was not significantly altered during growth cycle and was too low to account for the increase in glycogen. However, the activity of the glucose 6-phosphate (Glc6-P)-dependent synthase rose continuously during growth cycle, and intracellular Glc6-P-concentration increased about 10-fold in log phase cells to 0.72 mumol g-1 wet weight. A0.5 of synthase for Glc6-P was 0.79 mM. It was also found that in contrast to the enzyme from normal liver, glycogen phosphorylase a from C1I cells was inhibited by Glc6-P, the apparent Ki being 0.45 mM. It was concluded that glycogen accumulation in C1I cells was due to stimulation of synthase and inhibition of phosphorylase by Glc6-P. Findings from the glycogen-poor cell line which revealed similar specific activities of synthase and phosphorylase but only low Glc6-P (0.056 mumol g-1 wet weight) supported this conclusion. Addition of glucose to starved cells resulted in a transient activation of synthase in both cell lines. Net glycogen synthesis, was, however, only observed in the cells with a high Glc6-P-content. Thus, modulation of synthase and phosphorylase by Glc6-P and not activation/inactivation of the enzymes seems to play a predominant role in glycogen accumulation in this cell line.     

Study of carbohydrate metabolism in glycogen storing cell lines derived from cultured rat hepatocytes

Some aspects of carbohydrate metabolism were investigated in three non-malignant, glycogen storing, cell lines derived from a primary culture of rat hepatocytes, and in the Morris hepatoma 3924 cells. The three cell lines show biochemical alterations which are, to a large extent, similar to those found in the hepatoma cells: increased activity of glycolytic enzymes and decreased activity of gluconeogenetic enzymes. An increase of glucose-6-phosphate dehydrogenase activity is also found. The three cell lines, as the Morris hepatoma cells, actively convert glucose into lactate under the in vitro conditions of culture. Fructose is not taken up as quickly as glucose and galactose is not metabolized. As compared with normal hepatocytes, the three cell lines have altered metabolism and growth behaviour. They largely resemble the preneoplastic cells appearing in rat liver at the early stages of experimental carcinogenesis.     

Differential change of Ins-P3-Ca2+ signaling during culture of rat hepatocytes

Decrease of alpha-adrenergic responses during primary culture of rat hepatocytes was studied. Activation of glycogen phosphorylase by phenylephrine was decreased in the early stage of the culture (within 6 h), however, Ins-P3 production was almost intact until 12 h of the culture and then declined. alpha-Adrenoceptor-mediated Ca2+-mobilization and Ins-P3-induced Ca2+ release from microsomal fractions were decreased in the early stage of the culture, similar to the above change of phosphorylase activation. We found that decrease of Ins-P3-binding sites in the early stage of the culture was the cause of differential change of Ins-P3-Ca2+ signaling during the culture of hepatocytes. Similar changes described above were also observed in vasopressin-induced responses. However, the changes of Ins-P3-Ca2+ signaling did not occur in a high-cell density culture of rat hepatocytes. In conclusion, the loss of phenylephrine- and vasopressin-induced responses in cultured liver cells appear to be due to change of Ins-P3-binding sites as well as decreased Ins-P3 production due to reduction of receptor numbers.     

Promoting effect of feeder cells in maintenance of adult rat hepatocytes

A procedure is described for maintaining primary cultures of adult rat hepatocytes for prolonged periods of time on layer of irradiated mouse fibroblast cell line (C3H/1OT1/2) and on a secondary lung fibroblasts obtained from Sprague Dawley rats. Morphologically and ultrastructurally the cocultivated hepatocytes retained many characteristics of hepatocytes in vivo. Within 24 hours after seeding, the individual cells were attached on the feeder cell layer and the in vivo polarity of the liver cells reappeared. Electron microscope studies demonstrated the appearance of newly developed bile ducts and junctions between hepatocytes as well as between hepatocytes and feeder cells. Histochemically, these cells were positive for glucose-6-phosphatase and for glycogen. After 14 days in culture the hepatocytes could be reseeded onto fresh C3H1OT1/2 cells. In contrast, hepatocytes maintained on plastic substrate lost their glycogen content and the epithelial character of the liver cells after 5 days in culture, and by day 10 this culture became predominantly fibroblastic. It is suggested that hepatocytes maintained on an irradiated fibroblast feeder layer provide a valuable approach for studying the morphogenesis, cytotoxicity, or the metabolism of different chemicals in vitro.     

Primary rat hepatocyte culture on 3D nanofibrous polymer scaffolds for toxicology and pharmaceutical research

Primary rat hepatocytes are a widely used experimental model to estimate drug metabolism and toxicity. In currently used two‐dimensional (2D) cell culture systems, typical problems like morphological changes and the loss of liver cell‐specific functions occur. We hypothesize that the use of polymer scaffolds could overcome these problems and support the establishment of three‐dimensional (3D) culture systems in pharmaceutical research. Isolated primary rat hepatocytes were cultured on collagen‐coated nanofibrous scaffolds for 7 days. Cell loading efficiency was quantified via DNA content measurement. Cell viability and presence of liver‐cell‐specific functions (albumin secretion, glycogen storage capacity) were evaluated. The activity of liver‐specific factors was analyzed by immunofluorescent staining. RNA was isolated to establish quantitative real‐time PCR. Our results indicate that primary rat hepatocytes cultured on nanofibrous scaffolds revealed high viability and well‐preserved glycogen storage. Albumin secretion was existent during the entire culture period. Hepatocytes remain HNF‐4 positive, indicating highly preserved cell differentiation. Aggregated hepatocytes re‐established positive signaling for Connexin 32, a marker for differentiated hepatocyte interaction. ZO‐1‐positive hepatocytes were detected indicating formation of tight junctions. Expression of cytochrome isoenzymes was inducible. Altogether the data suggest that nanofibrous scaffolds provide a good in vitro microenvironment for neo tissue regeneration of primary rat hepatocytes. Biotechnol. Bioeng. 2011; 108:141–150. © 2010 Wiley Periodicals, Inc.     

Hormonal regulation of glycogen metabolism in neonatal rat liver

1. The development of active and inactive phosphorylase was determined in rat liver during the perinatal period. No inactive form could be found in tissues from animals less than 19 days gestation or older than the fifth postnatal day. 2. The regulation of phosphorylase in organ cultures of foetal rat liver was examined. None of the agents examined [glucagon, insulin or dibutyryl cyclic AMP (6-N,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate)] changed the amount of phosphorylase activity. 3. Glycogen concentration in these explants were nevertheless decreased more than twofold by 4h of incubation with glucagon or dibutyryl cyclic AMP. Incubation with insulin for 4h increased the glycogen content twofold. 4. Glycogen synthetase activity was examined in these explants. I-form activity (without glucose 6-phosphate) was found to decrease by a factor of two after 4h of incubation with dibutyryl cyclic AMP, whereas I+D activity (with glucose 6-phosphate) remained nearly constant. Incubation for 4h with insulin increased I-form activity threefold, with only a slight increase in I+D activity. 5. When explants were incubated with insulin followed by addition of dibutyryl cyclic AMP, the effects of insulin on glycogen concentration and glycogen synthetase activity were reversed. 6. These results indicate that the regulation of glycogen synthesis may be the major factor in the hormonal control of glycogen metabolism in neonatal rat liver.     

Critical evaluation of methods used for the isolation of rat liver hepatocytes for metabolic studies.

Hepatocytes were isolated from normal fed, fasted and alloxan diabetic animals. The best cell preparations were obtained by using low concentrations of collagenase (10–20 mg) and exposing the liver for a very short period of time (10–15 min). Addition of hyaluronidase significantly decreased the glycogen content of the isolated hepatocytes. Glucagon (10⁻¹²M) stimulated glycogenesis in hepatocytes containing high glycogen whereas, in cells containing low glycogen much higher concentration of glucagon was needed (10⁻⁹M). Addition of insulin (100 μunits) stimulated both glycogen and protein synthesis in isolated hepatocytes containing high glycogen. Under these conditions glycogen synthase activity was stimulated by 40%. Incorporation of ¹⁴C phenylalanine into protein was linear for only 3–4 hr in cells containing low glycogen whereas, in cells containing high glycogen incorporating was linear for 8–10 hr. These studies suggest that intracellular glycogen plays an important role in the hormonal regulation of metabolism in hepatocytes.     

Liver hyperplasia and paradoxical regulation of glycogen metabolism and glucose-sensitive gene expression in GLUT2-null hepatocytes. Further evidence for the existence of a membrane-based glucose release pathway

We investigated the impact of GLUT2 gene inactivation on the regulation of hepatic glucose metabolism during the fed to fast transition. In control and GLUT2-null mice, fasting was accompanied by a approximately 10-fold increase in plasma glucagon to insulin ratio, a similar activation of liver glycogen phosphorylase and inhibition of glycogen synthase and the same elevation in phosphoenolpyruvate carboxykinase and glucose-6-phosphatase mRNAs. In GLUT2-null mice, mobilization of glycogen stores was, however, strongly impaired. This was correlated with glucose-6-phosphate (G6P) levels, which remained at the fed values, indicating an important allosteric stimulation of glycogen synthase by G6P. These G6P levels were also accompanied by a paradoxical elevation of the mRNAs for L-pyruvate kinase. Re-expression of GLUT2 in liver corrected the abnormal regulation of glycogen and L-pyruvate kinase gene expression. Interestingly, GLUT2-null livers were hyperplasic, as revealed by a 40% increase in liver mass and 30% increase in liver DNA content. Together, these data indicate that in the absence of GLUT2, the G6P levels cannot decrease during a fasting period. This may be due to neosynthesized glucose entering the cytosol, being unable to diffuse into the extracellular space, and being phosphorylated back to G6P. Because hepatic glucose production is nevertheless quantitatively normal, glucose produced in the endoplasmic reticulum may also be exported out of the cell through an alternative, membrane traffic-based pathway, as previously reported (Guillam, M.-T., Burcelin, R., and Thorens, B. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 12317-12321). Therefore, in fasting, GLUT2 is not required for quantitative normal glucose output but is necessary to equilibrate cytosolic glucose with the extracellular space. In the absence of this equilibration, the control of hepatic glucose metabolism by G6P is dominant over that by plasma hormone concentrations.     

Metabolic effects after short-term sublethal cadmium exposure to dogfish (Scyliorhinus canicula)

1. Four groups of dogfish were subjected to 50 ppm of cadmium for 1, 2, 3 and 4 days and haematological and metabolic parameters analyzed from blood and tissue samples.2. A significant increase of blood glucose and lactate was observed.3. No differences were found in the majority of haematological parameters except for the red blood cell counts that showed a significant increase during the second and third day of treatment.4. Comparing liver and muscle metabolism a decrease of protein levels are observed in both tissues and reductions of glycogen in liver and lactate in muscle were detected.5. The results of the different parameters indicate a pattern of dual response depending on the time of exposure; a first phase of toxic impact during the first two days, followed by a second phase of recovery at the end of 4 days treatment.     

Effect of cobalt on the liver glycogen content in the streptozotocin-induced diabetic rats

Cobalt decreases blood glucose in diabetic rats but the mechanisms involved are unclear. To determine the contribution of glycogen metabolism to glycemia-lowering effect, glycogen contents of liver and muscle in the streptozotocin-induced diabetic rats were determined. The liver glycogen was depleted in diabetic rats. But when cobalt was administered to the rats, the glycogen returned to the level of healthy rats, concomitantly with the decrease in blood glucose. The cobalt treatment had no effect on the muscle glycogen in the diabetic rats. The tissue-specific responses of glycogen metabolism suggest the involvement of suppressed glucagon signaling due to cobalt treatment.     

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.