Glucose metabolism in the newborn rat. Hormonal effects in vivo

1. The concentrations of liver glycogen and plasma d-glucose were measured in caesarian-delivered newborn rats at time-intervals up to 3h after delivery after treatment of the neonatal rats with glucagon, dibutyryl cyclic AMP, cortisol or cortisol+dibutyryl cyclic AMP. Glycogenolysis was promoted by glucagon or dibutyryl cyclic AMP in the third hour after birth but not at earlier times. Cortisol and dibutyryl cyclic AMP together (but neither agent alone) promoted glycogenolysis in the second hour after birth, but no hormone combination was effective in the first postnatal hour. 2. The specific radioactivity of plasma d-glucose was measured as a function of time for up to 75 min after the intraperitoneal injection of d-[6-(14)C]glucose and d-[6-(3)H]glucose into newborn rats at delivery and after treatment with glucagon or actinomycin D. Glucagon-mediated hyperglycaemia at this time was due to an increased rate of glucose formation and a decreased rate of glucose utilization. Actinomycin D prevented glucose formation and accelerated the rate of postnatal hypoglycaemia. 3. The specific radioactivity of plasma l-lactate and the incorporation of (14)C into plasma d-glucose was measured as a function of time after the intraperitoneal injection of l-[U-(14)C]lactate into glucagon- or actinomycin D-treated rats immediately after delivery. The calculated rates of lactate formation were unchanged by either treatment, but lactate utilization was stimulated by glucagon administration. Glucagon stimulated and actinomycin D diminished (14)C incorporation into plasma d-glucose. 4. The factors involved in the initiation of glycogenolysis and gluconeogenesis in the rat immediately after birth are discussed.     

The effect of hormones on glucocorticoid binding capacity of rat liver cytosol.

The relationship between the glucocorticoid binding capacity of rat liver cytosol and the activity of tyrosine aminotransferase has been studied in adrenalectomized male rats. Bilateral adrenalectomy of male rats caused an increase within 3 days in the level of specific dexamethasone binding of liver cytosol accompanied by a rapid decrease in tyrosine aminotransferase activity. Known inducers of tyrosine aminotransferase were administered in vivo to test their effect on dexamethasone binding capacity, in order to determine whether the induction was by an indirect mechanism involving an increase in glucocorticoid binding capacity. Insulin, adrenalin, glucagon, dibutyryl cyclic AMP and oestradiol caused a significant increase in the activity of the enzyme, with no change in the specific dexamethasone binding. Tetracosactrin, a synthetic analogue of ACTH, had no effect on either parameter. It was concluded that the induction of tyrosine aminotransferase by the compounds tested was not mediated by an increase in glucocorticoid receptor activity.     

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

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

Regulation of tyrosine aminotransferase in foetal rat liver.

A specific tyrosine aminotransferase, separate from the aspartate aminotransferases, is present in low concentration in foetal rat liver at the 21st day of gestation. Intraperitoneal injections of tyrosine methyl ester into the foetuses in utero increase the activity 2-fold, whereas glucose injections decrease it. Tyrosine, dexamethasone and dibutyryl cyclic AMP induce the enzyme activity in organ culture to the same extent as in adult rat liver in vivo.     

Increase in hepatic tyrosine aminotransferase mRNA during enzyme induction by N6,O2'-dibutyryl cyclic AMP.

Tyrosine aminotransferase mRNA was quantitated by translation in a cell-free system derived from wheat germ followed by specific immunoprecipitation of the newly synthesized enzyme subunit. Hepatic poly(A)-containg RNA prepared from rats treated for 4 h with N6, O2'-dibutyryl cyclic AMP and theophylline was approximately 5.6 times more active in directing the synthesis of the tyrosine aminotransferase subunit relative to untreated controls. The overall template activity of the RNA prepared from control and cyclic AMP-treated animals was virtually identical, demonstrating that the cyclic nucleotide effect was specific for the tyrosine aminotransferase mRNA. At all times, after a single injection of dibutyryl cyclic AMP and theophylline, the increase in hepatic enzyme activity was accompanied by corresponding induction in the level of functional tyrosine aminotransferase mRNA. Other inducers of tyrosine aminotransferase, such as glucagon and hydrocortisone, also increased the level of tyrosine aminotransferase mRNA in proportion to their effect on enzyme activity. The RNA polymerase II inhibitor, alpha-amanitin, completely blocked the dibutyryl cyclic AMP-mediated increase in tyrosine aminotransferase mRNA activity. These studies demonstrate that, in intact animals, the induction of tyrosine aminotransferase activity by dibutyryl cyclic AMP can be completely accounted for by a corresponding increase in the level of functional mRNA coding for the enzyme.     

Glycogenolytic response to glucagon of cultured fetal hepatocytes. Refractoriness following prior exposure to glucagon.

The glycogenolytic effect of glucagon has been studied in fetal hepatocytes cultured for 3 to 4 days in the presence of cortisol (10 muM). The hepatocytes, when transplanted from young fetuses (15-day-old), contain only minute amounts of glycogen, whereas when cultured 3 to 4 days in the presence of cortisol, they contain high levels of stored glycogen. Glucagon induced a rapid but partial mobilization of glycogen, which was maximal after 2 hours. The half-maximal response was observed with about 0.1 nM glucagon. The glycogenolytic effect of glucagon in fetal hepatocytes is probably mediated by cyclic adenosine 3':5'-monophosphate (cyclic AMP) as in adult liver. This effect was mimicked by cyclic AMP and N-6, O-2-dibutyryl cyclic AMP, (dibutyryl cyclic AMP), and potentiated by theophylline. Glucagon addition was followed by accumulation of cyclic AMP in the cells within 2 min. Glucagon produces a marked stimulation of the rate of glycogen breakdown and an inhibition of the rate of incorporation of [14-C] glucose into glycogen. The glycogeneolytic effect of a single addition of glucagon was reversed within 4 hours. A second addition of glucagon at this time was unable to induce a new glycogenolytic response. A resistance to glucagon stimulation appeared in the cells after a first exposure to the hormone. This refractoriness was also shown by the loss of glucagon-dependent cyclic AMP accumulation and was not linked to the release by the cells of a "hormone antagonist" into the medium. The hepatocytes resistant to the action of glucagon retained their response to cyclic AMP, dibutyryl cyclic AMP, and norepinephrine. Finally, glycogenolytic concentrations of cyclic AMP and of its dibutyryl derivative failed to induce a refractoriness to glucagon.     

The adaptive behaviour of isoenzyme forms of rat liver alanine amino transferases during development

1. The activities of the mitochondrial and cytosol isoenzyme forms of l-alanine–glyoxylate and l-alanine–2-oxoglutarate aminotransferases were determined in rat liver during foetal and neonatal development. 2. The mitochondrial glyoxylate aminotransferase activity begins to develop in late-foetal liver, increases rapidly at birth to a peak during suckling and then decreases at weaning to the adult value. 3. The cytosol glyoxylate aminotransferase and the mitochondrial and cytosol 2-oxoglutarate aminotransferase activities first appear prenatally, increase further after birth and then rise to the adult values during weaning. 4. In foetal liver the mitochondrial glyoxylate aminotransferase and the cytosol 2-oxoglutarate aminotransferase activities are increased after injection in utero of glucagon, dibutyryl cyclic AMP (6-N,2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate) or thyroxine. The cytosol glyoxylate aminotransferase and the mitochondrial 2-oxoglutarate aminotransferase activities are increased after injection in utero of cortisol or thyroxine. 5. After birth the further normal increases in the mitochondrial and cytosol 2-oxoglutarate aminotransferase activities can be hastened by cortisol injection, whereas the increase in cytosol glyoxylate aminotransferase activity requires cortisol treatment together with the intragastric administration of casein. 6. The results are discussed with reference to the metabolic patterns and the changes in regulatory stimuli (hormonal and dietary) that occur during the period of development.     

Hormonal regulation of amino acid accumulation in human fetal liver explants Effects of dibutyryl cyclic AMP,glucagon and insulin

α-Aminoisobutyrate accumulation by human fetal liver explants in organ culture is stimulated by dibutyryl cyclic AMP ($\text{N}{}^6\text{,}{}^2\text{′O-}\text{dibutyryl}$ adenosine 3′–5′: cyclic monophosphate), glucagon or insulin. Theophylline increased the effect of submaximal concentrations of dibutyryl cyclic AMP or glucagon. Maximal concentrations of glucagon and dibutyryl cyclic AMP yielded the same results as either agent alone. A period of about 4–6 h was required to observe the stimulatory effect of dibutyryl cyclic AMP or insulin, which could be completely prevented by simultaneous incubation with cycloheximide. Maximal effects of either dibutyryl cyclic AMP or glucagon plus insulin produced additive results. These data support the hypothesis that insulin acts via a mechanism independent of the glucagon—cyclic AMP pathway in liver tissue.In addition, the pharmacologic receptor for glucagon was detected in liver explants from a 30-mm (crown - rump) specimen (6 weeks gestation). The liver had the competence to respond to dibutyryl cyclic AMP by the 36-mm stage. Tissue from a 36-mm specimen did not respond to insulin, but a clear response was elicited from a specimen at the 48-mm stage. These data demonstrate the ability of human fetal liver to respond to hormones at a very early stage in gestation.     

Hormonal regulation of four urea cycle enzymes in postnatal rat liver in organ culture

The administration of hydrocortisone to 3- to 15-day-old rats increased the levels of hepatic argininosuccinate synthetase (ASS) and arginase. In 13-day-old rat liver explants maintained in organ culture, ornithine carbamoyltransferase (OTC), carbamoylphosphate synthetase (CPS) and arginase were stimulated by betamethasone. Actinomycin D prevented the responses of the latter two enzymes. Dibutyryl cyclic AMP raised OTC, CPS, ASS and arginase in vitro. The responses of the latter three enzymes were blocked by cycloheximide and puromycin and partially inhibited by actinomycin D. The simultaneous presence of betamethasone and dibutyryl cyclic AMP in the culture medium raised CPS and OTC in an additive manner. The sequential treatment of the cultures with betamethasone followed by dibutyryl cyclic AMP increased CPS and arginase synergistically and amplified the response of ASS to dibutyryl cyclic AMP.     

Development of tyrosine aminotransferase in perinatal rat liver: changes in functional messenger RNA and the role of inducing hormones

Expression of the hepatic enzyme tyrosine aminotransferase was analyzed in the perinatal period of development in the rat, when this expression undergoes significant changes associated with hepatocyte differentiation. In late prenatal liver both enzyme and functional mRNA gene products are present at levels 10- to 15-fold below those in the fully differentiated adult liver. This low level of expression in fetal liver is refractory to induction by glucocorticoids, but both gene products are increased to a limited extent by cyclic AMP. This induction by cyclic AMP (cAMP) does not confer glucocorticoid-responsiveness on expression. By 3 hr after birth both functional mRNA and enzyme levels are significantly increased, an increase which continues until a peak is reached at 12 hr that is appreciably above the adult levels. Both gene products then decline until adult levels are reached by 24 hr. The postnatal shift in aminotransferase expression is accompanied by acquisition of the capacity to respond to glucocorticoids. Treatment of newborns with an antiglucocorticoid steroid or with glucose suppresses the postnatal overshoot of expression, but neither treatment affects the increase from fetal to adult levels of expression. The results indicate that prior to birth, expression of the aminotransferase gene is partially repressed, a repression that is lifted essentially immediately upon birth. The hormones capable of inducing aminotransferase synthesis have no apparent necessary role in this process.     

Stimulation by 6-N,2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate and glucocorticoids of phosphoenolpyruvate carboxykinase in the isolated perfused rat liver (Short Communication)

Dibutyryl cyclic AMP stimulated the activity of phosphoenolpyruvate carboxykinase in perfused livers of rats, fed on a low-protein diet, linearly over a 6h period. The enzyme activity was also significantly elevated by dexamethasone, the effect being considerably lower than that of the cyclic nucleotide. Since the time-course of phosphoenolpyruvate carboxykinase activity in response to dibutyryl cyclic AMP resembled that observed after dibutyryl cyclic AMP injection into intact animals, it is suggested that induction of the enzyme in vivo is due to a direct action of the cyclic nucleotide on the liver. Combined administration of dibutyryl cyclic AMP and glucocorticoids did not lead to an additive increase of liver phosphoenolpyruvate carboxykinase activity, either in vivo or in the perfused organ.     

Biochemical aspects of cardiac muscle differentiation. Possible control of deoxyribonucleic acid synthesis and cell differentiation by adrenergic innervation and cyclic adenosine 3':5'-monophosphate.

A single injection of either isoproternol or N6, O2'-dibutyryl adenosine 3':5'-monophosphate (dibutyryl cyclic AMP) results in an inhibition in the rate of [3H]thymidine incorporation into DNA of differentiating cardiac muscle of the neonatal rat. This inhibition is not due to substantially altered cellular uptake or catabolism of [3H]thymidine. Inhibition of [3H]thymidine incorporation by isoproterenol or dibutyryl cyclic AMP is potentiated by theophylline. Maximal inhibition (95%) is observed 24 h after administration of isoproterenol, and the rate of incorporation returns to a value 80% of control by 72 h. Norepinephrine also inhibits [3H]thymidine incorporation whereas cyclic GMP, N2, 02-Dibutyryl guanosine 3':5'-monophosphate (dibutyryl cyclic GMP), and phenylephrine have little effect. Equilibrium sedimentation analysis of cardiac muscle DNA in neutral and alkaline cesium chloride gradients using bromodeoxyuridine as a density label indicate that isoproterenol and dibutyryl cyclic AMP inhibit [3H]thymidine incorporation into DNA that is replicating semiconservatively. Administration of isoproterenol or dibutyryl cyclic AMP to neonatal rats inhibits by approximately 60% the incorporation of [3H]thymidine into DNA of tissue slices of cardiac muscle prepared 16 h later. [3H]Thymidine incorporation into DNA of tissue slices is into chains that were growing in vivo. This incorporation is linear for at least 4 h of incubation and is inhibited by isoproterenol and dibutyryl cyclic AMP. Inhibition is not due to altered cellular uptake of [3H]thymidine nor is it due to a cytotoxic action. Several other compounds which elevate intracellular levels of cyclic AMP (epinephrine, norepinephrine, glucagon, and prostaglandin E1) also inhibit [3H]thymidine incorporation into DNA or cardiac muscle tissue slices. Cyclic GMP, dibutyryl cyclic GMP, sodium butyrate, and phenylephrine have little effect. Isoproterenol administered together with theophylline to neonatal rats signficantly stimulates the in corporation of [3H]phenylalanine into total cardiac muscle protein and into myosin. This enhanced incorporation may be due in part to an increase in the cellular uptake of [3H]phenylalanine. DNA synthesis decreases progressively in differentiating cardiac muscle of the rat during postnatal development and essentially ceases by the middle of the third week (Claycomb, W. C. (1975) J. Biol. Chem. 250, 3229-3235). In reviewing the literature it was found that this decline in synthetic activity correlates temporally with a progressive increase in tissue concentrations of norepinephrine and cyclic AMP and with the anatomical and physiological development of the adrenergic nerves in this tissue. Because of these facts and data presented in this report it is proposed that cell proliferation and cell differentiation in cardiac muscle may be controlled by adrenergic innervation with norepinephrine and cyclic AMP serving as chemical mediators.     

Glutamine metabolism of isolated rat hepatocytes. Evidence for catecholamine activation of alpha-ketoglutarate dehydrogenase

Effects of norepinephrine on gluconeogenesis and ureogenesis from glutamine by hepatocytes from fasted rats were assessed. Comparisons were made to asparagine metabolism and to the effects of NH4Cl and dibutyryl cyclic AMP. With asparagine as substrate, aspartate content was very high but norepinephrine, dibutyryl cyclic AMP, or NH4Cl had little effect on gluconeogenesis or ureogenesis. Metabolism of asparagine could be greatly enhanced by the combination of oleate, ornithine, and NH4Cl. However, even under these conditions, asparatate content remained high, and norepinephrine and dibutyryl cyclic AMP had little influence on glucose or urea synthesis. With glutamine as substrate, aspartate content was much lower, but was greatly elevated by norepinephrine, dibutyryl cyclic AMP, or NH4Cl. Each of these effectors strongly stimulated glucose and urea formation from glutamine. NH4Cl stimulation was accompanied by an increased glutamate and decreased alpha-ketoglutarate content. This suggests the mechanism for NH4Cl stimulation is a near-equilibrium adjustment to ammonia by glutamate dehydrogenase and aspartate aminotransferase rather than a principal involvement of glutaminase. Although both norepinephrine and dibutyryl cyclic AMP lowered alpha-ketoglutarate to the same extent, norepinephrine more rapidly increased aspartate content and led to a smaller accumulation of glutamate than did dibutyryl cyclic AMP. Moreover, only norepinephrine led to a rapid increase in succinyl-CoA concentration. The catecholamine effect could not be explained by specific changes in cytosolic or mitochondrial redox states. The results suggest that alpha-ketoglutarate dehydrogenase is a site of catecholamine action in rat liver. Since purified alpha-ketoglutarate dehydrogenase is known to be Ca2+ stimulated and Ca2+ flux is involved in catecholamine action, these findings also suggest that mitochondrial Ca2+ is elevated by catecholamines.     

Regulation of coenzyme A biosynthesis by glucagon and glucocorticoid in adult rat liver parenchymal cells

We studied the effects of glucagon, dibutyryl cyclic AMP and dexamethasone on the rate of [(14)C]pantothenate conversion to CoA in adult rat liver parenchymal cells in primary culture. The presence of 30nm-glucagon increased the rate by about 1.5-fold relative to control cultures (range 1.4-2.3) and 2.4-fold relative to cultures containing 1-3m-i.u. of insulin/ml. The half-maximal effect was obtained at 3nm-glucagon. Dibutyryl cyclic AMP plus theophylline also enhanced the rate by about 1.5-fold. Dexamethasone acted synergistically with glucagon; glucagon at 0.3nm had no effect when added alone, but resulted in a 1.7-fold enhancement when added in the presence of dexamethasone (maximum effect at 50nm). The 1.4-fold enhancement caused by the addition of saturating glucagon concentrations was increased to a 3-fold overall enhancement by the addition of dexamethasone. However, dexamethasone added alone over the range 5nm to 5mum had no effect on the rate of [(14)C]pantothenate conversion to CoA. The stimulatory effect of dibutyryl cyclic AMP plus theophylline was also enhanced by the addition of dexamethasone. Changes in intracellular pantothenate concentration or radioactivity could not account for the stimulatory effects of glucagon, dibutyryl cyclic AMP or dexamethasone. Addition of 18mum-cycloheximide, an inhibitor of protein synthesis, decreased the rate of incorporation of [(14)C]pantothenate into CoA and the enhancement of this rate by glucagon and dibutyryl cyclic AMP plus theophylline in a reversible manner. These results demonstrate an influence of glucagon, dibutyryl cyclic AMP and glucocorticoids on the intracellular mechanism regulating total CoA concentrations in the liver.     

Regulation of the synthesis of the major surfactant apoprotein in fetal rabbit lung tissue

Antibodies directed against the major apoprotein of rabbit lung surfactant, a 29-36-kDa glycoprotein, were used to study changes in the levels of translatable surfactant apoprotein mRNA in rabbit lung tissue during development, as well as the effects of cortisol and cyclic AMP analogues on the levels of surfactant apoprotein and its mRNA in fetal rabbit lung tissue in organ culture. The major surfactant apoprotein and its mRNA were undetectable in lung tissues of 21-day gestational age fetal rabbits. Translatable mRNA specific for the major surfactant apoprotein was first detectable in lung tissues of 26-day fetuses, increased 25-fold on day 28, reached peak levels at day 31, and declined after birth. Incubation of 21-day fetal rabbit lung explants with cortisol in serum-free medium resulted in an increase in the specific content of the 29-36-kDa apoprotein. Cyclic AMP analogues and forskolin, an activator of adenylate cyclase, also caused a marked increase in the accumulation of surfactant apoprotein. When fetal lung explants were incubated with cortisol and dibutyryl cyclic AMP in combination, the specific content of the surfactant apoprotein was increased to levels greater than that of explants treated with either cortisol or dibutyryl cyclic AMP alone. These effects of dibutyryl cyclic AMP and cortisol on surfactant apoprotein accumulation were associated with comparable changes in the levels of translatable surfactant apoprotein mRNA. Thus, we have shown for the first time that the induction of pulmonary surfactant apoprotein synthesis during differentiation in vitro and in vivo is associated with an increase in the level of translatable mRNA and that cortisol and cyclic AMP increase both the accumulation of the major surfactant apoprotein and the corresponding mRNA in fetal rabbit lung tissue in vitro.