Josef P. Skala

Developmental changes in cyclic AMP, protein kinase, phosphorylase kinase, and phosphorylase in liver, heart, and skeletal muscle of the rat.

Abstract Changes in cyclic AMP, protein kinase, phosphorylase kinase, and phosphorylase levels were examined during development in the rat. In liver, cyclic AMP increased prenatally and for the first 10 postnatal days; protein kinase levels (both cyclic AMP-dependent and independent activities) were high prenatally and declined during the first 10 postnatal days. Both phosphorylase and phosphorylase kinase in liver increased rapidly prenatally and more slowly postnatally. In heart and skeletal muscle cyclic AMP increased prenatally and for the first 10 days after birth, then declined. Protein kinase in both these tissues was highest prenatally and declined perinatally. In heart and skeletal muscle phosphorylase and phosphorylase kinase activities were extremely low prenatally although both enzymes were largely in their activated forms. Postnatally the nonactive form of both enzymes increased greatly throughout 30 postnatal days. In all three tissues, particularly heart and skeletal muscle, these changes could not be correlated with levels of tissue glycogen.

Changes in interscapular brown adipose tissue of the rat during perinatal and early postnatal development and after cold acclimation. V. Adenyl cyclase, cyclic amp, protein kinase, phosphorylase, phosphorylase kinase, and glycogen

Abstract 1. 1. The glycogen content of rat brown adipose tissue decreased dramatically at birth. 2. 2. Cyclic AMP levels reached a peak just prior to birth and decreased postnatally. Adenyl cyclase activity increased steadily up to the 20th postnatal day. 3. 3. Cyclic AMP-dependent protein kinase activity increased perinatally and decreased after the 20th postnatal day. 4. 4. Phosphorylase and phosphorylase kinase were both present primarily in the activated form prenatally and both increased significantly immediately before birth; then declined rapidly. Non-active phosphorylase followed a similar developmental pattern but the non-activated form of phosphorylase kinase continued to increase for 20 postnatal days. 5. 5. The DNA and RNA content of the tissue was highest during prenatal life. 6. 6. Cold exposure of 20-day-old rats caused increases in total phosphorylase, protein kinase, adenyl cyclase and cyclic AMP levels when expressed on a tissue fresh-weight basis.

Changes in interscapular brown adipose tissue of the rat during perinatal and early postnatal development and after cold acclimation VI. Effect of hormones and ambient temperature

Abstract 1. 1. Administration of norepinephrine (NE) 1 day before birth and of cyclic AMP 4 days before birth caused an increase in respiratory enzyme activities in brown adipose tissue (BAT) I day after delivery. Cortisone or thyroxine had the opposite effect. 2. 2. Cold exposure and warm exposure of newboms for 5 days did not alter BAT. Cortisone had a detrimental effect on BAT in warm exposed animals; cold exposure prevented this effect. 3. 3. Cold exposure of 16-day-old rats for 14 days or daily NE injections resulted similarly in higher BAT: body-weight ratios, higher protein content of BAT, and higher respiratory activities.

The role of carnitine in brown adipose tissue of suckling rats

Abstract 1. 1. Brown adipose tissue of late fetal rats and suckling rats accumulates subcutaneously injected [14C]- dl -carnitine or [3H]- l -carnitine more rapidly than heart or muscle and this is not true for adult rats. 2. 2. Subcutaneous injection of deoxycarnitine (100 mg/100 g body weight) prevents the rise in oxygen consumption normally seen in suckling rats that are exposed to 30 or 25°C and also the large rise due to norepinephrine injection in adult cold adapted rats. 3. 3. Deoxycarnitine also prevents the rise in O2 consumption of brown fat in vitro due to addition of norepinephrine. 4. 4. Injection of norepinephrine 15 min after the administration of deoxycarnitine increases the free fatty acid level of the blood in suckling rats and usually decreases the level of β-hydroxybutyric acid. Norepinephrine alone has no such effect.

α2-Adrenergic receptors in brown adipose tissue of infant rats—II. Studies on function and regulation☆☆☆

Abstract 1. 1.Clonidine inhibited the forskolin- and MIX-induced rate oflipolysis in brown fat adipocytes isolated from interscapular brown fat of 7-day-old rats. Its effect could be prevented by the α 2 - antagonist yohimbine. 2. 2.Pertussis toxin prevented the above effect of clonidine, thus indicating that α 2 - adrenoceptors are linked with adenylate cyclase via the N i regulatory subunit. 3. 3.Che4mical sympathectomy of 5-day-old rats by 6-hydroxydopamine increased the number of low-affinity α 2 sites in brown fat. 4. 4.Chronic administration of yohimbine to 2-3-week-old rats also increased the density of α 2 - adrenoceptors in brown fat. 5. 5.It is suggested that brown fat of infant rats possesses functional α 2 - adrenoceptors .

Cyclic GMP and cyclic GMP-dependent protein kinase in brown adipose tissue of developing rats.

Abstract In order to ascertain the possible involvement of cyclic GMP in the physiological regulation of the function and development of brown fat of the rat, we have determined its tissue concentration in vivo under a variety of conditions. The steady-state concentration of cyclic GMP in interscapular brown adipose tissue of late foetus was about 80 pmol per g fresh weight. The concentration gradually declined during the first 2 weeks after birth to reach 40 pmol/g fresh weight and then remained constant into adulthood. The cyclic GMP content of brown fat was decreased by chemical sympathectomy and was increased after complete acclimatization of the animals to the cold. The activity of cyclic GMP-dependent protein kinase was also highest in tissue from newborn and cold-acclimatized rats. Both acute cold stress and injection of norepinephrine resulted in a significant but temporary increase in the concentration of cyclic GMP in brown fat, which was followed by a depression of the concentration below values in untreated animals. The concentration of cyclic AMP showed similar pattern of changes. Injection of phenylephrine was followed by a pronounced increase in the cyclic GMP content of brown fat, with little effect upon cyclic AMP. Injection of isoproterenol raised the content of cyclic AMP but not that of cyclic GMP. The ability of norepinephrine and phenylephrine to increase the concentration of cyclic GMP was abolished by pre-treatment of the animals with phenoxybenzamine, but not by pre-treatment with propranolol. Conversely, propranolol but not phenoxybenzamine abolished the effects of norepinephrine on the cyclic AMP content of the tissue. Thus we have established the responsiveness of the cyclic GMP content of brown fat to physiological and pharmacological stimuli and have evidence of the possible participation by cyclic GMP in the α-adrenergic stimulation and in the regulation of proliferative processes in the tissue.

Changes in interscapular brown adipose tissue of the rat during perinatal and early postnatal development and after cold acclimation—III. Some cytoplasmic enzymes

Abstract 1. 1. The activities of glucose-6-phosphate dehydrogenase, cytoplasmic α-glycerophosphate dehydrogenase and glyceraldehyde-phosphate dehydrogenase in brown adipose tissue of the rat decreased considerably between the first and fortieth postnatal day. In white adipose tissue no age changes were observed. 2. 2. Phosphofructokinase and pyruvate kinase activities in brown adipose tissue increased to a peak on about day 10 and then decreased. No changes were observed in white adipose tissue. 3. 3. Malic enzyme activity increased postnatally in white adipose tissue but remained unaltered up to day 40 in brown adipose tissue. 4. 4. Cold exposure of 21-day-old rats for 16 days increased the activites of glucose-6-P-dehydrogenase, α-glycerophosphate dehydrogenase and malic enzyme in brown adipose tissue.

α2-Adrenergic receptors in brown adipose tissue of infant rats—I. Identification and characteristics of binding sites in isolated plasma membranes

1. Alpha 2-Adrenoceptor antagonists [3H]yohimbine and [3H]RX 781094 and the partial alpha 2-agonist [3H]clonidine exhibited specific binding to plasma membrane fragments isolated from interscapular brown fat of 7-day-old rats. 2. Competition studies with epinephrine, yohimbine and practolol revealed that [3H]norepinephrine, the principal in vivo agonist acting upon brown adipocytes, can readily bind to alpha 2-adrenoceptors in brown fat of infant rats. 3. The presence of alpha 2-adrenoceptor subclass in brown fat of infant rats may play a role in the sympathetic regulation of this rapidly proliferating tissue.

The “Second Messenger” System in Brown Adipose Tissue of Developing Rats. Its Molecular Composition and Mechanism of Function

Our studies of the hormonal receptor system and of the sequence of enzymatic events interconnecting the initial hormonal stimulus to the brown adipocyte with its final subcellular effect are summarized here. The hormone-mediated regulatory pathway consists of the adenyl cyclase and the protein kinase systems; the former is composed of the receptor and catalytic sites, the latter of regulatory and catalytic subunits. Emphasis is given currently to the diversity and characteristics of the individual components of the protein kinase system, since it seems to carry out the ultimate unifying mechanism involved in a variety of hormone-mediated functions, i.e. the phosphorylation of a protein molecule.

Effect of insulin and prednisolone on cyclic nucleotides and phosphoenolpyruvate carboxykinase activity in brown fat and liver of developing rats

The concentrations of cyclic AMP and cyclic GMP in brown fat and liver of both suckling and adult rats at fixed times after injection of insulin (2.5 U/100 g body weight) or prednisolone (2.5 mg/100 g body weight) were compared with the activity of phosphoenolpyruvate carboxykinase assayed 24 h after the injections. A stimulus that produced an increase in cyclic AMP content also produced an increase in the enzyme activity. If the content of cyclic GMP was also increased there was no rise in phosphoenolpyruvate carboxykinase activity. A rise in the content of cyclic GMP alone was associated with a reduction in the activity of the enzyme. These preliminary results indicate that cyclic AMP could be involved in the induction of phosphoenolpyruvate carboxykinase and that cyclic GMP may somehow be related to its repression. The known differences in the response of phosphoenolpyruvate carboxykinase activity to insulin and prednisolone in different tissues and at different stages of ontogenic development may thus be linked to differences in the responsiveness of enzymes concerned with the metabolism of cyclic nucleotides.