Activation of adenosine 3',5'-monophosphate-dependent protein kinase and its relationship to cyclic AMP and lipolysis in hamster adipose tissue

The interrelationships among cAMP-dependent protein kinase activity, lipolysis, and cellular concentrations of cAMP were investigated in hamster epididymal adipose tissue. Isoproterenol, norepinephrine, and theophylline increased the protein kinase activity assayed in tissue extracts with no added cAMP, but not in the presence of added cyclic nucleotide. The maximum rate of lipolysis was associated with a nearly three-fold increase in cAMP levels and a protein kinase activity ratio of 0.8 (the ratio of activity assayed without cAMP to that assayed with cAMP). Rates of lipolysis less than maximum were associated with lesser degrees of protein kinase activity and lower levels of cAMP. The relatively pure alpha-adrenergic agent phenylephrine partially suppressed the isoproterenol-stimulated protein kinase activity, lipolysis, and cAMP levels. Conversely, the alpha-adrenergic blocking agent phentolamine increased the activity of protein kinase and cAMP levels in adipose tissues exposed to norepinephrine. These data are consistent with the primary role for cAMP and its dependent protein kinase in control of lipolysis in adipose tissue. Moreover, our data are consistent with the view that the antilipolytic action of alpha-adrenergic agents is mediated by a decrease in activity of protein kinase, caused by a decrease in cellular cAMP concentrations.     

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

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.     

Effect of pH on lipolysis, cAMP and cAMP-dependent protein kinase activity in isolated rat fat cells

The effect of acidosis and alkalosis on lipolysis, cAMP production and cAMP-dependent protein kinase activity in isolated rat fat cells incubated in the presence of norepinephrine and norepinephrine plus theophylline has been investigated. The pH of the incubation medium was adjusted to 6.8, 7.4 and 7.8 respectively. Acidosis inhibited both norepinephrine- and norepinephrine plus theophylline-induced release of glycerol whereas alkalosis led to slight stimulation. Norepinephrine produced an increase in cAMP and cAMP-dependent protein kinase activity. However, comparison of both parameters in acidosis and alkalosis with those at pH 7.4 indicates that they were higher at pH 7.8 and lower at pH 6.8. Addition of theophylline in combination with norepinephrine increases cAMP production within 5 min, under acidosis to values similar to those obtained at pH 7.4 with norepinephrine. The same effect on protein kinase activity was obtained. In spite of this increment in cAMP and protein kinase activity produced by addition of norepinephrine plus theophylline, lipolysis remains inhibited by acidosis. Addition of theophylline at pH 7.4 and 7.8 induced a much higher cAMP production and cAMP-dependent protein kinase activity although at pH 7.8 there was a statistically significant increase in protein kinase activity at 10 min it did not induce a significant increase in lipolysis. This is discussed and possible mechanisms are suggested to explain the effect of acidosis and alkalosis on the lipolysis induced by norepinephrine in rat fat cells.     

Protein kinases in brown adipose tissue of developing rats. Electrophoretic separation and assay of soluble protein kinases on polyacrylamide gels and a study of their properties and changes during development.

Protein kinase activity was detected and assayed directly on polyacrylamide gels after disc electrophoresis of the 100,000 X g supernatant fraction of brown adipose tissue of infant rats. Nine major bands of activity were detected, eight of which could be stimulated by cAMP or inhibited by the cAMP-dependent protein kinase inhibitor protein. This electrophoretic technique revealed heterogeneity in the cAMP-dependent protein kinase activity eluted from DEAE-cellulose by high concentrations of salt, but not in the peak of activity eluted by low concentrations of salt. The catalytic properties and substrate specificities of the kinases in the various bands were studied while the enzymes were still in the gels. The activity in each band differed from each of the others in at least one of these properties. The activities of the protein kinases in brown fat changed as the animals grew, and each band exhibited a distinct and unique developmental pattern. The major changes in kinase activities occurred in the immediate post-parturition period, then at 15 days after birth and at weaning. These developmental stages coincide with the periods during which the tissue undergoes changes in the rate of its proliferation, differentiation, and functional activity.     

Glycerokinase activity and lipolysis regulation in brown adipose tissue of cold acclimated rats.

Glycerol release by brown adipocytes from constant cold adapted rats was not stimulated by norepinephrine. On the contrary, the release was stimulated in rats adapted to a nycthemeral fluctuatiing temperature from 5 degrees to 28 degrees C. Glycerokinase activity was greatly increased in brown adipose tissue by cold adptation ; there was no change in the liver. However this increased activity cannot entirely explain the lack of norepinephrine stimulation of glycerol release in the brown adipose tissue of cold adapted rats.     

The concentration of cyclic AMP and the activity of cyclic AMP dependent protein kinase and an inhibitor in the adipose tissue of rats fed lard or glucose diets.

Measurements of tissue cyclic AMP (cAMP) concentration, the activity of cAMP-dependent protein kinase and the level of the enzyme's thermostable, macromolecular inhibitor were made on preparations of rat epididymal fat pad from animals fed high fat or high carbohydrate diets. The cAMP concentration from rats adapted to a high lard diet for 14-15 days was 153 +/- 17.8 pmoles/mg protein as opposed to 76 +/- 6.0 found with high glucose diet. No significant difference in total cAMP-dependent protein kinase activity was observed among rats fed high glucose, high lard or laboratory chow, although the enzyme's activity ratio (-cAMP)(+cAMP) was significantly elevated with lard feeding (0.49 +/- 0.02) as opposed to glucose feeding (0.43 +/- 0.01). Crude preparations from lard and glucose fed animals were equivalent in inhibitory activity when tested with enzyme from chow fed animals. Agarose column chromatography separated holoenzyme and C subunit forms of the protein kinase when 500 mM NaCl was present in the elution buffer. Absence of the salt allowed subunit reassociation to occur. Direct addition of NaCl greater than or equal to 75 mM significantly inhibited protein kinase activity. The results indicate that the adipose tissue of rats fed a high lard diet has a higher concentration of cAMP and an increased protein kinase activity ratio than tissue from rats fed a fat free, high glucose diet. Total cAMP-dependent protein kinase activity and the level of a thermostable macromolecular inhibitor remained unchanged.     

Physiological activation of brown adipose tissue destabilizes thermogenin mRNA

The amount of mRNA coding for the brown fat specific uncoupling protein thermogenin was followed in the brown adipose tissue of adult mice. As expected, cold exposure or norepinephrine injection caused an increase in the amount of thermogenin mRNA. However, contrary to expectation, the half-life of thermogenin mRNA was dramatically reduced, from about 18 h to about 3 h, when the mice were cold exposed. This destabilization of thermogenin mRNA was not related to the activity of protein synthesis. It was concluded that in brown adipose tissue an unusual mechanism operates which leads to a destabilization of thermogenin mRNA under the same physiological conditions which increase thermogenin gene expression.     

Activation of protein kinase in thyroid slices by thyroid-stimulating hormone.

Protein kinase activity in homogenates of control thyroid slices and those incubated with thyroid-stimulating hormone (TSH) and prostaglandin EI was assayed and correlated with changes in cyclic adenosine 3':5'-monophosphate (cAMP) concentrations and binding of [3H]cAMP. Both TSH and prostaglandin E1 (25 mug/ml) increased protein kinase activity and the activity ratio (expressed as activity - cAMP to activity plus cAMP). It is unlikely that such activation reflects effects of the increased cAMP liberated at the time of homogenization. Hormone-induced activation of protein kinase persisted even after the homogenate had been diluted so that its cAMP concentration would be insufficient to achieve maximal activation of the enzyme. In contrast to the previous results of J. D. Corbin, T. R. Soderling, and C. R. Park ((1973 J. Biol. Chem. 248, 1813) using adipose tissue, homogenization of thyroid tissue in 0.5 M NaCl and chromatography using Sephadex G-100 did not seem to stabilize dissociation of protein kinase into its receptor and catalytic subunits. However, increasing amounts of NaCl in the homogenizing buffer were associated with an increase in the cAMP independence of enzyme activity. Dilution of the homogenate did not change the protein kinase activity ratio whether the homogenizing buffer contained NcCl or not. Increasing concentrations of NaF inhibited protein kinase activity. Within 1 to 3 min of incubation of thyroid slices with TSH, protein kinase activity and the activity ratio were increased significantly. This correlated quite well with increased cAMP concentrations in the slices and inhibition of [3H]cAMP binding to the homogenates. Maximal activation of the enzyme was achieved by 10 min which corresponds to the time of maximal effect on cAMP concentrations. Activation of protein kinase was achieved by 0.125 milliunit/ml of TSH and maximal effects with 0.5 to 1.25 milliunits/ml. These amounts agree well with those required for other effects of TSH. Although larger amounts of TSH produced even greater increases in cAMP concentrations this was not always associated with augmented inhibition of [3H]cAMP binding. These results are compatible with the concept that the TSH-mediated increase in cAMP is associated with activation of protein kinase in the intact cell. They also suggest that not all of the intracellular cAMP is available for activation of protein kinase.     

In vivo brown fat response to hypothermia and norepinephrine in the ovine fetus

The goal of this study was to assess the response of fetal brown fat in vivo to hypothermia and norepinephrine infusion. In 10 unanaesthetized, chronically-prepared fetal sheep (133 +/- 2 days of gestation) cold water was passed through tubing encircling the fetus in utero and plasma glycerol concentration was measured as an indicator of brown fat activity. Following cooling for 60 min, amniotic fluid temperature fell 7.79 degrees C to 31.66 +/- 1.73 degrees C (n = 8, P less than 0.001) and maternal temperature fell 0.63 degree C to 38.63 +/- 0.08 degrees C (n = 9, P less than 0.001). Eight of the fetuses were subjected to a second experiment in which norepinephrine was infused intravenously for 15 min. During infusion fetal arterial temperature fell 0.38 degrees C to 39.05 +/- 0.25 degrees C (n = 7, P less than 0.05). Amniotic fluid temperature (n = 7, NS) and maternal arterial temperature (n = 7, NS) remained constant. Glycerol concentration during the infusion increased from 0.73 to 1.27 mg/dl, a 74% increase over control (n = 8, P less than 0.001). Although clearly detectable, these glycerol responses to hypothermia and norepinephrine stimulation are one-third or less of those achieved after birth, indicating that thermogenesis remains quiescent in the near-term fetal sheep, despite powerful stimuli for activation.     

Adenylyl cyclase pathway is involved in the regulation of intracellular calcium level regulation in brown preadipocytes

Mechanism of adrenergically activated calcium response in freshly isolated brown preadipocytes was studied with fluorescent probe Fura-2. Application of a direct activator of adenylylcyclase forskolin or cell permeable analog BrcAMP caused rise in the intracellular calcium level that was even higher than after the application of norepinephrine. Protein kinase A inhibitor H-89 in a dose-dependent manner reduced, while inhibitor of total phosphodiesterase activity IBMX, or protein phosphatase inhibitor ocadaic acid enhanced norepinephrine or isoproterenol initiated cellular calcium responses. It is concluded that cAMP and protein kinase A mediated phosphorylation play a crucial role in adrenergically initiated calcium signalling in brown preadipocytes.     

Variations of cyclic AMP and cyclic GMP contents of the liver and of brown and white adipose tissues of the rat during the first week of cold exposure.

The variation in the amounts of cyclic AMP and cyclic GMP were studied in white and brown adipose tissues and in the liver of rats during the first week of cold exposure (5 degrees C). In white fat, only a small increase in cAMP was observed on the first day. In brown fat, parallel decreases in cAMP and cGMP contents were induced which might be related to a large mobilization of tissue fatty acids. In the liver, cold exposure barely affected the cAMP content but the level of cGMP was markedly increased. These results are discussed with regard to the respective role of these different tissues in cold-induced energetic substrate mobilization.     

Chemical sympathetic denervation, suppression of myocardial transient outward potassium current, and ventricular fibrillation in the rat

Sympathetic denervation is frequently observed in heart disease. To investigate the linkage of sympathetic denervation and cardiac arrhythmia, we developed a rat model of chemical sympathectomy by subcutaneous injections of 6-hydroxydopamine (6-OHDA). Cardiac sympathetic innervation was visualized by means of a glyoxylic catecholaminergic histofluorescence method. Transient outward current (Ito) of ventricular myocytes was recorded with the whole-cell configuration of the patch clamp technique. We observed that sympathectomy (i) decreased cardiac sympathetic nerve density and norepinephrine level, (ii) reduced the protein expression of Kv4.2, Kv1.4, and Kv channel-interacting protein 2 (KChIP2), (iii) decreased current densities and delayed activation of Ito channels, (iv) reduced the phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and cAMP response element-binding protein (CREB), and (v) increased the severity of ventricular fibrillation induced by rapid pacing. Three weeks after 6-OHDA injections, which allowed time for sympathetic regeneration, we found cardiac sympathetic nerve density, norepinephrine levels, expression levels of Kv4.2 and KChIP2 proteins, and I(to) densities were partially normalized and ventricular fibrillation severity was decreased. We conclude that chemical sympathectomy downregulates the expression of selective Kv channel subunits and decreases myocardial I(to) channel activities, contributing to the elevated susceptibility to ventricular fibrillation.     

Differences in thermal sensitivities of the regulatory-catalytic and catalytic forms of cyclic AMP-dependent protein kinases from various tissues.

The cAMP-dependent protein kinase from various tissues was more thermally sensitive when activated by cAMP than the non-activated enzyme. For example, when the activity ratio (the activity of protein kinase assayed -cAMP/+cAMP) was 0.40, 80% and 76% of total hepatic cAMP dependent protein kinase activity was recoverable after incubations at 45 degrees C for 15 and 30 minutes, respectively. However, when the activity ratio was elevated to about 0.80 - 0.90 by increasing cAMP levels in vivo or adding exogenous cAMP to soluble liver extracts, the total protein kinase activity recoverable after incubations at 45 degrees C for 15 minutes was 34-44% and 19-22%, respectively. This observation was used to estimate the degree of activation of the enzyme in vivo and in vitro, since the loss of enzyme activity at 45 degrees C was directly related to the degree of activation of the enzyme in tissue extracts. The regulatory-catalytic form of cAMP-dependent protein kinase was thermally resistant at 45 degrees C unless activated by incubation with exogenous cAMP, histones or NaCl, while the catalytic form of the enzyme was highly thermally sensitive at this same temperature. These data describe a new property of the cAMP-dependent protein kinase and suggest an alternative method which measure the degree of activation of the enzyme.     

Beta-adrenergic, cAMP-mediated stimulation of proliferation of brown fat cells in primary culture. Mediation via beta 1 but not via beta 3 adrenoceptors.

The ability of adrenergic stimulation to affect the rate of DNA synthesis in mouse brown adipocyte precursor cells proliferating in primary culture was investigated. Addition of 1 microM norepinephrine to the cells at day 4 in culture (proliferating cells) significantly increased the rate of DNA synthesis, whereas no significant effect was seen at day 9 (confluent cells). The effect of norepinephrine could be mimicked by forskolin, cholera toxin, and by cAMP analogues. Specific [3H]thymidine incorporation (per unit of DNA) was reduced by norepinephrine stimulation, indicating saturation of the salvage pathway for dTTP synthesis already in unstimulated cells and implying a beta-adrenergic stimulation of dTTP synthesis. Pharmacological characterization of this effect indicated it was mediated by beta 1 receptors, with alpha 2 receptors exerting an opposing effect. Notably, the stimulation of DNA synthesis was not observed with the beta 3-specific agonist CGP-12177. In contrast, both norepinephrine and CGP-12177 were able to induce the expression of the uncoupling protein thermogenin in the confluent cultured cells. This coincided with a shift in the cAMP-elevating potential of CGP-12177, from being antagonistic to norepinephrine stimulation in proliferating cells to being itself a full agonist in confluent cells, implying occurrence of coupled beta 3 receptors as part of the differentiation process. It was concluded that brown fat precursor cells respond directly to norepinephrine stimulation with an increased DNA synthesis, and that this response is mediated via the classical beta 1 receptors. This probably represents the cellular basis for the hyperplasia observed in the tissue in physiologically recruited states.     

Comparative subcellular fractionation of control and cold-adapted rat brown and white adipose tissue with special reference to peroxisomal and mitochondrial distributions

A new technique for single-step subcellular fractionation of adipose tissue homogenates by analytical sucrose density gradient centrifugation in a vertical pocket reorientating rotor is described. The density gradient distributions of mitochondrial and peroxisomal marker enzymes in brown and white adipose tissue of control and cold exposed rats are compared. The equilibrium density of brown fat mitochondria was found to be significantly increased compared with white fat mitochondria. GDP binding activity was localized solely to the mitochondria in both control and cold-adapted brown adipose tissue. Brown and white fat mitochondria fractions were isolated by differential centrifugation and the specific activities of various enzymes in the homogenate and mitochondrial preparations determined. The specific activity of creatine kinase in brown adipose tissue was found to be ten-fold higher than in white fat and subcellular fractionation studies showed the activity to have an exclusively cytosolic distribution in both tissues. GDP binding activity and some of the mitochondrial enzymes showed, in brown adipose, a striking increase in total activity in cold adapted rats compared to control animals. For some enzyme activities there was a small increase when expressed per mg tissue or per mg mitochondrial protein. When expressed per mg DNA i.e. per cell, there was a reduced specific activity of the mitochondrial and peroxisomal enzymes in both brown and white adipose tissue on cold adaptation.     

Brown adipose tissue function in short-chain acyl-CoA dehydrogenase deficient mice

Brown adipose tissue is a highly specialized organ that uses mitochondrial fatty acid oxidation to fuel non-shivering thermogenesis. In mice, mutations in the acyl-CoA dehydrogenase family of fatty acid oxidation genes are associated with sensitivity to cold. Brown adipose tissue function has not previously been characterized in these knockout strains. Short-chain acyl-CoA dehydrogenase (SCAD) deficient mice were found to have increased brown adipose tissue mass as well as modest cardiac hypertrophy. Uncoupling protein-1 was reduced by 70% in brown adipose tissue and this was not due to a change in mitochondrial number, nor was it due to decreased signal transduction through protein kinase A which is known to be a major regulator of uncoupling protein-1 expression. PKA activity and in vitro lipolysis were normal in brown adipose tissue, although in white adipose tissue a modest increase in basal lipolysis was seen in SCAD−/− mice. Finally, an in vivo norepinephrine challenge of brown adipose tissue thermogenesis revealed normal heat production in SCAD−/− mice. These results suggest that reduced brown adipose tissue function is not the major factor causing cold sensitivity in acyl-CoA dehydrogenase knockout strains. We speculate that other mechanisms such as shivering capacity, cardiac function, and reduced hepatic glycogen stores are involved.     

Protein kinase A-mediated cell proliferation in brown preadipocytes is independent of Erk1/2, PI3K and mTOR

The physiological agonist norepinephrine promotes cell proliferation of brown preadipocytes during the process of tissue recruitment. In a primary culture system, cAMP mediates these adrenergic effects. In the present study, we demonstrated that, in contrast to other systems where the mitogenic effect of cAMP requires the synergistic action of (serum) growth factors, especially insulin/IGF, the cAMP effect in brown preadipocytes was independent of serum and insulin. Protein kinase A, rather than Epac, mediated the cAMP mitogenic effect. The Erk 1/2 family of MAPK, the PI₃K system and the mTOR complexes were all activated by cAMP, but these activations were not necessary for cAMP-induced cell proliferation; a protein kinase C isoform may be involved in mediating cAMP-activated cell proliferation. We conclude that the generally acknowledged cellular mediators for induction of cell proliferation are not involved in this process in the brown preadipocyte system; this conclusion may be of relevance both for examination of mechanisms for induction of brown adipose tissue recruitment but also for understanding the mechanism behind e.g. certain endocrine neoplasias.     

Sympathetic denervation of one white fat depot changes norepinephrine content and turnover in intact white and brown fat depots

It is well-established that the sympathetic nervous system (SNS) regulates adipocyte metabolism and recently it has been reported that sensory afferents from white fat overlap anatomically with sympathetic efferents to white fat. The studies described here characterize the response of intact fat pads to selective sympathectomy (local 6-hydroxydopamine (6OHDA) injections) of inguinal (ING) or epididymal (EPI) fat in male NIH Swiss mice and provide in vivo evidence for communication between individual white and brown fat depots. The contralateral ING pad, both EPI pads, perirenal (PR), and mesenteric (MES) pads were significantly enlarged 4 weeks after denervating one ING pad, but only intrascapular brown adipose tissue (IBAT) increased when both ING pads were denervated. Denervation of one or both EPI pad had no effect on fat depot weights. In an additional experiment, norepinephrine turnover (NETO) was inhibited in ING, retroperitoneal (RP), MES, and IBAT 2 days after denervation of both EPI or of both ING pads. NE content was reduced to 10-30% of control values in all fat depots. There was no relation between early changes in NETO and fat pad weight 4 weeks after denervation, even though the reduction in NE content of intact fat pads was maintained. These data demonstrate that there is communication among individual fat pads, presumably through central integration of activity of sensory afferent and sympathetic efferent fibers, that changes sympathetic drive to white adipose tissue in a unified manner. In specific situations, removal of sympathetic efferents to one pad induces a compensatory enlargement of other intact depots.     

UCP1-independent thermogenesis in white adipose tissue of cold-acclimated Ucp1-/- mice

Apart from UCP1-based nonshivering thermogenesis in brown adipocytes, the identity of thermogenic mechanisms that can be activated to reduce a positive energy balance is largely unknown. To identify potentially useful mechanisms, we have analyzed physiological and molecular mechanisms that enable mice, genetically deficient in UCP1 and sensitive to acute exposure to the cold at 4 degrees C, to adapt to long term exposure at 4 degrees C. UCP1-deficient mice that can adapt to the cold have increased oxygen consumption and show increased oxidation of both fat and glucose as indicated from serum metabolite levels and liver glycogen content. Enhanced energy metabolism in inguinal fat was also indicated by increased oxygen consumption and fat oxidation in tissue suspensions and increased AMP kinase activity in dissected tissues. Analysis of gene expression in skeletal muscle showed surprisingly little change between cold-adapted Ucp1+/+ and Ucp1-/- mice, whereas in inguinal fat a robust induction occurred for type 2 deiodinase, sarcoendoplasmic reticulum Ca2+-ATPase, mitochondrial glycerol 3-phosphate dehydrogenase, PGC1alpha, CoxII, and mitochondrial DNA content. Western blot analysis showed an induction of total phospholamban and its phosphorylated form in inguinal fat and other white fat depots, but no induction was apparent in muscle. We conclude that alternative thermogenic mechanisms, based in part upon the enhanced capacity for ion and substrate cycling associated with brown adipocytes in white fat depots, are induced in UCP1-deficient mice by gradual cold adaptation.