Differentiation of brown adipose tissue and biogenesis of thermogenic mitochondria in situ and in cell culture.

Differentiation and biogenesis of mitochondria in brown adipose tissue (BAT) was studied in situ and in cell culture by Western blotting, enzyme activity measurements, [35S]methionine incorporation and immunofluorescence microscopy. In different rodent species the perinatal development of BAT thermogenic function resulted from the formation of thermogenic mitochondria which replaced the preexisting nonthermogenic mitochondria. Their biogenesis was characterized by the sudden appearance and rapid increase of the uncoupling protein (UCP), increase of cytochrome oxidase (COX) and decrease of H(+)-ATPase. In primary cell culture, differentiation of precursor cells from mouse BAT to typical multilocular adipocytes was accompanied by increasing content of COX and H(+)-ATPase. A selective synthesis of UCP was induced by activation of beta-adrenergic receptors or by elevated levels of cellular cAMP. UCP was quantitatively incorporated into mitochondria and within 24 h after stimulation reached near physiological concentration. Both in situ and in cell culture, the conditions enabling the expression of UCP gene were accompanied by activation of intracellular thyroxine 5'-deiodinase.

Low content of mitochondrial ATPase in brown adipose tissue is the result of post-transcriptional regulation.

The mRNA levels of ATPase beta, ATPase 6, cytochrome oxidase (COX) VIb and COX I subunits were found to be 2.4-13.8-fold higher in brown adipose tissue (BAT) than in heart, skeletal muscle, brain and liver of mice. The comparison with tissue contents of ATPase and COX revealed that the selective, 5-11-fold reduction of ATPase in BAT is not caused by decreased transcription of ATPase genes. Likewise, the ATPase beta and COX VIb mRNA levels in cultured brown adipocytes were also not influenced by norepinephrine, which activated the expression of the UCP gene by two orders of magnitude. The results indicate that the biosynthesis of mitochondrial ATPase in BAT is post-transcriptionally regulated.

Induction of type II iodothyronine 5'-deiodinase and mitochondrial uncoupling protein in brown adipocytes differentiated in cell culture.

Brown adipocytes differentiated in primary cell culture were found to contain a type II iodothyronine 5'-deiodinase (5'D). Incubation of confluent cells with norepinephrine or dibutyryl-cAMP caused up to 17-fold increase in 5'D activity with a maximum after 8 h. Activation of 5'D required mRNA and protein synthesis and was accompanied by parallel, up to 5.8-fold increase in the amount of mitochondrial uncoupling protein with a maximum after 24 h. Analysis of adrenergic stimulation of 5'D suggested predominant involvement of the beta-receptors and increased intracellular cAMP levels, while the contribution of alpha 1-receptors was small.

Coordinate expression of beta-galactoside alpha 2,6-sialyltransferase mRNA and enzyme activity in suckling rat jejunum cultured in different media: transcriptional induction by dexamethasone.

In attempting to elucidate the molecular basis of the expression of alpha 2,6-sialyltransferase (alpha 2,6-ST) in jejunal explants of 7-day-old rats during cultivation, the total jejunal RNA was analysed by hybridization using a cDNA clone encoding rat liver alpha 2,6-ST. Under cultivation in both serum-free and serum-containing media jejunal alpha 2,6-ST mRNA closely paralleled the bound (100,000 g pellet) as well as the soluble (100,000 g supernatant) alpha 2,6-ST activity, the correlation coefficients being 0.976 and 0.816, respectively. Dexamethasone (Dx) treatment enhanced alpha 2,6-ST mRNA and membrane-bound alpha 2,6-ST activity in close correlation. Jejunal alpha 2,6-ST mRNA is sensitive to actinomycin D and is lost with apparently identical kinetics in Dx-stimulated and control explants, suggesting that regulation by Dx may be exerted by altering the rate of mRNA synthesis. Dx-dependent activation resulted in elevation of the 4.3-Kb mRNA and can be inhibited by the antiglucocorticoid onapristone, demonstrating the participation of the glucocorticoid receptor pathway.

Voltage-dependent chloride channels with several substates in excised patches from mouse neuroblastoma cells.

Single channels in mouse neuroblastoma cells with a high conductance of about 400 pS were described using the patch-clamp technique in the inside-out configuration. The channels were selective for Cl- as compared to cations and exhibited a linear I-V relationship between +40 and -40 mV. These Cl- channels were voltage-dependent and were activated by both depolarizing and hyperpolarizing potential steps from 0 mV to 10-40 mV. They closed, becoming inactivated, in tens of milliseconds (for depolarization) up to tens of seconds (for hyperpolarization) after each potential step. The typical feature of Cl- channels described was the dissipation of their conductance into several substates during the course of individual recordings.

Effect of age on kidney hyperplasia in the rat during cold acclimation.

In adult rats acclimated to 5 degrees C, the kidney weight increases with the length of the exposure. Kidney cells of younger rats survive explantation better than the cells of control rats of the same age. Kidney cells of older rats after cold acclimation and after unilateral nephrectomy have a lower vitality in vitro than corresponding control cells. Unilateral nephrectomy performed in rats aged 3 and 6 months, exposed previously to low temperature, leads to a better survival of explanted kidney cells than nephrectomy or cold only. Old rats do not survive exposure to 5 degrees C for more than 18 hr.

Proliferation and differentiation of chondrocytes in cell culture.

Chondrocytes freshly explanted from the sternal cartilage of 14- to 16-day-old chick embryos proliferate and differentiate in cell culture in a serum-free medium as well as in a medium containing 10% foetal calf serum. A comparable degree of multiplication and differentiation was found in chondrocytes cultivated in the serum-free medium containing native growth-promoting alpha-globulin (GPAG). The degree of proliferation was smaller in chondrocytes cultivated in a modified Eagle MEM which is fully chemically defined and contains low molecular weight substrates only. Since this medium does not contain either hormones or proteins, it is suitable for the cultivation of chondrocytes which should be employed when studying the mechanism of the effect of hormones which influence chondrocyte proliferation or chondrogenesis.

Pinocytosis of fluorescein-labelled growth-promoting alpha-globulin in human diploid cells.

Fluorescein isothiocyanate conjugated growth-promoting alpha-globulin (FITC-GPAG) binds to human diploid cells LEP 19 at 37 degrees C. After the initial binding step, labelled GPAG is progressively internalized by the cells. FITC-GPAG is present in pinocytic vesicles localized in the perinuclear region of the cells and its pinocytosis occurs without cell surface redistribution of GPAG.

Stimulation of DNA synthesis in rat liver and kidney by cold acclimation.

Exposure of adult Wistar female rats for 7 days to 4 degrees C leads to a marked increase in the weight of the liver and kidney, caused by an increased content of DNA and an increased number of cells in these organs. The weight as well as the DNA content of the cross-striated muscle do not change appreciably. Acclimation of the warmblooded rat to cold stimulates mitosis indirectly in cells capable of division, similarly as it stimulates directly the mitotic activity in mouse and human cells cultured and adapted to cold in vitro.

Complete replacement of serum in primary cultures of chick embryo brain cells by growth-promoting alpha-globulin.

Growth-promoting alpha-globulin (GPAG), a specific serum protein complex which induces mitotic activity in continually replicating metazoan cells in vitro, was shown in this study to support growth of astrocytes and mesenchymal cells as well as process formation of nerve cells isolated from cerebral hemispheres of chick embryos.

Vascular smooth muscle cells of male and female rats in culture, migration, proliferation and chromosome number.

The authors evaluated the migratory and proliferative properties and the chromosome number of cultivated male and female smooth muscle cells (SMC) obtained by the explanation method from the thoracic aorta of rats of a conventional and a specific pathogen-free (SPF) breed. It was found that male SMC, in most cases, began to migrate from the explants sooner than female SMC and that they migrated from a higher total number of explants. The time needed for the number of cells in the culture to double (doubling time) was practically the same for male and female SMC, but male SMC attained a higher maximum population density. Male SMC cultures (2nd passage) contained cells with a hyperploid chromosome number, whereas female SMC were diploid. It was also found that SMC from conventional rats, in which the presence of pathogens could be presumed, displayed higher migratory and proliferative capacity than the SMC of SPF rats. The capacity of the SMC of male rats for migration and proliferation could have been potentiated by the effect of a different composition of the intercellular matrix and a different chromosome number, and in conventional rats by the presence of pathogens.

Adrenergic control of induction of type II iodothyronine 5'-deiodinase activity in cultured mouse brown adipocytes.

Iodothyronine 5'-deiodinase (5'D) of mouse brown adipocytes differentiated in cell culture was characterized in detail with respect to the adrenergic control of its biosynthesis. The stimulation of 5'D required mRNA and protein synthesis and was dependent on the stage of differentiation of the cells. The maximum induction was observed around confluence (7-day-old cells), in pre- and post-confluent cells the 5'D activity was significantly less induced. The transient responsiveness of brown fat-cells to the stimulatory effect of adrenergic agents was reflected also in the time course of the induction of 5'D by different concentrations of agonists. The maximum response occurred regularly after an 8 h incubation and implicated a rather fast turnover of the induced enzyme. On the basis of the inhibitory effects of cycloheximide and actinomycin D, the half-life of the induced 5'D and its mRNA were estimated to be 1.5 and 3.3 h respectively. The noradrenaline-induced 5'D activity was shown to be that of the type II enzyme, insensitive to propylthiouracil (PTU). The estimated values of its apparent Km for thyroxine, Km for the co-substrate dithiothreitol, and Vmax. in the presence of 1 mM PTU were 2 nM, 2.6 mM, and 0.1 pmol of I-/h per mg of protein respectively. The 5'D activity was effectively induced by forskolin and dibutyryl cyclic AMP, as well as by isoprenaline, noradrenaline and CGP-12177, but not by phenylephrine, cirazoline or oxymetazoline. This indicates that, contrary to previous observations in vivo, stimulation of 5'D in cultured brown fat-cells involves elevated cyclic AMP levels and is mediated predominantly via beta-receptors, particularly via the so-called beta 3-adrenoceptors.

Fibronectin is not a serum spreading factor for HeLa cells.

The spreading of HeLa cells on plastic substratum is mediated by fibronectin-depleted foetal calf serum but not by fibronectin isolated by gelatin-Sepharose affinity chromatography. The same is true for freshly explanted chick embryonic chondrocytes. In contrast, BHK cell spreading exceeds 67% after 120 min at 37 degrees C in fibronectin-supplemented (10 micrograms ml-1) serum-free medium. Long-term cultivation of HeLa cells in Eagle's MEM supplemented with fibronectin-free serum is associated with the accumulation of cells in mitosis or before cytokinesis; many cells die and the remaining living cells, characterized by marked changes in morphology, multiply very slowly. It can be concluded therefore that fibronectin does not produce spreading in HeLa cells but forces them into mitosis.

Accumulation of boron-10 (10B) in cell cultures exposed to mercaptododecaborate (Na2H(11)10B12SH) used for the neutron capture therapy of brain tumors.

Toxicity of mercaptoundecahydro-closo-dodecaborate (MHB, Na2H(11)10B12SH) and accumulation of MHB-derived 10B were studied in E7 neuroblastoma, C6 glioma, HeLa cells and embryonic lung LEP 19 fibroblasts in culture in exponential and stationary phases of growth (2- and 7-day-old cultures, respectively). The pilot study of acute toxicity, performed on C6 glioma cells, showed good tolerance of the drug up to 1000 micrograms/ml (4.8 x 10(-3) M), when cell growth slowed and a small part of the population was lethally damaged (8.3%, 20-h incubation interval). The changes became more extensive and appeared sooner (toward 5 h) at 2000 micrograms MHB/ml (9.5 x 10(-3) M). None of the four cell lines used was found to be affected in gross morphology or growth by 200 micrograms MHB/ml within a 5-day culture interval. When exposed to this dose for 4 h, the amount of 10B accumulated in cell lines at the exponential growth phase ranged from 0.51 to 4.4 ng/micrograms protein; in the stationary cultures of the corresponding cell phenotype, the 10B values were 3 to 10 times lower (0.12-1.2 ng/micrograms protein). Irrespective of the growth phase, the values achieved in C6 glioma cells were several times higher than in the other cell lines. Furthermore, in the glioma cells, particularly in the exponential phase of growth, accumulation of 10B proceeded against the marked concentration gradient. The data provide a new indication for the use of MHB for boron neutron capture therapy of brain tumors.

Establishment of mouse neuroblastoma clone E 7 in serum-free medium.

Ninety-six clonal populations were derived from a wild mouse neuroblastoma cell population C 1300 in a serum-free medium containing commercially available serum growth-promoting proteins (GPP). From among these 96 lines the clonal population E 7 was chosen for further work because it displayed maximum spontaneous morphological differentiation. The neuroblastoma clonal population differs morphologically from the original population; it was defined both cytogenetically and by means of growth parameters. The cells of the neuroblastoma clone E 7 are hypertetraploid with two chromosome number modals - 88 and approximately 180-200. The majority of telocentric chromosomes in metaphases with a modal number of 88 chromosomes are identical with the chromosomes of mouse diploid cells. The cell generation time is 22 hours. The cells of the clonal population E 7 are highly sensitive to the action of ethanolamine, which induces morphological differentiation, so that the processes of 30% of the cells in the population are over 40 micron long. Electrophysiological studies showed that the cells of the neuroblastoma clonal population E 7 retain the character of excitable cells and they are thus suitable for studying some of the properties of nervous tissue cells.

Regulation of sialylation of intestinal brush-border enzymes and of sialyltransferase activity in organ cultures by dexamethasone.

To investigate the direct effect of glucocorticoids on sialylation of intestinal brush-border hydrolases, explants of fetal and suckling rat intestine were maintained in serum-free or serum-containing organ culture with or without dexamethasone (Dx). Glucoamylase and dipeptidyl peptidase IV developed in organ culture from 18-day-old fetuses persisted in highly sialylated forms for 8 days irrespective of Dx presence, parallel in vivo development leading to less sialylated forms at the age of 6 days. In postnatal cultures the Dx-stimulated glucoamylase appeared in a new highly sialylated form never seen after the hormone application in vivo. These findings are in agreement with the elevation of bound sialyltransferase (ST) of cultured intestine in protein-free media. In serum-containing medium Dx stimulated the formation and release of soluble ST into the culture medium.

Synthesis of mitochondrial uncoupling protein in brown adipocytes differentiated in cell culture.

In order to characterize the biogenesis of unique thermogenic mitochondria of brown adipose tissue, differentiation of precursor cells isolated from mouse brown adipose tissue was studied in cell culture. Synthesis of mitochondrial uncoupling protein (UCP), F1-ATPase, and cytochrome oxidase was examined by L-[35S]methionine labeling and immunoblotting. For the first time, synthesis of physiological amounts of the UCP, a key and tissue-specific component of thermogenic mitochondria, was observed in cultures at about confluence (day 6), indicating that a complete differentiation of brown adipocytes was achieved in vitro. In postconfluent cells (day 8) the content of UCP decreased rapidly, in contrast to some other mitochondrial proteins (beta subunit of F1-ATPase, cytochrome oxidase). In these cells, it was possible, by using norepinephrine, to induce specifically the synthesis of the UCP but not of F1-ATPase or cytochrome oxidase. The maximal response was observed at 0.1 microM norepinephrine and the synthesis of UCP remained activated for at least 24 h. Detailed analysis revealed a major role of the beta-adrenergic receptors and elevated intracellular concentration of cAMP in stimulation of UCP synthesis. A quantitative recovery of the newly synthesized UCP in the mitochondrial fraction indicated completed biogenesis of functionally competent thermogenic mitochondria.