Expression of specific white adipose tissue genes in denervation-induced skeletal muscle fatty degeneration.

Denervation of skeletal muscle results in rapid atrophy with loss of contractile mass and/or progressive degeneration of muscle fibers which are replaced to a greater or lesser degree by connective and fatty tissues. In this study, we show that denervated rabbit muscles are transformed into a white adipose tissue, depending on their fiber types. This tissue does express LPL, G3PDH and particularly the ob gene, a white adipose tissue-specific marker, and does not express the brown adipose tissue molecular marker UCP1 mRNA.

In vitro induction of UCP1 mRNA in preadipocytes from rabbit considered as a model of large mammals brown adipose tissue development: importance of PPARgamma agonists for cells isolated in the postnatal period.

UNLABELLED: In mammals with a lower mass-specific metabolic rate than small laboratory rodents, the brown adipose tissue (BAT) loses its thermogenic activity after birth and undergoes a transformation into white adipose tissue (WAT). Rabbit is a model of these mammals of larger body mass. Preadipocytes from cervical BAT of foetal or newborn rabbits differentiated in a chemically-defined medium and expressed low levels of uncoupled protein-1 (UCP1) mRNA, greatly increased by beta3-adrenergic or retinoic acid stimulations. On the contrary, preadipocytes from 1-month-old animals differentiated in the same conditions with no detectable,expression of UCP1. Peroxisome proliferator-activated receptor gamma (PPARgamma) agonists were necessary to induce UCP1 in these cells from older animals, a synergistic increase being noted in the presence of beta3-adrenergic agonists. In contrast to these results, preadipocytes from perirenal WAT stimulated by PPARgamma agonists never expressed UCPI. CONCLUSION: preadipocytes in the postnatal period are determined as brown or white preadipocytes. PPARgamma agonists induce UCP1 expression in brown postnatal preadipocytes, but they are unable to trigger the gene in white preadipocytes.

Expression of c-erbA alpha, c-erbA beta and Rev-erbA alpha mRNA during the conversion of brown adipose tissue into white adipose tissue.

The levels of mRNA encoding uncoupling protein (UCP), thyroid hormone receptors (c-erbA alpha, c-erbA beta) and a related protein Rev-erbA alpha have been studied in brown (pericervical) and white (perirenal) rabbit adipose tissues from birth to 180 days. The c-erbA alpha and c-erbA beta genes are expressed at similar levels in the two tissues. The alpha 1, alpha 2 and beta 1 transcripts do not change notably during development or during the conversion from brown to white phenotype which occurs in pericervical during postnatal life. Rev-erbA alpha mRNA is barely detectable at birth and dramatically increases between 7 and 30 days. However, this increase is not tissue-specific and is also observed in liver and heart. In conclusion, our results show that the decline in UCP expression during the transition from brown to white phenotype cannot be related to changes in the profiles of thyroid hormone receptors or Rev-erbA alpha mRNA expression. These profiles are not different between adipose tissue sites which are brown or white at birth.

The beta 3-adrenoceptor agonist ICI-D7114 is not as efficient on reinduction of uncoupling protein mRNA in sheep as it is in dogs and smaller species.

Adipose tissue in newborn lambs is brown, but within a few days it is transformed into white adipose tissue. In the same way, preadipocytes cultured in serum-free chemically defined medium achieve full differentiation and express uncoupling protein (UCP), a marker of brown adipose tissue, when isolated from perirenal adipose tissue of the newborn, whereas they no longer express UCP when isolated from older lambs. The effects of a chronic stimulation of adipose tissue by novel beta 3-adrenoceptor agonist (ICI D7114) on the maintenance after birth and on the reinduction in older lambs of UCP mRNA in adipose tissue were studied. Treatment of newborn lambs with this agonist for 25 d maintained a slight level of UCP mRNA in perirenal and pericardiac, but not in omental and inguinal, adipose tissue depots. Preadipocytes isolated from perirenal adipose tissue of treated animals differentiated, in vitro, into adipocytes, but no UCP mRNA could be detected either in the absence or in the presence of the beta 3-adrenoceptor agonist in the culture medium. Treatment of 1-mo-old lambs with ICI D7114 for 12 d restored UCP mRNA in perirenal and pericardiac adipose tissues of two of four treated lambs, but at a much lower level than in the same tissues at birth. In both experiments, the final BW and the ADG of lambs treated with ICI D7114 were not statistically different from controls. These results are quite different from those obtained with the same beta 3-adrenoceptor agonist in dogs and rodents.

Differentiation of adipocyte precursors in a serum-free medium is influenced by glucocorticoids and endogenously produced insulin-like growth factor-I.

Stromal vascular cells from rabbit perirenal adipose tissue differentiated at a high frequency in a chemically-defined serum-free medium containing insulin, transferrin, tri-iodothyronine and dexamethasone. The omission from the culture medium of dexamethasone resulted in a lack of adipose conversion. Addition of IGF-I increased glycerol-3-phosphate dehydrogenase (GPDH) activity. The conditioned media from adipocyte precursor cells contained measurable quantities of immunoreactive IGF-I as determined by RIA after neutralization of IGF binding proteins interference. Dexamethasone increased IGF-I secretion during the first seven days after plating and decreased IGF-I binding to conditioned media. Three molecular forms of IGF binding proteins (IGFBPs) were identified by Western ligand blots in conditioned media, with M(r) = 40,000, 29,000 and 25,000. The major form (M(r) = 29,000) was decreased by dexamethasone. In contrast, the M(r) = 24,000 form was increased. Specific binding of 125I-labelled IGF-I to rabbit adipocyte precursor cells was more effectively inhibited by unlabelled IGF-I than by unlabelled IGF-II or insulin. The electrophoretic migration of cross linked 125I-IGF-I to microsomal membranes revealed a complex with M(r) = 130,000 under reducing conditions corresponding to the alpha-subunit of the IGF-I receptor. The addition of IGF-I monoclonal antibody to rabbit adipocyte precursor cells cultured in serum-free medium significantly inhibited [3H]-thymidine incorporation and significantly decreased (50%) GPDH specific activities. This inhibitory effect was overcome by the addition of exogenous IGF-I. Thus stromal vascular cells isolated from perirenal adipose tissue secrete IGF-I and IGFBPs, possess IGF-I receptors and respond to exogenous and endogenous IGF-I.(ABSTRACT TRUNCATED AT 250 WORDS)

Transfer of genes for utilization of starch (sta2) and melibiose (mel) to industrial strains of Saccharomyces cerevisiae by single-chromosome transfer, using a kar1 mutant as vector.

A method has been developed for the transfer of genes from other yeast strains and species to industrial yeast strains, using a haploid, kar1-1 mutant strain of Saccharomyces cerevisiae as a vector. The sta2 gene, conferring the ability to metabolize starch was transferred from an auxotrophic haploid strain of S. cerevisiae (S. diastaticus) and the melibiose-metabolism (mel) gene(s), from S. kluyveri, to the kar1-1 mutant [K5-5A; (alpha ade2 his4 can1 gal) by normal mating and protoplast fusion. From this strain, the genes were transferred to baker's yeast and brewing yeast strains, which did not utilize starch, and to baker's yeast strains, which did not utilize melibiose, by protoplast fusion, spore-cell pairing, or rare-mating. Strains that utilized starch or melibiose were obtained by all three methods. Pulsed-field gel electrophoresis preparations showed little change in the mobility of the chromosomes of the hybrids. The most probable explanation for the results obtained is that single chromosomes were transferred, first, from the donor strains to the kar1-1 haploid mutant strain, and then from the kar1-1 vector to the recipient industrial strain of S. cerevisiae. The transfer of the genes is probably accomplished through formation of disomic strains and then, in the case of the hybrids that metabolize starch, by integration of the sta2 gene into the genome of the industrial yeast strains.

Differentiation of ovine brown adipocyte precursor cells in a chemically defined serum-free medium. Importance of glucocorticoids and age of animals.

The rapid apparent conversion of brown adipose tissue into white adipose tissue in newborn offspring of large mammals, such as sheep and cattle is not explained at the cellular level. To study the differentiation of lamb brown adipocyte, a genomic fragment corresponding to the uncoupling protein was cloned from an ovine DNA library. Stromal vascular fibroblasts isolated from the perirenal adipose tissue of newborn lambs completely differentiated into brown adipocytes expressing the uncoupling protein gene, in a chemically defined serum-free medium. Dexamethasone was necessary for the expression of the uncoupling protein gene. When stromal vascular fibroblasts were isolated from 3-week-old lambs, the glucocorticoid analog still promoted in vitro differentiation of adipocytes. However those adipocytes were unable to express uncoupling mRNA and could be considered as white adipocytes. The data indicate that dexamethasone is necessary but not sufficient clone for the complete differentiation of brown adipocytes, and that the preadipocytes are committed to differentiation into brown or white adipocytes before culture.

In vivo effects of a treatment with antibodies to adipocyte plasma membranes in the rabbit.

Antibodies against rabbit adipocyte plasma membranes were injected in 6-week-old rabbits. Controls received normal IgG. Animals were killed 1, 2, 5 or 9 weeks after treatment. Body weight and food intake were reduced significantly until the 7th week for the live weight and the 5th week for the intake. Whatever the anatomical location considered, adipose tissue was markedly reduced: -75% for week 1 and -20% for week 9 respectively for the total adipose mass. Cell volume and enzymatic activities of G3PDH, LPL and LDH were highly decreased during the first 2 weeks after treatment. Simultaneously the plasma levels of triglycerides and plasma free fatty acids were increased. As shown by others in the rat, it is possible to induce a long-term fatness reduction in the rabbit by treatment with antibodies to adipocyte plasma membranes. The cytotoxic effects of antibodies have also been discussed.

Differentiation of rabbit adipocyte precursor cells in a serum-free medium.

A serum-free, hormone-supplemented medium containing insulin, transferrin, and triiodothyronine (ITT medium), able to support differentiation of rat adipose precursor cells, has been used to study the regulation of the development of adipocytes in the rabbit. Adipose conversion was assessed by the appearance of glycerol-3-phosphate dehydrogenase activity. Stromal-vascular cells from rabbit perirenal adipose tissue differentiated to a very low extent or not at all in ITT medium. Supplementation of ITT medium with growth hormone or fibroblast growth factor did not increase the proportion of differentiated cells. In contrast, rabbit stromal-vascular cells were able to differentiate in ITT medium supplemented with glucocorticoids (dexamethasone, corticosterone) whereas sex steroids (beta-estradiol, testosterone, progesterone) did not affect the differentiation process. In the presence of both dexamethasone and insulin, 20 to 50% of rabbit stromal-vascular cells differentiated into adipocytes within 2 wk of culture. The stimulatory actions of dexamethasone or insulin were dose-dependent. Insulin-like growth Factor-I (IGF-I), did not replace insulin under our culture conditions and had only a slight effect when added along with dexamethasone (100 nM) and insulin (1.7 nM). The results suggest that glucocorticoids, in association with insulin, may play an important role in the development of adipocytes from rabbit precursor cells.

Differentiation of rabbit adipocyte precursors in primary culture.

A primary culture system was used to study the adipose conversion of adipocyte precursors derived from the stromal-vascular fraction of perirenal adipose tissue of rabbit fetuses Differentiation was assessed by the development of glycerol-3-phosphate dehydrogenase, Acid:CoA ligase and lipoprotein lipase activities. Stromal-vascular cells were not able to differentiate when maintained in a medium supplemented with fetal calf serum or with rabbit serum. In contrast, differentiation was induced when the medium was supplemented with rabbit plasma. It also occurred when the growth phase was performed in serum provided that the serum was replaced by plasma when the cultures reached confluence. Supplementation of the culture medium with mesenteric lymph or chylomicrons as lipid sources greatly enhanced both lipid accumulation and the level of enzymatic markers of adipocyte differentiation. Following confluence in serum, cell proliferation ceased almost completely. In contrast, cells in the presence of plasma continued to proliferate, leading to a higher cell density at the time of adipocyte differentiation. These results suggest a positive effect of plasma on the post-confluent mitoses of susceptible cells. To our knowledge, it is the first time that such a difference between plasma and serum has been shown for the differentiation of adipocytes, using an homologous system. These studies also demonstrate that rabbit adipocyte precursors differentiating in primary culture show both similarities to and differences from the adipocytes of cell lines or cell precursors obtained from other animal species.

Longitudinal study of adipose cell size in the dorsoscapular and perirenal depots of the growing rabbit.

The changes in fat cell size during normal growth of New Zealand rabbits were investigated longitudinally with serial dorsoscapular and perirenal fat biopsies. A remarkably complex pattern of changes appeared when individual evolutions were considered. About 50% of the rabbits were characterized by "significant drops" of mean diameter during fat tissue growth with shifting of distributions toward the smaller cells. These "drops" could not be attributed to regional variation observed within each depot or to growth or food intake disorders. Differences in behavior of perirenal and dorsoscapular depots were noted. The "drops" occurred earlier in perirenal than in dorsoscapular depots. The meaning of these "drops" remains unclear, but the results suggest that they may be due to a discontinuous recruitment of new observable cells. These results are discussed in relation to the hypothesis of a "critical size" of adipocytes.

Adipose tissue regeneration in 6-month-old and adult rabbits following lipectomy.

The effects of surgical ablation of adipose tissue were studied in male New Zealand rabbits. They were lipectomized or sham-operated either at 6 or 12 months, ages at which size and number of adipocytes are, respectively, stabilized in this species. The lipectomized animals were subjected to removal of about 80% of the perirenal and omental and to the totality of the dorsoscapular and inguinal fat tissues. Approximately 35 and 48% of the total body fat were, thus, surgically removed, respectively, in 6- and 12-month-old rabbits. All rabbits were killed 3 months after surgery and were carefully dissected. There was no significant difference in food consumption and body weight gain between lipectomized and sham-operated rabbits. Surgical removal of dorsoscapular, inguinal, and omental fat did not lead to regeneration whereas regeneration of the perirenal fat was substantial. At sacrifice the perirenal weight reached approximately 55% of the initial weight. Regeneration of perirenal adipose tissue in adults proceeded at roughly the same rate as after lipectomy in younger rabbits. These results suggest that adipose tissue regeneration in the rabbit is site dependent.

[Effect of rearing method on the storage of fats by adipose tissue in sheep]. / Influence d'un mode d'élevage sur la mise en réserve des graisses par le tissu adipeux chez les ovins.

Adipocyte synthesis de novo and lipoprotein lipase activity have been used simultaneously to measure the lipogenic activity of adipose tissue in sheep. Acetate and glucose were used as precursors of fatty acid synthesis. The sheep were raised either outdoors or in a sheepfold. They were slaughtered by lots at mean weights of 24 and 32.5 kg. Compared to lipoprotein lipase activity, de novo synthesis of fatty acids was the main way of constituting lipid depositions. Raising the sheep outdoors favored the use of glucose as precursor of lipid synthesis at the first slaughter stage at 24 kg. Later at 32.5 kg, glucose utilization was practically zero compared to acetate, whatever the mode of rearing. The NADPH production needed for fatty acid synthesis was almost entirely due to NADP isocitrate dehydrogenase activity. Variations in both de novo synthesis and in lipoprotein lipase activity in relation with rearing method and slaughter weight were especially evident in the group raised outdoors.

[Myosin isoenzymes during in vivo and in vitro differentiation of chick muscles]. / Isozymes de la myosine au cours de la différenciation in vivo et in vitro des muscles du poulet.

Muscle fibers differentiated, in vitro, from myoblasts of embryonic Pectoralis (presumptive fast) and embryonic Adductor magnus (presumptive slow) muscles synthesise the same type of myosin, which is identical to the type synthesised in day 10 embryonic muscles. This type of myosin comigrates with the third isozyme of the adult fast-twitch muscle. There is no change in the kind of myosin in cultures aged from 2 to 7 weeks, whereas during the in vivo differentiation of the same muscles, new isozymes appear which are different in the two muscles. With regard to myosin, the muscle fibers differentiated, in vitro, from myoblasts of fast or slow muscles expressed the same phenotype.

Influence of experimental hypothyroidism on chick myogenesis.

The influence on chick myogenesis of hypothyroid status induced in ovo by methimazole was histologically (number and types of muscle fibers) and biochemically studied. A single injection of methimazole induced an hypothyroid status during embryogenesis, as shown by the plasma T4 levels which were separately assayed in male and female controls and treated embryos from day 12 of incubation to day 4 after hatching. In both sexes, control plasma T4 reached a peak on day 20 of incubation, but the female values were significantly higher; plasma T4 in the treated embryos remained at a low level independently of sex. Only methimazole-treated males showed a significant increase (P less than 0.01) in the total number of fibers of the two muscles (tibialis posterior and flexor digitorum) studied. Moreover, the histochemical results on succinate dehydrogenase and myofibrillar ATPase evidenced that, of the three fiber types (alpha R, alpha W and beta R) constituting the skeletal muscles, only the alpha fibers increased significantly. Aldolase (glycolytic) and NADP isocitrate dehydrogenase (tricarboxylic acid cycle) activities, measured on the adductor and pectoralis muscles, showed a similar developmental pattern in control and treated animals, but was retarded in the latter due to a 5-day delay in hatching. It is not known whether the tissular differences were related directly to the hypothyroid status, to alterations in nervous system differentiation or, as suggested by intersexual differences, to modifications in hormonal balance.

[Sexual differences in the development of plasma thyroxine levels in the embryo and young chick]. / Différences sexuelles de l'évolution de la thyroxinémie chez l'embryon et le jeune poulet.

Plasma thyroxine (T4) levels were separately ascertained in male and female embryo and young chicken from the 12th day of incubation till the 4th day after hatching by the thyroxine binding globulin technique. In both sexes, plasma T4 reach a peak the 20th day of incubation, but values are significantly higher in females. A sharp decrease occurred thereafter, plasma T4 tending toward adult values the 4th day after hatching.

Enzymatic activities of muscle fibres differentiated, in vitro, from pectoralis major (white) and adductor magnus (red) muscles of chick embryos.

Specific activities of NADP isocitrate dehydrogenase and acetylcholinesterase were significantly higher in muscle fibres differentiated, in vitro, from myoblasts of adductor magnus (red) than pectoralis major (white) muscles 10-day-old chick embryos. This is evidence, as far as enzyme activities are concerned, that myoblasts from different types of skeletal muscles are able to give, in tissue culture, muscle fibres of different properties, even in the absence of nerve supply.

[The effect of hypophysectomy on the lipogenic activity of adipose tissue of rabbits]. / Effet de l'hypophysectomie sur l'activité lipogénique des tissus adipeux du Lapin

In vitro lipogenesis is always higher in adipocytes from hypophysectomized rabbit than from normal animals. Lipogenesis is estimated by the incorporation of labelled glucose and acetate into fatty acids. Insulin added to the culture medium slightly enhances lipogenesis. The ability of rabbit adipocytes to utilize acetate is clearly demonstrated. However, it seems different for different fatty tissues.