SFRP2 expression in rabbit myogenic progenitor cells and in adult skeletal muscles.

Satellite cells derived from fast- and slow-twitch muscles have different properties in culture. We have used the differential display technique to retrieve genes differentially expressed in fast- and slow-twitch muscle satellite cell cultures. Amongst these genes we have identified, cloned, sequenced and studied the expression in muscle of rabbit secreted frizzled related protein 2 (SFRP2) mRNA, whose importance in cell fate determination has been well documented. It has been shown that SFRP2 is widely expressed in the developing embryo but its expression in the adult is much more restricted. We show that primary cultures of satellite cells from adult rabbit fast- and slow-twitch muscles strongly and differentially express SFRP2 mRNA. Embryonic rabbit muscle cell primary cultures also strongly express SFRP2 mRNA whereas the myoblast C2.7 cell line shows little expression. We also studied SFRP2 mRNA expression in growing, regenerating and denervated muscles. Embryonic rabbit muscles express SFRP2 mRNA but this rapidly falls off after birth. In adult rabbit muscles SFRP2 mRNA is detected within 1 day of either muscle damage or denervation. Thereafter the SFRP2 mRNA expression profiles are different for fast- and slow-twitch muscle. The function of SFRP2 in muscle is unknown but its putative activity as a Wnt antagonist and its precocious expression after muscle damage suggest a role in satellite cell activation.

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)

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.

[Methodological approach to the circadian pattern of food intake in the growing domestic rabbit]. / Approche méthodologique de la répartition nycthémérale des prises d'aliment chez le lapin domestique en croissance.

We graphically recorded the feeding pattern of 9 New Zealand male rabbits during weeks 6, 9, 12, 15 and 18 of age. Feeding was considered as a periodic series of discrete events. We fitted a periodic Poisson process to each particular series. In order to determine if the model was a good representation of the time series, we compared the estimated density curves with non-parametric estimations of density. Approximation of the circadian pattern using the model was better from week 15 on. Before this age, 24-hour feeding activity was generally characterized by two peaks. The parameters of the model--average number of food intakes over a 24-hour period, time of highest feeding activity and scatter index--were easy to interpret. Age variations in circadian feeding pattern were mainly characterized by shortening of scatter and shifting of peak feeding activity from the beginning to the middle of the dark period between weeks 6 and 18.