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Adresse de correspondance: levin@supagro.inra.fr; International audience; Secreted Frizzled-related proteins (Sfrps) are extracellular regulators of Wnt signalling and play important roles in developmental and oncogenic processes. They are known to be upregulated in regenerating muscle and in myoblast cultures but their function is unknown. Here, we show that the addition of recombinant Sfrp1 or Sfrp2 to C2C12 cell line cultures or to primary cultures of satellite cells results in the inhibition of myotube formation with no significant effect on the cell cycle or apoptosis. Even though at confluence, treated and untreated cultures are identical in appearance, analyses have shown that, for maximum effect, the cells have to be treated while they are proliferating. Furthermore, removal of Sfrp from the culture medium during differentiation restores normal myotube formation. We conclude that Sfrp1 and Sfrp2 act to prevent myoblasts from entering the terminal differentiation process.

International audience; We previously showed that transfer of adult myoblasts (MB) into cardiotoxin-damaged muscle improved the properties of reinnervated tibialis anterior muscle of rabbits. However, this cell therapy protocol cannot be applied to humans because of the hazardous effects of the myotoxin. To circumvent this approach, we used the recently developed high-density injection technique to autotransplant cultured cells 1 mm from each other into the tibialis anterior muscle without previous cardiotoxin-induced damage. Two months after transection and immediate suture of the common peroneal nerve, we transferred by this technique two types of precursor cells, MB or cells isolated from the adipose tissue stromal vascular fraction. In contrast to our previous results, muscles studied at 4 months showed no benefits in terms of function or morphology, whatever the transferred cells. These results, together with the results of earlier studies, emphasize the importance of delivery methods and the muscle environment in supporting cell integration into host tissues.

International audience; The regenerating skeletal muscle environment is capable of inducing uncommitted progenitors to terminally differentiate. The aim of this work was to determine whether adipose tissue-derived stromal cells were able to participate in muscle regeneration and to characterize the effect on muscle mass and functional capacities after transplantation of these cells. Adipose tissue stromal cells labeled with Adv cyto LacZ from 3-day-old primary cultures (SVF1) were autotransplanted into damaged tibialis anterior muscles. Fifteen days later, beta-glactosidase staining of regenerated fibers was detected, showing participation of these cells in muscle regeneration. Two months after SVF1 cell transfer, muscles were heavier, showed a significantly larger fiber section area, and developed a significantly higher maximal force compared with damaged control muscles. These results are similar to those previously obtained after satellite cell transplantation. However, SVF1 transfer also generated a small amount of adipose tissue localized along the needle course. To minimize these adipose contaminants, we transferred cells from 7-day-old secondary cultures of the SVF1, containing only a small proportion of already engaged preadipocytes (SVF2). Under these conditions, no adipose tissue was observed in regenerated muscle but there was also no effect on muscle performances compared with damaged control muscles. This result provides further evidence for the existence of progenitor cells in the stromal fraction of freshly isolated adipose tissue cells, which, under our conditions, keep some of their pluripotent properties in primary cultures.

Skeletal muscle demonstrates a force deficit after repair of injured peripheral nerves. We tested the hypothesis that transplantation of satellite cells into reinnervated rabbit tibialis anterior (TA) muscles improves their properties. Adult rabbits underwent transection and immediate suture of the common peroneal nerve. In order to provide an environment favorable for cell transplantation, TA were then made to degenerate by cardiotoxin injection, either immediately or after a 2-month delay, which is sufficient for muscle reinnervation. In both cases, the injured TA were transplanted with cultured satellite cells 5 days after induction of muscle degeneration. When cells were transferred immediately after nerve repair, drastic morphological and functional muscle alterations were observed. However, when the muscles were allowed to become reinnervated before cell transplantation, muscles were heavier and developed a significantly higher maximal force compared to denervated-reinnervated muscles. Thus, application of the cell therapy protocol improved properties of denervated muscles only when they were allowed to become innervated. These results, which represent the application of cell therapy to improve force recovery of reinnervated muscles, will be of significant interest in certain clinical contexts, particularly after immediate or delayed muscle reinnervation.

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.

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 β3-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 (PPARγ) agonists were necessary to induce UCP1 in these cells from older animals, a synergistic increase being noted in the presence of β3-adrenergic agonists. In contrast to these results, preadipocytes from perirenal WAT stimulated by PPARγ agonists never expressed UCP1. Conclusion: preadipocytes in the postnatal period are determined as brown or white preadipocytes. PPARγ agonists induce UCP1 expression in brown postnatal preadipocytes, but they are unable to trigger the gene in white preadipocytes.

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.

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.

Reprint author: Bacou F. bacou@supagro.inra.fr; International audience; Skeletal muscle demonstrates a force deficit after repair of injured peripheral nerves. Data from the literature indicate that myoblast transfer enhances recovery of muscle function. Thus, we tested the hypothesis that transfer of adult myoblasts improves the properties of reinnervated rabbit tibialis anterior (TA) muscles in both the short term (4 months) and long term (14 months). Two months after transection and immediate suture of the common peroneal nerve, TA muscles were made to degenerate by cardiotoxin injection and then transplanted with adult myoblasts cultured for 13 days. Under these conditions, muscles studied at 4 months were heavier, contained larger fibers, and developed a significantly higher maximal force than muscles that had only been denervated-reinnervated. In the long term, although muscles made to degenerate were heavier and developed a significantly higher maximal force than denervated-reinnervated muscles, myoblast transfer failed to improve these parameters. However, the overall characteristics of long-term operated muscles tended clearly to approach those of the controls. Taken together, these results may have significant implications in certain orthopedic contexts, particularly after immediate or delayed muscle reinnervation.

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.

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.

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.