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This paper reports the metabolic and morphological characteristics of bovine adipose tissue (AT) at end of fetal life and its variability with breed and anatomical site of AT. Our hypothesis was that, in cattle, end-of-fetal-life differences in adipocyte number, size, and histology may account for differences in AT maturity. To address this question, perirenal and intermuscular AT were sampled from Charolais, Blond d'Aquitaine, and Holstein fetuses at 260 d postconception. Holstein fetuses showed greater leptin mRNA abundance, which is consistent with the greater perirenal AT weight (P= 0.03) than Blond d'Aquitaine fetuses. Compared with Blond d'Aquitaine or Charolais fetuses, Holstein fetuses had larger (P< 0.001) adipocytes, greater (P< 0.05) activities of enzymes involved in de novo fatty acid (FA) synthesis (FA synthase, glucose-6-phosphate dehydrogenase, malic enzyme) and FA esterification (glycerol-3-phosphate dehydrogenase), and greater (P= 0.06, P= 0.10, P< 0.001) mRNA abundance for lipolytic enzymes (hormone-sensitive lipase and adipose triglyceride lipase) and uncoupling protein 1 in both perirenal and intermuscular AT. This indicates increased FA turnover in Holstein adipocytes through FA storage, mobilization, and oxidation pathways. Whatever the breed, adipocytes were smaller in perirenal AT than intermuscular AT. Whatever the breed or anatomical site, bovine AT at 260 d postconception contained predominantly unilocular adipocytes believed to be white adipocytes together with a few multilocular brown adipocytes. We conclude that the greater metabolic and morphologic maturity of adipocytes from Holstein than Blond d'Aquitaine and Charolais fetuses may contribute to the greater thermogenic aptitude of Holstein newborns. Moreover, the presence of both white and brown adipocytes at the end of fetal life highlights the complexity of AT structure and may indicate that the cellular and functional heterogeneity of AT repeatedly observed postnatally has a developmental origin.

This work investigated the metabolic basis for the variability of carcass and i.m. adiposity in cattle. Our hypothesis was that the comparison of extreme breeds for adiposity might allow for the identification of some metabolic pathways determinant for carcass and i.m. adiposity. Thus, 23- to 28-mo-old steers of 3 breeds, 2 with high [Angus or Japanese Black × Angus (J. Black cross)] and 1 with low (Limousin) i.m. and carcass adiposity, were used to measure activities or mRNA levels, or both, of enzymes involved in de novo lipogenesis [acetyl-coA carboxylase, fatty acid synthase (FAS), glucose-6-phosphate dehydrogenase (G6PDH), malic enzyme], circulating triacylglycerol (TAG) uptake (lipoprotein lipase), and fatty acid esterification (glycerol-3-phosphate dehydrogenase), as well as the mRNA level of leptin, an adiposity-related factor. In a first study, enzyme activities were assayed in the s.c. adipose tissue (AT), the oxidative rectus abdominis, and the glycolytic semitendinosus muscles from steers finished for 6 mo. Compared with Angus or J. Black cross, Limousin steers had a 27% less (P= 0.003) rib fat thickness, and 23 and 29% less (P≤ 0.02) FAS and G6PDH activities in s.c. AT. In rectus abdominis and semitendinosus, the 75% less (P< 0.001) TAG content was concomitant with 50% less (P< 0.001) G6PDH activity. In a second study, enzyme activities plus mRNA levels were assayed in an oxido-glycolytic muscle, the longissimus thoracis (LT), in the i.m. AT dissected from LT, and in s.c. AT from the same Limousin steers and from Angus steers finished for 10 mo. Compared with Angus, the 50% less (P< 0.001) rib fat thickness in Limousin contrasted with the 1.1- to 5.8-fold greater (P≤ 0.02) mRNA levels or activities, or both, of acetyl-coA carboxylase, G6PDH, lipoprotein lipase, and glycerol-3-phosphate dehydrogenase in s.c. AT. Conversely, the 90% less (P< 0.001) TAG content in Limousin LT was concomitant to the 79 and 83% less (P≤ 0.002) G6PDH activity and leptin mRNA level. Such differences could arise from a greater number of adipocytes in LT from Angus steers because no difference was found between Limousin and Angus for G6PDH activity and leptin mRNA in i.m. AT. We conclude that FAS and G6PDH in s.c. AT could be involved in differences in carcass adiposity, but this relationship disappeared when the fatness increased strongly. Leptin and G6PDH are related to the expression of marbling whatever the body condition and thus could be relevant indicators of marbling in beef cattle.

Marbling is an important criterion for beef quality grading in many countries. The purpose of the current study was to utilize the natural genetic variation to identify major metabolic indicators of marbling in cattle differing in genotypes. Rectus abdominis (RA, oxidative), semitendinosus (glycolytic), and longissimus thoracis (LT, oxido-glycolytic) muscles were taken from steers of different genotypes that expressed high [Angus, n = 16; and crossbred (Angus × Japanese Black), n = 10] or low (Limousin, n = 12) levels of marbling in their meat. Muscles from Angus and crossbred steers were characterized, as expected, by a greater triacylglycerol (TAG) content (P< 0.001) and also by greater protein contents of fatty acid-binding protein specific for heart and muscles (H-FABP; P< 0.001 for RA and P< 0.05 for LT muscle) or for adipocytes (A-FABP; P< 0.001 for RA and LT muscles). Moreover, oxidative enzyme activities (β-hydroxyacyl-CoA dehydrogenase, citrate synthase, isocitrate dehydrogenase, cytochrome-coxidase) were greater (P< 0.01 to 0.001) in the 3 muscles studied, whereas glycolytic enzyme activities (phosphofructokinase and lactate dehydrogenase) were lower (P< 0.001) in RA muscle in Angus and crossbred steers compared with Limousin steers. Significant correlations were observed between TAG content and H- and A-FABP protein contents, and oxidative (r ≥ +0.55, P< 0.001) or glycolytic enzyme activities (r ≥ −0.47, P< 0.001), when the 3 genotypes and muscles studied were considered as a whole. In addition, A-FABP protein content and some oxidative enzyme activities were significantly correlated with TAG content independently of the genotype and muscle effects. In conclusion, A-FABP protein content, as well as oxidative enzyme activities, may be used as indicators of the ability of steers from extreme genotypes to deposit intramuscular fat.

The aim of the present study was to precise the origin of the particular muscle characteristics of double-muscled cattle by comparing muscle properties of Holstein and double-muscled Belgian Blue (BB) foetuses. Ten 100-day-old foetuses of each genotype were studied. The weight and length of foetuses and the length, weight and area of the Semitendinosus(ST) muscle were analysed. Contractile differentiation of the different fibre types was studied by immunohistochemistry using several monoclonal antibodies raised against different myosin heavy chain isoforms (MHC slow, fast, foetal) and by electrophoresis. Proliferation phase of myoblasts from each genotype was analysed in primary culture. On 100 days of foetal life, the foetuses of both genotypes did not show any significant differences in their weight and length. However, BB cattle already present muscle hypertrophy, which seems to originate from a higher myoblast proliferation observed in primary culture. The use of anti-MHC antibodies shows that ST muscle of BB contained a smaller proportion of primary fibres and a higher proportion of secondary fibres which will give principally fast fibres in adult muscle. Electrophoresis analysis confirms a lower proportion of slow MHC in ST of BB.

An experiment was conducted with 42 growing Montbéliard steers to study the effect of feed restriction, followed by refeeding, on circulating concentrations of thyroxine (T4) and triiodothyronine (T3) and on hepatic and muscle activities of 5′-deiodinase (5′D). At 9 mo of age, 21 steers were diet-restricted for 3 mo (ADG, 641 g/d), prior to a 4-mo compensatory growth period with ad libitum access to the same diet (ADG, 1,240 g/d). They were compared to 21 control steers continuously gaining 1,100 g/d between 9 and 16 mo of age. Blood samples were collected every 14 d and samples of liver and semitendinosus and triceps brachii (triceps) muscles were obtained at slaughter at the end of the restriction and refeeding periods (12 and 16 mo of age, respectively). Compared to control steers, feed restriction decreased plasma concentrations of T4after 56 to 83 d of feed restriction (P< 0.05), whereas T3concentration decreased only after 83 d of feed restriction (P< 0.05). No differences in hepatic and muscle 5′D activities were observed after 87 d of feed restriction and decreased growth rate (12 mo of age). During the refeeding period (compensatory growth), circulating concentrations of T4and T3were restored to control levels within 14 d. Moreover, T3concentration rose above that of control steers after 56 d of refeeding and remained higher for the duration of the experiment (P< 0.05). Hepatic 5′D activity was higher (P= 0.07) in compensated than in control steers at the end of refeeding period (16 mo of age) and higher (P< 0.01) after compensation at 16 mo than during restriction at 12 mo. Activities of 5′D in semitendinosus and triceps muscles were higher (P< 0.001) in 16-mo-old than in 12-mo-old steers, but no differences were observed due to feed restriction or compensatory growth. These results indicate that nutritional status regulates both thyroidal secretion and extrathyroidal T3production in cattle. The data also suggest that extrathyroidal T3production may be involved in the mechanism of compensatory growth in cattle.

To characterize the regulatory pathways involved in the inhibition of cell differentiation induced by the impairment of mitochondrial activity, we investigated the relationships occurring between organelle activity and myogenesis using an avian myoblast cell line (QM7). The inhibition of mitochondrial translation by chloramphenicol led to a potent block of myoblast differentiation. Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone and oligomycin, which affect the organelle at different levels, exerted a similar influence. In addition, we provided evidence that this phenomenon was not the result of an alteration in cell viability. Conversely, overexpression of the mitochondrial T3 receptor (p43) stimulated organelle activity and strongly potentiated myoblast differentiation. The involvement of mitochondrial activity in an actual regulation of myogenesis is further supported by results demonstrating that the muscle regulatory gene myogenin, in contrast to CMD1 (chicken MyoD) and myf5, is a specific transcriptional target of mitochondrial activity. Whereas myogenin mRNA and protein levels were down-regulated by chloramphenicol treatment, they were up-regulated by p43 overexpression, in a positive relationship with the expression level of the transgene. We also found that myogenin or CMD1 overexpression in chloramphenicol-treated myoblasts did not restore differentiation, thus indicating that an alteration in mitochondrial activity interferes with the ability of myogenic factors to induce terminal differentiation.

We have previously shown that c-Erb A and v-Erb A display a cell-specific activity in avian myoblasts. In this work, we have compared the molecular basis of thyroid hormone action in HeLa cells and in QM7 myoblasts. The transcriptional activity of c-Erb A alpha 1 through a palindromic thyroid hormone response element (TRE) was similar in both cell types. However, c-Erb A did not activate gene transcription through a direct repeat sequence (DR) 4 TRE in myoblasts in contrast to results obtained in HeLa cells. Moreover, whereas retinoic acid receptor-AP-1 interactions were functional in both cell types, thyroid hormone receptor (T3R)-AP-1 interactions were only functional in HeLa cells. Using electrophoretic mobility shift assays, functional tests, and Northern blot experiments, we observed that RXR isoforms are not expressed in proliferating myoblasts. Expression of RXR gamma in these cells did not influence T3R transcriptional activity through a palindromic TRE but induced such an activity through a DR4 TRE. Moreover, it restored c-Erb A-AP-1 functionality in QM7 myoblasts and enhanced the myogenic influence of T3. We also observed that c-Jun overexpression in proliferating QM7 cells restored T3R transcriptional activity through a DR4 TRE. Therefore, alternative mechanisms are involved in the induction of T3R transcriptional activity according to the cell status (proliferation: c-Jun; differentiation: RXR). In addition we provide the first evidence that RXR is required to allow inhibition of AP-1 activity by ligand-activated T3R. Lastly, we demonstrate the importance of RXR in the regulation of myoblast differentiation by T3.

In order to characterize Sterling's triiodothyronine (T3) mitochondrial receptor using photoaffinity labeling, we observed two specific T3-binding proteins in the inner membrane (28 kDa) and in the matrix (43 kDa) of rat liver mitochondria. Western blots and immunoprecipitation using antibodies raised against the T3-binding domain of the T3 nuclear receptor c-Erb A alpha 1 indicated that at least the 43-kDa protein was c-Erb A alpha 1-related. In addition, gel mobility shift assays demonstrated the occurrence of a c-Erb A alpha 1-related mitochondrial protein that specifically binds to a natural or a palindromic thyroid-responsive element. Moreover, this protein specifically binds to a direct repeat 2 sequence located in the D-loop of the mitochondrial genome. Furthermore, electron microscopy studies allowed the direct observation of a c-Erb A-related protein in mitochondria. Lastly, the relative amounts of the 43-kDa protein related to c-Erb A alpha 1 were in good correlation with the known mitochondrial mass in three typical tissues. Interestingly, expression of a truncated form of the c-Erb A alpha 1 nuclear receptor in CV1 cells was associated with a mitochondrial localization and a stimulation of mitochondrial activity. These results supply evidence of the localization of a member of the nuclear receptor superfamily in the mitochondrial matrix involved in the regulation of mitochondrial activity that could act as a mitochondrial T3-dependent transcription factor.