Localization and abundance of early markers of fat cell differentiation in the skeletal muscle of cattle during growth--are DLK1-positive cells the origin of marbling flecks?

Localization and abundance of early markers of adipogenic differentiation were investigated in bovine muscle tissue to verify their association with marbling development. Bovine skeletal muscle samples were used for immunohistochemical localization and measurement of mRNA and protein abundance of delta-like homolog 1 (DLK1) and CCAAT/enhancer-binding protein beta (CEBPB). The localization of DLK1-positive cells confirmed the position of small clusters of adipocytes which can be considered as the origin of marbling flecks. The results indicated higher DLK1 expression in the less marbled muscle of Holstein (HS) steers (P < 0.05) and a trend to higher CEBPB expression in Japanese Black (JB) steers (P < 0.1) at slaughter. The number of DLK1-positive cells and fat content were negatively correlated. The lower expression of DLK1 together with higher CEBPB abundance during fattening in JB may have contributed to the development of more adipocytes in the skeletal muscle of JB.

Modulation of vH+-ATPase is part of the functional adaptation of sheep rumen epithelium to high-energy diet.

Ruminal vacuolar H(+)-ATPase (vH(+)-ATPase) activity is regulated by metabolic signals. Thus, we tested whether its localization, expression, and activity were changed by different feeding. Young male sheep (n = 12) were either fed hay ad libitum (h) or hay ad libitum plus additional concentrate (h/c) for 2 wk. The vH(+)-ATPase B subunit signal was predominantly found in the cell membrane and cytosol of rumen epithelial cells (REC) with basal/parabasal phenotype. The elevated number (threefold) of these cells in rumen mucosa of h/c-fed sheep reflects a high proliferative capacity and, explains the 2.3-fold increase of the total number of vH(+)-ATPase-expressing REC. However, in accordance with a 58% reduction of the vH(+)-ATPase B subunit mRNA expression in h/c-fed sheep, its protein amount per single REC was decreased. Using the fluorescent probe BCECF and selective inhibitors (foliomycin, amiloride), the contribution of vH(+)-ATPase and Na(+)/H(+) exchanger to intracellular pH (pH(i)) regulation was investigated. REC isolated from h/c-fed sheep keep their pH(i) at a significantly higher level (6.91 ± 0.03 vs. 6.74 ± 0.05 in h-fed sheep). Foliomycin or amiloride decreased pH(i) by 0.16 ± 0.02 and 0.57 ± 0.04 pH units when applied to REC from h-fed sheep, but the effects were markedly reduced (-88 and -33%) after concentrate feeding. Nevertheless, we found that REC proliferation rate and [cAMP](i) were reduced after foliomycin-induced vH(+)-ATPase inhibition. Our results provide the first evidence for a role of vH(+)-ATPase in regulation of REC proliferation, most probably by linking metabolically induced pH(i) changes to signaling pathways regulating this process.

Agouti revisited: transcript quantification of the ASIP gene in bovine tissues related to protein expression and localization.

Beside its role in melanogenesis, the agouti signaling protein (ASIP) has been related to obesity. The potentially crucial role in adipocyte development makes it a tempting candidate for economic relevant, fat related traits in farm animals. The objective of our study was to characterize the mRNA expression of different ASIP transcripts and of putative targets in different bovine tissues, as well as to study consequences on protein abundance and localization. ASIP mRNA abundance was determined by RT-qPCR in adipose and further tissues of cattle representing different breeds and crosses. ASIP mRNA was up-regulated more than 9-fold in intramuscular fat of Japanese Black cattle compared to Holstein (p<0.001). Further analyses revealed that a transposon-derived transcript was solely responsible for the increased ASIP mRNA abundance. This transcript was observed in single individuals of different breeds indicating a wide spread occurrence of this insertion at the ASIP locus in cattle. The protein was detected in different adipose tissues, skin, lung and liver, but not in skeletal muscle by Western blot with a bovine-specific ASIP antibody. However, the protein abundance was not related to the observed ASIP mRNA over-expression. Immuno-histochemical analyses revealed a putative nuclear localization of ASIP additionally to the expected cytosolic signal in different cell types. The expression of melanocortin receptors (MCR) 1 to 5 as potential targets for ASIP was analyzed by RT-PCR in subcutaneous fat. Only MC1R and MC4R were detected indicating a similar receptor expression like in human adipose tissue. Our results provide evidence for a widespread expression of ASIP in bovine tissues at mRNA and, for the first time, at protein level. ASIP protein is detectable in adipocytes as well as in further cells of adipose tissue. We generated a basis for a more detailed investigation of ASIP function in peripheral tissues of various mammalian species.

Effects of leptin and adiponectin on proliferation and protein metabolism of porcine myoblasts.

The aim of this study was to show the abundance of leptin and adiponectin receptors (LEPR, ADIPOR1, ADIPOR2) and to determine the direct effects of leptin and adiponectin on the in vitro growth of porcine skeletal muscle cells. ADIPOR1 and ADIPOR2 were abundant at mRNA and protein level in proliferating and differentiating myoblast cultures derived from semimembranosus and semitendinosus muscles of newborn piglets, whereas LEPR expression was close to the detection limit. Adiponectin (10, 20, 40 µg/ml) attenuated the proliferation of porcine myoblasts, measured as [(3)H]-thymidine incorporation and real-time monitoring of the cells in response to 24- and 48-h exposure, in a dose-dependent manner. This effect resulted from suppressed basic fibroblast growth factor (bFGF)-mediated stimulation of DNA synthesis in serum-free medium (SFM) containing bFGF. No effects of leptin (5, 10, 20, 40, 80 ng/ml) on myoblast proliferation in SFM were detectable. Neither leptin nor adiponectin altered protein synthesis and degradation in differentiating porcine myoblasts cultured in SFM. The results on receptor abundance suggest that porcine skeletal muscle cells may be sensitive to adiponectin and leptin. However, except via inhibitory interaction of adiponectin with bFGF, these adipokines appear not to affect in vitro proliferation and protein metabolism of porcine muscle cells directly under serum-free culture conditions.

Efficient, global-scale quantification of absolute protein amounts by integration of targeted mass spectrometry and two-dimensional gel-based proteomics.

Knowledge on absolute protein concentrations is mandatory for the simulation of biological processes in the context of systems biology. A novel approach for the absolute quantification of proteins at a global scale has been developed and its applicability demonstrated using glucose starvation of the Gram-positive model bacterium Bacillus subtilis and the pathogen Staphylococcus aureus as proof-of-principle examples. Absolute intracellular protein concentrations were initially determined for a preselected set of anchor proteins by employing a targeted mass spectrometric method and isotopically labeled internal standard peptides. Known concentrations of these anchor proteins were then used to calibrate two-dimensional (2-D) gels allowing the calculation of absolute abundance of all detectable proteins on the 2-D gels. Using this approach, concentrations of the majority of metabolic enzymes were determined, and thus a quantification of the players of metabolism was achieved. This new strategy is fast, cost-effective, applicable to any cell type, and thus of value for a broad community of laboratories with experience in 2-D gel-based proteomics and interest in quantitative approaches. Particularly, this approach could also be utilized to quantify existing data sets with the aid of a few standard anchor proteins.

The vacuolar-type H-ATPase in ovine rumen epithelium is regulated by metabolic signals.

In this study, the effect of metabolic inhibition (MI) by glucose substitution with 2-deoxyglucose (2-DOG) and/or application of antimycin A on ovine rumen epithelial cells (REC) vacuolar-type H(+)-ATPase (vH(+)-ATPase) activity was investigated. Using fluorescent spectroscopy, basal pH(i) of REC was measured to be 7.3 +/- 0.1 in HCO(3) (-)-free, glucose-containing NaCl medium. MI induced a strong pH(i) reduction (-0.44 +/- 0.04 pH units) with a more pronounced effect of 2-DOG compared to antimycin A (-0.30 +/- 0.03 versus -0.21 +/- 0.03 pH units). Treatment with foliomycin, a specific vH(+)-ATPase inhibitor, decreased REC pH(i) by 0.21 +/- 0.05 pH units. After MI induction, this effect was nearly abolished (-0.03 +/- 0.02 pH units). In addition, membrane-associated localization of vH(+)-ATPase B subunit disappeared. Metabolic control of vH(+)-ATPase involving regulation of its assembly state by elements of the glycolytic pathway could provide a means to adapt REC ATP consumption according to energy availability.

Rumen epithelial cells adapt magnesium transport to high and low extracellular magnesium conditions.

A protein of approximately 70-kDa was identified as a candidate Na+/Mg2+ exchanger in rumen epithelial cells (REC). Melastatin-related Transient Receptor Potential 7 (TRPM7) and Magnesium Transporter 1 (MagT1) transcripts and, from them, encoded proteins were also detected. The regulation of these Mg transport pathways by extracellular [Mg] changes was the main focus of this study. Therefore, a 24-h pre-incubation of ovine REC in control (1.2 mM), low (0.12 mM)-Mg, and high (5 mM)-Mg medium was performed. Na+/Mg2+ exchangers, TRPM7 and MagT1 abundance and activity were investigated by Western blot analysis, flow cytometry, immunocytochemistry and fluorescence spectroscopic measurements of [Mg2+]i changes. Inhibitors were employed to differentiate Na+/Mg2+ exchanger-mediated (imipramine) and channel-mediated (cobalt(III)hexaammine, nitrendipine) Mg transport. Basal [Mg2+]i (0.40 +/- 0.02 mM) was not influenced by pre-incubation in low- or high-Mg medium. However, compared with control REC (4.1 +/- 0.7 microM/min), such cells showed reduced (2.8 +/- 0.6 microM/min) or elevated (6.4 +/- 0.9 microM/min) Mg extrusion rates that correlated with a decreased (25%) and increased (38%) expression of the putative Na+/Mg2+ exchanger protein, respectively. Low- and high-Mg pre-incubated REC were both characterized by an increased (30-40%) influx capacity. In low-Mg REC, the latter resulted mainly from a strong activation of the TRPM7-related transport component. The data thus clearly demonstrate the intrinsic regulation of REC transmembrane Mg transport.

Expression and functional activity of the Na/Mg exchanger, TRPM7 and MagT1 are changed to regulate Mg homeostasis and transport in rumen epithelial cells.

The present study was performed to show the molecular identity of functionally characterized Mg transport pathways in rumen epithelial cells (REC) and to investigate the effects of extracellular [Mg] changes on their expression and activity. By using RT- PCR, Western blot, flow cytometry and immunocytochemistry, TRPM7, MagT1 and a Na+/Mg2+ exchanger were found in REC. Compared with control conditions ([Mg]e = 1.2 mM), a decreased or increased MagT1 (-30%; 20%) and Na+/Mg2+ exchanger (-25%; 40%) protein abundance was observed after a 24-h incubation of REC in low (0.12 mM)- and high (5 mM)-Mg medium, respectively. To determine the Mg transport capacity, [Mg2+]i changes were measured by use of mag-fura 2. The basal [Mg2]i (0.43 +/- 0.03 mM) was not influenced by the [Mg] of the pre-incubation medium. However, compared to control cells, REC incubated in low- or high-Mg medium showed significantly reduced (59%) and elevated (57%) Mg extrusion rates, respectively. In addition, they were characterized by an increased influx capacity (30-40%). In low-Mg cells the latter results mainly from a strong TRPM7 related transport component whereas in high-Mg cells the imipramine-sensitive, the Na+/Mg2+ exchanger-mediated transport component causes this effect. In conclusion, TRPM7, MagT1 and a Na+/Mg2+ exchanger are shown to be the main Mg transport proteins in REC and their expression and functional activity is influenced by the cellular Mg status. The latter responses permit adaptation of epithelial Mg absorption and enable REC to maintain a physiological [Mg2+]i which is a prerequisite for various cell functions.