Regulatory expression of uncoupling protein 1 and its related genes by endogenous activity of the transforming growth factor‐β family in bovine myogenic cells

Abstract

Uncoupling protein 1 (UCP1) is responsible for non‐shivering thermogenesis, with restricted expression in brown/beige adipocytes in humans and rodents. We have previously shown an unexpected expression of UCP1 in bovine skeletal muscles. This study evaluated factors affecting Ucp1 gene expression in cultured bovine myogenic cells. Myosatellite cells, which were isolated from the bovine musculus longissimus cervicis, were induced to differentiate into myotubes in the presence of 2% horse serum. Previous studies using murine brown/beige adipocytes revealed that Ucp1 expression levels are directly increased by forskolin and all‐trans retinoic acid (RA). The transforming growth factor‐β (TGF‐β)/activin pathway negatively regulated Ucp1 expression, whereas activation of the bone morphogenetic protein (BMP) pathway indirectly increases Ucp1 expression through the stimulation of brown/beige adipogenesis. Neither forskolin nor RA significantly affected Ucp1 mRNA levels in bovine myogenic cells. A‐83‐01, an inhibitor of the TGF‐β/activin pathway, stimulated myogenesis in these cells. A‐83‐01 significantly increased the expression of some brown fat signature genes such as Pgc‐1α, Cox7a1, and Dio2, with a quantitative but not significant increase in the expression of Ucp1. Treatment with LDN‐193189, an inhibitor of the BMP pathway, did not affect the differentiation of bovine myosatellite cells. Rather, LDN‐193189 increased Ucp1 mRNA levels without modulating the levels of other brown/beige adipocyte‐related genes. The current results indicate that the regulation of Ucp1 expression in bovine myogenic cells is distinct from that in murine brown/beige adipocytes, which has been more intensely characterized. Significance of the study We previously reported unexpected expression of Ucp1 in bovine muscle tissues; Ucp1 expression has been known to be detected predominantly in brown/beige adipocytes. This study examined regulatory expression of bovine Ucp1 in myogenic cells. Consistent with the changes in expression levels of brown/beige adipocyte‐selective genes, Ucp1 expression tended to be increased by inhibition of endogenous TGF‐β activity. In contrast, inhibition of endogenous BMP significantly increased Ucp1 expression without affecting brown/beige adipocyte‐selective gene expression. The current results indicate that regulatory expression of Ucp1 in bovine myogenic cells is distinct from that in murine brown/beige adipocytes that is more intensely characterized.

Weak response of bovine hepcidin induction to iron through decreased expression of Smad4

“…Western blotting was performed as previously described 29 . Briefly, the cells were lysed in lysis buffer (20 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% (v/v) Triton X‐100, 1 mM PMSF, 1% (v/v) aprotinin, and 1 mM Na 3 VO 4 ).…”

“…Western blotting was performed as previously described. 29 Briefly, the cells were lysed in lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% (v/v) Triton X-100, 1 mM PMSF, 1% (v/v) aprotinin, and 1 mM Na 3 VO 4 ). After heating for 10 min at 98°C, the proteins were electrophoresed in 10% or 5%-20% sodium dodecyl sulfate-polyacrylamide gels under reducing conditions in the presence of a protein marker and transferred onto a membrane.…”

Isolation of the canine inhibin βB subunit gene and characterization of signalling mediated by canine inhibin βB

“…Western blot analyses were performed as previously described. 24 The immunoreactive proteins were visualized using Chemi-Lumi OneUltra (Nacalai Tesque, Kyoto, Japan) following the manufacturer's protocol.…”

Possibility of uncoupling protein 1 expression in bovine fast-twitch muscle fibers

“…Although regulation of Ucp1 expression is well characterized in brown/beige adipocytes of rodents [ 6 ], it may not be applicable to the regulation of bovine muscles. Our previous in vitro study using bovine myosatellite cells revealed that Ucp1 expression did not increase in response to several factors known to stimulate Ucp1 transcription in rodents [ 6 ]; forskolin (a protein kinase A activator) and retinoic acid did not increase Ucp1 expression in bovine myosatellite cells and myotubes [ 2 ]. In addition, the bone morphogenetic protein (BMP) pathway increased Ucp1 expression through stimulation of murine brown adipogenesis [ 20 ], but inhibition of the BMP pathway increased Ucp1 expression without affecting myogenesis in bovine muscular cells [ 2 ].…”

“…Our previous in vitro study using bovine myosatellite cells revealed that Ucp1 expression did not increase in response to several factors known to stimulate Ucp1 transcription in rodents [ 6 ]; forskolin (a protein kinase A activator) and retinoic acid did not increase Ucp1 expression in bovine myosatellite cells and myotubes [ 2 ]. In addition, the bone morphogenetic protein (BMP) pathway increased Ucp1 expression through stimulation of murine brown adipogenesis [ 20 ], but inhibition of the BMP pathway increased Ucp1 expression without affecting myogenesis in bovine muscular cells [ 2 ]. These inconsistent regulations between rodent adipocytes and bovine muscular cells in cell culture systems suggest the necessity of evaluating regulatory Ucp1 expression in bovine skeletal muscle tissue.…”