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

Hepcidin negatively regulates systemic iron levels by inhibiting iron entry into the circulation. Hepcidin production is increased in response to an increase in systemic iron via the activation of the bone morphogenetic protein (BMP) pathway. Regulation of hepcidin expression by iron status has been proposed on the basis of evidence mainly from rodents and humans. We evaluated the effect of iron administration on plasma hepcidin concentrations in calves and the expression of bovine hepcidin by the BMP pathway in a cell culture study. Hematocrit as well as levels of blood hemoglobin and plasma iron were lower than the reference level in calves aged 1-4 weeks. Although intramuscular administration of iron increased iron-related parameters, plasma hepcidin concentrations were unaffected. Treatment with BMP6 increased hepcidin expression in human liver-derived cells but not in bovine liver-derived cells. A luciferase-based reporter assay revealed that Smad4 was required for hepcidin reporter transcription induced by Smad1. The reporter activity of hepcidin was lower in the cells transfected with bovine Smad4 than in those transfected with murine Smad4. The lower expression levels of bovine Smad4 were responsible for the lower activity of the hepcidin reporter, which might be due to the instability of bovine Smad4 mRNA. In fact, the endogenous Smad4 protein levels were lower in bovine cells than in human and murine cells. Smad4 also confers TGF-ß/activin-mediated signaling. Induction of TGF-ß-responsive genes was also lower after treatment with TGF-ß1 in bovine hepatocytes than in human hepatoma cells. We revealed the unique regulation of bovine hepcidin expression and the characteristic TGF-ß family signaling mediated by bovine Smad4. The present study suggests that knowledge of the regulatory expression of hepcidin as well as TGF-ß family signaling obtained in murine and human cells is not always applicable to bovine cells.

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

Uncoupling protein 1 (UCP1) is responsible for non-shivering thermogenesis in brown/beige adipocytes in humans and rodents. Previously, we showed unexpected expression of UCP1 in bovine skeletal muscles. Here we evaluated Ucp1 mRNA levels in the muscle tissue of Japanese Black steers. Expression of Ucp1 was higher in 30-month-old cattle than in 26-month-old cattle. Levels of myosin heavy chain (Myh)1, an MYH predominantly expressed in fast-twitch muscles, were also significantly higher in cattle aged 30 months. A similar tendency was observed in the expression of other Myhs that are highly expressed in fast-twitch muscles, Myh2 and Myh4. Ucp1 expression was positively correlated with expression of Myh1, Myh2, and Myh4. Our results indicate the possibility of Ucp1 expression in fast-twitch muscle fibers.

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

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-ß (TGF-ß)/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-ß/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-ß 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.

Expression levels of brown/beige adipocyte-related genes in fat depots of vitamin A-restricted fattening cattle1.

Brown/beige adipocytes dissipate energy as heat. We previously showed that brown/beige adipocytes are present in white adipose tissue (WAT) of fattening cattle. The present study examined the effect of vitamin A restriction on mRNA expression of brown/beige adipocyte-related genes. In Japan, fattening cattle are conventionally fed a vitamin A-restricted diet to improve beef marbling. Twelve Japanese Black steers aged 10 mo were fed control feed (n = 6) or vitamin A-restricted feed (n = 6) for 20 mo. Subcutaneous WAT (scWAT) and mesenteric WAT (mesWAT) were collected, and mRNA expression levels of molecules related to the function of brown/beige adipocytes (Ucp1, Cidea, Dio2, Cox7a, and Cox8b) as well as transcriptional regulators related to brown/beige adipogenesis (Zfp516, Nfia, Prdm16, and Pgc-1α) were evaluated. The vitamin A restriction significantly increased or tended to increase expression levels of Cidea and Pgc-1α in scWAT, and Cidea, Dio2, and Nfia in mesWAT. Previous studies revealed that the bone morphogenetic protein (Bmp) pathway was responsible for commitment of mesenchymal stem cells to brown/beige adipocyte-lineage cells. The vitamin A restriction increased expression of Bmp7 and some Bmp receptors in WAT. The interrelationship between gene expression levels indicated that expression levels of Nfia, Prdm16, and Pgc-1α were closely related to those of genes related to the function of brown/beige adipocytes in scWAT. Also, expression levels of Nfia, Prdm16, and Pgc-1α were highly correlated with those of Alk3 in scWAT. In summary, the present results suggest that the vitamin A restriction increases the number or activity of brown/beige adipocytes through regulatory expression of transcriptional regulators to induce brown/beige adipogenesis, especially in scWAT of fattening cattle, which may be governed by the Bmp pathway.

Relationships between immunoglobulin M and immunoglobulin G or A in colostrum of Japanese black multiparous cows.

This study was conducted to clarify the relationships among immunoglobulin (Ig)M, IgG, IgA, ß-carotene, vitamin A and α-tocopherol contents in colostrum of 24 Japanese Black multiparous cows in order to evaluate the role of IgM on colostral IgG and IgA production. Compared with colostral IgG, colostral IgM and IgA were very low but varied widely. There was positive correlation between colostral IgM and IgG, but colostral IgM was not related with colostral IgA. There was no relationship between colostral IgM and age of cows, although colostral IgG was increased with aging. There were positive correlations among colostral ß-carotene, vitamin A and α-tocopherol and these vitamins were positively correlated with colostral IgM and IgG. These results indicate that fat-soluble vitamins may affect colostral IgG and IgM in cows and colostral IgG increases with the increase of colostral IgM.

Relationships between immunoglobulin and fat-soluble vitamins in colostrum of Japanese Black multiparous cows.

Data from 19 Japanese Black multiparous cows were collected to clarify the relationships among immunoglobulin (Ig) G, IgA, ß-carotene, vitamin A and α-tocopherol contents in colostrum of cows in order to evaluate the role of fat-soluble vitamins on colostral IgG and IgA production. Mean colostral IgG was 141 mg/mL, ranging from 65 to 208 mg/mL, whereas mean colostral IgA was 8.7 mg/mL, ranging from 1.0 to 34.6 mg/mL. Colostral IgG increased with aging in multiparous cows. There were positive correlations between colostral IgG and colostral vitamin A or colostral α-tocopherol in cows, and the higher adjusted R(2) was obtained in the prediction model of colostral IgG from age and colostral vitamin A. Colostral vitamin A was positively correlated with colostral ß-carotene or colostral α-tocopherol in cows, but there were no relationships between colostral IgA and colostral IgG or colostral fat-soluble vitamins. These results indicate that fat-soluble vitamin contents in colostrum of cows may change in similar patterns and high colostral vitamin A is related with high colostral IgG.