For adipocyte-related genes,
KLF2 (p<0.001) and
ERK2 (p = 0.004) levels were higher in the treatment group than in the control group (
Figure 2). The
KLF2 and
ERK2 genes are critical in the embryogenesis stage and adipogenesis.
KLF2 has also been demonstrated to maintain the preadipocyte condition rather than promote adipogenic commitment [
28]. In addition, 3T3-L1 cells overexpressing KLF2 showed reduced gene expression of
PPARγ and
C/EBPα, which are related to adipogenesis [
28]. However, in the current study, both
KLF2 and
PPARγ were expressed a higher level (p<0.05) in the VA treatment group than in the control group.
KLF2 is highly expressed in the preadipocyte period and is rapidly downregulated upon the induction of adipogenic differentiation [
29]. Based on a previous study, we considered that
KLF2 gene expression indicates cells to maintain the preadipocyte state.
PPARγ has been reported to play an essential role in adipogenesis in adipocytes [
30] and a key role in the development of muscle by regulating of the insulin sensitivity and the transition of myosin heavy chain isoforms [
31].
Longissimus dorsi muscle contains many types of cells, including muscle cells, preadipocytes, progenitor cells and immunocytes. Especially in the early growth stage in calves,
longissimus dorsi muscle contains more muscle cells than other cell types.
PPARγ is expressed in human skeletal muscle, although this expression level is two-thirds of that in adipocytes [
32]. Amin et al [
33] reported that the overexpression of
PPARγ in murine muscle cells affects muscle fiber-type composition, lipid metabolism, and the synthesis and secretion of adiponectin (such as fatty acid oxidation), as well as improves muscle insulin sensitivity. Therefore, the upregulated
PPARγ expression in the VA treatment group seemed to be related more to muscle development (corresponding to the increased gene expression of
MyoD,
MYF5, and
MYF6, p<0.05) than to terminal adipogenic differentiation. Taken together, these data showing increased
KLF2 and
PPARγ gene expression levels in the VA treatment group suggest that VA supplementation may help maintain the preadipocyte status and promote muscle development in the calf. Activation of the
ERK pathway (
ERK1 and
ERK2) promots adipogenic commitment in mouse embryonic stem cells upon retinoic acid supplementation [
34], indicating that additional VA supplementation at 78,000 IU/d in pregnant cattle may increase the preadipocyte development of offspring. However, no differences were observed in the expression of
Wnt10B (p = 0.054),
β-catenin (p = 0.131),
SOX9 (p = 0.683),
Pref-1 (p = 0.275),
ERK1 (p = 0.097), or
Zfp423 (p = 0.201;
Figure 2B). In a previous study,
Pref-1 and
Wnt10B were used as potential markers of preadipocytes [
35]. In addition, Berry et al [
36] reported that high VA supplementation in 8-week-old male mice for 8 weeks and retinoic acid treatment in preadipocytes resulted in increased
Pref-1,
Sox9, and
KLF2 gene expression levels both
in vivo and
in vitro. Moreover,
Zfp423 was reported to be highly expressed in preadipocytes (within adipogenic epithelial cells) and mature adipocytes (brown adipocytes and white adipocytes), which is also related to preadipocyte proliferation and adipogenic differentiation [
6]. Furthermore, retinoic acid reduces DNA methylation at CpG-rich promoters, such as the
Zfp423 promotor, that are primarily regulated by epigenetic modifications [
6]. However, in the current study, we did not find any significant changes in the expression of
Pref-1,
Sox9,
ERK1, or
Zfp423.
The mRNA expression of a genes related to late adipogen esis, such as
LPL,
C/EBPα, and
SCD, was not show different between the two groups (p>0.05,
Figure 2B). C/EBPα, LPL, and SCD are used as marker genes for mature adipocytes and are considered as important factors for stimulating adipogenic differentiation and lipid accumulation in mature adipocytes [
3]. We hypothesized that VA supplementation inhibited adipogenic differentiation, but
longissimus dorsi muscle in calves contain few mature adipocytes.