Studies on the regulatory expression of uncoupling protein 1 in bovine skeletal muscle

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Studies on the regulatory expression of uncoupling protein 1 in bovine skeletal muscle
（ウシ骨格筋における脱共役タンパク質 1 発現調節に関する研究）
DIAO ZHICHENG
Uncoupling protein 1 (UCP1) is a brown adipocyte-specific protein located in the
internal mitochondrial membrane. Through uncoupling oxidative phosphorylation from
ATP synthesis, UCP1 leads to the dissipation of chemical energy in the form of heat
instead of ATP in a non-shivering thermogenesis (NST) manner. Unexpected Ucp1
expression was found in bovine skeletal muscle. Immunohistochemical analyses
showed that UCP1 surrounded muscle fibers, but not adipocytes residing in skeletal
muscle. In this thesis, bovine Ucp1, especially muscular Ucp1 regulatory expression
was explored. In this thesis, I clarified the following points:

1. Four alternative splice Ucp1 variants were identified in bovine brown adipose
tissue. mRNAs of Ucp1 variant 2/4 are less stable than those of Ucp1 variant 1/3 with
longer 3ʹ- untranslated region (UTR). Variant 1 is the main form of Ucp1 because
variant 2-4 are efficiently degraded by the proteasome system. Variants differing in the
3ʹ-UTR were previously shown in murine Ucp1, but the spliced regions in bovine Ucp1
gene are distinct from those in murine Ucp1 gene.

2. Ucp1 expression in bovine skeletal muscle tissue was analyzed. The skeletal
muscle consists of two types of muscle fibers (fast and slow)- the slow muscle
predominantly generates ATP through aerobic metabolism, while ATP is mainly
generated through anaerobic metabolism in the fast muscle. Considering the metabolism
characteristics, the relationship between Ucp1 expression and muscle fiber type was
evaluated in this chapter. The results indicate that Ucp1 prefers to express in fast-twitch
muscles or the requirements of muscular Ucp1 activation is similar to those of
fast-twitch muscle growth.

3. I established a method to stimulated myogenesis especially fast-twitch myosin
heavy chain (Myh) expression so that the relation between Ucp1 and Myhs could be

studied in this model. Myogenesis method was explored in C2C12, a mouse myogenic
cell line. In this chapter, a combination treatment of capsaicin, an Endoplasmic
reticulum stress (ER stress) inducer, and vitamin C synergistically stimulated C2C12
myogenesis; fast-twitch Myh1 and MYH1/2 expression was dramatically increased by
this treatment. Capsaicin stimulated the ER stress marker gene expression in C2C12.
After 1 day of ER stress induction by capsaicin, vitamin C stimulated myogenin
(MYOG) and MYH1 expression, accompanied by increased collagen (Col)1 expression.
In this experiment, capsaicin treatment amplified effects of vitamin C on collagen
synthesis and myogenesis in C2C12.

4. I performed capsaicin and vitamin C treatment in primary bovine myosatellite
cells. Capsaicin also stimulated the expression of ER stress marker genes. However,
vitamin C could not further stimulate myogenesis in bovine myogenic cells after
capsaicin treatment. In the following experiments, insulin and triiodothyronine were
supplemented in differentiation medium to stimulate myogenesis and Ucp1 expression.
I treated bovine myosatellite cells with 4-Phenylbutyric acid (4-PBA), a histone
Deacetylase (HDAC) inhibitor, during myogenic differentiation. Vitamin C treatment
was also conducted because of its condition-dependent effects on myogenesis. 4-PBA
stimulated expression of fast-twitch Myhs and myogenic regulatory factors (Mrfs)
except Myog. 4-PBA also stimulated the expression of Ucp1 and brown/beige adipocyte
markers. The p38 mitogen-activated protein kinase seems to be one of the main
signaling pathways that are involved in 4-PBA induced Ucp1 expression. Unlike the
data in chapter 3, Ucp1 was not positively related to fast-twitch Myhs. This is because
that vitamin C stimulated Myhs expression but had a negative effect on Ucp1
expression. Anyway, 4-PBA inhibited Myh7 expression, which indicates the
requirements for activation of Ucp1 in bovine myosatellite cells may suppress slow
twitch Myh7 expression. This data supports the hypothesis that bovine Ucp1 prefers to
express in fast-twitch muscle fibers from another perspective.

In conclusion, these results indicate that bovine muscular Ucp1 is related to
myofiber type, prone to express in fast-twitch muscle. ...