The Effects Of Tail-interacting Protein Of 47 KDa (TIP47) On The Lipid Metabolism In Mouse Skeletal Muscle Myoblasts

Organism life activities rely on the energy supply. The skeletal muscle is important locomotorium that consumes most energy supply in human. Therefore, the skeletal muscle is animportant organ for energy metabolism. Lipid is used as energy storagemolecule in the skeletal muscle, it can be hydrolyzed to release the free fatty acids fo β-oxidation which will then generate ATP for energy supply. The ectopic distribution of lipids in skeletal muscle will lead to insulin resistance, which may deteriorate into type Ⅱ diabetes. Thus, it is important to study the dynamic of lipid metabolism in the skeletal muscle.Skeletal muscle cells store lipids in lipid droplets (LDs) the latter of which are comprised of a phospholipid monolayer that surrounds a core of neutral lipids, such as cholesterol esters (CE) and triacylglycerols (TAG). Besides, plenty of proteins localize on the surface of LDs, such as the PAT protein family which is originally named for perilipin, ADRP, TIP47 and then two other PAT proteins, S3-12 and OXPAT, are found. These proteins decorated surfaces of LDs grant LDs multiple biology functions. LDs are involved not only in lipid metabolism but also in interorganellar communication, development, apoptosis and immunity. LDs are likely linked to ER-mediated protein degradation and essential for replication of viruses. Except perilipin, other members of PAT protein family all express on the LDs in the skeletal muscle cells. Adipose differentiation related protein (ADRP) is most abundant followed by tail-interacting protein of 47 kDa (TIP47), S3-12 and the oxidative tissue-enriched PAT protein (OXPAT).TIP47 is also named M6PRBP1, which is first identified by a yeast two-hybrid screen using the cytosolic domain of the cation-dependent mannose 6-phosphate receptor (M6PR) as a bait. Because it is 47 kDa and interacts with the cytosolic tails of the M6PRs, it is named the tail-interacting protein of 47 kDa. TIP47 binds to M6PR, and transports M6PR from the late endosome back to the trans-Golgi network. TIP47 can bind directly to the small molecule guanosine triphosphatase (GTPase) Rab9 to promote the affinity of TIP47 with the M6PR and the transport. TIP47 is expressed widely in placenta, ovary, adrenal gland, brain, skin, fat, muscle and heart. Unexpectedly, TIP47 is expressed highly in the cervix carcinoma tissue. So far, TIP47 has been used as the serum marker of the cervix carcinoma and as a biomarker of cervical dysplasia and invasive carcinoma。 TIP47 is also expressed in eyes and engaged in the light/dark-adapted eyes reaction. In addition, TIP47 has an effect on the hepatic steatosis, virus infection and the tumor progression.There is 43% identity throughout the length of the amino acid sequence between TIP47 and ADRP. In absence of ADFP, Tip47 may compensate functionally for ADRP. TIP47 can be recruited to the LDs surface and promote nascent LDs maturation together with ADRP. The research about the ADRP is more plentiful. Furthermore. ADRP knockout mice show resistance to high fat induced fatty liver. However, there is less research on the TIP47 and the function relation between LDs and TIP47 is poorly understood and there’s no TIP47 knockout mice in the world. Thus, we will knock out TIP47 in the mouse C2C12 myoblasts by newly developed CRISPR/Cas9 technology to give an insight into the role of TIP47 in the lipid metabolism in skeletal muscle cells.CRISPR/Cas9, a type II CRISPR/Cas, is a RNA-mediated adaptive defense system which is evolved by the bacteria to protect organisms from invading phages and plasmids. The artificial modified CRISPR/Cas9 can be easily and efficiently used for gene-targeting and genome-editing applications. Now this technology has been used in animal model establishment, gene therapy, crop and livestock breeding. The foundmental principle as described below, the Cas9 is guided by a sequence-specific single guide RNA (sgRNA) to the double-strand DNA target site by Watson-Crick base pairing, then the endonuclease in Cas9 cleave the double-strand DNA to introduce double-strand breaks in target DNA that trigger the cellular DNA repair mechanisms, including error-prone nonhomologous end joining (NHEJ) and homology-directed repair (HDR), finally it will lead to base pairs deletions or insertions mutation at the cleavage site.This study successfully knocked out the TIP47 gene in the mouse C2C12 myoblasts by using artificial modified CRISPR/Cas9 technology, and got some TIP47 knockout (TIP47 KO) C2C12 cell lines. We used the laser scanning confocal microscope to observe LDs stained by LipidTOXTM Red, under normal circumstances LDs in TIP47 KO cells showed no changes compared with the control group.We inferred that TIP47 did not affect the neutral lipid synthesis and LDs formation in C2C12 cells under normal circumstances. However, the number of LDs in TIP47 KO cells become morethan the control group when cells in both groups were treated with sodium oleate. It suggested that TIP47 may be involved in lipid synthesis stimulated by sodium oleate. We used the 3H labled sodium oleate to trace the TAG metabolism, the result showed that the amount of TAG synthesis and hydrolysis in TIP47 KO cells was increased. It suggested that TIP47 play an important role in TAG metabolism. What’s more, the results from Western blot showed the ADRP protein expression was increased in TIP47 KO cells, it suggested that ADRP might compensate functionally for TIP47in TIP47 knocked out C2C12 cells.In conclusion, our study showed that TIP47 has important effects on lipid metabolism in C2C12 cells.