Theresia Galuh Wandita1, Joseph dela Cruz2, Seong Gu Hwang1* This work was carried out by the support of the Rural Development Administration Grant (Project No. PJ01268703). Abstract No. 9410 Program No. LB813 The effects of acetate on enhancing the proliferation and preadipocyte differentiation of stromal vascular cells isolated from Hanwoo beef cattle Theresia Galuh Wandita1, Joseph dela Cruz2, Seong Gu Hwang1* Department of Animal Life and Environmental Science, Hankyong National University, South Korea1, Department of Basic Veterinary Sciences, College of Veterinary Medicine, University of the Philippines Los Banos, Philippines2 *Corresponding author: sghwang@hknu.ac.kr bstract We investigated the effect of acetate on the proliferation and differentiation of bovine stromal-vascular cells (SVC). SVC was isolated from the longissimus muscle collected directly from approximately 30-month-old Hanwoo beef cattle steers. After confluence, the differentiation was induced by 1-methyl-3-isobutyl-xanthine, dexamethasone, insulin, and rosiglitazone for 2 days, and then subsequently cultured for 14 days. The cells were treated with different concentrations of acetate (0.125 to 2 mMol) throughout the differentiation period. Cell viability assay revealed a dose-dependent increase in the proliferation of SVC. Monitoring of the differentiation through Oil Red-O staining showed that treatment with acetate up to 1mMol significantly increased cell differentiation and fat accumulation. Supplementation of acetate in the media throughout the differentiation period also enhanced the induction of all differentiation markers. Acetate treatment unregulated the mRNA and protein expression of peroxisome proliferator-activated receptor gamma (PPAR-γ), CCAAT/enhancer binding protein alpha (C/EBP-α), Sterol regulatory element-binding protein 1c (SREBP-1c), and adipocyte protein 2 (aP2). These data suggests that acetate can enhance adipocyte differentiation from SVC isolated from Hanwoo beef cattle and possibly improve meat palatability through increased intramuscular fat deposition and reduce meat toughness through decreased fibrogenic differentiation.   ntroduction Muscle cells, adipocytes, and fibroblasts are originated from the same pool of mesenchymal progenitor cells, myoblast progenitor cells and fibroblast progenitor cells; diverge early during development (Du et al., 2013). Stromal vascular cells from fat tissue include adult mesenchymal stem cells, often referred to as preadipocytes and in addition various other cell types (De Ugarte et al., 2003). It has been established that stromal vascular cells are major sources of adipogenic cells in skeletal muscle. Enhancing adipogenesis of progenitor cells increases intramuscular fat (marbling) and improving palatability of meat (dela Cruz et al., 2015). Manipulating the by-products of rumen fermentation can alter fat deposition in cattle. The 3 main VFAs produced are acetate, propionate, and butyrate. A high quality of roughage which is very rich in cellulose was identified to produce acetate in the high concentration. Glucose and acetate are known as a carbon source for fatty acid synthesis. Fiber-utilizing bacteria produce more acetate as a by-product whereas starch-utilizing bacteria produce more propionate. Furthermore, Parnes and Wertheimer (1949) proved that acetate increase glycogen synthesis and decrease glucose utilization. Hence, this experiment was designed to examine the effect of acetate on enhancing proliferation and differentiation of stromal vascular cells isolated from Hanwoo beef cattle's adipose tissue. Oil red O staining and triglyceride content measurement was undertaken to observe the effect of acetate in the adipogenic differentiation of SVC cells. Oil red O staining of cells on day 14 showed that increasing concentrations of acetate until 1.0 mM enhances adipocyte differentiation (Figure 2a). To support the subjective micrograph observations in oil red O staining, elution of oil red O stain with isopropanol (Figure 2b) showed triglyceride content of differentiated adipocytes increased along with the increase in the cooncentration of acetate till 1.0 mM/ml then declined thereafter. intramuscular fat (i.e., marbling). Acetate besides being an important source of energy is also an excellent glycogen percursor for SVCs differentiation. Glycogen synthesis of stromal vascular cells of Hanwoo beef cattle was markedly increased by the addition of 0.125 until 1.0 mM acetate and then decreased. According to the result, acetate enhances proliferation and differentiation of Hanwoo beef cattle perirenal adipose stromal vascular cells until 1.0 mM in concentration and then declines. aterials and methods The expression of PPAR-γ, C/EBP-α, and SREBP-1c is very important for adipogenesis. The mRNA expression with densitometry of several transcription factors associated with adipocyte differentiation such as PPAR-γ, C/EBP-α, SREBP-1c, and aP2 were analyzed to determine the molecular mechanism governing how increasing concentrations of acetate enhances adipocyte differentiation till 1.0 mM then declines as clearly shown in Figure 3a. During the conversion of preadipocyte into mature adipocyte, PPAR-γ and C/EBP have critical regulatory roles. Those markers reinforce each other to induce genes specific to adipocytes. As a result, SVC accumulate lipid droplets and become mature adipocytes. Acetate have role on the expression of adipogenic markers in SVC, which induces differentiation of preadipocytes into adipocytes. onclusion Our current study has shown that exposure of primary isolates of IM SVCs to high acetate levels decrease adipogenesis of the cells as seen in the results of the RT-PCR and Western blot analyses. The SVCs are differentiating in ascending order along with the concentration till 1.0 mM. Data derived from this study shows that in vivo, increasing the acetate levels in the microenvironment of fat precursor cells leads to a higher percentage of adipogenesis. The expressions of several proteins associated with adipocyte differentiation were analyzed to determine the molecular mechanism, at the level of protein synthesis, which is the final product of gene expression and cells differentiation. As shown in the Figure 4a, protein expression of adipogenic markers such as PPAR-γ, C/EBPα, SREBP-1c, and aP2 in SVC cells was induced by increase in acetate until 1.0 mM. esult and discussion eferences Cell viability assay was done to evaluate the effect of acetate concentrations (0.125, 0.25, 0.5, 1.0 and 2.0 mM) on the cell viability of stromal vascular cells. Acetate promotes cell proliferation for both 12 and 24 hours as exhibited by the dose dependent increase till 1.0 mM then declines in the cell viability percentage presented in Figure 1. dela Cruz, J. F., Oh, Y. K., & Hwang, S. G. (2015). The Control of Stromal Vascular Cell Differetiation by Retinoic Acid and Calcium in Hanwoo Beef Cattle Adipose Tissue. Journal of Animal Production Advances, 5(11), 835-844. De Ugarte, D. A., Alfonso, Z., Zuk, P. A., Elbarbary, A., Zhu, M., Ashjian, P., ... & Fraser, J. K. (2003). Differential expression of stem cell mobilization- associated molecules on multi-lineage cells from adipose tissue and bone marrow. Immunology l etters, 89(2), 267-270. Du, M., Huang, Y., Das, A. K., Yang, Q., Duarte, M. S., Dodson, M. V., & Zhu, M. J. (2013). Manipulating mesenchymal progenitor cell differentiation to optimize performance and carcass value of beef cattle. J. Anim. Sci, 91, 1419-1427. Parnes, I., & Wertheimer, E. (1950). Effect of acetate on the glycogen synthesis in vivo. Biochemical Journal, 46(5), 520. Because adipogenic and fibrogenic cells share immediate common progenitor cells, this provides an opportunity to manipulate the differentiation of progenitor cells to favor adipogenesis. Enhancing adipogenesis in developing muscle increases