Regulation of proliferation and differentiation of Luxi bovine intramuscular preadipocytes by propionate

The purpose of the present study was to investigate the regulation of propionate on the proliferation and differentiation of cultured intramuscular preadipocytes from three adult Luxi Yellow steers. The fl ow cytometry, lipid droplets stained by oil red O, and cytoplasmic fat content were detected after cultured cells were treated with different concentrations of propionate (0, 1, 3, 6 mM). The results showed that the separated cells were highly homogeneous and proliferative. Propionate (1 mM) did not signifi cantly affect (P>0.05) the cell proportion of phase G0/G1, but remarkably (P<0.05) increased the proportion of phase S+G2/M. However, the cell proportion signifi cantly increased (P<0.05) in the phase G0/G1, and decreased (P<0.05) in the phase S+G2/M when the concentration of propionate reached to 6 mM. When the confl uent intramuscular preadipocytes were treated with insulin (10 μg/ml) and dexamethasone (0.25 μM), small lipid droplets began to appear, and their number rapidly increased around the nuclei on day 6. Subsequently, when the confl uent intramuscular preadipocytes were treated with propionate, signifi cant accumulation of lipid droplets was observed on day 6, and lipid accumulation extent was dependent on the dose of propionate. In addition, cytoplasmic fat contents of cultured preadipocytes treated with propionate (1, 3, 6 mM) for 6 days were 1.5, 2, and 2 times higher than those of control group (without propionate addition). In conclusion, the present study confi rmed that there were indeed functionally active preadipocytes existing in the muscle of Chinese adult Luxi steers. Propionate may play an important physiological role in regulation of adipogenesis and stimulation of proliferation and differentiation of Luxi bovine intramuscular preadipocytes.


INTRODUCTION
Beef marbling is characterized as intramuscular fat in a cross section of musculus longissimus.High levels of marbling improve the palatability and acceptability of beef by affecting the taste and tenderness of the meat (Nishimura et al., 1999).Intramuscular fat deposition involves a series of events that initiate and maintain preadipocyte proliferation, differentiation of in musculus longissimus (Albrecht et al., 2006).
Ruminant animals ferment dietary carbohydrates into VFA by ruminal microorganisms, then use propionate as a primary precursor of gluconeogenesis.Therefore, propionate from rumen fermentation is largely associated with body fat deposition.Because little literature is available concerning the regulation of bovine intramuscular preadipocyte proliferation and differentiation by propionate, we design the present experiment to examine the effect of propionate on proliferation and differentiation of intramuscular preadipocytes from Chinese adult Luxi steers.

MATERIAL AND METHODS
Three Luxi adult Yellow steers (18 months of age) were used in this study.All procedures involving animals were conducted under the approval of the China Agricultural University Animal Care and Use Committee.Isolated preadipocytes were obtained using modifi ed Aso's method (Aso et al., 1995).All preparations were performed under aseptic conditions, and all experiments were performed between the third and sixth passage and cultured in a 5% CO 2 humidifi ed chamber at 37°C.
Bovine intramuscular preadipocytes treated with propionate were analysed in fl ow cytometry.Briefl y, cells were seeded in T-25 fl ask at the density of 5×10 4 cells/ cm 2 in growth medium supplemented with propionate at the concentration of 1, 3, and 6 mM.Media were changed every 2 days.Six days later, the treated cells were collected and suspended in the sterile tubes, and counted with a haemocytometer to determine cell number.Subsequently, the cells were permeabilized with 0.1% Triton X-100 in DPBS for 30 min at room temperature.Permeabilized cells were treated with 40 µg of RNAse/ml and then the DNA was immediately labelled with 100 µg/ml propidium iodide (PI)/ml before analysis.WAN R. ET AL.
When cultured bovine intramuscular preadipocytes reached confl uency (referred to day 0), the cells were further cultured in DMEM containing 10 µg/ml insulin, 0.25 µmol/ l dexamethasone, and serum-free (differentiation medium) for 2 days, and subsequently in fresh differentiation medium in the absence or presence of increasing concentrations of propionate.In order to determine the extent of preadipocytes differentiation at day 6, both oil red O staining lipid droplets and cytoplasmic fat contents were determined using modifi ed Ramirez-Zacarias' method (Ramirez-Zacarias et al., 1992).
The data were subjected to a one-way analysis of variance using the GLM procedure of SAS (version 8.0, SAS Institute, Cary, NC) and the differences among the treatment means were tested with the procedure of STDERR and TDIFF.

RESULTS
To examine the effect of propionate on bovine intramuscular preadipocytes proliferation, the cells were treated with propionate for 6 days, and then the treated cells were collected for analysis by fl ow cytometry.Propionate at 1 mM did not signifi cantly (P>0.05)affect the cell proportion of phase G0/G1, but remarkably (P<0.05)increased the proportion of phase S+G2/M compared with control group (0 mM propionate).However, the cell proportion signifi cantly increased (P<0.05) in the phase G0/G1, and decreased (P<0.05) in the phase S+G2/M when the concentration of propionate reached to 6 mM (Table 1).Furthermore, almost 90% bovine preadipocytes treated with propionate remained in phase G0/G1 (result not shown).The cell cycle is generally considered to be composed of four phases, namely the gap prior to DNA replication (G1) , the synthetic phase (S) , the gap after DNA (G2) and mitosis (M), and quiescent or noncycling cells would be considered in G0 (Hartwell et al., 1989) To investigate the effect of propionate on bovine intramuscular preadipocytes differentiation, we determined cytoplasmic fat contents of the cells treated with propionate for 6 days and stained cytoplasmic lipid droplets with oil red O.After the confl uent cells were treated with 10 µg/ml insulin and 0.25 µM dexamethasone, small lipid droplets began to appear on day 2 (results not shown) and the number of lipid droplets rapidly increased around the nuclei on day 6 (Figure 1A,B,C).When the cells were treated with propionate, signifi cant accumulation of cytoplasmic lipid droplets was observed at day 6 (Figure 1D,E,F), and lipid accumulation extent was dependent on the dose of propionate.In addition, cytoplasmic fat contents of cultured preadipocytes with the treatment of propionate (1, 3, 6 mM) for 6 days were 1.5, 2, and 2 times higher than that of propionate at 0 mM (Table 2).

DISCUSSION
In the present study, we demonstrated the effects of propionate on the proliferation and differentiation of bovine preadipocytes in vitro.
In our experiments, propionate at 1 mM did not signifi cantly affect the cell proportion of phase S+G2/M, and almost 90% bovine preadipocytes treated with propionate remained in the phase G0/G1.S+G2/M refers to the extent of cell proliferation.The above results indicated that propionate at the appropriate concentration is in favour of preadipocytes proliferation.In addition, adipose tissue is mainly composed of matured adipocytes.So it indirectly enhances the potential of intramuscular fat deposition.When the confl uent cells were treated with propionate for 6 days, 1 mM propionate signifi cantly increased intracellular fat contents of bovine intramuscular preadipocytes.Nevertheless, intracellular fat contents almost failed to rise when the cells were treated with propionate at the concentration of 3 or 6 mM.Short-chain fatty acids stimulate leptin production in adipocytes (Xiong et al., 2004) and fatty acids with a chain length superior or equal to eight carbons effectively inhibited insulin-stimulated leptin secretion (Cammisotto et al., 2003).Leptin mainly produced in adipose tissue.These data suggest that volatile fatty acids within their physiological levels (total ∼1 mM) may only promote adipogenesis (Quigley et al., 1991).Plasma leptin levels are likely to regulate propionate homeostasis in ruminants by a feedback mechanism.The above results also remind us that the production of VFA, especially propionic acid, is important to ruminants.So it is essential for us to regulate the production of VFA and proportion of VFA.Moreover, it is useful for us to further understand the mechanism by which the intramuscular adipose is deposed in the tissues, and improve the beef quality in local beef cattle breed.

CONCLUSIONS
Propionate signifi cantly promoted bovine intramuscular preadipocyte proliferation and differentiation.With incremental propionate concentration, cytoplasmic fat contents of cultured preadipocytes were increased, indicating a dose dependent manner with propionate addition.Further studies are required to clarify a signal transduction mechanism of propionate in adipogenesis.

Figure 1 .
Figure 1.Morphological changes of bovine intramuscular preadipocyte differentiation stimulated by propionate.(A) The cells were not treated with propionate.Bovine intramuscular preadipocytes were treated with insulin (10 µg/ml) and dexamethasone (0.25 µmol/l) for 6 days after confl uence, intracellular lipid was not (B) or stained (C) with oil red O. Cells on day 6 after being induced to differentiation in the presence of 1 mM (D), 3 mM (E), and 6 mM (F) propionate.Bar= 100 µm

Table 1 .
Infl uence of propionate on the proliferation of Luxi bovine intramuscular preadipocyte

Table 2 .
Changes of cytoplasmic fat content in Luxi bovine intramuscular preadipocyte differentiation by propionate indicated the cells treated with propionate were always seeded in 24-well plates at the density of 5×10 4 cells/cm 2 , and OD Value was determined to collect the number of cells per well *