Low and high concentrations of butyrate regulate fat accumulation in chicken adipocytes via different mechanisms

Figures & data

Table 1. The primers and siRNAs used in this study

Genes	Sequence 5ʹto 3ʹ
GAPDH	Forward-CTACACACGGACACTTCAAG Reverse-ACAAACATGGGGGCATCAG
PPARG	Forward-AGACACCCTTTCACCAGCATCC Reverse-AACCCTTACAACCTTCACAAGCA
FAS	Forward-TCCTTGGTGTTCGTGACG Reverse-CGCAGTTTGTTGATGGTGAG
ADPN	Forward-TCACCTACGACCAGTTCCA Reverse-CCCGTTGTTGTTGCCCTC
FABP4	Forward-TGAAGCAGGTGCAGAAGT Reverse-CAGTCCCACATGAAGACG
LPL	Forward-CAGTGCAACTTCAACCATACCA Reverse-AACCAGCCAGTCCACAACAA
UCP-3	Forward- GCAGCGGCAGATGAGCTT Reverse-AGAGCTGCTTCACAGAGTCGTAGA
FFAR2	Forward-AACGCCAACCTCAACAAGTC Reverse-TGGGAGAAGTCATCGTAGCA
FFAR3	Forward-GAAGGTGGTTTGGGAGTGAA Reverse-CAGAGGATTTGAGGCTGGAG
ACC	Forward-AATGGCAGCTTTGGAGGTGT Reverse-TCTGTTTGGGTGGGAGGTG
SREBP-1 c	Forward-GCCCTCTGTGCCTTTGTCTTC Reverse–ACTCAGCCATGATGCTTCTTCC
SiRNA-FFAR2	Forward-GCUUCUUCUCCAGCAUCUATT Reverse-UAGAUGCUGGAGAAGAAGCTT
SiRNA-FFAR3	Forward-CCCACUGUUCCAUCAUCUUTT Reverse-AAGAUGAUGGAACAGUGGGTT

Figure 1. Effect of serial concentrations of sodium butyrate (SB) on fat accumulation in chicken adipocytes. Chicken preadipocytes were incubated for 8 days with 0.01, 0.1, 0.5, 1, or 2 mM SB in the presence of adipogenic stimuli (AS). (a) Lipid droplets visualized by confocal microscopy in differentiated adipocytes upon bodipy (green)-staining, colocalizing with DAPI (blue)-stained nuclei. Scale bars represent 100 μm. (b) Lipid droplets visualized in differentiated adipocytes upon oil red O (red)-staining. The nuclei were stained with haematoxylin (purple). Scale bars represent 100 px. (c) The stained oil red O was quantified after isopropanol extraction, which was shown as mmol/g total protein. (d) The TG contents from differentiated adipocytes upon different treatments, which were shown as mmol/g total protein. (e) The pure TG contents in differentiated adipocytes upon different treatments, which were shown as mmol/well. (f) Relative mRNA levels of lipogenic markers and FFARs determined by qRT-PCR in the treated cells on day 8. The mRNA levels were normalized to GAPDH. (g) Representative images of western blots and quantitative analysis of the expression of lipogenic markers on day 8 post treatment (n = 3). GAPDH serves as a loading control. Data are the means ± SEM of at least 3 independent experiments. *p < 0.05, **p < 0.01 vs. the control (0 mM SB)

Figure 2. SB inhibits preadipocyte proliferation. (a) EdU assay was performed after the cells were incubated for 24 h with serial concentrations of SB. Confocal microscopy of preadipocytes perfused with EdU (red) and counterstained with Hoechst 33,342 (blue). The EdU/Hoechst 33,342 ratio represents the cell proliferation rate. Scale bars represent 100 μm. (b) CCK-8 assay was conducted to detect cell viability after butyrate treatment. The data are the means ± SEM (n = 6). *p < 0.05, **p < 0.01 vs. the control (0 mM SB)

Figure 3. Signalling elucidation of the role of 0.01 mM SB in fat accumulation

(a)The effect of specific siRNAs on the mRNA expression of FFAR2 and FFAR3, determined by qRT-PCR. (b) Oil red O staining was conducted in differentiated adipocytes treated with 0.01 mM SB alone or together with siRNA-FFARs. The nuclei were stained with haematoxylin (purple). (c) Relative mRNA levels of lipogenic markers were determined by qRT-PCR in the treated cells on day 8. (d) Representative images of western blots and quantitative analysis of the expression of p-ERK, ERK, p-AMPK and AMPK after the cells treated with 0.01 mM SB or vehicle (n = 3). GAPDH serves as a loading control. The data are the means ± SEM (n = 3_6). * p < 0.05, ** p < 0.01 vs. the control (0 mM SB).

Figure 4. Signalling elucidation of the role of 1 mM SB in fat accumulation

(a)Effect of 1 mM SB on phosphorylation of ERK and AMPK at the indicated time points, determined by western blotting and quantification analysis. GAPDH serves as a loading control. This experiment was repeated 3 times. (b) HDAC activity in the treated cells, determined using a commercial assay kit. (c) Oil red O staining (red) was performed in differentiated adipocytes treated with SB or TSA for 8 days. The nuclei were stained with haematoxylin (purple). (d) Quantification of the accumulated TG based on the same protein content. (e) Relative mRNA expression levels of PPARG, FAS, APN, LPL and FABP4 in treated cells. (f) Relative mRNA expression levels of FFAR2 and FFAR3 in treated cells. (g) Representative images of western blots and quantitative analysis of PPARG, FABP4, histone H3 and acetyl-histone H3 in the treated cells (n = 3). GAPDH was used as an internal control. The data are the means ± SEM (n = 3_6). *p < 0.05, ** p < 0.01 vs. the control; # p < 0.05, ## p < 0.01 indicate differences between the 0.01 mM and 1 mM treatments.

Figure 5. Dietary SB supplementation (0.1%) reduces fat deposition in broilers

(a) Liver indexes in the control- and SB-treated broilers. (b) H&E staining of the liver slides at d 21 and d 42. Scale bars represent 50 μm. (c) TG and TCH contents in liver tissues at d 21. (d) Abdominal fat ratios in the control- and SB-treated broilers. (e) H&E staining of the abdominal fat slides at d 21 and d 42. Images are representative pictures for each treatment. scale bars represent 50 μm. (f) Quntification of the adipocyte area in abdominal fat tissues. (g) The relative mRNA levels of fat synthesis associating genes and FFARs in liver tissues of 21-day-old broilers. (h) The relative mRNA levels of fat deposition associating genes and FFARs in adipose tissues of 21-day-old broilers. (i) Serum TG content in the control- and SB-treated broilers at d 21. (j) Serum TCH content in the control- and SB-treated broilers at d 21. The data are the means ± SEM of n = 5_8. *p < 0.05 vs. the control.