Regulatory roles of adiponectin receptor 1 and 2 in sheep preadipocytes during adipocyte differentiation

Figures & data

Table 1. siRNA sequence.

Gene	Sense sequence 5′–3′	Antisense sequence 5′–3′
AdipoR1-siRNA 1	GCGGGCGGUCACCAGCCCA	UGGGCUGGUGACCGCCCGC
AdipoR1-siRNA 2	GUCUCAUCAGAUCUUCCAU	AUGGAAGAUCUGAUGAGAC
AdipoR1-siRNA 3	AGAUGGAGGAGUUCGUGUA	UACACGAACUCCUCCAUCU
AdipoR2-siRNA 1	CUGGUAUUGCUCUUCUGAU	AUCAGAAGAGCAAUACCAG
AdipoR2-siRNA 2	GUCUCGAACCUCCAGGAGU	ACUCCUGGAGGUUCGAGAC
AdipoR2-siRNA 3	CACACUGAGACAGGCAACA	UGUUGCCUGUCUCAGUGUG

Abbreviations: AdipoR1, adiponectin receptor 1; AdipoR2, adiponectin receptor 2.

Table 2. Primers used for real-time PCR.

Gene	Forward 5′–3′	Reverse 5′–3′
LPL	CCGCCGACAGGATTACAAGA	AGCCACAGATTCCGTCACTC
HSL	GCCTTACCTCAAGAGCTGGG	GCAGTAGGCGTAGAAGCACT
FAS	AGACTTGGGACCAGACCTTCA	GGGCGGCCTCAGTGATAAG
PPARα	TGAACGACCAAGTCACCCTG	GTCGAACTTGGGCTCCATGA
PPARγ	AAGAGCTGACCCGATGGTTG	TGAGGGAGTTGGAAGGCTCT
Adiponectin	CTCGAGGAGAAGGGCAAGC	CATCTTCTCCATGGCGTGGT
AdipoR1	CTCTTGTAGAGGCGTCTGGC	ATCACTCGCCATCGACCTTC
AdipoR2	CCACTATGACGGCACCACTG	CTGGTCCACGTTCTGGTTCT
GAPDH	TCCGTTGTGGATCTGACCTG	CCCTGTTGCTGTAGCCGAAT

Abbreviations: LPL, Lipoprotein lipase; HSL, Hormone-sensitive lipase; FAS, Fatty acid synthase; PPARα/PPARγ, Peroxisome proliferator-activated receptor alpha/gamma; AdipoR1, Adiponectin receptor 1; AdipoR2, Adiponectin receptor 2; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase.

Figure 1. Intracellular lipid accumulation and mRNA expression of adipogenesis-related genes in sheep preadipocytes during adipocyte differentiation. (A–C) Morphological characteristics of sheep preadipocytes derived from muscle adipose tissues. The morphology of sheep preadipocytes was cultured in growth medium for 1 day (d) (A), 3 days (B), and 7 days (C). Bars = 100 μm. (D–F) Intracellular lipid accumulation of the undifferentiated (D), differentiated 3 days (E), and differentiated 7 days (F) preadipocytes by Oil Red O staining. (G) Quantitative analysis of Oil Red O stained cells in preadipocytes. Bars = 25 μm. (H) Quantitative analysis of the expression level of eight adipogenesis-related genes in differentiated preadipocytes (differentiation days-7). Values are means ± SEM of duplicate independent analyses. *p < .05; **p < .01.

Figure 2. Quantitative analysis of the mRNA and protein expression levels of AdipoR1 or AdipoR2 in its silencing and overexpressing preadipocyte. (A) FAM-siRNA were transfected into the sheep preadipocytes. Bar = 50 μM. (B and C) The expression of AdipoR1-GFP and AdipoR2-GFP in preadipocytes. Bars = 100 μm. (D) The mRNA and protein expression levels of AdipoR1 in its silencing preadipocytes. (E) The mRNA and protein expression levels of AdipoR2 in its silencing preadipocytes. (F) The mRNA and protein expression levels of AdipoR1 in its overexpressed preadipocytes. (G) The mRNA and protein expression levels of AdipoR2 in its overexpressed preadipocytes. The relative expression abundance of a given gene was calculated after normalisation to GAPDH mRNA expression. Values are means ± SEM of duplicate independent analyses. *p < .05; **p < .01.

Figure 3. Intracellular lipid accumulation in AdipoR1 and AdipoR2 silencing and overexpressing preadipocyte. (A) NC-siRNA preadipocytes; (B) AdipoR1- siRNA1 preadipocytes; (C) AdipoR2- siRNA2 preadipocytes; (D) AdipoR1-GFP overexpressed preadipocytes; (E) AdipoR2-GFP overexpressed preadipocytes; Bars = 25 μm. (F) Quantitative analysis of Oil Red O stained cells in preadipocytes during differentiation 7 days. Values are means ± SEM of duplicate independent analyses. *p < .05; **p < .01.

Figure 4. Quantitative analysis of the mRNA expression level of FAS (A), HSL (B), PPARα (C), and PPARγ (D) in sheep preadipocyte after silencing and overexpressing of AdipoR1 or AdipoR2. The fold change in expression of each gene was calculated relative to in undifferentiated preadipocytes (day 0). The relative expression abundance of a given gene was calculated after normalisation to GAPDH mRNA expression. Values are means ± SEM of duplicate independent analyses. *p < .05; **p < .01.