C-type natriuretic peptide regulates lipid metabolism through a NPRB-PPAR pathway in the intramuscular and subcutaneous adipocytes in chickens

Abstract

Natriuretic peptides (NPs) have an important role in lipid metabolism in skeletal muscle and adipose tissue in animals. C-type natriuretic peptide (CNP) is an important NP, but the molecular mechanisms that underlie its activity are not completely understood. Treatment of intramuscular fat (IMF) and subcutaneous fat (SCF) adipocytes with CNP led to decreased differentiation, promoted proliferation and lipolysis, and increased the expression of natriuretic peptide receptor B (NPRB) mRNA. Silencing natriuretic peptide C (NPPC) had the opposite results in IMF and SCF adipocytes. Transcriptome analysis found 665 differentially expressed genes (DEGs) in IMF adipocytes and 991 in SCF adipocytes. Seven genes in IMF adipocytes (FABP4, APOA1, ACOX2, ADIPOQ, CD36, FABP5, and LPL) and eight genes in SCF adipocytes (ACOX3, ACSL1, APOA1, CPT1A, CPT2, FABP4, PDPK1 and PPARα) are related to fat metabolism. Fifteen genes were found to be enriched in the peroxisome proliferator-activated receptor (PPAR) pathway. Integrated analysis identified 113 intersection genes in IMF and SCF adipocytes, two of which (APOA1 and FABP4) were enriched in the PPAR pathway. In conclusion, CNP may regulated lipid metabolism through the NPRB-PPAR pathway in both IMF and SCF adipocytes, FABP4 and APOA1 may be the key genes that mediated CNP regulation of fat deposition.

Keywords: C-type natriuretic peptide; Intramuscular adipocytes; PPAR pathway; Subcutaneous adipocytes.

Fig. 1

Morphological changes of intramuscular preadipocytes (inverted microscope, 100×). (A–C) Cellular morphology of IMF preadipocytes 2, 5, and 7 days after isolation. (D) Morphology of IMF adipocytes 24 h after passage. (E) Differentiation of IMF adipocytes cultured with oleic acid for 3 days. (F) IMF adipocytes stained with Oil red O after induced differentiation for 3 days.

Fig. 2

Effect of CNP on the proliferation of intramuscular preadipocyte. (A) The number of preadipocytes was significantly increased by CNP (Cell Counting Kit-8, n = 8, *p < 0.05 vs. control group). (B) EdU staining of intramuscular preadipocytes induced by CNP for 24 h and 72 h (n = 3). EdU, number of proliferative cells; Hoechst, number of cells before proliferation; Overlay, superimposition of the two images shows the location of proliferating cells.

Fig. 3

Effect of CNP on differentiation of intramuscular adipocytes. (A) CNP 10⁻⁷ mol/L significantly decreased the differentiation of adipocytes (n = 3; **p < 0.01 vs. control). (B) Morphologic changes and lipid deposition induced by 10⁻⁷ mol/L CNP in adipocytes in vitro (inverted microscope, 400× magnification). Oil Red O staining shows that CNP 10⁻⁷ mol/L decreased both the size and number of the droplets in each accumulation compared with cells not treated with CNP.

Fig. 4

Change of the glycerol content at 6 days after CNP treatment in intramuscular adipocytes (n = 3), (**p < 0.01 vs. control).

Fig. 5

Change of NPPC mRNA expression after 24 h transfection with NPPC siRNA in the intramuscular adipocytes.

Fig. 6

Effect of NPPC interference on the proliferation of intramuscular preadipocytes. (A) Number of preadipocytes after transfection with NPPC siRNA (CCK8 kit, n = 8, *p < 0.05 vs. control group).); B. EdU staining of chicken preadipocytes after transfection with NPPC siRNA. EdU, number of proliferative cells; Hoechst, number of cells before proliferation; Overlay, superimposition of the two images shows the location of proliferating cells.

Fig. 7

Effect of NPPC interference on the differentiation of intramuscular adipocytes. (A) Adipocyte differentiation increased significantly after transfection with NPPC siRNA (n = 3) (**p < 0.01 vs. control). (B) Morphologic changes and lipid deposition after transfection with NPPC siRNA in adipocytes in vitro (inverted microscope, 400× magnification). Lipid droplets (stained with Oil Red O) accumulated as larger clusters and in greater numbers in cells transfected with NPPC siRNA compared with the negative control vector.

Fig. 8

Change of the glycerol content at 4 d after transfection with NPPC siRNA in intramuscular adipocytes. (n = 3), (**p < 0.01 vs. control).

Fig. 9

Change of NPRB expression in intramuscular adipocytes at 6 d after CNP treatment ( n = 3), (*p < 0.05 and **p < 0.01 vs. control).

Fig. 10

PPAR pathway from the KEGG database^–.

Fig. 11

Validation of differentially expressed genes by qPCR (n = 3), (**p < 0.01).

Fig. 12

Effect of CNP on the proliferation of subcutaneous preadipocytes.(A) Proliferation of preadipocytes induced for 24 h and 48 h by CNP (n = 8, * p < 0.05 vs. control group). (B) EdU staining of intramuscular preadipocytes induced for 24 h and 72 h by CNP (n = 3). EdU, number of proliferative cells; Hoechst, number of cells before proliferation; Overlay, superimposition of the two images shows the location of proliferating cells. *p < 0.05 vs. control group.

Fig. 13

Effect of CNP on the differentiation of subcutaneous adipocytes. A, Morphologic changes and lipid deposition induced by 10⁻⁷mol/L CNP in adipocytes in vitro (inverted microscope, 200× magnification). Lipid droplets (stained with Oil Red O) were smaller and accumulated in fewer groups in cells exposed to 10⁻⁷mol/L CNP compared with untreated cells. B, Differentiation of adipocytes was significantly decreased by 10⁻⁷ mol/L CNP. (n = 3, **p < 0.01 vs. control).

Fig. 14

Change of the glycerol content at 6 days after CNP treatment in subcutaneous adipocytes. (n = 3), (*p < 0.05 vs. control).

Fig. 15

Change of NPPC mRNA expression in subcutaneous adipocytes after transfection with NPPC siRNA for 24 h. (n = 3) (**p < 0.01).

Fig. 16

Effect of NPPC interference on the proliferation of subcutaneous preadipocytes (n = 5) (**p < 0.01 vs. control). (A) The number of preadipocytes assayed by CCK8 kits after NPPC interference; (B) EdU staining assay of chicken preadipocytes transfected with NPPC siRNA. EdU, number of proliferative cells; Hoechst, number of cells before proliferation; Overlay, superimposition of the two images shows the location of proliferating cells.

Fig. 17

Effect of NPPC interference on the differentiation of subcutaneous adipocytes (A) The differentiation of adipocytes was significantly increased after transfection with NPPC siRNA (n = 3) (**p < 0.01 vs. control). (B) Morphologic changes and lipid deposition after transfection with NPPC siRNA in adipocytes in vitro (inverted microscope, 400× magnification). Lipid droplets (stained with Oil Red O) accumulated in greater numbers and in larger groups in cells transfected with NPPC siRNA compared with the negative control vector.

Fig. 18

Change of the glycerol content at 4 d after transfection with NPPC siRNA in subcutaneous adipocytes. (n = 3), (**p < 0.01 vs. control).

Fig. 19

Change of NPRB expression in subcutaneous adipocytes at 6 d after CNP treatment ( n = 3), (*p < 0.05 and **p < 0.01 vs. control).

Fig. 20

Validation of differentially expressed genes by qPCR (n = 3) (**p < 0.01 and ***p < 0.001).

Fig. 21

GO analysis of intersection genes in both IMF and SCF adipocytes.

Fig. 22

CNP regulated lipid metabolism through the NPRB-PPAR pathway in both IMF and SCF adipocytes. In IMF adipocytes, metabolism was regulated mainly by FABP4, FABP5, APOA1, ACOX2, ADIPOQ, CD36, and LPL enriched PPAR pathways, and by ACSL1, APOA1, CPT1A, CPT2, FABP4, PDPK1, ACOX3, and PPARα enriched PPAR pathways in SCF adipocytes.