Involvement of guanylin and GC-C in rat mesenteric macrophages in resistance to a high-fat diet

Abstract

A high-fat diet (HFD) is a well-known contributing factor in the development of obesity. Most rats fed HFDs become obese. Those that avoid obesity when fed HFDs are considered diet resistant (DR). We performed a microarray screen to identify genes specific to the mesenteric fat of DR rats and revealed high expression of guanylin and guanylyl cyclase C (GC-C) in some subjects. Our histologic studies revealed that the cellular source of guanylin and GC-C is macrophages. Therefore, we developed double-transgenic (Tg) rats overexpressing guanylin and GC-C in macrophages and found that they were resistant to the effects of HFDs. In the mesenteric fat of HFD-fed Tg rats, Fas and perilipin mRNAs were downregulated, and those of genes involved in fatty acid oxidation were upregulated, compared with the levels in HFD-fed wild-type rats. In vitro studies demonstrated that lipid accumulation was markedly inhibited in adipocytes cocultured with macrophages expressing guanylin and GC-C and that this inhibition was reduced after treatment with guanylin- and GC-C-specific siRNAs. Our results suggest that the macrophagic guanylin-GC-C system contributes to the altered expression of genes involved in lipid metabolism, leading to resistance to obesity.

Fig. 1.

GC-C mRNA expression in mesenteric fat tissue. (A) Quantitative PCR for guanylin or GC-C-mRNA. Cont, control rats fed standard chow; DIO, rats with HFD-induced obesity; DR, rats with resistance to an HFD. (B) Double immunostaining of mesenteric fat with anti-guanylin or anti-GC-C plus anti-CD68 antibodies in DR rats. Left panel, guanylin (brown) and CD68 (blue); middle panel, GC-C (brown) and CD68 (blue); right panel, control IgG. Scale bar, 50 μm.

Fig. 2.

Generation of guanylin-GC-C BAC transgenic (Tg) rats. Structures of the (A) rat SR-A (CH230-56C20) and (B) human guanylin (RP11-627G14) BAC clones. Each BAC clone contains the full-length coding sequence and the 5- and 3-flanking sequences. (C, D) Schematic diagram of transgene construction. By using Red/ET recombination, the coding region of the rat SR-A gene was replaced with either (C) the entire human guanylin gene or (D) the human GC-C cDNA fragment. Ex, exon. (E) Southern blot analysis of guanylin-GC-C double-Tg lines. Hybridization with a radioactive probe showed that the BAC-transduced human guanylin and GC-C sequences were detected as a 1.7 kb PstI fragment and a 1.9 kb Dra I fragment, respectively. The copy number of each integrated transgene was determined through comparison with the intensity of a control signal (right panel). (F) Western blot analysis of the protein levels of guanylin and GC-C in the mesenteric fat tissues of male Tg and WT rats fed standard chow. β-actin was used as a loading control.

Fig. 3.

Parameters of energy metabolism in guanylin-GC-C double-Tg rats. (A‑D) Body weight (A, C) and food intake (B, D) of WT and Tg rats that were fed standard chow (STD; A, B) or an HFD (C, D). (E) Macroscopic appearance of 21-week-old HFD WT and HFD Tg rats. (F) Weight of the mesenteric fat of WT and Tg rats fed an STD or an HFD. (G) Hematoxylin-eosin staining of sections of mesenteric fat. Scale bar, 100 μm. (H) Diameters of adipocytes from HFD Tg and WT rats. Data are presented as means ± SEM (n = 3). ***P < 0.001 versus WT rats. (I) Plasma concentrations of free fatty acid (FFA), glycerol, and total cholesterol. All data are presented as means ± SEM (n = 4 or 5). *P < 0.05 compared with value for WT rats on the same diet.

Fig. 4.

Gene expression in the mesenteric fat of WT and guanylin-GC-C double-Tg rats. (A, B) Quantitative RT-PCR analysis of mRNA transcripts of genes associated with (A) adipocyte maturation and droplet formation and (B) fatty acid oxidation in mesenteric adipose tissue. Data were normalized to the GAPDH mRNA levels and are presented as means ± SEM (n = 4 or 5). *P < 0.05 compared with value for WT rats on the same diet. Acadam, acyl-CoA dehydrogenase C4 to C12 straight-chain; Acadl, acyl-CoA dehydrogenase long chain; Acox1, acyl-CoA oxidase 1 palmitoyl; Adipoq, adiponectin; C/EBPα, CCAAT-enhancer-binding protein α; Cpt1a, carnitine palmitoyltransferase 1a; GLUT4, glucose transporter 4; PPARγ, peroxisome proliferator-activated receptor γ.

Fig. 5.

(A) Macroscopic appearance of the liver. (B) Hematoxylin-eosin staining of liver sections. Scale bar, 50 μm. (C) Triacylglycerol content of the livers of Tg and WT rats. (D) Blood glucose and plasma insulin levels in Tg and WT rats. Data are presented as means ± SEM (n = 4 or 5). **P < 0.01 compared with value for WT rats on the same diet.

Fig. 6.

Coculture of primary adipocytes with guanylin- and GC-C-expressing macrophages. (A) Lipid accumulation in adipocytes cocultured with peritoneal macrophages of WT and Tg rats. Neutral lipids were stained with Oil Red O on day 8 after the induction of adipogenesis. Scale bar, 20 μm. (B) Quantitative PCR of genes involved in adipocyte maturation and droplet formation. Data were normalized against the mRNA levels of ribosomal protein 36B4 and are expressed as fold induction relative to that of adipocytes with peritoneal macrophage of WT rats. Data are presented as means ± SEM (n = 16). *P < 0.05, **P < 0.01, ***P < 0.001 compared with the value for adipocytes cocultured with peritoneal macrophages of WT rats. (C) Lipid accumulation in adipocytes cocultured with nontargeting control mock-transfected macrophages or cocultured with macrophages transfected with guanylin and GC-C siRNAs. Neutral lipids were stained with Oil Red O six days after the induction of adipogenesis. Scale bar, 20 μm. (D) Quantitative PCR of genes involved in adipocyte maturation and droplet formation. Data were normalized against the amount of 36B4 mRNA and were expressed as fold induction relative to that of adipocytes cocultured with mock-transfected macrophages. Data are presented as means ± SEM (n = 5). ***P < 0.001 compared with the value for adipocytes cocultured with mock-transfected macrophages. (E) mRNA levels of GC-C and guanylin in macrophages 24 h after their transfection with specific siRNAs against GC-C or guanylin or mock siRNA. ***P < 0.001 compared with value for mock-transfected macrophages (n = 7). (F) Western blot analysis of GC-C and immnunohistochemical analysis of guanylin in macrophages 6 days after transfection with GC-C, guanylin, or mock siRNA. β-actin was used as a loading control. For immunohistochemical analysis, cultured cells were fixed with paraformaldehyde and stained with anti-guanylin antibody and Alexa-594-conjugated anti-rabbit IgG. Scale bar, 50 μm.