Adipocyte endothelin B receptor activation inhibits adiponectin production and causes insulin resistance in obese mice

Abstract

Aims: Endothelin-1 (ET-1) is elevated in patients with obesity and adipose tissue of obese mice fed high-fat diet (HFD); however, its contribution to the pathophysiology of obesity is not fully understood. Genetic loss of endothelin type B receptors (ET_B) improves insulin sensitivity in rats and leads to increased circulating adiponectin, suggesting that ET_B activation on adipocytes may contribute to obesity pathophysiology. We hypothesized that elevated ET-1 in obesity promotes insulin resistance by reducing the secretion of insulin sensitizing adipokines, via ET_B receptor.

Methods: Male adipocyte-specific ET_B receptor knockout (adET_BKO), overexpression (adET_BOX), or control littermates were fed either normal diet (NMD) or high-fat diet (HFD) for 8 weeks.

Results: RNA-sequencing of epididymal adipose (eWAT) indicated differential expression of over 5500 genes (p < 0.05) in HFD compared to NMD controls, and changes in 1077 of these genes were attenuated in HFD adET_BKO mice. KEGG analysis indicated significant increase in metabolic signaling pathway. HFD adET_BKO mice had significantly improved glucose and insulin tolerance compared to HFD control. In addition, adET_BKO attenuated changes in plasma adiponectin, insulin, and leptin that is observed in HFD versus NMD control mice. Treatment of primary adipocytes with ET-1 caused a reduction in adiponectin production that was attenuated in cells pretreated with an ET_B antagonist.

Conclusion: These data indicate elevated ET-1 in adipose tissue of mice fed HFD inhibits adiponectin production and causes insulin resistance through activation of the ET_B receptor on adipocytes.

Keywords: adipocytes; endothelin‐1; insulin sensitivity; obesity.

Figure 1:

Generation and detection of adET_BKO or adET_BOX mice. A and B) ET_B receptor western blot of membrane and cytosolic fraction of visceral adipose from floxed control, adipocyte ET_B overexpression, and adipocyte knockout mice (A&B). C) PCR product using primers around the floxed region of the EDNRB gene. Recombination was found only in epididymal, subcutaneous, and brown adipose tissue of adiponectin Cre⁺ EDNRB^flox/flox mice. E) Schematic of human EDNRB cloned into intron 1 of the ROSA26 gene and inserted using Crispr/Cas9. Upstream of the EDNRB gene is a floxed PolyA site to stop transcription. In the presence of Cre recombinase, the PolyA site will be removed leading to transcription and overexpression of the hEDNRB gene tagged with an mCherry reporter. F and G) mCherry florescence in various tissues (A-brain, B-lung, C-heart, D-inguinal adipose, E-liver, F-spleen, G-kidney, H-gonadal adipose, I-skeletal muscle) or live animals to detect recombination in either adiponectin Cre- or Cre+ transgenic mice. mCherry reporter was only detected in adipose tissue of Cre+ hEDNRB transgenic mice (2,5, and 7).

Figure 2.

Endothelin-1 decreases peroxisome proliferator-activated receptor gamma (Pparg) and adiponectin production in mouse primary adipocytes via ET_B receptor. Droplet digital PCR of Pparg, Adiponectin, insulin receptor substrate-1 (Irs-1), insulin receptor substrate-2 (Irs-2), and glucose transporter 4 (SLC2A4) from epididymal white adipose (eWAT) primary adipocytes of floxed control and adET_BKO mice (n=6) treated with ET-1 for 5 days (A-D) and from eWAT primary adipocytes of floxed control mice (n=3) treated with ET-1 in the presence or absence of BQ-788, a specific ET_B inhibitor, for 5 days (E-H). Adiponectin release by primary adipocytes from eWAT of Floxed control mice (n=6) treated with ET-1 and BQ-788 for 5 days (I). Data are expressed as mean ± SEM. * = p<0.05, † = p<0.01.

Figure 3.

Adipocyte ET_B receptor knockout or overexpression does not alter body composition in HFD fed mice. Body weight (A&B), percent fat mass (C&D), measured by echo MRI in adET_BKO and adET_BOX and the respective controls at 0, 4, and 8 weeks of NMD or HFD. E) H&E stain of eWAT and quantification of adipocyte size. NMD=normal diet; HFD=High fat diet; Ad-ET_BKO=Adipocyte ET_B knockout; Ad-ET_BOX=Adipocyte ET_B overexpression. Data were analyzed by three-way RM ANOVA. P values represent probability that a given variable or interaction of variables contributes to variation. If p value is not presented, the interaction was ns.

Figure 4.

High-fat diet increases ET-1 and Hif1α in adipose tissue. Droplet digital PCR of Hif1α, ET-1, EdnrA, and EdnrB in eWAT (n=5) (A,C,E, and G respectively), and iWAT (n=5) (B, D, F, and H respectively) of NMD control, HFD control and HFD adET_BKO mice after 8 weeks on diet, respectively. Data are expressed as mean ± SEM. * = p<0.05, † = p<0.01 following Tukey’s post hoc test.

Figure 5:

Adipocyte ET_B receptor knockout improves expression of genes enriched in metabolic and insulin signaling pathways. A) RNA sequencing heat map of differentially expressed genes (HFD vs. HFD adET_BKO) within metabolic pathways identified by KEGG analysis. B) Principal component analysis plot of top 500 expressed genes from NMD control (n=5), HFD control (n=4), HFD adET_BKO (n=5), and HFD adET_BOX (n=4). C) List of top five upregulated (red) and top 5 downregulated (blue) pathways between HFD control and HFD adET_BKO.

Figure 6.

Adipocyte ET_B receptor knockout attenuates HFD induced reduction in major mediators of insulin signaling. Droplet digital PCR of Irs-1, Irs-2, and SLC2A4 from iWAT (n=5) (A-C), eWAT (n=5) (D-F), and Adipocytes from eWAT (n=3; G-I), of NMD FLOXED, HFD FLOXED, HFD adET_BKO, and HFD adET_BOX mice after 8 weeks on diet. Data are expressed as mean ± SEM. * = p<0.05, † = p<0.01 following Tukey’s post hoc test.

Figure 7.

Adipocyte ET_B receptor knockout improves insulin sensitivity in HFD-fed mice. Intraperitoneal insulin tolerance (ITT, 0.75 IU/kg) and oral glucose tolerance (GTT, 2 g/kg) in adET_BKO (A & C) and adET_BOX (E and G) and respective controls after 8 weeks on NMD or HFD. Area under the curve for each mouse was analyzed for ITT (B & F) and GTT (D & H). Data were analyzed by two-way ANOVA and are expressed as mean ± SEM. * = p<0.05, † = p<0.01following Tukey’s post hoc test.

Figure 8.

Adipocyte ET_B receptor knockout improves adipokine expression profile in HFD-fed mice. Analysis of fasting plasma adiponectin and plasma leptin (A & B) in NMD floxed (n=10), HFD floxed (n=10), HFD adET_BKO (n=12), and HFD adET_BOX mice after 8 weeks on diet. Droplet digital PCR of Adiponectin, Adipsin, Leptin and Resistin in iWAT (n=5) (C-F), eWAT (n=5) (G-J), and adipocytes from eWAT (n=3) (K-N), of NMD and HFD adET_BKO adET_BOX mice after 8 weeks on diet, respectively. Data are expressed as mean ± SEM; * = p<0.05, † = p<0.01.