Tripeptide IRW Improves AMPK/eNOS Signaling Pathway via Activating ACE2 in the Aorta of High-Fat-Diet-Fed C57BL/6 Mice

Abstract

This study aims to investigate the effect of tripeptide IRW on the local renin-angiotensin system (RAS), particularly angiotensin-converting enzyme 2 (ACE2), and their association with signaling pathways in the aorta of a high-fat-diet (HFD)-induced insulin-resistant mouse model. C57BL/6 mice were fed HFD (45% of the total calories) for six weeks, and then IRW was added to the diet (45 mg/kg body weight (BW)) for another eight weeks. ACE2 mRNA expression and protein level(s) were increased (p < 0.05), while angiotensin II receptor (AT1R) and angiotensin-converting enzyme (ACE) protein abundance was significantly reduced (p < 0.05) in the aorta of HFD mice treated by IRW. IRW supplementation also improved glucose transporter 4 (GLUT4) abundance (p < 0.05) alongside AMP-activated protein kinase (AMPK) (p < 0.05), Sirtuin 1 (SIRT1) (p < 0.05), and endothelial nitric oxide synthase (eNOS) (p < 0.05) expression. IRW downregulated the levels of endothelin 1 (ET-1) and p38 mitogen-activated protein kinases (p38 MAPK, p < 0.05). Furthermore, the levels of AMPK and eNOS in vascular smooth muscle cells (VSMCs) were significantly reduced in ACE2 knockdown cells treated with or without IRW (p < 0.01). In conclusion, this study provided new evidence of the regulatory role of IRW on the aortic ACE2 against metabolic syndrome (MetS) in an HFD-induced insulin-resistant model.

Keywords: ACE2; GLUT4; IRW; eNOS; insulin resistance; peptides.

Figure 1

Effect of IRW on protein and RNA expression of ACE2 and protein expression of ACE and AT1R in aorta of HFD mice. (a) Quantification and Western blots of ACE2. (b) q-PCR quantification of ACE2. Quantification and Western blots of (c) ACE and (d) AT1R. ACE2, ACE, and AT1R were normalized to GAPDH. Data expressed as mean ± SEM of n = 6 mice. *, p < 0.05, **, p < 0.01, and ****, p < 0.0001 versus HFD. (Fold change with regard to (w.r.t) HFD).

Figure 2

Effect of IRW on expression of p-AMPK, p-eNOS, Sirtuin1, and PPARγ in aorta of HFD mice. Quantification and Western blots of (a) p-AMPK, (b) p-eNOS, (c) Sirtuin 1, and (d) PPARγ. P-AMPK, Sirtuin1, and PPARγ were normalized to GAPDH. p-eNOS was normalized to Total eNOS. Data expressed as mean ± SEM of n = 4 for p-eNOS, Sirtin 1, and p-AMPK, and n = 6 for PPARγ. *, p < 0.05, ***, p < 0.001 versus HFD. (Fold change with regard to (w.r.t) HFD).

Figure 3

Effect of IRW on expression of ACE2, p-eNOS, p- AMPK, and Total eNOS in vehicle, IRW-treated, and ACE2 knockdown (KD) VSMCs. Quantification and Western blots of (a) ACE2, (b) p-eNOS, (c) p-AMPK, and (d) Total eNOS. ACE2, p-AMPK, p-AMPK, and Total eNOS were normalized to GAPDH. Data expressed as mean ± SEM of four independent experiments. *, p < 0.05, **, p < 0.01, and ****, p < 0.0001 versus IRW-treated group.

Figure 4

Effect of IRW on expression of GLUT4 in aorta of HFD mice. Quantification and Western blots of GLUT4. GLUT4 was normalized to GAPDH. Data expressed as mean ± SEM of n = 6 mice. **, p < 0.01 and ***, p < 0.001 versus HFD. (Fold change with regard to (w.r.t) HFD).

Figure 5

Effect of IRW on expression of ET-1, MAPK P38, and P-ERK1/2 in aorta of HFD mice. (a) Quantification and Western blots of ET-1, (b) MAPK P38, and (c) p-ERK. ET-1, MAPK P38, and p-ERK were normalized to GAPDH. Data expressed as mean ± SEM of n = 4 mice. *, p < 0.05, **, p < 0.01 versus HFD. (Fold change with regard to (w.r.t) HFD).