High Glucose Attenuates Cardioprotective Effects of Glucagon-Like Peptide-1 Through Induction of Mitochondria Dysfunction via Inhibition of β-Arrestin-Signaling

Abstract

An increased vulnerability has been detected after ischemia/reperfusion injury in cardiomyocytes in diabetic patients. Glucagon-like peptide-1 (GLP-1) has been proven to have a notable cardioprotective effect in cardiomyocytes. However, in diabetic patients, the cardioprotective effects of GLP-1 are compromised, which is called GLP-1 resistance. β-arrestin is one of the two main downstream effectors of GLP-1 and β-arrestin signaling pathway exerts cardioprotective effects upon activation of GLP-1R. Our hypothesis is that the increased vulnerability of cardiomyocytes in diabetic patients is partly due to disruption of the β-arrestin signaling pathway. To test this, we analyzed cardiomyocyte viability and survival in high glucose and normal glucose condition after hypoxia/reoxygenation injury in vitro, additional GLP-1 was used to determine whether β-arrestin signaling pathway was involved. We also investigated the role of mitochondrial dysfunction in GLP-1 resistance. Our results showed that cardioprotective effects of GLP-1 were reduced in high glucose cultured H9C2 cells compared to normal glucose cultured H9C2, verifying the existence of GLP-1 resistance in high glucose cultured H9C2 cells. Further study suggested that β-arrestin plays a key role in GLP-1 resistance: β-arrestin expression is notably downregulated in high glucose condition and cardioprotective effects of GLP-1 can be diminished by downregulation of β-arrestin in normal glucose condition while upregulation of β-arrestin can restore cardioprotective effects of GLP-1 in high glucose condition. Then we explore how β-arrestin affects the cardioprotective effects of GLP-1 and found that β-arrestin exerts cardioprotective effects by improving mitochondria quality control via the PI3K/Akt signaling pathway. Thus, our study found out a new mechanism of GLP-1 resistance of cardiomyocytes in high glucose conditions that impaired β-arrestin expression, caused mitochondria dysfunction and eventually cell death. Our study provided a new perspective in treating myocardial ischemia/reperfusion injury in diabetic patients.

Keywords: GLP-1; PI3K/Akt; diabetic cardiomyocyte; mitochondria dysfunction; β-arrestin.

FIGURE 1

High glucose condition attenuates cardioprotective effects of glucagon-like peptide-1. (A,B) TUNEL staining was conducted and apoptosis index (%) was calculated. (C,D) Anti-apoptotic Bcl-2 and pro-apoptotic BAX were measured using qRT-PCR. (E) Caspase-3 activity was measured using caspase-3 activity kit. (F) Cell viability was measured via CCK-8 assay. *P < 0.05.

FIGURE 2

Increased susceptibility of mitochondrial dysfunction attenuated cardioprotective effects of GLP-1 in high glucose cultured H9C2 cells. (A,B) Measurement of mitochondrial function using ROS staining. (C,D) Detection of mitochondrial fission level by qRT-PCR measurement of Drp1 and Mff. (E,F) Detection of mitophagy level by qRT-PCR measurement of Atg5 and Beclin1. *P < 0.05.

FIGURE 3

β-arrestin overexpression restored cardioprotective effects of GLP-1 in high glucose cultured H9C2 cells. (A) qRT-PCR measurement of β-arrestin transcription. (B–D) Exploring the changes in cardioprotective effects of Exendin-4 while utilizing β-arrestin siRNA and adenovirus transfection. *P < 0.05.

FIGURE 4

High glucose attenuates cardioprotective effects of GLP-1 through induction of mitochondrial dysfunction via inhibition of β-arrestin-signaling. (A,B) Mitochondrial fission level measured by qRT-PCR analysis of Drp1 and Mff. (C,D) Mitophagy level measured by qRT-PCR analysis of Atg5 and Beclin1. (E–G) Detection of mitochondria morphology using mitochondria staining. *P < 0.05.

FIGURE 5

Upregulation of β-arrestin attenuates mitochondrial dysfunction via PI3K/Akt signaling pathway. (A) qRT-PCR analysis of apoptotic biomarkers Bcl-2 and BAX. (B) qRT-PCR analysis of mitochondrial fission biomarkers Drp1 and Mff. (C) qRT-PCR analysis of mitophagy biomarkers Atg5 and Beclin1. *P < 0.05.