Adiponectin Protects Against Cerebral Ischemic Injury Through AdipoR1/AMPK Pathways

Abstract

Excitotoxicity induced by excessive N-methyl-D-aspartate (NMDA) receptor activation underlies the pathology of ischemic injury. Adiponectin (APN) is an adipocyte-derived protein hormone that modulates a number of metabolic processes. APN exerts a wide range of biological functions in the central nervous system. However, the role of APN and its receptors in cerebral ischemia/reperfusion (I/R)-induced injury and the related mechanisms remain to be clarified. Here, we found that APN and APN receptor agonist AdipoRon (APR) were protective against excitotoxicity induced by oxygen and glucose deprivation/reperfusion (OGD/R) and NMDA in primary neurons. Adiponectin receptor 1 (AdipoR1) knockdown reversed the protection conferred by either APN or APR. Moreover, the protective effects offered by both APN and APR were compromised by compound C, an inhibitor of amp-activated protein kinase (AMPK) phosphorylation. Both APN and APR protected the dissipation of the ΔΨm caused by OGD/R. They also up-regulated the PGC-1α expression, which was reversed by compound C. Furthermore, both APN and APR ameliorated but APN knockout aggravated the infarct volume and neurological deficient induced by transient middle cerebral artery occlusion (tMCAO) in vivo. Taken together, these findings show that APN and APR protect against ischemic injury in vitro and in vivo. The protective mechanism is mainly related to AdipoR1-dependent AMPK phosphorylation and PGC-1α up-regulation.

Keywords: AdipoRon; PGC-1α; adiponectin; adiponectin receptor 1; amp-activated protein kinase; ischemia; mitochondrial.

Figure 1

Effects of APN and APR on OGD/R-induced and NMDA-induced cell viability in promary neurons. (A) When APN (0.5 h before OGD treatment) was administered, cell viability was tested in primary neurons by MTT assay after OGD/R-induced injury (n = 6 per condition; **P <0.01, ***P <0.001 with ANOVAs followed by Tukey’s post hoc test). Data are presented as mean ± SEM. (B) When APR (0.5 h before OGD treatment) was administered, cell viability was tested in primary neurons by MTT assay after OGD/R-induced injury (n = 8-14 per condition; **P <0.01, ***P <0.001 with ANOVAs followed by Tukey’s post hoc test). Data are presented as mean ± SEM. (C) When APN (0.5 h before NMDA treatment) was administered, cell viability was tested in primary neurons by MTT assay after NMDA-induced (200 μM, 2 h) (n = 9-14 per condition; *P < 0.05, ***P < 0.001 with ANOVAs followed by Tukey’s post hoc test). Data are presented as mean ± SEM. (D) When APR (0.5 h before NMDA treatment) was administered, cell viability was tested in primary neurons by MTT assay after NMDA-induced (200 μM, 2 h) injury (n = 7 per condition; *P <0.05, **P <0.01, ***P <0.001 with ANOVAs followed by Tukey’s post hoc test). Data are presented as mean ± SEM.

Figure 2

Effects of APN and APR on OGD/R-induced apoptosis and dendritic morphology in promary neurons. (A) Effects of APN (1 µg/ml) and APR (1 µM) on apoptotic cells stained by TUNEL in primary neurons under OGD/R. TUNEL-positive cells are green, and all cells are stained with DAPI (blue). Scale bar: 250 µm. (B) The bar graph indicated the effects of APN and APR on the percentage of TUNEL-positive apoptotic neurons under OGD/R (n = 4 per condition; ***P <0.001 with ANOVAs followed by Tukey’s post hoc test). Data are presented as mean ± SEM. (C) Effects of APN (1 µg/ml) and APR(1 µM) on MAP-2-positive (red) neuronal morphology under OGD/R. Scale bar: 50 µm. (D) The bar graph indicated the effects of APN and APR on the total dendritic length under OGD/R in primary neurons. (n = 4 per condition; **P <0.01, ***P <0.001 with ANOVAs followed by Tukey’s post hoc test). Data are presented as mean ± SEM. (E) Sholl analysis of the effects of APN and APR on the dendritic intersections under OGD/R in primary neurons (n = 9 per condition; **P <0.01 with ANOVAs followed by Tukey’s post hoc test). Data are presented as mean ± SEM.

Figure 3

Involvement of AdipoR1 in the protective effects of APN and APR against OGD/R-induced injury in primary neurons. (A) Representative Western blots and the bar graph showing the AdipoR1 expression in different brain regions under tMCAO-induced cerebral injury (n = 3 per condition; *P <0.05, ***P <0.001 with ANOVAs followed by Tukey’s post hoc test). Data are presented as mean ± SEM. (B) Representative Western blots showing the AdipoR2 expression in the liver and brain. (C) Representative Western blots and the bar graph showing the AdipoR1 expression interfered by three different siRNA sequences cultured cortical neurons (n = 3 per condition; **P <0.01 vs. negative control group with ANOVAs followed by Tukey’s post hoc test). Data are presented as mean ± SEM. (D) The cell viability tested by MTT assay showing the effect of AdipoR1 siRNA on the protections of APN and APR against OGD/R in primary neurons (n = 8–9 per condition; *P <0.05, **P <0.01, ***P <0.001 with ANOVAs followed by Tukey’s post hoc test). Data are presented as mean ± SEM. (E) The cell viability tested by MTT assay showing the effect of AdipoR1 siRNA on the protection of APN and APR against NMDA in primary neurons (n = 9 per condition; *P <0.05, **P <0.01, ***P <0.001 with ANOVAs followed by Tukey’s post hoc test). Data are presented as mean ± SEM.

Figure 4

Involvement of AMPK pathway in the protective effects of APN and APR against OGD/R-induced injury. (A) Representative Western blots and bar graph showing the effects of APN (1 µg/ml, 0.5 h before OGD), APR (1 µM, 0.5 h before OGD) and compound C (10 µM, 1 h before OGD) on AMPK phosphorylation under OGD/R in primary neurons (n = 3 per condition; *P <0.05, **P <0.01 with ANOVAs followed by Tukey’s post hoc test). Data are presented as mean ± SEM. (B) Effects of compound C (10 µM, 1 h before OGD) on the protections of APN (1 µg/ml, 0.5 h before OGD) and APR (1 µM, 0.5 h before OGD) against OGD/R-induced injury in primary neurons by MTT assay (n = 8 per condition; *P <0.05, ***P <0.001 with ANOVAs followed by Tukey’s post hoc test). Data are presented as mean ± SEM. (C) Representative JC-1 fluorescence images showing the effects of APN (1 µg/ml, 0.5 h before OGD) and APR (1 µM, 0.5 h before OGD) on OGD/R-induced injury in primary neurons. Red fluorescence indicates a polarized state and green fluorescence indicates a depolarized state. Scale bar: 50 µm. (D) Representative Western blots and the bar graph showing the PGC-1α expression treated by compound C (10 µM, 1 h before OGD), APN (1 µg/ml, 0.5 h before OGD), and APR (1 µM, 0.5 h before OGD) in primary neurons under OGD/R-induced injury (n = 4 per condition; *P <0.05, **P <0.01,***P <0.001 with ANOVAs followed by Tukey’s post hoc test). Data are presented as mean ± SEM.

Figure 5

APN and APR alleviate the tMCAO-induced injury through AMPK pathway. (A) Brain sections stained by 2,3,5-Triphenyltetrazolium chloride (TTC) from WT and APN-/- mice showing the infarct area in those receiving saline, APN (i.c.v., 0.3 µg/mouse, 0.5 h before tMCAO), APR (i.c.v., 1 µg/mouse, 0.5 h before tMCAO), and compound C (i.c.v., 40 µg/mouse, 1 h before tMCAO). The bar graph showing the infarct volume (B) and neurological scores (C) (n = 4–5 per condition; *P <0.05, **P <0.01, ***P <0.001 with ANOVAs followed by Tukey’s post hoc test). Data are presented as mean ± SEM.