Evidence that adiponectin receptor 1 activation exacerbates ischemic neuronal death

Abstract

Background: Adiponectin is a hormone produced in and released from adipose cells, which has been shown to have anti-diabetic and anti-inflammatory actions in peripheral cells. Two cell surface adiponectin receptors (ADRs) mediate the majority of the known biological actions of adiponectin. Thus far, ADR expression in the brain has been demonstrated in the arcuate and the paraventricular nucleus of hypothalamus, where its activation affects food intake. Recent findings suggest that levels of circulating adiponectin increase after an ischemic stroke, but the role of adiponectin receptor activation in stroke pathogenesis and its functional outcome is unclear.

Methods: Ischemic stroke was induced in C57BL/6 mice by middle cerebral artery occlusion (MCAO) for 1 h, followed by reperfusion. Primary cortical neuronal cultures were established from individual embryonic neocortex. For glucose deprivation (GD), cultured neurons were incubated in glucose-free Locke's medium for 6, 12 or 24 h. For combined oxygen and glucose deprivation (OGD), neurons were incubated in glucose-free Locke's medium in an oxygen-free chamber with 95% N2/5% CO2 atmosphere for either 3, 6, 9, 12 or 24 h. Primary neurons and brain tissues were analysed for Adiponectin and ADRs using reverse transcriptase polymerase chain reaction (RT-PCR), immunoblot and immunochemistry methods.

Results: Cortical neurons express ADR1 and ADR2, and that the levels of ADR1 are increased in neurons in response to in vitro or in vivo ischemic conditions. Neurons treated with either globular or trimeric adiponectin exhibited increased vulnerability to oxygen and glucose deprivation which was associated with increased activation of a pro-apoptotic signaling cascade involving p38 mitogen-activated protein kinase (p38MAPK) and AMP-activated protein kinase (AMPK).

Conclusions: This study reveals a novel pathogenic role for adiponectin and adiponectin receptor activation in ischemic stroke. We show that cortical neurons express ADRs and reveal a pro-apoptotic role for ADR1 activation in neurons, which may render them vulnerable to ischemic death.

Figures

Figure 1

Neurons express ADR1 and ADR2 and respond to oxygen and glucose deprivation. (A) ADR1 and ADR2 mRNA are present in cultured cortical neurons determined by single-cell RT-PCR analysis. The numbers indicate the number of neurons from which RNA was amplified; 1 and 3 cortical neurons consistently yielded a positive PCR signal for the ADR1 and ADR2 with exactly predicted size. (B) Cortical neurons subjected to OGD for the indicated times show increased levels of ADR1 and ADR2 mRNA in a time-dependent manner. (C) Immunoblot analysis of proteins in cell lysates of neurons in control cultures and cultures subjected to OGD for 3-24 h. OGD resulted in increased levels of ADR1 and ADR2 (D) ADR1 immunoreactivity (red) in cultured neurons; cells were counterstained with DAPI (blue) to label all nuclei. Arrow points to the axon of a neuron and arrowheads point to dendrites of the same neuron. (E) Cortical neurons subjected to OGD following globular adiponectin treatment show increased levels of ADR1.

Figure 2

Cerebral ischemia increases ADRs immunoreactivity in the brain. (A) Immunoblot analysis of protein samples from the cerebral cortex of sham operated control mice and mice subjected to 1 h cerebral ischemia and 1-24 h reperfusion. Ischemia resulted in rapid increases in the levels of ADRs immunoreactivities in neurons in the penumbra area (P). (Scale bars: 50 μM). (B) Images of brain sections showing ADR1 immunoreactivities (green) and NeuN (neuronal marker) in mice subjected to cerebral ischemia (1 h) and reperfusion (24 h). (C) Adiponectin accumulates in vessels like structures (large yellow arrow) and in parenchyma (small yellow arrow) in the human ischemic brain. (D) Control brain tissue stained with secondary antibody shows no staining.

Figure 3

ADR activation mediates neuronal cell death following in vitro ischemia-like conditions. (A, B) Neuronal cultures were treated with 10 μg of the globular adiponectin (gAd) and then subjected to OGD for 12 h (A) or GD for 24 h (B). Proteins in cell lysates were then subjected to immunoblot analysis by using the indicated antibodies. The gAd treatment enhanced OGD or GD-induced increases in levels of p-AMPK, p-38 MAPK and activated caspase-3. (C-D) Globular adiponectin (gAd) and trimeric adiponectin (tAd) treatment exacerbates OGD induced death of cultured primary neurons. Neuronal cell death was quantified 12 h later. Values are mean ± s.e.m. (n = 6-10 cultures). ***P < 0.0001 compared to OGD or vehicle treated OGD value, *P < 0.05 compared to OGD or vehicle treated OGD value. (E) Globular adiponectin (gAd) treatment exacerbates GD induced death of cultured primary neurons. Neuronal cell death was quantified 24 h later. Values are mean ± s.e.m. (n = 6-10 cultures). ***P < 0.0001 compared to GD or vehicle treated GD value.