Elsevier

Brain Research Bulletin

Volume 169, April 2021, Pages 145-155
Brain Research Bulletin

Exendin-4 induces a novel extended effect of ischemic tolerance via crosstalk with IGF-1R

https://doi.org/10.1016/j.brainresbull.2020.11.008Get rights and content

Highlights

  • Sustained Ex-4 administration confers cerebral ischemic tolerance against focal ischemia.

  • Ex-4 activates the PI3K-AKT-mTOR-HIF-1 signaling pathway after MCAO.

  • Ex-4 augments downstream mediators of HIF-1, leading to inhibition of neuronal apoptosis after MCAO.

  • Crosstalk between GLP-1R and IGF-1R system contributes to Ex-4-induced long-term ischemic tolerance.

Abstract

Glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) agonist exendin-4 (Ex-4), a drug that has been used in the clinical treatment of type 2 diabetes mellitus, also confers a neuroprotective effect against stroke. Although GLP-1 analogs were reported to induce sustained insulin secretion and glucose tolerance improved after cessation of treatment, no study has revealed whether Ex-4 exerts sustained neuroprotection against stroke and the underlying mechanism after treatment cessation. In this study, mice were pretreated with Ex-4 for 7 days, and middle cerebral artery occlusion (MCAO) was performed on different days after cessation of Ex-4 treatment. Ex-4 ameliorated neurological dysfunction and reduced the infarct volume induced by MCAO. These protective effects lasted for 6 days after the cessation of Ex-4 treatment and were associated with sustained upregulation of PI3K, AKT, mTOR, and HIF-1α levels, as well as HIF-1α downstream genes. Knockdown of GLP-1R or HIF-1α in the brain by short hairpin RNA abolished Ex-4 treatment-mediated neuroprotection. In normal mice, Ex-4 treatment led to instant upregulation of p-PI3K, p-AKT, p-mTOR, and HIF-1α expression levels, which quickly returned to normal after cessation of Ex-4 treatment, while the expression levels of insulin growth factor-1 receptor (IGF-1R) remained high for 6 days after Ex-4 cessation. Additionally, Ex-4 did not directly induce IGF-1 production, which was only induced by MCAO. Ex-4 induces extended cerebral ischemic tolerance. This neuroprotective effect is associated with activation of GLP-1R and upregulation of IGF-1R in the brain, and the latter then activates the PI3K/AKT/mTOR/HIF-1 signaling pathway via binding to IGF-1 secreted from the ischemic brain.

Introduction

Ischemic stroke is a devastating disease that affects more than 10 million people per year worldwide and is the second most common cause of death according to the World Health Organization (Feigin et al., 2014). Millions of major surgical procedures, such as coronary artery bypass grafting, cardiac valve replacements, carotid endarterectomies, and brain surgeries, are performed every year worldwide, with an increasing frequency of perioperative cerebral ischemia and leading to profound neuronal defects (Weiser et al., 2008). Diabetes mellitus (DM) is a common and well-established risk factor of ischemic stroke (Lau et al., 2019). Stroke patients with DM have poorer short- and long-term prognoses than stroke patients without DM (Boehme et al., 2017). Furthermore, many patients with ischemic stroke exhibit diabetes or pre-diabetes symptoms upon admission to the hospital (Zinman et al., 2017). For patients at high risk of diabetic stroke, although several glucose-lowering agents such as rosiglitazone and pioglitazone may have advantages in stroke prevention, effective preventive strategies remain a major clinical challenge (Woo et al., 2019). The number of effective therapeutics for cerebral ischemia is limited and, to date, the only approved acute treatment for ischemic stroke is tissue plasminogen activator (tPA) reperfusion. However, because of the limited time window for treatment and the rigorous inclusion criteria, a minority of patients with stroke are able to benefit from this treatment (Fisher and Saver, 2015; Prabhakaran et al., 2015; Wardlaw et al., 2012). Additionally, thrombolytic treatments increase the risk of hemorrhage, the incidence of which is typically elevated when patients have common stroke comorbidities, such as atrial fibrillation, hypertension, and DM (Whiteley et al., 2012; Yaghi et al., 2014). Therefore, it is important to develop therapeutic approaches that can salvage the ischemic brain from widespread neuronal damage. Considering that millions of individuals are at an increased risk of global or focal cerebral ischemia and that our ability to predict those who are predisposed to or are at risk of cerebral ischemia has significantly improved in recent years, it is extremely important to develop a prophylactic neuroprotective intervention that can be delivered before blood flow to the brain is stopped (Dirnagl et al., 2009; T.V. N et al., 2015).

Glucagon-like peptide 1 (GLP-1) is a gut-derived hormone that facilitates insulin release under high blood sugar conditions by activating the GLP-1 receptor (GLP-1R). Recently, the GLP-1R agonists exendin-4 (Ex-4), liraglutide, and lixisenatide have been developed for the treatment of type 2 DM (Eng et al., 2014; Lindamood and Taylor, 2015). The beneficial effects of GLP-1R agonists are not limited to the treatment of diabetes but also include significant neuroprotection in animal models of cerebral ischemia by reducing apoptosis, suppressing oxidative stress, and inhibiting inflammation (Darsalia et al., 2014b, 2015). Interestingly, previous studies, including ours, have demonstrated that GLP-1R agonists can mediate a “memory effect” after treatment cessation. Hui et al. found that although GLP-1 is rapidly degraded and its circulating levels return to normal within 1 day of treatment cessation, its beneficial effects on insulin secretion lasted up to 3 weeks after its infusion was stopped in Zucker diabetic fatty rats (Hui et al., 2002). A sustained improvement in glucose tolerance was also observed in diabetic mice for weeks after cessation of treatment with DPP VI-resistant analogs of GLP-1 and nonpeptidic GLP-1R agonists (Chen et al., 2007; Ma et al., 2009; Su et al., 2008; Xu et al., 1999). In a clinical trial, insulin secretion was sustained in patients treated with exenatide after discontinuing the drug for 4 weeks (Bunck et al., 2011). However, the molecular mechanisms of these long-term beneficial effects of GLP-1R agonists remain unclear. Because type 2 DM is a major risk factor for ischemic stroke, the “memory effect” provides a potential clinical use of GLP-1R agonists for the treatment of stroke in patients with type 2 DM or individuals at high risk of stroke; however, to the best of our knowledge, no report has revealed a similar “memory effect” of neuroprotection induced by GLP-1R agonists. The goal of this study, therefore, was to investigate whether Ex-4 elicits long-term neuroprotection against focal cerebral ischemia in mice and explore the molecular mechanisms involved.

Section snippets

Experimental animals

Age-matched (8–10 weeks) male C57BL/6 mice weighing 18–22 g were acquired from the Experimental Animal Center of the Fourth Military Medical University (Xi’an, China). The animals were housed in a conventional state under controlled lighting and were provided with food and water ad libitum. All experimental procedures performed were approved by the Animal Care Committee of the Fourth Military Medical University. In all experiments, mice were transcardially perfused at the time of killing to

Sustained Ex-4 administration confers cerebral ischemic tolerance against focal ischemia

First, we investigated whether Ex-4 treatment induced ischemic tolerance in a mouse model of focal cerebral ischemia and established the optimal treatment conditions (dose and time) of Ex-4. Mice were treated with Ex-4 (5 μg/kg/day intraperitoneally) or vehicle every day for 7 days, and then mice were subjected to MCAO at day 7 of Ex-4 administration (E7D) or at day 2, 4, 6, 8, or 10 after cessation of Ex-4 (W2D, W4D, W6D, W8D, and W10D, respectively) (Fig. 1A). The stroke outcomes were

Discussion

GLP-1R is widely expressed in the brain (Timper et al., 2020), mainly in neurons (Fortin et al., 2020). In recent years, several studies have provided evidence of concept data showing that GLP-1R mediates neuroprotection in rodent models of neurological diseases. Within the research field of ischemic stroke, experimental evidence supporting the potential administration of GLP-1R agonists as therapeutics is growing. GLP-1R activation has been reported to be beneficial for behavioral recovery and

Conclusions

Our results indicate that Ex-4 can induce 6-day ischemic tolerance in the brain and that this state can protect against cerebral ischemia through the upregulation of IGF-1R and lead to the activation of the IGF-1R-mediated PI3K/AKT/mTOR/HIF-1 signaling pathway via binding to IGF-1 induced by MCAO.

CRediT authorship contribution statement

Huinan Zhang: Writing - original draft, Conceptualization, Methodology. Yunhan Liu: Data curation, Software. Liusiyuan Cheng: Visualization, Investigation. Xue Ma: Supervision, Writing - review & editing. Xiaoxing Luo: Writing - review & editing.

Declaration of Competing Interest

The authors declare no conflict of interest.

Acknowledgement

This work was supported financially by ProvinceNatural Science Foundation of Shaanxi 2020SF-088.

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