Upregulated hepatokine fetuin B aggravates myocardial ischemia/reperfusion injury through inhibiting insulin signaling in diabetic mice

https://doi.org/10.1016/j.yjmcc.2020.03.002Get rights and content

Highlights

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    Hepatokine fetuin-B is significantly upregulated in diabetes.

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    Upregulated fetuin-B exacerbates MI/R injury in diabetes and suppression of fetuin-B exerts cardiac protective effects.

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    Fetuin-B interacts with insulin receptor-β subunit, impairs cardiac insulin signaling and consequently causes MI/R injury.

Abstract

Patients with type 2 diabetes mellitus (T2DM) are more susceptible to acute myocardial ischemia/reperfusion (MI/R) injury. However, the mechanism remains largely elusive. Clinical observation showed that high levels of hepatokine fetuin-B (FetB) in plasma are significantly associated with both diabetes and coronary artery diseases. This study was aimed to determine whether FetB mostly derived from liver exacerbates MI/R-induced injury and the underlying mechanisms in T2DM. Mice were given high-fat diet and streptozotocin to induce T2DM model and subjected to 30 min MI followed by reperfusion. Diabetes caused increased hepatic FetB expression and greater myocardial injury as evidenced by increased apoptosis and myocardial enzymes release following MI/R. In T2DM hearts, insulin-induced phosphorylations of insulin receptor substrate 1 at Tyr608 site and Akt at Ser473 site and glucose transporter 4 membrane translocation were markedly reduced. Interaction between FetB and insulin receptor-β subunit (IRβ) was enhanced assessed by immunoprecipitation analysis. More importantly, FetB knockdown via AAV9 alleviated MI/R injury and improved cardiac insulin-induced signaling in T2DM mice. Conversely, upregulation of FetB in normal mice caused exacerbated MI/R injury and impairment of insulin-mediated signaling. In cultured neonatal mouse cardiomyocytes, incubation of FetB significantly reduced tyrosine kinase activity of IR and insulin-induced glucose uptake, and increased hypoxia/reoxygenation-induced apoptosis. Furthermore, FoxO1 knockdown by siRNA suppressed FetB expressions in hepatocytes treated with palmitic acid. In conclusion, upregulated FetB in diabetic liver contributes to increased MI/R injury and cardiac dysfunction via directly interacting with IRβ and consequently impairing cardiac insulin signaling.

Introduction

Coronary artery disease (CAD) is one of the major life-threatening diseases and has emerged as a major cause of death worldwide. More than 400 million adults suffer from diabetes globally [1] and diabetic patients are at higher risk of CVD including myocardial ischemia [2]. Researchers have revealed that heart in type 2 diabetes mellitus (T2DM) is more susceptible to acute myocardial ischemia/reperfusion injury and diabetes influences the function of ischemic preconditioning and some cardioprotective pharmacologic agents [3]. However, the mechanisms of increased vulnerability of diabetic myocardium to ischemia/reperfusion are not fully understood.

In T2DM, many bioactive factors release into circulation from adipose tissue, skeletal muscle and liver. Some of these factors exert protective or adverse effects in diabetic cardiovascular health. Recently, much attention has been focused on fetuin-B (FetB) as it might be one of the most important hepatokines regulating metabolism. FetB is one of the fetuins family produced primarily in the liver that circulates at high levels [4,5]. Patients with gestational diabetes mellitus have significantly higher FetB levels as compared to control [6]. FetB causes glucose intolerance in both mice and patients with hepatic steatosis and diabetes. Silencing of FetB in obese mice improves glucose tolerance without any effect on body weight [7]. Importantly, serum levels of FetB have been found increased in patients with CAD [8]. in vivo administration of FetB weaken the stability of the plaque in mice [9], indicate the possible role of FetB on cardiovascular disorders. However, whether FetB plays a role in the increased myocardial susceptibility to ischemia/reperfusion injury in T2DM is unknown. Most interestingly, the secretory fetuin-A (FetA), another fetuins member, binds several types of FFAs such as palmitic acid and serves as an adaptor protein for saturated fatty acid-mediated activation of Toll-like receptor 4 (TLR4) [10]. The FetA-FFA complex might be a potential driving force behind T2DM and CVD by inducing inflammatory signaling and insulin resistance. FetA might also induce insulin resistance by itself since it inhibits the insulin receptor at the tyrosine kinase level [11]. However, whether and how FetB regulates insulin signaling are remain largely elusive.

Therefore, we hypothesized that FetB may participate in cardiac vulnerability in T2DM, which is attributable to impaired myocardial insulin signaling. This study was aimed to evaluate whether and how FetB regulates myocardial insulin signaling and MI/R injury in T2DM.

Section snippets

Animals

This study was performed in adherence with the National Institutes of Health Guidelines for the Use of Laboratory Animals, and was approved by the Fourth Military Medical University Committee on Animal Care. Male C57BL6J mice which were 6–7 wks old weighing 18–20 g were provided by the Experimental Animal Center of the Fourth Military Medical University (Xi'an, China). All animals were allowed free access to food and water, and were maintained at 22–24 °C under a 12:12 h light-dark cycle.

T2DM models

To

Cardiac dysfunction was aggravated following MI/R injury in diabetic mice

T2DM mice showed significant increase in blood glucose (P < .01, Fig. S1). Ejection fraction was markedly decreased after MI/R surgery (P < .01, Fig. 1A and B). MI/R surgery caused myocardial injury as evidenced by significantly increased apoptosis (TUNEL-positive staining and caspase-3 activation) as well as myocardial enzymes (CK-MB and LDH, the marker enzymes of myocardial injury) activities (Fig. 1C–G). Unsurprisingly, diabetic MI/R mice showed smaller ejection fraction, more MI/R-induced

Discussion

The major findings of the present study are as follows. First, FetB expression is enhanced in diabetic livers, which is mediated by increased FoxO1. Second, increased FetB levels contribute to aggravated myocardial vulnerability to ischemia/reperfusion in T2DM mice. Third, FetB directly communicates with insulin receptor-β subunit and inhibits its phosphorylation at tyrosine site and the downstream signaling pathway, which is at least partly responsible for the increased myocardial

Sources of funding

This work was supported by grants from the National Natural Science Foundation of China (Nos. 81700342, 81870280, 81670449 and 31600938), National Key Research and Development Project (No. 2019YFF0301603), Shaanxi Science and Technology Research and Development Program Project (No. 2019JQ-330), Young Talent Fund of University Association for Science and Technology in Shaanxi, China (No. 20180302) and Chuying Project of FMMU and School of Aerospace Medicine (No. 2019HBRC06).

Declaration of Competing Interest

None.

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