# NEUROENDOCRINE RESEARCH IN HEALTH AND DISEASE

EDITED BY : Yu-Feng Wang, Xue Qun Chen, Lei Sha, Leo T. O. Lee, Keith Maurice Kendrick and Christopher H. K. Cheng PUBLISHED IN : Frontiers in Neuroscience and Frontiers in Endocrinology

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ISSN 1664-8714 ISBN 978-2-88963-714-0 DOI 10.3389/978-2-88963-714-0

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# NEUROENDOCRINE RESEARCH IN HEALTH AND DISEASE

Topic Editors: Yu-Feng Wang, Harbin Medical University, China Xue Qun Chen, Zhejiang University, China Lei Sha, China Medical University, China Leo T. O. Lee, University of Macau, China Keith Maurice Kendrick, University of Electronic Science and Technology of China, China Christopher H. K. Cheng, The Chinese University of Hong Kong, China

Citation: Wang, Y.-F., Chen, X. Q., Sha, L., Lee, L. T. O., Kendrick, K. M., Cheng, C. H. K., eds. (2020). Neuroendocrine Research in Health and Disease. Lausanne: Frontiers Media SA. doi: 10.3389/978-2-88963-714-0

# Table of Contents

Di Xiao, Hao Kou, Shuxia Gui, Zhenyu Ji, Yu Guo, Yin Wu and Hui Wang

*44 Intrapancreatic Ganglia and Neural Regulation of Pancreatic Endocrine Secretion*

Wenjing Li, Guangjiao Yu, Yudan Liu and Lei Sha

*54 Metabolite Profiles of the Cerebrospinal Fluid in Neurosyphilis Patients Determined by Untargeted Metabolomics Analysis*

Li-Li Liu, Yong Lin, Wei Chen, Man-Li Tong, Xi Luo, Li-Rong Lin, Hui-Lin Zhang, Jiang-Hua Yan, Jian-Jun Niu and Tian-Ci Yang

*64 Effects of Intranasal Oxytocin on Pup Deprivation-Evoked Aberrant Maternal Behavior and Hypogalactia in Rat Dams and the Underlying Mechanisms*

Xiao Yu Liu, Dongyang Li, Tong Li, Haitao Liu, Dan Cui, Yang Liu, Shuwei Jia, Xiaoran Wang, Runsheng Jiao, Hui Zhu, Fengmin Zhang, Danian Qin and Yu-Feng Wang

*75 Chronic Glucocorticoid Exposure Induces Depression-Like Phenotype in Rhesus Macaque (Macaca Mulatta)*

Dongdong Qin, Zhifei Li, Zhaoxia Li, Limin Wang, Zhengfei Hu, Longbao Lü, Zhengbo Wang, Yun Liu, Yong Yin, Zhaofu Li and Xintian Hu

*84 Expression of Dopamine Receptors in the Lateral Hypothalamic Nucleus and Their Potential Regulation of Gastric Motility in Rats With Lesions of Bilateral Substantia Nigra*

Yan-Li Yang, Xue-Rui Ran, Yong Li, Li Zhou, Li-Fei Zheng, Yu Han, Qing-Qing Cai, Zhi-Yong Wang and Jin-Xia Zhu

*92 Treadmill Exercise Ameliorates Depression-Like Behavior in the Rats With Prenatal Dexamethasone Exposure: The Role of Hippocampal Mitochondria*

Tianwen Wu, Yan Huang, Yuxiang Gong, Yongjun Xu, Jianqiang Lu, Hui Sheng and Xin Ni

*102 Voluntary Wheel Running Reverses Deficits in Social Behavior Induced by Chronic Social Defeat Stress in Mice: Involvement of the Dopamine System*

Jing Zhang, Zhi-xiong He, Li-min Wang, Wei Yuan, Lai-fu Li, Wen-juan Hou, Yang Yang, Qian-qian Guo, Xue-ni Zhang, Wen-qi Cai, Shu-cheng An and Fa-dao Tai

Juan Cheng, Haojuan Wu, Huawei Liu, Hua Li, Hua Zhu, Yongmei Zhou, Hongxia Li, Wenming Xu and Jiang Xie

*132 Therapeutic Potential of Oxytocin in Atherosclerotic Cardiovascular Disease: Mechanisms and Signaling Pathways*

Ping Wang, Stephani C. Wang, Haipeng Yang, Chunmei Lv, Shuwei Jia, Xiaoyu Liu, Xiaoran Wang, Dexin Meng, Danian Qin, Hui Zhu and Yu-Feng Wang

Li Zhou, Xue-Rui Ran, Feng Hong, Guang-Wen Li and Jin-Xia Zhu

Hongyan Lin, Yang Yuan, Sai Tian, Jing Han, Rong Huang, Dan Guo, Jiaqi Wang, Ke An and Shaohua Wang

Hongbo Yang, Kang Li, Xinyu Liang, Bin Gu, Linjie Wang, Gaolang Gong, Feng Feng, Hui You, Bo Hou, Fengying Gong, Huijuan Zhu and Hui Pan

*212 Oxytocin Inhibition of Metastatic Colorectal Cancer by Suppressing the Expression of Fibroblast Activation Protein-*a

Mingxing Ma, Li Li, He Chen and Yong Feng

*221 Activation of the G Protein-Coupled Estrogen Receptor Elicits Store Calcium Release and Phosphorylation of the Mu-Opioid Receptors in the Human Neuroblastoma SH-SY5Y Cells*

Xiaowei Ding, Ting Gao, Po Gao, Youqiang Meng, Yi Zheng, Li Dong, Ping Luo, Guohua Zhang, Xueyin Shi and Weifang Rong

# Editorial: Neuroendocrine Research in Health and Disease

Yu-Feng Wang1 \*, Xuequn Chen2 \*, Lei Sha3 \*, Keith M. Kendrick 4 \*, Leo T. O. Lee5 \* and Christopher H. K. Cheng6 \*

1 Department of Physiology, Harbin Medical University, Harbin, China, 2 Division of Neurobiology and Physiology, Department of Basic Medical Sciences, School of Medicine, Zhejiang University, Hangzhou, China, 3 Department of Neuroendocrine Pharmacology, School of Pharmacy, China Medical University, Shenyang, China, 4 Key Laboratory for Neuroinformation, The Clinical Hospital of Chengdu Brain Science Institute, University of Electronic Science and Technology of China, Chengdu, China, 5 Faculty of Health Sciences, University of Macau, Shanghai, China, 6 School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong, China

Keywords: hormone, hypothalamus, innervation, neuropeptide, receptor

Edited and reviewed by: Hubert Vaudry, Université de Rouen, France

#### \*Correspondence:

Yu-Feng Wang<EMAIL_ADDRESS>Xuequn Chen<EMAIL_ADDRESS>Lei Sha<EMAIL_ADDRESS>Keith M. Kendrick<EMAIL_ADDRESS>Leo T. O. Lee<EMAIL_ADDRESS>Christopher H. K. Cheng<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Neuroscience

Received: 25 December 2019 Accepted: 17 February 2020 Published: 24 March 2020

#### Citation:

Wang Y-F, Chen X, Sha L, Kendrick KM, Lee LTO and Cheng CHK (2020) Editorial: Neuroendocrine Research in Health and Disease. Front. Neurosci. 14:176. doi: 10.3389/fnins.2020.00176 **Editorial on the Research Topic**

#### **Neuroendocrine Research in Health and Disease**

To facilitate interactions between Chinese neuroendocrine researchers and the international neuroendocrine community, promote the growth of neuroendocrine research, and explore optimal treatment strategies for neuroendocrine diseases, we opened this topic on February 10, 2018. At the end of this topic, we had reviewed 31 manuscripts and accepted 22 for publication in the Neuroendocrine Science Section of the Journals of Frontiers in Neuroscience and Frontiers in Endocrinology. Among these papers, there were 19 original research contributions and three reviews. The research reported in these papers covered neuroendocrine regulation of mental activity, novel neuroendocrine pathways, peripheral effects of neuropeptides, receptor signaling, neuroendocrine diseases and treatments, and other associated areas.

#### NEUROENDOCRINE REGULATION OF MENTAL ACTIVITY

Kendrick and colleagues initiated the publications by presenting findings that stated "oxytocin (OT) facilitates empathic- and self-embarrassment ratings through attenuating amygdala and anterior insula responses" (Geng et al.). OT, a hypothalamic neuropeptide, can enhance emotional empathy in association with reduced amygdala activation. By using a randomized, double-blind placebo controlled functional MRI experiment on 70 male and female subjects, the authors investigated the effects of intranasal OT (40 IU) on behavioral and neural responses to embarrassment experienced by the self or others. The results demonstrated that OT increases ratings of self- and other embarrassment and that this is associated with reduced physiological arousal and activity in neural circuitry involved in emotional arousal. Interestingly, the neural effects of OT are stronger in individuals with high trait anxiety, suggesting that they may particularly reduce their anxiety in embarrassing situations.

Next, Lin et al. reported that high plasma levels of irisin, a humoral factor important for metabolism and homeostasis of energy balance, are associated with early cognitive deficits in Chinese patients with Type 2 diabetes mellitus (T2DM). In this study on 133 Chinese patients with T2DM, the authors found that a higher level of irisin in the plasma is associated with impaired overall cognition, specifically in executive function. It is likely that irisin functions both as "a sports pill" and as a signal of impaired cognition.

In a morphometric MRI study, Yang H. et al. reported alterations in cortical thickness in young male patients with childhood-onset adult growth hormone deficiency. They revealed a close correlation between serum growth hormone (GH)/insulin-like growth factor-1 (IGF-1) levels and age-related cognitive function. This correlation is based on alterations in cortical thickness in different brain lobes/regions.

In another study, Liu X. Y. et al. reported that intranasal OT alleviated maternal postpartum depression while improving milk production. Findings demonstrated that pup deprivation can evoke aberrant maternal behavior and hypogalactia in rat dams and that OT significantly improves these symptoms. In the supraoptic nucleus (SON), a major source of brain OT, intranasal administration can reverse pup deprivation -evoked reduction of c-Fos and increases glial fibrillary acidic protein (GFAP) filaments. Notably, OT also increases plasma levels of adrenocorticotropic hormone and corticosterone in pupdeprived dams. These Liu X. Y. et al. findings highlight the therapeutic potential of OT in pup-deprived dams by restoring the activity of the hypothalamic OT-secreting system involving modulation of astrocytic plasticity. However, OT's activation of the hypothalamic pituitary adrenal (HPA) axis likely compromises the facilitatory effects of intranasal administration.

Involvement of the HPA axis in mental activity has been well-known for decades. In the report by Zhou et al., the causal association between cortisol levels and depression is verified through a Mendelian randomization approach in a CORNET consortium of 12,597 participants (Zhou and Qiao). The result confirmed that a genetic predisposition to higher serum morning cortisol levels is associated with an increased depression score. Moreover, Wu R. et al. reported that prenatal dexamethasone exposure causes depression-like behavior in adult rats due to mitochondrial dysfunction. Intervention with treadmill exercise in early life can reverse this depression through improving mitochondrial function in the hippocampus (Wu T. et al.). Consistent with these latter findings, Qin et al. show that in rhesus macaques, a non-human primate, chronic glucocorticoid exposure leads to depression-like behavior and reduces dopamine (DA) levels in cerebrospinal fluid (CSF). Paradoxically, this treatment decreases cortisol concentration in the blood but increases them in hair. Thus, chronic glucocorticoid exposure may disturb both acute and chronic HPA axis reactivity, which eventually disturbs neurotransmitter systems and leads to a depressionlike phenotype.

In addition to OT and corticosteroid hormones, sex steroid hormones are also involved in the regulation of mental activity. A paper by Cheng et al. demonstrated that androgens and androgenic signaling are involved in the occurrence of depression and anxiety. Following exposure to androgen during pregnancy, rats manifest depressive, anxious, and stereotypical behaviors in the adolescent period. These phenotypes are possibly associated with changes in neurogenesis in the dentate gyrus of the hippocampus.

#### NOVEL NEUROENDOCRINE PATHWAYS

Neuroendocrine regulation of bodily activity can be influenced by direct neural innervation along with the actions of circulating neurohormones. In studying the mechanism underlying gastroparesis in patients with Parkinson's disease (PD), Yang Y.-L. et al. demonstrated that DA neurons in the substantia nigra (SN) project to and activate the lateral hypothalamic nucleus (LH) via the D1 receptor. LH neurons expressing orexin A also innervate the dorsal motor nucleus of the vagus (DMV). By activating the orexin receptor 1 of DMV, orexin A from the LH can alter gastric motility. Reduction of DA in the SN of PD patients decreases the activity of this SN-LH-DMV pathway, which ultimately leads to gastric dysfunction via the vagal nerve (Yang Y.-L. et al.).

Zhou et al. also reported that dopaminergic neurons in the SN are involved in the regulation of glucose metabolism through corticotropin-releasing hormone (CRH) neurons that express DA receptor 2 (D2) in the hypothalamic paraventricular nucleus (PVN). Bilateral SN lesions decrease glucose tolerance in rats by down-regulating the D2 receptor and up-regulating CRH in the PVN (Zhou et al.). These findings highlight that high prevalence of glucose metabolism abnormalities in PD patients associates with down-regulation of D2 and up-regulation of CRH in the PVN.

The review by Li et al. systematically demonstrated how pancreatic endocrine secretion is under the regulation of extrapancreatic nerves projecting to the islet directly, or converging on intrapancreatic ganglia, innervated by sympathetic, parasympathetic, enteric, sensory nerve fibers, and other intrapancreatic ganglia. It also highlighted the necessity of clarifying the roles and the mechanisms of intrapancreatic ganglia in physiological and disease states of the pancreas (Li et al.).

#### PERIPHERAL EFFECTS OF NEUROPEPTIDES

Neuropeptides have extensive influences on bodily functions and this view is illustrated further in the current topic. Zhang J. et al. or demonstrated that the neuropeptide substance P released from primary sensory fibers promotes proliferation of adult pancreatic ductal cells—one of the important sources of pancreatic islet β-cell neogenesis—but not their differentiation into β-cells. The observed effect of substance P involves the NK-1 receptor and Wnt signaling pathway. This finding indicates that lack of substance P may be a possible risk factor for diabetes development (Zhang N. et al.).

The review by Wang et al., comprehensively demonstrated the therapeutic potential of OT in atherosclerotic cardiovascular disease and the underlying mechanisms and signaling pathways. This review first linked atherosclerosis with varieties of immunometabolic disorders that can suppress OT receptor (OTR) signaling in the cardiovascular system (CVS). Next, the authors discussed evidence for this and the cellular and molecular mechanisms underlying CVS protective effects of OT. Finally, the authors also presented evidence for OT-evoked cardiovascular disturbance and the strategy for applying OT safely in clinical practice.

#### RECEPTOR SIGNALING

Different hormones can have multiple receptors and the effects of activating a specific receptor rely on specific signaling events. The report by Ding et al. showed that activation of the G protein-coupled estrogen receptor (GPER) elicits the release from calcium stores and phosphorylation of the mu-opioid receptor by regulating reproduction and metabolism in human neuroblastoma cells. Activation of GPER is followed by rapid calcium mobilization, translocation, and activation of protein kinase C-α and -ε at the plasma membrane leading to mu-opioid receptor phosphorylation. The GPER-mediated rapid calcium signal may also transmit to the nucleus to impact on gene transcription (Ding et al.). Such a signaling cascade may play an important role in the regulation of opioid signaling in the brain.

The review by Wang et al., discussed cellular and molecular mechanisms underlying cardiovascular protective effects of OT. The OT receptor signals mainly belong to the reperfusion injury salvage kinase pathway composed of PI3K-Akt-endothelial nitric oxide synthase cascades and extracellular signal-regulated protein kinase 1/2 (ERK 1/2). Additionally, AMP-activated protein kinase, Ca2+/calmodulin-dependent protein kinase signaling, and many others can influence OTR signaling related to cardiovascular protection. These signaling events coordinate at many levels to suppress metabolic disorders, reduce the production of inflammatory cytokines, and inhibit the activation of apoptotic pathways, particularly endoplasmic reticulum stress and mitochondrial oxidative stress. This comprehensive review expands our understanding of the immunoregulatory functions of OT.

# DISEASE AND TREATMENT

Neuroendocrine studies have established the involvement and treatment effects of neuropeptides in a variety of diseases. In their study, Ma et al. found for the first time that the expression of OT and OTR declines in human colorectal tissues as malignancy of colorectal cancer (CRC) increases. They reported that OT can suppress the expression of tumor-associated immunosuppressive proteins fibroblast activation protein-α (FAPα) and chemokine (C-C motif) ligand 2 (CCL-2). The reduction in OT-OTR expression can unleash the expression of FAPα and CCL-2 and, thus, facilitates CRC metastasis. Importantly, OT can reduce the invasion ability of human CRC cells. Although the authors limited their findings to colorectal adenocarcinoma, the therapeutic potential of directly applying OT to inhibit CRC migration is worthy of further exploration.

The report by Zhu et al., studied the spatial distribution patterns of primary age-related tauopathy (PART) in subcortical nuclei, including the neuroendocrine hypothalamus of postmortem human brains. They found that the prevalence and severity of tau pathology in subcortical nuclei of PART and AD positively correlate with the stage of NFT Braak neurofibrillary tangles, suggesting that these nuclei are increasingly involved as PART and AD progress. Thus, these subcortical nuclei are probably the sites initially affected by aging associated tau pathology, especially brainstem nuclei.

Zou et al., reviewed the role of leptin in mood disorders and neurodegenerative diseases and summarized findings that leptin can improve learning and memory, affect hippocampal synaptic plasticity, exert neuroprotective efficacy, and reduce the risk of several neuropsychiatric diseases, including depression. These effects are associated with leptin regulation of the release of neurotransmitters and neurotrophic factors and can reverse dysfunction in the HPA axis.

Sheng et al. demonstrated an involvement of mitogenic signaling in the regulation of thyroid proliferation by insulin glargine and human insulin. Their results show that therapeutic doses of glargine do not stimulate thyroid cell proliferation, despite longer-lasting hypoglycemic control than human insulin. However, high doses of these insulin products can stimulate thyroid cell proliferation, which should therefore be a consideration in the clinical use of insulin glargine.

# OTHERS

The topic also includes neuroendocrine studies in other associated research areas. Liu L. L. et al. reported CSF metabolite profiles in neurosyphilis patients following an untargeted metabolomics analysis. These differential metabolites may potentially improve neurosyphilis diagnostics in the future and deserve further exploration. Xiao et al. demonstrated that prenatal ethanol exposure produces age-dependent changes in glucose metabolism, pancreatic morphology, and function in male offspring rats. This intrauterine programming alteration in the GC-IGF1 axis may contribute to prenatal and postnatal pancreatic dysplasia and impaired insulin biosynthesis in male offspring.

In addition, Wu R. et al. presented research suggesting that brief mindfulness meditation can significantly improve emotion processing. The beneficial effect correlates with reduced activity of the HPA-axis and stress responses (Wu R. et al.). The report by Zhang J. et al. revealed that voluntary wheel running reverses deficits in social behavior following chronic social defeat stress in mice due to the reversal of reduced levels of tyrosine hydroxylase in the ventral tegmental area and the D2 receptor in the nucleus accumbens shell.

Taken together, our topic submissions demonstrate that Chinese neuroendocrine researchers are contributing significantly to the field of neuroendocrinology. While many challenges remain, the growth of a new generation of Chinese neuroendocrine researchers will, we hope, result in even stronger contributions to novel approaches, concepts, and principles in both fundamental and clinical neuroendocrine research.

# AUTHOR CONTRIBUTIONS

All six authors contributed to organizing the Research Topic, in which XC has taken the most editorial assignments. Y-FW wrote the first draft, all participated in the correction of this editorial and KK made the final revision.

# ACKNOWLEDGMENTS

We thank Drs. Pierrette Gaudreau, Sebastien G. Bouret, Lee E. Eiden, and James A. Carr for participation in the editorial process and Dr. Benjamin C. Nephew and the many other reviewers for their valuable contributions to the quality of our accepted papers.

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Wang, Chen, Sha, Kendrick, Lee and Cheng. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Oxytocin Facilitates Empathic- and Self-embarrassment Ratings by Attenuating Amygdala and Anterior Insula Responses

YaYuan Geng, Weihua Zhao, Feng Zhou, Xiaole Ma, Shuxia Yao, Benjamin Becker\* and Keith M. Kendrick\*

*MOE Key Laboratory for Neuroinformation, Clinical Hospital of Chengdu Brain Science Institute, University of Electronic Science and Technology of China, Chengdu, China*

The hypothalamic neuropeptide oxytocin has been reported to enhance emotional empathy in association with reduced amygdala activation, although to date studies have not investigated empathy for individuals expressing self-conscious, moral emotions which engage mentalizing as well as emotion processing networks. In the current randomized, double-blind placebo controlled functional MRI experiment in 70 male and female subjects we have therefore investigated the effects of intranasal oxytocin (40 IU) on behavioral and neural responses to embarrassment experienced by others or by self. Results showed that oxytocin significantly increased ratings of both empathic and self-embarrassment and concomitantly decreased skin conductance response, activation in the right amygdala and insula but not in the medial prefrontal cortex. The amygdala effects of oxytocin were associated with the magnitude of the skin conductance response and trait anxiety scores. Overall our results demonstrate that oxytocin increases ratings of self- and other embarrassment and that this is associated with reduced physiological arousal and activity in neural circuits involved in emotional arousal. The neural effects of oxytocin were more pronounced stronger in individuals with high trait anxiety suggesting that it may particularly reduce their anxiety in embarrassing situations.

#### Edited by:

*James A. Carr, Texas Tech University, United States*

#### Reviewed by:

*Ben Nephew, Worcester Polytechnic Institute, United States Aldo Lucion, Universidade Federal do Rio Grande do Sul (UFRGS), Brazil*

#### \*Correspondence:

*Benjamin Becker<EMAIL_ADDRESS>Keith M. Kendrick<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Endocrinology*

Received: *12 June 2018* Accepted: *06 September 2018* Published: *25 September 2018*

#### Citation:

*Geng Y, Zhao W, Zhou F, Ma X, Yao S, Becker B and Kendrick KM (2018) Oxytocin Facilitates Empathic- and Self-embarrassment Ratings by Attenuating Amygdala and Anterior Insula Responses. Front. Endocrinol. 9:572. doi: 10.3389/fendo.2018.00572* Keywords: oxytocin, empathy, embarrassment, anxiety, insula, amygdala, prefrontal cortex, sex differences

# INTRODUCTION

Our ability to empathize with others is a core feature influencing our social behavior through allowing us to understand both what others are thinking and feeling and thereby promoting our social interactions with them. As such, impaired ability to empathize with others is often a core feature of disorders where social communication and interactions are dysfunctional, such as autism spectrum disorders (1), depression (2) and psychopathy (3).

Even though empathy has been extensively investigated, its sub-components and associated neural mechanisms are still not fully established (4). The most prevalent view considers empathy as a multidimensional construct including both cognitive (identifying emotions expressed by another person) and emotional components (being aroused by or feeling the same emotion expressed by another person) (5). Meta-analytic data has suggested that a network including left orbital frontal cortex, left anterior mid-cingulate cortex, left anterior insula and left dorsal medial thalamus is

**10**

involved in cognitive-evaluative empathy, whereas another network including the dorsal anterior cingulate cortex, bilateral anterior insula, right dorsal medial thalamus and midbrain is involved in affective-perceptual empathy (6). Studies that directly compared the two empathy components additionally proposed that the inferior frontal gyrus is essential for emotional empathy, whereas the superior and middle frontal gyrus and the orbital gyrus are specifically engaged in cognitive empathy (7). However, the derived empathy network can differ dependent upon the paradigm used (4) thus making it important to establish a core network that is maintained across different paradigms.

The hypothalamic neuropeptide oxytocin (OXT) has been implicated in a number of crucial aspects of social cognition and emotional bonds (8, 9) and importantly studies have reported that it can enhance components of empathy (10–12). The most consistent findings have been in studies using paradigms that distinguish cognitive from emotional empathy components. For example, in male Caucasian subjects OXT was found to enhance both direct and indirect aspects of emotional empathy, but not cognitive empathy per se, in the Multifaceted Empathy Task (MET) (12). Urbach-Wiethe disorder patients with selective and complete bilateral amygdala-lesions also exhibited deficits in emotional but not cognitive empathy in the MET suggesting that the emotional empathy enhancing effects of OXT might be mediated by the amygdala (12). We recently confirmed these behavioral findings in Chinese male, as well as female subjects using a Chinese version of the MET and demonstrated that OXT effects on emotional empathy were associated with decreased amygdala activity and increased physiological arousal (10). Finally, another study using dynamic, empathy-inducing video clips in which protagonists expressed sadness, happiness, pain or fear demonstrated that OXT exerted no effects on cognitive empathy but selectively enhanced emotional empathy for fear (11). Thus, OXT may particularly enhance emotional empathy, although in the context of the strong modulatory influence of personal and social contextual factors on the specific effects of OXT (8, 13) it is important to extend these observations using other paradigms and contexts where empathic responses are evoked.

Some initial support for OXT also influencing aspects of cognitive empathy has been reported using the reading the mind in the eyes test (RMET) where participants identify from visual cues restricted to the eye regions which of four different complex emotions is being experienced by subjects. Thus, in one study OXT was reported to increase accuracy, particularly for more difficult items (14). However, subsequent studies found that OXT either only increased RMET accuracy for difficult items in individuals with low empathy scores (15) or generally only in individuals with lower socially proficiency associated with higher levels of maternal love withdrawal (16). Furthermore, a final comprehensive study failed to observe any effects of OXT on RMET performance even taking into account item difficulty, gender and valence and subject traits (17). Thus, evidence for OXT enhancing cognitive empathy using the RMET paradigm remains equivocal.

In the current double-blind, placebo-controlled study we therefore investigated whether OXT would enhance empathy in social situations involving more complex moral, "self-conscious," emotions such as embarrassment. Embarrassment is primarily a self-reflection and self-evaluative process which serves as a way to help humans adapt their behavior to social norms by punishing non-compliance to such norms with a negative emotional state (18). The neural pathways involved in embarrassment include both those controlling mentalizing and emotional arousal (19). Oxytocin has previously been shown to modulate activity in both of these networks in the context of processing self-referential information (mPFC–(20, 21)) and emotional stimuli [amygdala and anterior insula–(22–27)].

We can experience empathic-embarrassment (EE) toward others we see in embarrassing situations irrespective of whether they are familiar or not (28), and so in the current paradigm we investigated OXT effects on behavioral and neural responses to pictures depicting others in embarrassing situations. Additionally, we also asked subjects to imagine their own feelings if they experienced a similar situation themselves (i.e., self-embarrassment–SE). We reasoned that the latter selfrelated context would involve a stronger mentalizing component, although both contexts should have strong arousal components. On the behavioral level, we hypothesized that, in line with its enhancement of emotional empathy (10, 12), OXT would increase both EE and SE ratings. At the neural level we hypothesized that in line with previous findings, both in the context of emotional empathy (10) and responses to fear and other arousing stimuli (8, 24), OXT would decrease amygdala activity and also change that of the insula (22, 25–27). We additionally hypothesized that given the mentalizing component associated with embarrassment there would be involvement of the mPFC which could also be modulated by OXT given previous findings (20, 21), but in view of the greater self-orientation and mentalizing component in SE we additionally hypothesized that the effects of OXT on mPFC activation would be greater in this condition. We have previously shown that some behavioral and neural effects of OXT on empathy and other behaviors are associated with increased physiological arousal and modulated by autistic traits (10, 29). The neural effects of embarrassment are also modulated by anxiety (30) and autism (19). Thus, in the current study we also investigated the effects of OXT on physiological arousal as assessed by electrodermal activity and associations of its effects with autism and anxiety traits.

#### MATERIALS AND METHODS

#### Participants

A total of n = 70 participants were enrolled in the present randomized double-blind, placebo-controlled between-subject experiment. Participants were randomly assigned to receive OXT (40 International Units, IU) or placebo (PLC) intranasal treatment resulting in 35 participants (female n = 17) in the OXT treatment group and 35 (female n = 15) in the PLC treatment group. Both groups were of comparable age [mean ± STD, OXT: 22.03 ± 2.15; PLC: 21.86 ± 1.97; p = 0.73,t(70) = −0.35], education [OXT: 16.09 ± 1.65; PLC: 15.89 ± 1.64; p = 0.61, t(70) = −0.51], and gender distribution [χ 2 (1) = 0.23, p = 0.63]. All participants reported having no past / current physical,

psychiatric or neurological disorders or regular/current use of medication or tobacco. Subjects were required to refrain from nicotine, alcohol or caffeine intake for at least 12 h before the experiment. None of the female participants were taking oral contraceptives or were in their menstrual period. The distribution of females estimated to be in their follicular or luteal phases did not differ significantly between the groups (χ 2 = 0.06, p = 1.00). Written informed consent was obtained from each participant before the experiment. The study was approved by the ethical committee of the University of Electronic Science and Technology of China, and all procedures were in accordance with the latest revision of the declaration of Helsinki.

#### Experimental Paradigm

40 pictures depicting male or female protagonists (Chinese protagonists; 20 male, 20 female) in everyday embarrassing situations were evaluated by an independent sample (n = 29, 14 females) prior to the experiment to balance mean ratings for empathic embarrassment (EE–how embarrassed do you feel for the person in the picture?) and self-embarrassment (SE–if you were in the same situation, how embarrassed would you feel?) in response to the pictures and the gender of the protagonist (EE: females, 6.54 ± 1.14, males, 6.65 ± 0.89; SE: females, 7.91 ± 0.55, males, 7.58 ± 0.67). No significant differences were found with regard to both EE and SE ratings between male and female participants (EE, p = 0.77, t = −0.30; SE, p = 0.17, t = 1.41). For the fMRI experiment stimuli were presented in a mixed block/event related design during four subsequent runs. Each run containing one block of EE and one block of SE trials, with 10 trials of stimuli presented during each block. Each block started with a 3 s cue presentation indicating whether the subject was required to rate EE or SE. Within each block stimuli were presented for 3 s, followed by a jittered low-level baseline during which a fixation cross was presented for 4 s (3–5 s). After each stimulus, subjects were given 5 s to rate EE or SE using a 1–9 Likert rating scale (1 = not at all, 9 = very strong) followed by another 5 s (4–6 s) jittered inter-trial interval. An ABBA blockdesign was used to counterbalance the order of conditions (see **Figure 1** for paradigm details).

#### Procedure

To control for potential confounders, all participants completed a test battery of Chinese versions of mood and trait questionnaires before intranasal administration: Beck's Depression Inventory (BDI) (31), Emotional Intelligence Scale (Wleis-C) (32), Empathy Quotient (EQ) (33), Liebowitz Social Anxiety Scale (LSAS) (34) and Positive and Negative Affect Scale (PANAS) (35). Based on previous research reporting that individual variations in autism and anxiety influence (1) embarrassment-associated neural

activity (30), as well as (2) effects of OXT on embarrassmentrelated functional domains (e.g., emotional arousal, empathy and self-appraisal) and neural activity in embarrassment-related regions, including the insula (10, 29, 36–38), levels of anxiety and autism were assessed in the present sample. To this end, participants additionally completed the State Trait Anxiety Inventory (STAI) (39) and Autism Spectrum Quotient (ASQ) (40). Next, subjects self-administered intranasal spray (either 40IU of OXT or PLC lacking the neuropeptide, both supplied by the Sichuan Meike Pharmaceutical Co., Ltd, Sichuan, China). The PLC spray had identical packing and ingredients as the OXT spray (sodium chloride and glycerine, minus the peptide). In accordance with previous recommendations for the intranasal administration of OXT in humans (8, 41), the experimental paradigm started 45 min after treatment. In post-experiment interviews participants were unable to guess better than chance which treatment they had received (34 subjects guessed correctly; χ 2 = 0.22, p = 0.64), confirming successful blinding.

During the experiment electrodermal activity was also measured to assess skin conductance responses (SCR) to the stimuli as an index of autonomic sympathetic activity (42) using the same approach as previously described (10). For the SCR data an event-related analysis approach was employed focusing on SCR responses associated with the presentation of SE and EE stimuli (procedures for preprocessing and event-related analysis of the SCR data were identical to our previous study, details provided in Geng et al. (10).

#### fMRI Acquisition

MRI data was acquired using a GE (General Electric Medical System, Milwaukee, WI, USA) 3T Discovery 750 MRI system with a standard head coil. fMRI time series were acquired using a T2∗ -weighted echo planar imaging pulse sequence (repetition time, 2,000 ms; echo time, 30 ms; slices, 39; thickness, 3.4 mm; gap, 0.6 mm; field of view, 240 × 240 mm2 ; resolution, 64 × 64; flip angle, 90◦ ). Additionally, a high resolution T1-weighted structural image was acquired using a 3D spoiled gradient recalled (SPGR) sequence (repetition time, 6 ms; echo time, 2 ms; flip angle 9◦ ; field of view, 256 × 256 mm2 ; acquisition matrix, 256 × 256; thickness, 1 mm without gap) to exclude subjects with apparent brain pathologies and to improve normalization of the fMRI data.

#### fMRI Data Processing

fMRI data were analyzed using SPM12 (Wellcome Trust Center of Neuroimaging, University College London, London, United Kingdom). The first five volumes were discarded from further analyses and images were realigned to the first image to correct for head motion using a six-parameter rigid body algorithm and unwarping. Tissue segmentation, bias-correction and skull-stripping were performed for the high-resolution structural images. Functional images were further corrected for slice-acquisition time differences, co-registered to the anatomical scan and subsequently spatially normalized to the standard Montreal Neurological Institute (MNI) template. The normalized functional volumes were written out at a 3 × 3 × 3 mm voxel size and were finally smoothed with an 8 mm full-width-at-halfmaximum (FWHM) isotropic Gaussian kernel.

On the first level, separate event-related regressors for the EE and SE conditions were included as main regressors of interest. Additionally, separate regressors for the rating phases, the cue phase and the six head motion parameters were included. The regressors were convolved with the standard hemodynamic response function (HRF). To evaluate the sex-dependent effects of OXT on embarrassment an ANOVA including treatment (OXT, PLC), sex (male, female) as between-subject factors and embarrassment type (EE, SE) as a within-subject factor was conducted on the second level.

Based on previous studies indicating that regions involved in arousal (amygdala, anterior insula, AI) and mentalizing (mPFC) neurally underpin embarrassment processing a regionof-interest (ROI) analysis specifically focused on these regions. The amygdala was structurally defined based on masks from the Automated Anatomical Labeling (AAL) atlas (43). Given the size and functional heterogeneity of these regions, the ROIs for the mPFC and anterior insula (specifically dorsal AI, dAI) were defined using 6 mm spheres centered at peak coordinates of these regions reported in a previous study examining the neural basis of embarrassment (30). For the five a priori defined ROIs the first eigenvariate was extracted using Marsbar (44). The individual activity estimates were subsequently subjected to mixed ANOVAs with the between-subject factors treatment (OXT, PLC) and sex (male, female) and the within-subject factor embarrassment type (EE and SE) in SPSS (Statistical Package for the Social Sciences, Version 22). Multiple comparisons for the ROI analysis were controlled for by applying False Discovery Rate (FDR) correction. To explore effects in regions beyond the predefined ROIs an exploratory whole brain analysis was conducted in SPM using cluster-level Family-Wise Error (FWE) correction for multiple comparisons. In line with recent recommendations (45) for the control of false positives using cluster based methods (46) an initial cluster defining threshold of p <.001 (uncorrected) was applied to the data resampled at 3 mm voxel resolution. This voxel-wise analysis employed a flexible factorial design to model the mixed ANOVA computed on the extracted regional estimates and included the within-subject factor embarrassment type (EE and SE) and the between subject factors sex (male, female) and treatment (OXT, PLC). Due to limitations of the flexible factorial design to estimate between-subject main effects, the corresponding main effects of the between-subject factors were additionally examined using separate independent sample t-tests and appropriate first level contrasts. Significance level for the corrected p-values was p < 0.05.

# RESULTS

No significant trait and mood differences were observed between participants in the OXT and PLC group (see **Supplementary Table S1**).

#### Behavioral and SCR Results

An ANOVA analysis with treatment and sex as betweensubject factors, embarrassment context as a within-subject factor and embarrassment rating as dependent variable, revealed a significant treatment main effect [PLC: 6.45 ± 0.11, OXT: 6.77 ± 0.11, F(1,66) = 4.30, p = 0.04, η 2 p = 0.06] with OXT

enhancing embarrassment ratings in both contexts (**Figure 2**). No significant interaction effects involving treatment were found [Sex ∗ Treatment, F(1,66) = 2.20, p = 0.14, η 2 p = 0.03; Sex ∗ Treatment ∗ embarrassment context, F(1,66) = 0.09, p = 0.76, η 2 p = 0.001].

As a result of scanner-induced noise SCR data from n = 12 subjects had to be excluded (baseline readings could not be reliably obtained in >50% of trials) leading to a final sample size of n = 58 subjects for the SCR analysis (n = 33, OXT; n = 25, PLC). An ANOVA analysis including the same factors as for the behavioral analysis and SCR response as dependent variable revealed a significant main effect of treatment [F(1,54) = 7.35, p = 0.009, η 2 p = 0.12] with OXT decreasing the SCR magnitude in both EE and SE conditions (see **Figure 3**).

#### fMRI Results

For the ROI-based analysis we initially explored whether there were either effects of embarrassment type or gender in the PLC group. This revealed a main effect of embarrassment type in the mPFC [F(1,33) = 5.54, PFDR = 0.04, η 2 p = 0.14] and in the amygdala [F(1,33) = 7.21, PFDR = 0.01, η 2 p = 0.18] but not in the dAI. This confirmed our expectation that there would be a difference in activation in mentalizing in the SE compared to the EE condition and additionally that there was greater activation in the amygdala during EE compared to SE trials. Examination of OXT by comparing the OXT and the PLC treated groups revealed significant main effect of treatment on right amygdala [F(1,66) = 9.97, PFDR = 0.01, η 2 p = 0.10] and right dAI [F(1,66) = 5.82, PFDR = 0.03, η 2 p = 0.08] responses, with OXT reducing activity in both EE and SE contexts (see **Figure 4**). No interactions between treatment and gender and embarrassment type were found in these regions. The exploratory whole-brain analysis did

task. Histograms show mean SCR magnitude for EE and SE trials combined (\**P* < 0.05).

not reveal significant main or interaction effects of treatment on the neural level (PFWE < 0.05).

#### Correlation Between Behavioral, Physiological, and Neural Data and Trait Questionnaires

There was a negative association between overall embarrassment ratings (average of EE and SE) and STAI Trait scores in the OXT but not the PLC group (STAI Trait: PLC, r = 0.06, p = 0.75, OXT, r = −0.34, p = 0.05), suggesting that ratings were particularly increased under OXT in more anxious individuals. However, the correlation difference did not achieve significance (Fisher's Z-Test, z = 1.66, p =0.098). For the neural data there was a similar negative association between right amygdala activation and STAI trait scores in the OXT group but not the PLC group (STAI Trait: PLC, r = 0.13, p = 0.44, OXT, r = −0.35, p = 0.04) and in this case the correlation difference was significant (Fisher's Z-Test, z = 1.99, p = 0.05) (see **Figure 5**). There was also a negative association between right amygdala activation and the magnitude of the SCR in the OXT group whereas in the PLC group there was a positive association (PLC, r = 0.32, p = 0.12, OXT, r = −0.33, p = 0.06; Fisher's Z-Test, z = 2.40, p = 0.016) (see **Figure 5**). In contrast, no significant associations between levels of autism (ASQ scores) and behavioral, SCR or neural effects were observed (all ps > 0.15).

#### DISCUSSION

The current experiment demonstrated for the first time that OXT increases both empathic- and self-embarrassment ratings in male and female subjects and that its behavioral effects are associated with decreased responses in the right amygdala and

FIGURE 4 | fMRI analysis showing that oxytocin significantly reduced right amygdala and right dAI responses during EE and SE trials. Histograms show parameter estimates for EE and SE trials combined (\**P* < 0.05).

and PLC groups is significant (Fisher's *Z*-test *p* < 0.05).

in the right dAI and the magnitude of physiological arousal (SCR). On the other hand, OXT appeared to have no effects on responses in the mentalizing network (mPFC) during either of the embarrassment rating contexts. Furthermore, in contrast with the PLC group the effects of OXT in reducing amygdala responses were negatively correlated with both STAI trait scores and SCR magnitude, implying that it had an anxiolytic effect and particularly in individuals with higher trait anxiety.

Our ROI-based findings in the PLC group that empathic embarrassment primarily increases activation in both mentalizing (mPFC) and emotion processing (amygdala and insula) brain regions is consistent with previous studies (18, 19, 30). Our expectation that the mentalizing component in self- compared to empathic embarrassment would be different was also supported in terms of differential mPFC activation in the PLC group. The effects of OXT in both embarrassment contexts were however restricted to emotion processing regions with reduced amygdala activation, similar to our previous findings for emotional empathy (10) and additionally decreased insula activation. Thus, although a number of studies have reported effects of OXT on both dorsal and ventral mPFC activation in the context of self-vs. other referential and ownership contexts (20, 21) it would appear that this does not occur in self-vs. otherembarrassment. However, these previous studies demonstrating OXT effects on the mPFC in self-vs. other processing have shown that in both behavioral and neural term's it reduced the normal self-bias. Thus, it is possible that OXT had no effects on the mPFC since EE and SE ratings were similar and there was therefore no indication of self-bias for embarrassment experience in the present paradigm.

In contrast to the lack of effects of OXT on self-vs. other processing in the mPFC it increased embarrassment across both, EE and SE in the context of attenuated amygdala and insula reactivity. Although EE trials produced stronger amygdala responses than SE ones following PLC, OXT suppressed them equivalently in both conditions. In line with the social salience hypothesis of OXT (47) the general enhancement of embarrassment may reflect an increased awareness and impact of the social context. However, previous studies reporting OXTenhanced salience of external social stimuli commonly report decreased amygdala and concomitantly increased insula activity (9, 25, 48) which would be difficult to reconcile with the observed OXT-induced attenuation in both regions in the present study. Decreased insula activity following OXT has been previously observed in response to pain empathy (22), negative social feedback (49) and approach behavior (48). Moreover, in concert with concomitantly decreased amygdala activity attenuated stress in response to negative interactions (50) and reduced anxiety during social sharing (in women) have been reported (51). In line with these previous findings the suppression of both regions may thus rather reflect the stress-buffering or anxiolytic effects of OXT. Together with the hippocampus, the amygdala and insula are considered core nodes of the threat and anxiety circuitry (52, 53) and are hyperactive across anxiety disorders (52)– particularly social anxiety disorder (54). Moreover, successful non-pharmacological therapy for exaggerated anxiety has been accompanied by decreased hyperactivity in these regions (55, 56). The amygdala is also critically involvemed in emotional modulation of the SCR (57) and during OXT-enhanced empathy and protective responses suppression of amygdala reactivity was accompanied by increased SCR (10) and acoustic startle response (26). On the other hand, OXT decreased the SCR in response toward conditioned threat stimuli (58) and decreased autonomic stress reactivity (59). Moreover, increased activity in the dAI has been specifically associated with increased arousal during embarrassing social situations (30), however not embarrassment-related schadenfreude (60). Together, the pattern of decreased reactivity in the two threat nodes as well as the autonomic arousal measures during exposure to embarrassing, social-stressful situations may thus reflect anxiolytic effects of OXT. This interpretation is further supported by a negative association between amygdala activation and trait anxiety scores and the magnitude of the SCR in the OXT group suggesting that it reduced anxiety and particularly in more anxious individuals.

It seems paradoxical therefore that in the context or empathic and self-embarrassment OXT-reduced amygdala responses were associated with a decreased SCR and yet ratings were still increased. This demonstrates that OXT-induced reduction of amygdala activity per se is not sufficient to infer anxiolytic effects of OXT, and indeed another study has also reported that OXT can promote an anxiogenic response in the context of concomitantly reduced amygdala activation (25). It thus may be hypothesized that the insula plays an important role in shaping the specific behavioral effects of an unspecific OXT-attenuation of the amygdala. Whereas concomitantly increased insula activity may promote the salience-enhancing effects of OXT (25, 26, 48), a concomitant reduction may reflect anxiogenic or arousal reducing effects of OXT (22, 48, 49) which is in line with the contribution of the anterior insula to several functional domains including salience, and arousal (61, 62). The observed pattern of neural effects of OXT in the present study may thus favor an anxiolytic interpretation of its action during embarrassment processing. This may also reflect a reduction of the aversive and threatening affect evoked by the embarrassment stimuli and facilitate a more cognitive appraisal of the social contexts and their implications thus enhancing embarrassment levels across situations experienced both by others or by self.

Thus, OXT may have reduced the feelings of both pain and anxiety experienced during embarrassment, leading to a more cognitive assessment such that subjects rated the level of embarrassment experienced as higher. In this respect it should be noted that subjects in our task were asked to rate the level of embarrassment being experienced in a particular situation and not specifically to rate the intensity of their feelings for others in embarrassing situations, or of their own personal feelings in the self-condition. It is possible therefore that in the context of moral self-conscious emotions such as embarrassment, OXT may actually reduce emotional empathy toward others in order to promote a more accurate cognitive assessment of what someone is feeling. This might in turn promote more efficient avoidance of potential embarrassing situations in the future. Further experiments are required to disentangle these cognitive and emotional factors.

In our previous experiment using the MET task we observed that the OXT enhancement of emotional empathy ratings and reduced amygdala activity showed some associations with trait autism, however in the current study we did not find this. Previous studies have reported decreased responses in the anterior insula to social pain in adults with autism spectrum disorder (19), although not for empathic neural responses toward physical pain (63). However, we found no associations between neural responses to embarrassment and autistic traits in healthy subjects. On the other hand, we did find a negative association between trait anxiety and the effect of OXT on the amygdala and SCR, which contrasted with no such association in the PLC group. This suggested that OXT was particularly attenuating neural and behavioral indices of anxious arousal in subjects with high trait anxiety, and resonates with previous findings suggesting that OXT particularly reduced negative appraisal following social stress induction in high trait anxiety subjects (36). These observations conflict with a recent report on OXT-induced increased startle responsivity in high anxious subjects (38). However, in this previous study OXT specifically increased startle responsivity toward non-social stimuli, further emphasizing how complex interactions between personal and social contextual factors may moderate the specific effects of OXT.

No sex-differential effects were observed in the behavioral effects of OXT on embarrassment ratings or on right amygdala or right insula responses, which is consistent with our previous observations that OXT enhanced emotional empathy in the MET in both sexes (10). These findings contrast with other studies reporting sex-dependent responses in amygdala and insula (23, 64) in the context of the impact of positive and negative personal characteristics on face attraction and sub-liminal processing of emotional faces. However, this may reflect the fact that exhibiting empathic-embarrassment and self-embarrassment are of equal adaptive importance in males and females and that OXT only tends to promote or amplify sex-differences in behavioral and neural responses associated with sex-specific priorities in social salience and social preference processing (see 22).

It is still unclear how intranasal administration of OXT exerts its neural and behavioral effects. While it has been questioned whether OXT administered via an intranasal spray does actually enter the cerebrospinal fluid (CSF) and influence brain OXT receptors (65) it is now established from work using labeled peptides in monkeys that it does enter into the CSF as well as the blood (66). In humans intranasal OXT does also increase CSF as well as blood OXT concentrations although with different time courses (67). It has still to be demonstrated however whether it can both enter the brain across the blood brain barrier from increased concentrations in the peripheral vasculature (66) and directly via the established lymphatic or other transport mechanisms at the back of the nasal cavity [see (8)]. Importantly however, despite some early studies in autistic subjects reporting behavioral effects of intravenous oxytocin (68, 69) recent studies have failed to find any behavioral or neural effects of intravenous compared with intranasal routes of administration (70, 71). Intranasal administration of OXT in humans also results in extensive increases in regional cerebral blood flow in the majority of brain regions containing OXT receptors at the same time course as routinely used in the current as well as most previous intranasal treatment studies (i.e., around 45 min)(72). Thus, in the context of the findings in the current experiment it is likely that functional effects of OXT on embarrassment and reduced physiological arousal, as well as those on the amygdala and insula, are caused primarily by its actions following entry into the brain, although we cannot rule out some contributions from indirect actions via receptors in peripheral organs.

Findings of the present study need to be interpreted in the context of limitations. Firstly, compared to previous studies that determined sex-differential neural effects of OXT during

#### REFERENCES

evaluation social-emotional stimuli the present sample was smaller [n = 70, previous studies enrolled slightly higher samples between around n = 80–90 subjects, (23, 64)]. The lack of sex-differential effects of OXT on embarrassment therefore needs to be replicated in larger samples. Secondly, our task did not adequately separate cognitive from emotional components of embarrassment in order to provide a better understanding of how OXT attenuation of both amygdala and insula responses resulted in increased ratings of embarrassment.

Overall therefore we have demonstrated for the first time that OXT can enhance ratings of both empathic and selfembarrassment in males and females, showing that it also influences moral, self-conscious emotional responses. These behavioral effects of OXT are associated with decreased physiological arousal and decreased responses in both the right amygdala and anterior insula, but not in mentalizing networks (mPFC). Furthermore, OXT effects on the amygdala are strongest in individuals with high trait anxiety. Thus, OXT in this context may be promoting an anxiolytic effect resulting in a more cognitive rather than emotional appraisal of embarrassment levels.

#### AUTHOR CONTRIBUTIONS

YG and KK designed this experiment, YG collected the data, YG, WZ, FZ, KK, and BB analyzed the data, YG, WZ, XM, SY, BB, and KK interpreted the results. YG, BB, and KK wrote the paper.

#### ACKNOWLEDGMENTS

This study was supported by the National Natural Science Foundation of China (NSFC) grant 563 (grants 31530032; 91632117), the Fundamental Research Funds for the Central Universities of China (ZYGX2015Z002) and the Sichuan Science and Technology Department (2018JY0001).

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fendo. 2018.00572/full#supplementary-material

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Geng, Zhao, Zhou, Ma, Yao, Becker and Kendrick. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Effects of Neuropeptide Substance P on Proliferation and β-Cell Differentiation of Adult Pancreatic Ductal Cells

Nan Zhang, Di Gao, Yudan Liu, Sihan Ji and Lei Sha\*

Department of Neuroendocrine Pharmacology, School of Pharmacy, China Medical University, Shenyang, China

Purpose: The pancreas is innervated by sensory nerves, parasympathetic and sympathetic nerves. The classical neurotransmitters, acetylcholine and noradrenaline, and some kind of neuropeptides are contained in the terminals of these nerves. Neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) co-released from the primary sensory fibers have been identified as the key neurotransmitters in pancreas. Pancreatic ductal epithelium cells are one of the important sources of the pancreatic islet β-cell neogenesis. We hypothesized that SP and CGRP might play a role on proliferation of ductal cells and differentiation of ductal cells toward the β-cell neogenesis.

#### Edited by:

Lee E. Eiden, National Institutes of Health (NIH), United States

#### Reviewed by:

Eberhard Weihe, Philipps-Universität Marburg, Germany Terry Moody, National Cancer Institute (NCI), United States

> \*Correspondence: Lei Sha<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Neuroscience

Received: 30 June 2018 Accepted: 16 October 2018 Published: 05 November 2018

#### Citation:

Zhang N, Gao D, Liu Y, Ji S and Sha L (2018) Effects of Neuropeptide Substance P on Proliferation and β-Cell Differentiation of Adult Pancreatic Ductal Cells. Front. Neurosci. 12:806. doi: 10.3389/fnins.2018.00806

Methods: Primary ductal cells of rat pancreas at the third passage (P3) were used. The identification of P3 cells were confirmed with flow cytometry analysis and immunostaining by CK19 (the ductal cell marker). Proliferation of ductal cells was verified by CCK-8 assay and Ki67 immunostaining. Differentiation of ductal cells was determined with immunostaining and flow cytometry. Possible mechanism was explored by testing the key proteins of Wnt signaling using Western blot analysis.

Results: Our data showed that SP but not CGRP promoted proliferation of ductal cells. Moreover, NK-1 receptor antagonist L-703,606 blocked the SP-induced stimulation of proliferation. The results of Western blot analysis showed that L-703,606 attenuated the effects of substance P on NK1R, GSK-3β, and β-catenin expression. However, SP did not directly induce the differentiation of ductal cells into β-cells, and did not promote the progression of ductal cells to differentiate into more insulin-produced cells in induction medium.

Conclusions: These findings suggested that SP but not CGRP promoted proliferation of adult pancreatic ductal cells. SP promoted proliferation of ductal cells but not differentiation into β-cells. NK1R and Wnt signaling pathway might be involved in the mechanism of promoting the proliferation of ductal cells by SP. Findings in this study indicated the lack of SP might be a possible indicator for the initial of diabetes. SP could also be used as a drug candidate for the treatment of diabetes.

Keywords: Substance P, calcitonin gene-related peptide, pancreatic ductal cells, proliferation, β-cell differentiation, NK-1 receptor

# INTRODUCTION

The β-cell mass is dynamic and is regulated to maintain glucose homeostasis in response to the changes in physiological and pathological demands (Bonner-Weir, 2000; Halban et al., 2010; Juhl et al., 2010). When the demand for insulin is increased, the β-cell mass is expanded by replication and/or hypertrophy of pre-existing β-cells (Dor, 2006), and β-cell neogenesis from pancreatic progenitor cells located in duct epithelial cells or surrounding centroacinar cells/terminal ductules (Bonner-Weir et al., 2010). Therefore, strategies targeted to improve proliferation or differentiation of ductal cells may lead to improvements in the alleviation of β-cell loss pathology.

 In injured adult mouse pancreas, β-cell progenitors located in the ductal lining were activated (Xu et al., 2008). Several in vitro studies have shown that adult pancreatic ductal cells can differentiate into insulinproducing cells (Fukazawa et al., 2006; Seeberger et al., 2006; Li et al., 2011). Proliferating pancreatic ductal epithelium cells were induced to differentiate into β-cells with TNF-like weak inducer of apoptosis (Wu et al., 2013). Expanded pancreatic ductal cells also differentiated into insulin-producing β-cells in an appropriate environment (Rovira et al., 2010). Capacity of self-renewal and pluripotency is an important feature of stem cells. Despite the differentiation capability of ductal cells has been demonstrated, the proliferation potential and the possible factors controlling of growth in these cells is not wellunderstood.

The importance of the nervous system in maintaining body homeostasis has previously been described, and it is suggested that organogenesis and tissue repair are under neural control (Besedovsky and del Rey, 1996). There is increasing evidence that neuroendocrine-remodeling does take place in the pancreatic islets of diabetic disease models (Persson-Sjögren et al., 2005; Razavi et al., 2006). Two neuropeptide substance P (SP) and calcitonin gene-related peptide (CGRP) have been found to tightly link to the development of diabetes. SP mediates insulin secretion and plays an important role in the development of type I diabetes (Razavi et al., 2006). CGRP is also involved in the activity of insulin secretion and contributes to the development of type II diabetes (Gram et al., 2007; Tanaka et al., 2011). SP and CGRP fibers not only innervate islets, but also innervate pancreatic ducts (Razavi et al., 2006; Gram et al., 2007), suggesting that SP and CGRP might modulate the activity of pancreatic ducts. We hypothesized that the innervations of the primary sensory fibers to the pancreatic ducts play a role on ductal epithelium cells proliferation and differentiation toward the β-cell neogenesis.

In the present study we investigated the effects of SP and CGRP on primary cultured ductal cells of rat pancreas. We examined the effects of SP and CGRP on proliferation of pancreatic ductal cells, and further the effect of SP on differentiation of ductal cells toward β-cells. Moreover, we investigated the possible mechanism of the proliferative promotion effects of SP.

# MATERIALS AND METHODS

#### Animals

Sprague Dawley rats (male, 2 months old) were purchased from the Animal Center of China Medical University. All animal protocols were approved by the Animal Care Commitee in China Medical University (Shenyang, China) and performed according to institutional guidelines.

# Preparation of Substance P (SP) and Calcitonin Gene Related Peptide (CGRP)

SP was purchased from Millipore Co. (Catalog number: 05-23- 0600-1MGCN, Billerica, MA, USA), CGRP was gained from Bioss Co. (Catalog number: bs-0791P, Beijing, China), and both kept protected from light during the experiments. Stock solution of SP (1 mg/ml) and CGRP (1 mg/ml) were dissolved in distilled water and stored at −20◦C.

# Primary Culture and Identification of Pancreatic Ductal Cells

Adult Sprague Dawley rats were sacrificed and the pancreases were rapidly removed. Pancreases were then dissociated with V collagenase (1.5 g/L). The digested tissues were triturated through 600µm cell strainer to obtain primary ductal cells. The cultures were grown in the complete medium containing DMEM/F12 supplemented with 10% fetal bovine serum (FBS), 100 U/ml Penicillin and 100µg/ml Streptomycin (all from Gibco) at 37◦C in a humidified atmosphere with 5% CO2. The medium was changed 24 h after, and the non-adherent cells were discarded. The attached cells were labeled P0, and the medium was changed again every 3 days. When cells became 90% confluent, cultures were dissociated with trypsin (1 ml 0.25% trypsin) at 37◦C 2– 3 min, and serum was used to inactivate the enzyme. Cells were then expanded at the third passage (P3) for purification. Cells were characterized for the ductal cell marker cytokeratin19 (CK19) by immunocytochemistry and flow cytometry.

Cells were plated at a density of 1.0 × 105 cells/ml and cultured. At day 1, 3, 5, 7, and 9. Cells in each well were collected and counted by hemocytometer. At least 6 wells were assessed at each time point. Growth curve was designed to reveal the self-renewal capacity of the cells.

# Assay for Proliferation of Ductal Cells

Disassociated ductal cells at 3rd passage (P3) were plated in 96 well-plates at a density of around 1.0 × 105 cells per well. The cells were divided into groups randomly and treated with saline (1 mM) or serial concentrations of SP (10−6 , 10−5 , 10−4 , 10−3 , 10−2 , 10−1 , 1, and 10µM; n = 6 wells for each) or CGRP (10−6 , 10−5 , 10−4 , 10−3 , 10−2 , 10−1 , 1, and 10µM; n = 6 wells for each) for 3 days. In the other experiment, cells were treated with SP or SP plus L-703,606 (1, 2, 4, 6, 8, 10µM) for 3 days. The medium containing SP or CGRP was changed every 24 h to ensure the concentration of neuropeptides in the medium. After treatments, cell viability was determined by CCK-8 test. Briefly, the cells were incubated with 10 µl CCK-8 (Invitrogen) at 37◦C for 4 h. Absorbance at 490 nm was determined with a microplate reader (Thermo Fisher Scientific Inc., USA). The results were presented as percentages of the value of normal control cells.

The proliferation potential of ductal cells was tested by Ki67 immunostaining after treated with serial doses of SP (10−3 , 10−2 , 10−1 , and 1µM n = 6 wells for each) with or without L-703,606 (2µM) for 3 days. The medium containing SP was changed every 24 h. Cells were fixed with 4% paraformaldehyde and then incubated with primary antibody at 4◦C overnight. The primary antibodies used were as follows: mouse anti CK19 (1:100; Proteintech, Wuhan, China) and rabbit anti Ki67 (1:50, Abcam, Cambridge, UK), rabbit anti NK1R (1:100) and mouse anti Ki67 (1:100, All from Bioss, Beijing, China). After rinsing with PBS, cells were incubated with Cy3 or FITC-conjugated secondary antibodies (1:200; all from Jackson ImmunoResearch Lab, West GroveCity, PA, USA) at room temperature for 1 h. Immunofluorescence controls were performed without primary antibodies. The cells were covered with mounting medium (Vector Laboratories, Burlingame, CA, USA) containing DAPI (0.5µg/mL; Sigma, St. Louis, MO, USA). Results were visualized with fluorescence microscopy (Nikon Eclipse E600; Nikon Corporation, Tokyo, Japan). Cells were counted in triplicate cultures in ten randomly chosen areas under the microscope. NIH ImageJ was used to count Ki67+/CK19+ cells, and the mean numbers were used for analysis.

### Detection of Differentiation and Immunocytochemistry

P3 ductal cells were directly treated with SP (10−2 , 10−1 , and 1µM) or treated with SP and insulin-produced cell induction medium (DMEM/F12 plus 2% FBS, 10 mmol/L Niacinamide, 20 ng/ml HGF, 20 µg/L bFGF, and 10 nmol/L exendin-4) for 28 days and immunostained with CK19 and Pdx-1, or Pdx-1 and insulin antibodies to detect differentiation. The medium containing SP was changed every 24 h. Cells were fixed with 4% paraformaldehyde and then were permeabilized with 0.5 ml of 0.1% Triton X-100. After blocking with 3% BSA, cells were incubated with primary antibody at 4◦C overnight. The following primary antibodies were used: mouse anti-CK19 (1:100; Proteintech, Wuhan, China), rabbit anti-Pdx-1(1:100; Proteintech, Wuhan, China) and mouse antiinsulin (1:100; NovusBio, Littleton CO, USA). Cells were then stained with FITC- or Cy3-labeled secondary antibodies (Jackson ImmunoResearch Lab, West Grove, PA at 1:200 dilution). Nuclei were stained with DAPI. Results were visualized with fluorescence microscopy (Nikon Eclipse E600; Nikon Corporation, Tokyo, Japan). The proportion of cells labeling with specific marker in total number of DAPI+ cells was accessed and expressed as the mean percentage of specific differentiation.

# Flow Cytometry Analysis

To determine the percentage of CK19+ cells in total ductal cells, ductal cells at 3rd passage were stained with CK19. Cells were fixed and permeabilized using Cytofix/Cytoperm system (BD Bioscience, San Diego, CA, USA). After permeabilization, cells were stained with mouse anti Cytokeratin 19 (CK19) antibody for 30 min at 4◦C. Then cells were incubated with FITClabeled secondary antibody for 30 min at room temperature. To detect the differeciation of ductal cells, intracellular staining was performed for insulin production and CK19 expression. After cell permeabilization, cells were stained with mouse anti CK19 and PE-labeled insulin antibodies, following incubation of FITC-labeled secondary antibody. Mouse anti CK19 and PE-labeled anti-insulin antibodies were purchased from Novus Biologicals(Littleton, CO, USA). FITC-labeled antimouse secondary antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Data were analyzed by FACScan flow cytometer (BD Biosciences) using the CellQuest software (BD Biosciences).

# Western Blot Analysis

Cells were lysed by pre-cooling cell lysis buffer for Western and IP (Beyotime biotechnology, Shanghai, China) supplemented with 1 mM PMSF (Beyotime biotechnology, Shanghai, China) on ice for 15 min and centrifuged at 12,000 × g for 15 min at 4◦C. Protein qualification was measured by using a BCA kit (Beyotime biotechnology, Shanghai, China). Forty micrograms total proteins in each lane were electrophoretically separated by 10% SDS-PAGE (Beyotime biotechnology, Shanghai, China) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA). Membranes were blocked with 3% BSA in TBST buffer (50 mM Tris•HCl, pH 7.4, 150 mM NaCl, 0.1% Tween-20) for 1 h at room temperature. After rinsing with TBST buffer three times, membranes were incubated with primary antibodies overnight at 4◦C.  After washing with TBST, membranes were incubated by horseradish peroxidase (HRP)-conjugated anti-rabbit or antimouse secondary antibodies for 1 h at room temperature. ECL chemiluminescence solution (Bio-Rad Laboratories, Hercules, CA) was added after rinsing with TBST. Signal intensity of immunoreactive bands was analyzed using Image Lab software (Bio-Rad Laboratories, Hercules, CA) and ImageJ software (Version 1.48v, National Institutes of Health, Bethesda, MD). For the Western blot assay, the optical density of the protein bands was normalized to the density of β-actin band to yield the densitometric ratio value.

# Statistical Analysis

Statistical analysis was performed with SPSS version 16.0 (2007; Chicago, IL, USA). Most data are expressed as the mean ± SD, data of Western blot are expressed as the mean ± SEM, and one-way ANOVA with Bonferroni correction for multiple comparisons was used to analyze the differences between the groups. p < 0.05 was determined as significance.

# RESULTS

# Generation and Identification of Rat Pancreatic Ductal Cells

The ductal cells obtained from rat pancreas were expanded at 3rd passage (P3) for purification and characterized phenotypically and molecularly. Cells at P3 were adherent in serum containing medium, and were long, spindle shaped cells in morphology. Majority of the P3 cells were positive for CK19 (**Figure 1A**), a ductal cell marker. Flow cytometry results showed that more than 80% of single P3 cells were positive for CK19 (**Figure 1B**), indicating that the isolated cells were ductal cells and qualified for subsequent experiments.

Cell growth was measured at day 1, 3, 5, 7, and 9 to assess the self-renewal capacity of ductal cells. The result showed that the cells expanded rapidly between day 1 and day 5 and plateaued after day 5 (**Figure 1C**).

#### Substance P but Not CGRP Enhanced Self-Renewal Capacity of Ductal Cells

Proliferation is tightly linked to self-renewal capabilities of ductal cells. To determine the effects of SP or CGRP on proliferation, P3 ductal cells were treated with series concentrations of SP (10−6 , 10−5 , 10−4 , 10−3 , 10−2 , 10−1 , 1, and 10µM) or CGRP (10−6 , 10−5 , 10−4 , 10−3 , 10−2 , 10−1 , 1, and 10µM) in growth medium for 3 days. Cell viability was assessed by CCK-8 test. Compared with control cells, SP (10−6 , 10−5 , 10−3 , 10−2 , 10−1 , and 1µM) treated cells exhibited a stronger proliferation capacity (**Figure 2A**). However, there was not a significant effect of CGRP on cell viability (**Figure 2B**), indicating that SP but not CGRP promoted cell proliferation of pancreatic ductal cells.

To further confirm the effect of SP on ductal cell proliferation, the proliferation of ductal cells was tested with Ki67 (a marker for cell proliferation) immunostaining. After incubation with SP (10−6 , 10−3 , 10−2 , 10−1 , and 1µM) for 3 days, ductal cells were immunostained with Ki67 followed by image analysis. As shown in **Figures 3A,B**, significantly higher levels of Ki67 positive cells were found in SP treated groups compared with control (P < 0.05, P < 0.05, P < 0.05, P < 0.01, P < 0.05, respectively). Moreover, growth curves of day 1, 3, 5, 7, and 9 were plotted to assay the self-renewal capacity of ductal cells

FIGURE 2 | Effects of substance P or CGRP on self-renewal capacity of adult pancreatic ductal cells. (A) Cell viability assay with treatment of substance P. Cells were treated with a serial of concentrations of substance P (10−6 , 10−5 , 10−4 , 10−3 , 10−2 , 10−1 , 1, and 10µM; n = 6 wells for each) for 72 h, and the cell viability was measured by CCK-8 assay. (B) Cell viability assay with treatment of CGRP. Cells were treated with different concentrations of CGRP (10−6 , 10−5 , 10−4 , 10−3 , 10−2 , 10−1 , 1, and 10µM; n = 6 wells for each) for 72 h, following assayed by CCK-8 test. \*P < 0.05, compared with control groups.

point. \*P < 0.05, \*\*P < 0.01, compared with control groups.

treated with different concentrations of SP (10−6 , 10−3 , and 10−1 µM). The results indicated that cells incubated with SP expanded more rapidly on day 3, 5, 7, and day 9 (**Figure 3C**). These findings indicated that SP promoted proliferation of ductal cells.

#### Substance P Did Not Induce Differentiation of Ductal Cells Into β-Cells

Above results showed ductal cells were sensitive to SP, and cell proliferation was promoted at a concentration of 10−6 µM. Next, we tested whether SP further promotes differentiation on ductal cells. To determine the effects of SP on differentiation of ductal cells, cells were treated with serial doses of SP (10−6 , 10−1, and 1µM) for 28 days. In substance P treated groups, cells positive for CK19 and Pdx-1 were negative for insulin (**Figures 4A,B**), as verified by triple immunostaining, indicating that SP dose not directly induce differentiation of adult pancreatic ductal cells into β-cells.

Further, cell culture medium was replaced by insulinproduced induction medium with or without SP (10−6 , 10−1 , and 1µM). After 28 days, ductal cells developed into insulinproduced cells (insulin+) (**Figure 4C**). Flow cytometric analysis showed that SP did not promote ductal cells differentiated into greater numbers of insulin-produced cells (P > 0.05, P > 0.05, respectively, **Figure 4D**) as compared with induction medium control group. All these results indicated that SP did not affect the differentiation potential of ductal cells into β-cells.

# Blockade of SP-Induced Stimulation of Proliferation With NK-1 Receptor Antagonist L-703,606

To evaluate the influence of NK-1 receptor (NK1R) antagonist L-703,606 on the effects of SP on duct cells, cells were incubated with SP (10−1 µM) plus L-703,606 (1, 2, 4, 6, 8, 10 µM) for 72 h. After 72 h incubation, cell viability was measured by CCK8 assay. Exposure of SP-treated cells to L-703,606 (2, 4, 6, 8, 10µM) significantly inhibited the cell proliferation induced by SP (P < 0.01, P < 0.01, P < 0.01, P < 0.01, P < 0.01, respectively, **Figure 5A**). The dose of 2µM of L-703,606 was used to block the stimulatory effects of SP on duct cells in further experiments.

To investigate whether SP could mediate ductal cell proliferation via NK-1 receptor, we added NK1R antagonist L-703,606 (2µM) into the culture medium containing different doses of SP (10−2 , 10−1 , and 1µM). In L-703,606 plus SP-treated groups, L-703,606 treatment decreased cell viability of ductal cells compared with SP-treated groups (percentage of control without SP or L-703,606, 108.02 ± 19.65 vs. 136.10 ± 20.59 in 10−2 µM, P < 0.05; 128.50 ± 16.33 vs. 172.81 ± 24.15 in 10−1 µM, P < 0.01; 110.95 ± 10.78 vs. 137.14 ± 17.06 in 1µM, P < 0.01; **Figure 5B**).

Ki67 immunostaining was performed to verify the inhibition of L-703,606 on proliferation effects of SP on ductal cells. Compared with control groups, the SP-treated cells showed a significantly higher number of cells positive for Ki67 (21.34 ± 1.89% vs. 16.17 ± 1.04%, P < 0.05, **Figures 5C,D**). Compared with SP-treated groups (10−1 µM), L-703,606 treatment decreased the number of Ki67 positive cells (16.17±1.04% vs.

21.34 ± 1.89%, P < 0.05, **Figures 5C, D**). These results mean that SP plays its role in proliferation of ductal cell via NK-1 receptor.

#### Substance P Promotes NK1R Expression, Decreases GSK-3β and Increased β-Catenin in Ductal Cells

We performed double immunostaining of NK1R and ki67 expression on ductal cells. Similar to Garland's finding (Garland et al., 1994), we found that NK1R was expressed in cytoplasm after SP treatment (**Figure 6A**). To investigate whether SP affect NK1R, and GSK-3β/β-catenin pathway, cells were treated with SP (10−1 µM), SP (10−1 µM)+L-703,606 (2µM) and L-703,606 (2µM) alone for 72 h, total protein was harvested and Western blot analysis was performed to test expression of NK1R, GSK-3β and β-catenin (**Figures 6B,D**). Quantitative analysis revealed that NK1R protein levels were significantly increased in SP treated cells as compared with control groups (**Figure 6C**, P < 0.01), but L-703,606 treatment significantly decreased NK1R levels (**Figure 6C**, P < 0.05). Quantitative analysis of GSK-3β and βcatenin protein showed that SP treatment significantly decreased GSK-3β levels (**Figure 6E**, P < 0.01) and significantly increased β-catenin levels (**Figure 6E**, P < 0.01) compared with control group, whereas L-703,606 treatment decreased these effects of SP on ductal cells (SP+L-703,606 group vs. SP group, P < 0.05, P < 0.05, respectively).

# DISCCUSION

In the present study, we demonstrated that SP but not CGRP promoted cell viability and proliferation of ductal cells derived from rat pancreas, which is inhibited by the NK1 receptor antagonist. SP did not exert the effects of differentiation toward β-cell of these cells. Our experiments also revealed that SP treatment enhanced NK1R expression, reduced GSK-3β levels and increased β-catenin levels. However, treatment with NK1R inhibitor L-703,606 attenuated the effects of SP on NK1R, GSK-3β and β-catenin levels. Collectively, these findings suggest that SP promotes pancreatic duct cell proliferation via NK1R, and the Wnt/β-catenin pathway is involved in the mechanisms of SP-induced proliferation of ductal cells.

SP and CGRP have been showed to be capable to promote proliferation in some other tissues. SP has been reported to promote proliferation of adult neural progenitor cells (NPCs) (Park et al., 2007), cord blood CD34+ hematopoietic stem cells (Shahrokhi et al., 2010), and fibroblast-like cells derived from bile duct (Tian et al., 2014). CGRP stimulates cell proliferation and inhibits cell apoptosis of bone-marrow MSCs in short term culture, and promotes osteogenetic differentiation in long term culture (Xu and Jiang, 2014; Liang et al., 2015). It has been reported that CGRP overexpressed rat adipose-derived stem cells (ADSCs) exerted more potency of proliferation, neurosphere

\*P < 0.05, \*\*P < 0.01, as compared with substance P treated groups.

formation (Yang et al., 2014), and osteoblastic differentiation (Fang et al., 2013). Moreover, CGRP and SP treatment promoted human skin keratinocyte proliferation (Shi et al., 2013). Besides promoting proliferation, SP has also been shown to stimulate differentiation of mesenchymal stem cells (MSCs) into osteoblastic cells through neurokinin (NK)-1 receptor (Sun et al., 2010).

Our results showed that SP stimulated proliferation of pancreatic ductal cells and pancreatic ductal cells were very sensitive to SP on proliferation. Opolka et al. (2012) reported that SP at the concentration of 10−4 µM promoted proliferation of murine chondrocytes. SP at 10−3 µM induces pro-proliferation effects on fibroblast-like cells from bile duct and hematopoietic stem cells (Shahrokhi et al., 2010; Tian et al., 2014). In bone marrow-derived mesenchymal stem cell-like cells and bone marrow stromal stem cells, 10−2 µM SP is needed to promote cell proliferation (Mei et al., 2013; Dubon and Park, 2015; Liu et al., 2016). SP concentration as high as 10−1 µM is needed in ARPE-19 cells, a human retinal pigmented epithelial (RPE) cell line (Baek et al., 2016), and neural stem/progenitor cells derived from spinal cord to show its effect (Kim et al., 2015). In bone marrow mesenchymal stem cells (MSCs), SP at as low as 10−6 µM enhances osteoblast differentiation of MSCs (Wang et al., 2009) and protects against apoptosis induced by serum deprivation (Fu et al., 2015). Our results indicated that SP at as low as 10−6 µM had pro-proliferation effect on ductal cells. Thus, pancreatic ductal cells are among the most sensitive cells to SP, similar to MSCs.

blot. (E) Quantitative analysis of GSK-3β and β-catenin. Substance P reduced GSK-3β levels and increased β-catenin levels, whereas L-703,606 blocked the effects of substance P on GSK-3β and β-catenin. Densitometric ratios are the mean ± SEM of three experiments. \*\*P < 0.01, \*P < 0.05 as compared with control groups. ##P < 0.01, #P < 0.05 as compared with substance P treated groups.

The actions of SP are mediated by three G protein–coupled receptors, NK-1, NK-2, and NK-3 receptors; among these, the NK-1 receptor (NK1R) has the highest affinity for SP (Takeda et al., 1991). In Kim's (Kim et al., 2015) and Park's (Park et al., 2007) reports, NK1R inhibitor L-703,606 (1–10 µM) decreased SP-induced proliferation of spinal cord-derived neural stem/progenitor cells (SC-NSPCs). We examined the inhibitory effect of L-703,606 (1–10µM) on SP-stimulated cell proliferation. Our results indicated that L-703,606 at dose of 2–10µM effectively blocked the proliferative effect of SP. Our results showed that L-703,606 inhibited the proliferation effects of SP on pancreatic ductal cells, indicating the proliferation stimulating effect of SP on ductal cells through NK1R. Notably, several studies are consistent with our results. Glaser et al. (2011) reported that knockout of the NK1R reduced cholangiocyte proliferation in bile duct-ligated mice, indicating a specific mitotic effect of SP on bile ductal cells. In Liu's study (Liu et al., 2016), they demonstrated that SP enhanced proliferation of bone marrow mesenchymal stem cell derived osteoblasts (BMSC-OB), and this effect would be inhibited by adding NK1R antagonist. In has been shown that pretreatment with the NK1R antagonist reduced the effect of SP on proliferation of ARPE-19 cells (Baek et al., 2016).

It has been reported that SP at concentration of 1µM increased NK1R mRNA expression in pancreatic acinar cells (Koh et al., 2012). In our study, we performed double immunostaining of NK1R and ki67 expression on ductal cells. The images showed that the ductal cells had stronger NK1R immune staining in SP-treated cells by compared with control cells. Garland et al. (1994) reported that NK1R was located in intracellular vesicles, rather than aggregated in the plasma membrane in epithelial cells after incubation with SP. Similar to Garland's finding, we found that NK1R was expressed in cytoplasm after SP treatment. Consistent with the results of staining, our results of Western blotting showed that the level of NK1R was significantly higher in SP treated group, compared with control group.

It is interesting that CGRP did not show any effect on proliferation of ductal cells. SP and CGRP are two important neurotransmitters released from primary sensory fibers of dorsal root ganglia. CGRP and SP are colocalized and coreleased from these fibers (Gibbins et al., 1985). Besides primary sensory fibers of dorsal root ganglia, SP was also from another important source which is intrapancreatic ganglia. Neurons in pancreatic ganglia innervate islets as well as pancreatic ducts (Sha et al., 2001; Love et al., 2007). Our study (Shen et al., 2016) revealed that intrapancreatic ganglia contain SP neurons and contribute around 50% of SP in pancreas. Our results suggested that SP released from primary sensory fiber and fibers of pancreatic ganglia stimulate proliferation of pancreatic ducts. Pancreatic ganglia don't contain any CGRP neurons (our unpublished data) and all CGRP fibers appear coming from primary sensory neurons (Russell et al., 2014). Thus, via release of CGRP, primary sensory fibers might uniquely modulate activity other than proliferation of pancreatic duct.

Pancreatic ductal cells play as pancreatic stem cells that exhibit stem cell properties, including proliferation and differentiation potential. Even though SP showed pro-proliferation activity, we did not find SP exert any promoting effect on the ductal cells differentiating toward β-cells. Our results showed that SP did not induce pancreatic ductal cells differentiate into insulin-positive cells directly, although the ductal cells all expressed Pdx-1, a pancreatic progenitor cell marker (Murtaugh, 2007). Further, in insulin-produced cell induction medium, SP did not promote pancreatic ductal cells differentiating into greater numbers of insulin-produced cells either.

Activation of Wnt signaling has been shown to be essential for proliferation of multiple cells (Mah et al., 2016; Arrigoni et al., 2018; Majidinia et al., 2018). Glycogen synthase kinase-3β (GSK-3β), a key molecule of the Wnt pathway, induces phosphorylation of β-catenin, causing the degradation of βcatenin. GSK-3β activity is inhibited following activation of the Wnt pathway, stimulating the accumulation of β-catenin in the cytoplasm, and β-catenin transferred into the nucleus, serving as a transcription factor to induce the activation of downstream target genes (Wang et al., 2018). We propose that SP regulates ductal cell proliferation via activation of Wnt pathway. Our results of western blot showed that SP treatment enhanced NK1R expression, decreased GSK-3β levels and increased β-catenin levels. Moreover, L-703,606, a NK1R antagonist, decreased NK1R expression and attenuated the effects of SP on GSK-3β and β-catenin levels, indicating that SP may promote proliferation of ductal cells via augmenting NK1R and GSK-3β/β-catenin signaling.

Our study was designed to understand the possible effects of neuropeptides released by primary sensory nerve fibers on pancreatic ductal cells. However, we found that only SP promoted the proliferation of pancreatic ductal cells. Moreover, SP did not induce or promote the differentiation of ductal cells into β-cells. The onset of diabetes is related to the lack of SP. Razavi et al. (2006) reported that reduced release of substance P initiates immune stress response of islet β-cells, leading to inflammation of islet cells and invasion of lymphocytes. On top of that, the ductal cells in the pancreas have stem cell properties and are one of the sources of β-cell neogenesis (Xu et al., 2008; Bonner-Weir et al., 2010). The proliferative effect of SP on ductal cells may explain another possible pathological course in the developmental stage of diabetes. The stabilization of SP levels might indicate that the pancreatic endogenous β-cell pool is abundant and is one of the conditions for maintaining β-cell homeostasis. Therefore, the lack of SP might be an indicator of the initial of diabetes. In addition, SP can also be used as a drug candidate for the treatment of diabetes.

In summary, SP but not CGRP stimulates the proliferation of pancreatic ductal cells. SP does not induce or promote the differentiation of ductal cells toward β-cells. Moreover, SP may promote proliferation of pancreatic ductal cells through enhancing NK1R expression, decreasing GSK-3β and increasing β-catenin levels.

#### INFORMED CONSENT

Informed consent was obtained from all individual participants included in the study.

# AUTHOR CONTRIBUTIONS

NZ, DG, YL, and SJ participated as investigators and reviewed, edited, and approved of the final version of the manuscript. LS initiated the study of neuropeptides substance P and CGRP in diabetes, is the guarantor of this work, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. NZ and LS wrote the manuscript.

#### ACKNOWLEDGMENTS

This work was supported by the National Natural Science Foundation of China (grant NO. 81270900; 81503273; 81670492).

#### REFERENCES

ductal cells into islet beta-cells mediated by hepatocyte growth factor. Biochem. Biophys. Res. Commun. 404, 711–716. doi: 10.1016/j.bbrc.2010.12.048

neogenesis from pancreatic ductal epithelium in adult mice. PLoS ONE 8:e72132. doi: 10.1371/journal.pone.0072132

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Zhang, Gao, Liu, Ji and Sha. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Age-Characteristic Changes of Glucose Metabolism, Pancreatic Morphology and Function in Male Offspring Rats Induced by Prenatal Ethanol Exposure

#### Di Xiao1†, Hao Kou2,3†, Shuxia Gui 1 , Zhenyu Ji 1 , Yu Guo1,3, Yin Wu1,3 and Hui Wang1,3 \*

*1 Department of Pharmacology, School of Basic Medical Sciences of Wuhan University, Wuhan, China, 2 Department of Pharmacy, Zhongnan Hospital, Wuhan University, Wuhan, China, 3 Hubei Provincial Key Laboratory of Developmentally Originated Diseases, Wuhan, China*

#### Edited by:

*Yu-Feng Wang, Harbin Medical University, China*

#### Reviewed by:

*Wen Xie, University of Pittsburgh, United States Yuping Wang, Louisiana State University Health Sciences Center Shreveport, United States*

> \*Correspondence: *Hui Wang<EMAIL_ADDRESS>
*†These authors have contributed equally to this work*

#### Specialty section:

*This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Endocrinology*

Received: *09 August 2018* Accepted: *16 January 2019* Published: *04 February 2019*

#### Citation:

*Xiao D, Kou H, Gui S, Ji Z, Guo Y, Wu Y and Wang H (2019) Age-Characteristic Changes of Glucose Metabolism, Pancreatic Morphology and Function in Male Offspring Rats Induced by Prenatal Ethanol Exposure. Front. Endocrinol. 10:34. doi: 10.3389/fendo.2019.00034* Intrauterine growth restricted offspring suffer from abnormal glucose homeostasis and β cell dysfunction. In this study, we observed the dynamic changes of glucose metabolic phenotype, pancreatic morphology, and insulin synthesis in prenatal ethanol exposure (PEE) male offspring rats, and to explore the potential intrauterine programming mechanism of the glucocorticoid-insulin-like growth factor 1 (GC-IGF1) axis. Ethanol (4 g/kg·d) was administered through oral gavage during gestational day (GD) 9–20. Serum glucose and insulin levels, pancreatic β cell mass, and expression of glucocorticoid receptor (GR), IGF1 and insulin were determined on GD20, postnatal week (PW) 6, PW12 with/without chronic stress (CS), and PW24, respectively. Both intraperitoneal glucose and insulin tolerance tests were conducted at PW12 and PW24. Results showed that the serum glucose and insulin levels as well as pancreatic β cell mass were reduced on GD20 in PEE males compared with the controls, while pancreatic GR expression was enhanced but IGF1 and INS1/2 expression were suppressed. After birth, compared with the controls, β cell mass in the PEE males was initially decreased at PW6 and gradually recovered from PW12 to PW24, which was accompanied by increased serum glucose/insulin levels and insulin resistance index (IRI) at PW6 and decreased serum glucose contents at PW12, as well as unchanged serum glucose/insulin concentrations at PW24. In addition, both improved glucose tolerance and impaired insulin sensitivity of the PEE males at PW12 were inversed at PW24. Moreover, at PW6 and PW12, pancreatic GR expression in the PEE group was decreased, while IGF1 expression was reversely increased, resulting in a compensatory increase of insulin expression. Moreover, CS induced pancreatic GR activation and inhibited IGF1 expression, resulting in impaired insulin biosynthesis. Conclusively, the above changes were associated with age and the intrauterine programming alteration of GC-IGF1 axis may be involved in prenatal and postnatal pancreatic dysplasia and impaired insulin biosynthesis in PEE male offspring.

Keywords: prenatal ethanol exposure, intrauterine growth restriction, pancreatic β cell development, insulin expression, glucocorticoids-insulin-like growth factor 1 axis

# INTRODUCTION

Alcohol consumption is common in both developing and developed countries. Epidemiological studies have shown that the alcoholism rate in young women has increased in recent years (1), and some of female alcoholics could not quit drinking during pregnancy (2). Prenatal alcohol exposure can lead to a range of adverse developmental outcomes in offspring, which are collectively termed fetal alcohol spectrum disorder (FASD) (3). In European countries, the morbidity of FASD is 0.97 per 1,000 births, whereas the morbidity of FASD in U.S. is 1.95 per 1,000 births (4). As one of the primary symptoms of FASD, intrauterine growth restriction (IUGR) is usually diagnosed with a birth weight and/or length below the 10th percentile for gestational age and an abdominal circumference that is less than the 2.5th percentile, with pathologic restriction of fetal growth (5). Human surveys and animal studies indicate that IUGR offspring induced by prenatal ethanol exposure (PEE) are characterized by altered glucose homeostasis and an increased risk of type 2 diabetes (T2DM) in adulthood (6, 7). It has been suggested that PEE induces glucose intolerance and hyperinsulinemia in IUGR male rats on postnatal day (PD) 91 (6, 8) and that the abnormality of glucose metabolism is more substantial in PEE male offspring (9, 10).

Intrauterine environmental challenges can "program" pancreatic β-cell structure and function (11, 12), which lead to glucose-insulin metabolic dysfunction and increased T2DM risk in adult offspring (13). The postnatal dysfunction of metabolic phenotype and abnormal pancreatic morphology induced by PEE have been reported in some studies (6–8, 14). However, these studies have some contradictory results, which might be attributed to a single selected time point for the investigations (15). Therefore, the dynamic changes of pancreatic development and glucose metabolism in PEE offspring, as well as the intrauterine programming mechanism, remains to be clarified.

It has been documented that glucocorticoids (GC) could affect pancreatic differentiation in utero (16, 17). High levels of glucocorticoids might induce an imbalance of pancreatic differentiation, reduction of β cell mass, and deceleration of insulin expression and secretion (16, 18). Insulin-like growth factor 1 (IGF1) is one of the key factors regulating pancreatic development (19). IGF1 not only promotes rapid division and proliferation of pancreatic β cells (20, 21) but also suppresses their apoptosis (20, 22). Furthermore, the IGF1 signaling pathway could regulate the proliferation of pancreatic precursor cells to affect the directional differentiation of β cells (23). It has been documented that glucocorticoids could reduce IGF1 expression in various cells via glucocorticoid receptor (GR) activation (24, 25). The evidence mentioned above suggests that the effect of glucocorticoids on IGF1 expression may play an important role in pancreatic development and β cell mass.

Previously, we have demonstrated that ethanol administered though oral gavage on gestational day (GD) 9–20 caused IUGR in rats (26). Furthermore, PEE can induce fetal rat over-exposure to maternal GC, which program IUGR offspring to be susceptible to multiple adult diseases (26– 28). It is still unknown whether high levels of glucocorticoids in fetal blood can change the expression of IGF1 in the pancreas and further affect pre- and postnatal pancreatic development and insulin synthesis. In previous studies, we found a negative relationship between serum corticosterone (CORT) levels and IGF1 contents in PEE offspring before and after birth; this negative relationship also correlates to the increased susceptibility to metabolic diseases. Based on these findings, we proposed a programming alteration mechanism of the glucocorticoid-insulin-like growth factor 1 (GC-IGF1) axis to explain multi-organ development toxicity and disease susceptibility in PEE offspring (26, 28).

Researches reveal that IUGR is associated with glucose intolerance in both men and women during adulthood (29), while the female offspring of undernourished rats do not develop glucose intolerance (30) or develop insulin resistance only in old age (31), suggesting gender-specific programming of glucose metabolism in these animals. Evidence also reveals that males are more sensitive to early life programming of insulin action than females (32). In this study, we observed dynamic changes in the glucose metabolic phenotype, pancreatic morphology and insulin synthesis in the PEE male offspring. The selected time points include GD20, postnatal week (PW) 6, 12, and 24, which are approximately equivalent to the fetus, childhood, early, and late stages of adulthood in humans (33). Moreover, a previously described chronic stress (CS) test, induced by a 2-week ice-water swimming test from PW10 (33, 34), was used to confirm the involvement of the GC-IGF1 axis in the alteration of pancreatic development and insulin synthesis induced by PEE. This study is significant for clarifying the developmental toxicity of ethanol and seeking early prevention and treatment strategies for fetaloriginated adult diabetes.

#### MATERIALS AND METHODS

#### Chemicals and Reagents

Ethanol (analytical pure grade) was purchased from Zhen Xin Co., Ltd. (Shanghai, China). Isoflurane was obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Ultrasensitive Rat insulin ELISA kits, Rat insulin ELISA kits and Rat/Mouse Proinsulin ELISA kits were provided by Mercodia (Uppsala, Sweden). Glucose oxidase assay kits were purchased from Mind Bioengineering Co. Ltd. (Shanghai, China). TRIzol reagent was purchased from Invitrogen Co. (Carlsbad, CA, USA). Reverse transcription and real-time quantitative polymerase chain reaction (RT-qPCR) kits were offered by TaKaRa Biotechnology Co., Ltd. (Dalian, China). Oligonucleotide primers for rat RT-qPCR genes (PAGE purification) were custom synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). RNAlater was provided by Qiagen (Düsseldorf, North Rhine-Westphalia, Germany). Glucocorticoid receptor (GR), IGF1, and insulin gene enhancer protein isl1 (ISL1) antibodies were obtained from Abcam (San Francisco, California, USA). Insulin antibody was provided by Sigma-Aldrich (St. Louis, Missouri, USA). Immunohistochemical staining agents were purchased from Zhongshan Golden Bridge Biotechnology Co. Ltd. (Beijing, China).

#### Animals and Treatment

This project was performed at the Center for Animal Experiment of Wuhan University (Wuhan, China), which is recognized and designated by the Association for Assessment and Accreditation of International Laboratory Animal Care. This study was carried out in accordance with the Guidelines for the Care and Use of Laboratory Animals of the Chinese Animal Welfare Committee (AAALAC International). The protocol was approved by the Committee on the Ethics of Animal Experiments of the Wuhan University School of Medicine (permit number: 14016). Specific pathogen free Wistar rats (12∼13-week-old, with weights of 197– 221 g for females and 241–275 g for males) were purchased from the Experimental Center of Hubei Medical Scientific Academy (No. 2012–2014, certification number:<PHONE_NUMBER>2258, license number: SCXK [Hubei]).

Animals were fed in metal wire cages in temperaturecontrolled conditions and allowed ad libitum access to tap water and standard chow at all times (room temperature: 18–22◦C; humidity: 40–60%, light cycle: 12 h light-dark cycle; 10–15 air changes per hour). After 7 days of acclimation, each two female rats and one male rat were mated from 7:00 p.m. to 7:00 a.m. The appearance of a vaginal plug or vaginal smear with sperm cells was confirmed as successful mating. We designated that day as GD0. Pregnant rats were then transferred to individual cages. From GD9 to GD20, the pregnant rats were given ethanol (4 g/kg·d, 40%) or distilled water through oral gavage at 8:00 a.m. every day as described in previous studies (26, 35). The animal processing schedule was shown in **Figure 1**.

For fetal rat experiment, a portion of pregnant rats were anesthetized with isoflurane and decapitated, and fetal rats were obtained through cesarean section on GD20. To eliminate the interferences induced by litter size, only pregnant rats with 8∼14 pups were included and each group finally contained 14 pregnant rats. The fetal rats were quickly picked and weighed. Fetal rats were decapitated, and the pancreases were weighed. The fetal blood or pancreases (per litter) were collected and pooled into one independent sample, respectively. Fetal blood was centrifuged at 2263 × g for 15 min, and the serum was stored at −80◦C. Some intact fetal pancreases (including the head, body and tail) were weighed and put into RNAlater (1 mg: 100 µl) overnight at 4◦C, and then transferred to the −80◦C refrigerator for gene expression analysis. Other intact fetal pancreases (head, body, and tail) were selected and fixed per litter (n = 5) for histology observation or fixed for electron microscope observation.

For postnatal rat experiment, the remaining pregnant rats (including the control and PEE groups) were subjected to delivery. The pups remained with their original mothers after birth and throughout lactation. To ensure adequate and relatively equal intake of nutrients during the suckling period, only litters with 8∼14 pups were chosen for the following experiment and normalized to 8 pups per litter (male: female = 1:1) (36). Finally, a total of 16 litters (8 controls & 8 PEEs) were included and all pups were weaned until postnatal week (PW) 4. Four male offspring rats from each litter were selected and assigned to the four studies at different postnatal time points or condition (i.e., PW6, PW12, PW12 with CS and PW24), respectively. The CS test was achieved by forced 5-min ice-water swimming per day (5–7◦C) from PW10 to PW12 (33, 34). Intraperitoneal glucose tolerance tests (IPGTT) and insulin tolerance tests (ITT) were performed at PW12 and PW24. Pups were anesthetized with isoflurane and then decapitated. Blood and pancreases were collected. Intact pancreases (including head, body, and tail) were weighed. Blood was centrifuged at 2263 × g for 15 min, and the serum was stored in an −80◦C refrigerator for gene expression analysis. Some pancreases were weighed and put into RNAlater (1 mg: 100 µl) overnight at 4◦C, and then they were restored at −80◦C. Other pancreases (n = 5) were fixed in a 10% neutral formalin solution for histology observation.

#### IPGTT, ITT, and Area Under the Curve (AUC)

Based on the reference (37), rats were pre-fasted for 12 h before the IPGTTs, which started at 8:00 a.m. Each rat was intraperitoneally administered glucose (2 g/kg). The interval time of administration to each animal was 2 min, and the total operating time was limited to within 30 min. Blood glucose levels which measured 0, 15, 30, 60, and 120 min after the glucose challenge were determined using a glucometer (ACCU-CHEK Performa, Roche). The AUCs were calculated using the trapezoidal rule (38). ITTs were administered after a 5 h fast (8:00 a.m.−1:00 p.m.) (39), and insulin (1 unit/kg) was intraperitoneally injected. The measurement of blood glucose levels which measured 0, 15, 30, 60, and 120 min after the insulin challenge and calculation of the corresponding AUCs was the same as that described for the IPGTTs. Con. N min represented serum glucose at each time point. Time N min-N min represented the time gap between two time points.

Con. Nmin (%) = Con. Nmin / Con. 0min

AUC = (Con. 0min+Con. 15min)/2×Time 0min-15min+ (Con. 15min+Con. 30min)/2×Time 15min-3min+(Con. 30min+Con. 60min)/2×Time 30min-60min+(Con. 60min+Con. 120min)/2×Time 60min-120min

#### Serum Glucose/Insulin Concentrations and Pancreatic Insulin/Proinsulin Contents Determination

The serum insulin and glucose levels were measured through ELISA and biochemical assay kits, respectively, following the manufacturer's protocol. The fasting glucose (FG), fasting insulin (FI) and insulin resistance index (IRI) was acronymized as b. Pancreatic insulin and proinsulin were extracted with acidic ethanol (1.5% [vol/vol] HCl in 75% [vol/vol] ethanol) and assayed using ELISA kits.

IRI = FG (mmol/L) × FI (mIU/L)/22.5

#### Pancreatic Morphometric Analysis

Pancreases at GD20, PW6, PW12, and PW24 were weighed and fixed in 10% neutral buffered formalin overnight, dehydrated, and embedded in paraffin. Each analytical result of the pathological indices was obtained from a total of five animals per group, with five complete longitudinal sections (5µm) per embedded pancreas (i.e., at their maximum widths) in (at minimum) 200µm intervals. The hematoxylin-eosin (HE) and insulin immunohistochemical staining were subjected to the

GD20, PW6, PW12, PW12 with chronic stress, and PW24.

*ISL1, insulin gene enhancer protein isl1; INS1, insulin 1; INS2, insulin2; IGF1, insulin-like growth factor 1; GR, glucocorticoid receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.*

pancreases of GD20 to PW24. As a negative control, pancreatic sections underwent similar treatment, and no positive reactions to the antibody against insulin were observed (data not shown). Sections were viewed at a magnification of ×40. Approximately 3–4 fields per fetal pancreas section and 30–40 fields per postnatal pancreas section were acquired, and we used NIS-Elements Br 4.20 (Nikon, USA) to analyze the total pancreas areas and β-cell areas (insulin-positive cells). The size of the insulin-positive cell area and the pancreatic tissue area were measured (average value, five sections per animal). The β cell fraction and mass were calculated according to the formula provided below (40). The unit of β cell mass/body weight was µg/g (39). Fetal pancreases were cut and fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 2 h at 4 ◦C and post fixed with 1% osmium tetroxide. We dehydrated these tissues with a graded series of ethanol and then embedded them in Epon 812. Ultrathin sections (∼50 nm) were cut with LKB-V ultramicrotome (Bromma, Sweden), dual stained with uranyl acetate and lead citrate and observed with a Hitachi H600 transmission electron microscope (EM) (Hitachi, Tokyo, Japan).

β cell fraction (%) = β cell area/total pancreas area × 100 β cell mass (mg) = β cell fraction × pancreas weight.

#### Pancreatic mRNA and Protein Expression Analysis

GD20 and PW6 pancreases (including the head, body, and tail) were grinded and mixed in liquid nitrogen. We used 25 mg of pancreatic tissue to extract total RNA, based on the manufacturer's protocol (RNA-Solv Reagent). The concentrations and purity of total RNA were measured in A260nm and A280nm, and the rates of A260nm /A280nm were kept between 1.8 and 2.0. The total RNA concentrations were adjusted to 1 µg/µL. cDNA synthesis and RT-qPCR amplification were performed using the manufacturer's protocol. Relative standard curves were constructed for the following target genes: GR, IGF1, ISL1, insulin 1 (INS1), and INS2. The oligonucleotide primers and annealing temperatures in RT-qPCR are listed in **Table 1**. To precisely quantify the gene transcripts, the mRNA level of the housekeeping gene glyceraldehyde phosphate dehydrogenase (GAPDH) was measured and used as the quantitative control (26, 35).

The relative protein expressions of GR, IGF1, ISL1, and insulin in PW12 and PW24 were semi-quantified by immunohistochemistry (IHC) and the area of these protein expression in the pancreatic tissues also could be observed. PW12 and PW24 pancreases were embedded and sliced as

immunohistochemical staining (×40 and ×400) were shown in Supplementary Figures 1A–D.

mentioned above. As negative controls, pancreatic sections underwent similar treatment, and no positive reactions to the antibody against GR, IGF1, ISL1, and insulin were observed (data not shown). The mean density for pancreases in the same group was taken as the expression level for the protein of interest. Sections were viewed at a magnification of ×400. Approximately 200 fields per postnatal pancreas section were acquired, and we used NIS-Elements Br 4.20 (Nikon, USA) to analyze the mean density of GR, IGF1, ISL1, and insulin staining.

#### Statistical Analysis

All data presented were expressed as the mean ± S.E.M., and the statistical analysis was performed with SPSS for Windows version 19 (SPSS Science Inc., Chicago, IL, USA). The normality and homogeneity of the variances were analyzed using the onesample Kolmogorov–Smirnov test and Levene's test, respectively. Differences between the control and PEE groups were compared using a repeated measures ANOVA (IPGTT and ITT) or two-tail Student's test. Groups which were assumed to have normal distribution, and equal variances were compared through repeated measures ANOVA and Student's two-tailed t-test. When data failed tests for normal distribution and homogeneity of variance, we chose the Mann–Whitney U test (equivalent to Wilcoxon W). Statistical significance was designated at P < 0.05 and P < 0.01.

# RESULTS

#### In Male Fetal Rats

#### Islet Function and Pancreatic Morphological Changes

In our previous study, a decrease bodyweight and a high IUGR rate were observed in PEE male fetal rats (26). Based on this batch of animals, we further observed changes in islet function and pancreatic morphology of fetal rats. Compared with the controls, serum glucose and insulin levels of the PEE fetal rats were decreased (P < 0.01, **Figures 2A,B**), while serum proinsulin level was unchanged (**Figure 2C**), as a result, the ratio of serum proinsulin and insulin presented increasing trend in the PEE offspring (P = 0.06, **Figure 2D**). Pancreas weight (P < 0.01, **Figure 2E**), β cell mass (P < 0.05, **Figure 2G**), and β cells mass/body weight (P = 0.06, **Figure 2H**) were all decreased or showed a decreasing trend in the PEE fetal rats, whereas the β cell fraction (**Figure 2F**) did not change. The typical β cell ultrastructure could be observed in the fetal pancreases from the control group (**Supplementary Figures 1E,F**): the mature dense-core β-granules were highly populated, and there were halo spaces between the core and β-granule membrane. However, there was lower electron density and a lack of the characteristic halos of mature β-granules in the PEE group. The above data suggest that islet function was reduced, and pancreatic morphology was impaired in the PEE male fetal rats.

#### Pancreatic Insulin Biosynthesis, GR and IGF1 Expression Changes

The results indicated that the contents of pancreatic insulin and proinsulin in the PEE group were lower than those in the controls (P < 0.05, P < 0.01, **Figures 3A,B**), whereas the ratio of proinsulin/insulin was unchanged (**Figure 3C**). Furthermore, compared with the controls, the expression levels of ISL1, INS1, and INS2 mRNA were decreased in the PEE fetal pancreases (P < 0.01, **Figure 3D**). Meanwhile, the GR mRNA expression level was increased, while the expression level of the IGF1 gene was decreased (P < 0.01, **Figure 3E**). These results suggest that pancreatic insulin biosynthesis was inhibited and that this inhibition was accompanied by increased Gr expression and decreased IGF1 expression.

# In Male Offspring Rats at Different Time Points After Birth

#### Glucose Metabolic Phenotype Changes at Different Time Points

At PW6, serum glucose, insulin levels and IRI were increased in the PEE group compared with the controls (P < 0.05, P < 0.01, **Figures 4A–C**). However, at PW12, the basal level of serum glucose was significantly decreased (P < 0.01, **Figure 4D**), while the serum insulin concentration and IRI did not change in the PEE group (**Figures 4E,F**). In the IPGTT of the PEE group, when compared with the controls, the serum glucose concentration at 30 min was lower in the PEE male pups (P < 0.05, **Figure 4G**), while the corresponding AUC was not obviously changed. In the ITT of the PEE group, the 15 min serum glucose concentration was higher than that of the control (P < 0.01), whereas the corresponding AUC in the PEE group was 1.13-fold higher than that of the controls (P = 0.08, **Figure 4H**). These results suggest that the glucose tolerance was increased, while the insulin sensitivity was decreased in the PEE male pups of PW12. At PW24, the basal serum glucose and insulin levels, as well as the IRI of the PEE male rats, were not altered (**Figures 4I–K**). However, the glucose tolerance of the PEE offspring was remarkably weakened compared with the control, as evidenced by the conspicuous increase in the concentration of blood glucose at 30 min and the increased corresponding AUC after adding the glycemic load (P < 0.01, **Figure 4L**). Furthermore, in the ITT, the serum glucose levels of the PEE males at 30 and 120 min, as well as the corresponding AUC, were decreased (P < 0.01, P < 0.05, **Figure 4M**). These data suggest that a weakened glucose intolerance and enhanced insulin sensitivity existed, although there was no significant change in the glucose metabolic phenotype in the PEE male offspring rats for PW24.

#### Pancreatic Morphological Changes at Different Time Points

At PW6, compared with the controls, the pancreas weight (P < 0.01, **Figure 5A**), β cell fraction (P = 0.088, **Figure 5B**), β cell mass (P < 0.05, **Figure 5C**), and β cell mass/body weight rate (P < 0.05, **Figure 5D**) were all decreased or showed a decreasing trend in the PEE offspring. However, at PW12, the β cell fraction (**Figure 5F**), β cell mass and β cell mass/body weight rate (**Figures 5G,H**) showed no obvious changes, except for the lower pancreas weight (P < 0.01, **Figure 5E**). At PW24, the pancreas weight (P < 0.05, **Figure 5I**) was still decreased, while the β cell mass (P = 0.055, **Figure 5K**) and β cell mass/body weight rate (P = 0.058, **Figure 5L**) only showed a decreasing tendency in the PEE group, and the β cell fraction did not show any obvious changes (**Figure 5J**). The above data suggest that β cells demonstrate a "catch-up" growth pattern in the PEE male offspring.

#### Pancreatic Insulin Biosynthesis, GR and IGF1 Expression Changes at Different Time Points

At PW6, when compared with the respective controls, the GR mRNA expression was unchanged while the IGF1 mRNA expression was increased (P<0.05, **Figure 6A**), meanwhile, the expression levels of ISL1, INS1 and INS2 mRNA were significantly increased (P < 0.01, **Figure 6B**). At PW12 (**Figures 6C,D**), the GR protein expression level was decreased, however, the IGF1 protein expression level was increased in the PEE offspring rats (P < 0.01, P <0.05), and the

FIGURE 4 | Effects of prenatal ethanol exposure (PEE) on the glucose metabolism phenotype at postnatal week (PW) 6, PW12, and PW24 male offspring rats. (A–C) Serum glucose and insulin levels, as well as insulin resistance index (IRI) at PW6, (D–F) serum glucose and insulin levels, as well as IRI at PW12, (G) blood glucose concentrations during intraperitoneal glucose tolerance test (IPGTT) and area under the curve (AUC) at PW12, (H) blood glucose concentrations during insulin tolerance test (ITT) and the AUC at PW12, (I–K) serum glucose and insulin levels as well as IRI at PW24, (L) blood glucose concentrations during IPGTT and AUC at PW12, and (M) blood glucose concentrations during ITT and AUC at PW24. Mean ± S.E.M., *n* = 8. \**P* < 0.05, \*\**P* < 0.01 vs. control.

expression levels of ISL1 and insulin protein were increased (P < 0.01, P < 0.05). At PW24 (**Figures 6E,F**), the GR protein expression level was decreased, while that of the IGF1 protein was still increased (P < 0.05, P < 0.05), however, the protein expression levels of ISL1 and insulin were not significantly changed. These results suggest that the insulin synthesis was enhanced after birth in the PEE male offspring and that this enhancement was accompanied by the downregulated expression of GR and the up-regulated expression of IGF1 and ISL1. Finally, the expression levels of ISL1 and insulin decreased to the control levels during the late stage of adulthood.

#### Glucose Metabolic Phenotype, Pancreatic GR/IGF1 Expression, and Insulin Biosynthesis Changes at PW12 After a Chronic Stress Test

Furthermore, we aimed to confirm the involvement of the GC-IGF1 axis in pancreatic development and insulin synthesis in the PEE offspring. The results showed that, in the PEE group subjected to the CS, the serum glucose level was higher, while the serum insulin level was lower (P < 0.01, P < 0.05, **Figures 7A,B**), and the IRI was unchanged (**Figure 7C**). Meanwhile, the protein expression level of GR was higher, and those of IGF1, ISL1 and insulin were all decreased, compared with the controls (P < 0.05, P < 0.01, **Figures 7D,E**).

# DISCUSSIONS

# Age-Characteristic Changes of Glucose Metabolic Abnormalities in Male PEE Offspring Rats