Oral Supplementation of L-Carnosine Attenuates Acute Stress-Induced Corticosterone Release and Mitigates Anxiety in CD157 Knockout Mice

1. Introduction

Organisms adapt to stress by stimulating the hypothalamic–pituitary–adrenal (HPA) axis to cope with environmental changes. The adrenal glands secrete corticosterone, the primary glucocorticoid in humans, which increases pulse and blood pressure, raises blood sugar levels, and suppresses excessive immune responses. However, excessive corticosterone levels weaken the prefrontal cortex and cause neuronal death in the hippocampus, leading to anxiety, depression, and other neurological and psychiatric disorders. Therefore, attempts have been made to reduce surplus corticosterone levels [1].

Several nutrients, probiotics, and prebiotics are potential therapeutic agents for mitigating excessive stress responses [2]. L-carnosine is an imidazole dipeptide composed of beta-alanine and L-histidine, and its concentration is very high in the skeletal muscle and brain of mammals. It can cross the blood–brain barrier or can be synthesized from beta-alanine and histidine in the brain. In practice, L-carnosine has been marketed as a nutritional supplement for its anti-aging and fatigue-relieving properties and ameliorating effects on lifestyle-related diseases (e.g., diabetes, hypertension, and atherosclerosis) through its antioxidant and pH-buffering properties [3,4]. In the brain, it has beneficial effects on various neuropsychiatric disorders such as ischemic stroke, cognitive impairment [5,6], autism spectrum syndrome [7], schizophrenia [8,9], Alzheimer’s disease and dementia [10,11], attention deficit hyperactivity disorder [12], and Gulf War syndrome [13]. L-carnosine is involved in stress-induced corticosterone responses and anxiety behaviors in rodents. L-carnosine-administered mice subjected to restraint stress showed suppressed elevation of plasma corticosterone levels compared to that in the control Kunming mice [14,15]. Administration of L-carnosine to rats induces anxiolytic-like behavior in a dose-dependent manner.

Bone marrow stromal cell antigen (BST-1), also known as CD157, was first cloned as a glycosyl phosphatidylinositol-anchored protein involved in the growth of pre-B cells [16]. CD157/BST-1 is a glycosyl phosphatidylinositol-anchored membrane protein that functions as an ADP ribosyl cyclase, and the loss of CD157 expression in mice results in anxiety-like behaviors and social behavioral deficits. It is a paralog of CD38 that catalyzes cyclic ADP-ribose to regulate intracellular Ca2+ [17]. CD157/BST-1 is constitutively expressed in the myeloid cells of peripheral blood mononuclear cells and regulates humoral immune response [18]. Moreover, CD157/BST-1 is associated with neuropsychiatric disorders, such as Parkinson’s disease, autism spectrum disorder (ASD), rapid eye movement sleep behavior disorder, major depressive disorder, restless leg syndrome/Willis–Ekbom disease, and Alzheimer’s disease [19]. Although the physiological role of CD157 in the brain remains largely unexplored, an association between CD157/BST1 and ASD has been reported [20,21]. Homozygous CD157 knockout (CD157 KO) mice display social behavioral impairments and anxiety-related and depression-like behaviors, which can be restored by treatment with antidepressants or oxytocin [22,23,24,25]. These findings suggest that CD157 KO mice may be useful as a model of ASD with regard to modeling the behaviors associated with ASD symptoms. We have previously observed that chronic administration of L-carnosine ameliorates social behavioral deficits, which is a core symptom of ASD, in CD157 KO mice [26]. However, the effects of L-carnosine on other comorbid symptoms of ASD, such as anxiety-related behaviors and altered stress responses, have not yet been investigated in CD157 KO mice. In this study, we examined the effect of chronically administered L-carnosine on corticosterone response induced by acute stress and anxiety-like behavior in CD157 KO mice.

2. Materials and Methods

2.1. Animals

C57BL6/N wild-type (WT) mice were obtained from Japan SLC Inc. (Hamamatsu, Japan) via the Sankyo Laboratory Service Corporation (Toyama, Japan). CD157 KO mice were developed as previously described [18]. Homozygous CD157 KO mice were used in this study. WT and CD157 KO mice were housed at the Institute for Experimental Animals, Advanced Science Research Center, Kanazawa University, under standard conditions (22 °C; 12-h light/dark cycle, lights on at 8:45 a.m.) in standard mouse cages (300 × 160 × 110 mm) with sawdust bedding and access to food and water ad libitum. Mice weaned at 21–28 days of age were housed in same-sex groups of three to five animals until 11 weeks of age. Male mice were single-housed for 14 days before acute stress, and behavioral tests were conducted. Carnosine-treated mice were maintained on a steady dose of L-carnosine (Phytopharma Co., Ltd., Kanagawa, Japan) diluted in drinking water (0.09 g/100 mL) from weaning up till the behavioral test. Water intake and body weight were measured during the experiment. This study was conducted in accordance with the Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology of Japan. The protocol was approved by the Committee on Animal Experimentation of Kanazawa University (AP-143261). We divided into three groups, wild-type (WT) as control, CD157 KO mice with or without L- carnosine administration (KO-Car or KO-Water).

2.2. Acute Stress

Forced swimming was performed as previously described [22]. Briefly, Mice were placed individually in a cylinder (height 25 cm, diameter 15 cm) filled up to a 10-cm depth with water (25 ± 1 °C) for 6 min. A restraint stress study was performed as mice were placed in a 50-mL polypropylene conical tube (Eppendorf, Hamburg, Germany) with air holes for 10 min.

2.3. Plasma Sampling and Enzymatic Detection of Corticosterone

Male mice were anesthetized immediately after acute stress by an intraperitoneal injection of pentobarbital (35 mg/kg). Blood samples (0.8–1 mL) were collected by cardiac puncture, and 8–10 μL of 0.1 g/mL ethylenediaminetetraacetic acid was added. The samples were centrifuged at 1,600 ×g for 15 min at 4 °C. Plasma samples (200–400 μL/mouse) were collected and stored at -80 °C until use.

2.4. Enzyme Immunoassay of Corticosterone

Immunoreactivity of plasma corticosterone was analyzed using a corticosterone EIA kit (Enzo Life Sciences, NY, USA), following the manufacturer’s instructions. The plasma samples (5 μL) were thawed and diluted to 1:80 in assay buffer. Fifty microliters of the sample were used for the assay. Blood samples were assayed without protein extraction as previously described [25]. The assay had two linear ranges, covering a concentration range of 30–1000 pg/mL. The inter- and intra-assay coefficients of variation were < 5%.

2.5. Elevated Plus Maze

The mice in the homecage was placed in the experiment room for at least one hour for the habituation. Duration of elevated plus maze is five minutes. Behavior was measured using digital video system and ANY-maze software (Sloelting Co, Wood Dale, IL, USA).

2.6. Statistical Analysis

Statistical analysis was performed using Prism v.8 (GraphPad Software Inc., San Diego, CA, USA). The data are presented as mean ± standard error. The induction of corticosterone in the blood after the stress tests were compared with the baseline blood corticosterone levels in each group using an unpaired t-test. One-way analysis of variance (ANOVA) was used to assess the differences in blood corticosterone levels among groups. Subsequently, Tukey’s post hoc multiple comparison test was performed. We did not observe any effect of L-carnosine on blood corticosterone levels in WT mice; therefore, no further behavioral examination of WT mice was conducted. For the behavioral examination, an unpaired t-test was conducted to compare each index between the KO-Water, KO-Car, and WT-Water groups.