Regulation of AKT activity prevents autonomic nervous system imbalance

Tsubasa Furuhashi and Kazuichi Sakamoto


Autonomic nervous system (ANS) imbalances are involved in the etiology of cancer, allergy, and collagen diseases. Previously, we hypothesized that FoxO and HSF-1 limit autonomic stress responses via negative feedback on the ANS. Here, we evaluated the role of AKT, a negative regulator of FoxO, during activation of the ANS by loneliness stress in mice. Spontaneous motility was increased during loneliness stress and decreased after release from stress. The AKT activator SC79 attenuated stress-induced spontaneous motility, whereas the AKT inhibitor API-2 prevented decreases in motility after stress release. Our results show that AKT activity regulates ANS responses to loneliness stress.

Keywords: AKT; Autonomic nervous system; FoxO; loneliness stress; spontaneous motility.

1. Introduction

The autonomic nervous system (ANS), which consists of the sympathetic nervous system (SNS) and parasympathetic nervous system (PNS), responds to many kinds of stressors and maintains physiological homeostasis [1]. However, chronic or excessive activation of the ANS can have adverse effects on health, and has been related to the etiologies of some cancers, allergy, and collagen diseases [2]. To investigate possible treatments for ANS imbalances, we have previously considered controlling the activity of genes that are regulated by the ANS during the stress response as well as longevity genes such as FoxO and HSF-1.
In previous work, we demonstrated that activation of FoxO/DAF-16 or HSF-1 alleviates or prevents heat stress damage in C. elegans, respectively [3, 4]. In addition, we found that neurotransmitters including octopamine and acetylcholine promote thermotolerance via DAF-16 and increase the mRNA expression of hsp-16.2 and hsp-70 [5], which are positively regulated by HSF-1 [6, 7]. These results suggest that the activities of FoxO/DAF-16 and HSF-1 are regulated by the central nervous system, and specifically that alterations in DAF-16 and HSF-1 provide negative feedback on the ANS. Based on these findings, we hypothesized FoxO and HSF-1 limit autonomic stress responses via negative feedback phenomena.
Previously, it was reported that AKT negatively regulates the activity of FoxO [8]. Moreover, HSF-1 is negatively regulated by insulin-like signaling in C. elegans [6, 7]. The availability of pharmacological inhibitors and activators of AKT have permitted several in vivo experiments. API-2 is known as AKT specific inhibitor. API-2 inhibits phosphorylation of AKT and its kinetic activity [9]. Furthermore, API-2 has no effects on phosphorylation of activity of PI3K, PDK and ACG kinase family [9]. API-2 showed anti-tumor effects in mice [9, 10]. SC79 is suggested as AKT activator. SC79 specifically binds to PH domain of AKT and promotes its phosphorylation at both the Thr308 and S473 sites, required for membrane translocation of AKT [11]. Furthermore, SC79 was demonstrated to enhance the phosphorylation of both AKT and FoxO in vivo [11]. Therefore, we studied AKT as regulator of FoxO and HSF-1 and analyzed its function in the autonomic stress response in mice.
We used spontaneous motility as an index for ANS activation. In a previous study, knockout of the β-adrenergic receptor decreased spontaneous motility in mice [12], suggesting that spontaneous motility is regulated by the ANS. In addition, we used loneliness stress to study ANS imbalances because loneliness a known mental stressor that activates the SNS [13] and can cause lasting disease such depression in mice [14]. In this study, we analyzed the function of AKT in autonomic responses to loneliness stress in mice by analyzing alterations in spontaneous motility. Our data suggest that regulating AKT activity can prevent imbalances of ANS activation.

2. Materials and methods

2.1. Strain and culture

The care and use of animals in this study was in accordance with the animal care guidelines of the University of Tsukuba. C57BL/6Jjcl male mice (6–20 weeks of age) (CLEA Japan, Tokyo, Japan) were housed in groups of 3 per cage on a 12-h light/dark cycle in a temperature-controlled (22–25 °C) environment with ad libitum access to CE-2 food (CLEA Japan) and tap water. To reduce the number of animals used in our study, mice were reused 1–3 times at an interval of 2–5 weeks between each experiment.

2.2. Spontaneous motility assay under loneliness stress

For the loneliness stress condition, mice were transferred to new cages 0–2 h prior to the dark period and housed separately. Spontaneous motility was recorded using the Locomo system (LC-8; MELQUEST, Toyama, Japan) every 30 min after the beginning of the dark period.

2.3. Spontaneous motility assay after loneliness stress

Mice were transferred to new cages 0–2 h before the dark period and housed separately for 72 h. After the loneliness stress period, mice were collected in one cage and spontaneous motility was recorded using the Locomo system (LC-8; MELQUEST, Toyama, Japan) every 30 min after the beginning of the dark period.

2.4. Drug treatment

Labetalol hydrochloride (Sigma Aldrich, Missouri, USA), an α1 and β1/2 adrenergic receptor antagonist, was mixed into drinking water during spontaneous motility measurements as previously described [15]. Triciribine hydrate (API-2; Sigma Aldrich) was dissolved in DMSO to a concentration of 5 mg/ml. SC79 (Merck Millipore, Darmstadt, Germany) was dissolved in DMSO to a concentration of 40–50 mg/ml. These stock solutions were then diluted in PBS to yield a final vehicle concentration of 5% DMSO. Mice were treated with 5% DMSO vehicle control, 1 mg/kg API-2 [9, 10], or 8–10 mg/kg SC79 by i.p. injection in a volume of 100 µL [11].

2.5. Statistical analysis

Significant differences were analyzed using Student’s t-tests or Tukey’s test.

3. Results

3.1. Loneliness stress increases spontaneous motility via SNS activation

The spontaneous motility of mice exposed to loneliness stress for 72 h was significantly greater than that of grouped mice (Fig. 1A). To indirectly study the role of the SNS in loneliness stress-induced spontaneous motility, we measured spontaneous motility in mice treated with labetalol, an α1 and β1/2 adrenergic receptor antagonist, or vehicle control. Stress-induced spontaneous motility in labetalol-treated mice was lower than that in vehicle-treated mice (Fig. 1B). These results suggested that activation of the SNS was responsible for loneliness stress-induced increases in spontaneous motility.

3.2. AKT activation prevents excess SNS activation caused by loneliness stress

Next, we analyzed the role of AKT in loneliness stress. Mice were treated with the AKT inhibitor API-2 (1 mg/kg, i.p.) and spontaneous motility was measured during loneliness stress. API-2 remarkably increased spontaneous motility of grouped mice (Fig. 2A). And, API-2 injection slightly increased spontaneous condition under loneliness stress during 48 to 60 h (Fig. 2A). We then treated mice with the AKT activator SC79 (8– 10 mg/kg, i.p.) and measured spontaneous motility during loneliness stress. Although no differences in spontaneous motility were observed between groups during the first dark (active) period, SC79 attenuated loneliness stress-induced spontaneous motility during the second dark period (Fig. 2B). Therefore, it was hypothesized that AKT activation attenuated the response of the SNS to loneliness stress.

3.3. Release from loneliness stress decreases spontaneous motility through a negative feedback mechanism

Because imbalances in the ANS are maintained by negative feedback phenomena, we hypothesized that release from loneliness stress would also alter spontaneous motility. After 72 h of exposure to loneliness stress, mice were collected in one cage (group-housing) and spontaneous motility was measured. Mice exposed to loneliness stress showed lower spontaneous motility than control mice (Fig. 3). Based on this result, we postulated that release from stress enhances negative feedback on the SNS, possibly through activation of the PNS.

3.4. AKT inhibition prevents negative feedback on the ANS after loneliness stress

Based on the observation that API-2 increased spontaneous motility in control mice, we evaluated the effect of API-2 on mice after exposure to loneliness stress. Mice were more active during exposure to loneliness stress than during group housing in our manipulation. The spontaneous motility of mice released from loneliness stress was decreased in the vehicle group but not in the API-2 group (Fig. 4). Therefore, it was concluded that inhibition of AKT may prevent PNS activation induced by release from loneliness stress.

4. Discussion

In the present study, we found that loneliness stress increased spontaneous motility through activation of the SNS. Furthermore, release from loneliness stress decreased spontaneous motility, suggesting that release from stress activated the PNS to provide negative feedback on the SNS. Our findings indicated that AKT activation limited the sympathetic response to loneliness stress, whereas AKT inhibition prevented negative feedback on the ANS after release from loneliness stress. Therefore, it is expected that control of AKT activity may prevent imbalances of ANS activation due to physiological or environmental stress. Our study is the first research to address control of the ANS by the pharmacological manipulation of negative feedback phenomena. Generally, it is known that ANS imbalances can be caused by the change of seasons and release from busy period. Our findings indicate that activation of AKT immediately prior to stress or inactivation after release from stress can mitigate ANS imbalances.
API-2 increased spontaneous motility of mice under both conditions of standard and loneliness (Fig. 2A). These results suggested that API-2 enhances spontaneous motility via different pathway of loneliness stress, which activates SNS (Fig. 1B). Previously, it has been reported that adrenergic pathway regulates AKT activity. In fact, treatment of isoproterenol, an agonist of β-adrenoreceptor, phosphorylates AKT in cardiomyocytes, eosinophils and area CA1 of hippocampus [16-18]. Because loneliness stress increased spontaneous motility via SNS activation (Fig. 1B), it is expected that AKT is activated under loneliness stress, suggesting that SNS is activated by loneliness stress to activate AKT pathway. This suggestion expects that AKT inactivation by API-2 injection has a potential to activate SNS and inactivate PNS as negative feedback phenomena. Therefore, probably API-2 injection increases spontaneous motility via PNS activation.
Based on our previous work, we hypothesize that the activity of FoxO and HSF serve to activate negative feedback on the ANS. Moreover, it was recently found that acetylcholine treatment decreases phosphorylated FoxO3a in H9c2 cells [19]. The transcriptional activity of FoxO enhances hibernation [20] and is maintained by the PNS in squirrels [21]. And, FoxO is phosphorylated by isoproterenol in eosinophils [17]. Therefore, one possible mechanism for the observed effect of AKT on ANS responses to loneliness stress is via FoxO. It is expected that drug discovery of activators and/or inhibitors for not only AKT but also FoxO will demonstrate utility in conditions of ANS imbalance after stress exposure.
Our study has possible applications in the field of therapeutic cancer research. API-2 has previously demonstrated anti-tumor effects in mice [9, 10]. In addition, a previous study indicated that an enriched environment suppresses tumor growth in mice through SNS activation [22], such that the effect of AKT activation on tumor growth may be mimicked by SNS activation. Furthermore, AKT activity may be useful for the treatment and management of allergies: oral administration of LY294002, an inhibitor of PI3K, suppresses allergy responses to the ovalbumin (OVA) challenge [23]. In this context, it is possible that AKT activity alters the allergy response through modulation of the ANS. However, the translatability of these studies is limited by the use controlled experimental conditions; in human society, individuals are constantly exposed to a variety of psychological, psychological, and environmental stressors. Therefore, a better understanding of the role of AKT modulation on cancer or allergy responses in more relevant contexts (e.g., under loneliness stress) is required.


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