Insulin level regulators may affect cognitive ability caused by motion sickness: an experimental study

doi:10.21203/rs.3.rs-4469299/v1

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Insulin level regulators may affect cognitive ability caused by motion sickness: an experimental study

https://doi.org/10.21203/rs.3.rs-4469299/v1

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Abnormal acceleration induced motion sickness (MS) and elevated blood glucose levels, showing obviously cognitive impairments. The mechanism of cognitive impairment caused by MS is still unclear. Here, blood metabolite detection, insulin level regulators, stress hormones, cytokines and MS assessment were conducted for the population and MS model rats, correlation analysis of motion sickness index (MSI) and above factors were conducted by Pearson correlation analysis. We found glucose after acceleration was positively correlated with Graybiel’s score. Insulin and leptin levels decreased, while ghrelin level increased after acceleration in both human and rat groups. We injected insulin level regulators into rats before being exposed to acceleration, the results showed that MSI of the insulin group (INS) was significantly lower than rotation group (ROT), streptozotocin group (STZ) and streptozotocin & insulin group (SINS). MSI in STZ was higher than ROT and INS. Rats injected with ghrelin showed higher MSI than the control group and (D-LYS3)-GHRP-6 (ghrelin antagonist) group. Acceleration stimulation induced phosphorylation of insulin receptor substrate 1 (IRS1) and expression of synaptic protein in hippocampus. We also found that the insulin microinjection into hippocampus prevented MS symptoms and cognitive ability as measured by the MSI, the total distance of the Open Field Test and correct choice of T-maze. Our study indicates that insulin and insulin level regulators can affect MS symptoms and cognitive ability.

motion sickness

cognitive impairment

insulin

metabolite

Abnormal acceleration induces Motion sickness (MS), which is characterized by autonomic nervous system symptoms, including pallor, restlessness, cold sweat, nausea, and vomiting (Zheng et al. 2014). MS occurs in a variety of conditions such as in cars, ships, airplanes, or spacecraft, and may cause inconvenience and distress (Lackner and Dizio 2006). MS poses a great challenge to the operation safety which causes spatial orientation obstacle in flight, affect navigation operation in navigation, and even threaten the safety of diving operations. Due to the development of automated vehicles and virtual reality, MS is also receiving more and more attention (Keshavarz B 2022). Many theories (e.g., the sensory conflict and neural mismatch theory, the toxin detector hypothesis, the neurotransmitter hypothesis, and the referred visceral discomfort hypothesis) have been proposed to explain the development of MS (Lackner and Graybiel 1983; Reason 1978; Wood and Graybiel 1970; Balaban 1999). Researchers have also suggested that unpredictable motion and vestibular morphological ssymmetry are also causes of MS (Kuiper et al. 2020; Harada et al. 2021). However, the etiology of MS has not been clearly studied.

Metabonomics has been widely used to measure organismal metabolic responses to stimuli. Researchers have also found that stress hormones and arginine vasopressin (AVP) may be related to MS (Li et al. 2005). Blood insulin, and cortisol concentrations markedly changed after acceleration (Dong et al. 2011). After repeated-acceleration stimuli, males and females showed different IL-6 production (Farrar et al. 1989). Therefore, stress hormones and cytokines as well as insulin level regulators may play important roles in MS. To verify that blood insulin level regulators play a regulatory role in cognitive impairment caused by MS, we detected metabolites and evaluated MS symptoms in MS population and rats, then conducted molecular and behavioral experiments on rats.

2.1. Animals and Human Subjects

A total of 50 healthy males were recruited from the Naval Medical University. All volunteers read and signed informed consent forms. This protocol was approved by Committee on Ethics of Biomedicine, Naval Medical University (Reference no: 2009LL010).

A total of 201 Male Sprague-Dawley (SD) rats weighing 250–300 g were obtained from Sino-British SIPPR/BK Lab Animal Ltd (Shanghai, China). The protocol was approved by the Committee on Ethics of Biomedicine, Naval Medical University (Shanghai, China). (Reference no: 2009LL010). All animal experiments took place at Naval Medical University (Shanghai, China). Animals were housed at an ambient temperature of 22 ± 2°C and relative humidity of 50–70%, maintained under a normal 12-hour light/dark cycle, and allowed access to food and water ad libitum. Rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.). After the rats were anesthetized, washed brain blood-free via aortic perfusion with PBS, and killed by cervical dislocation.

2.2. Human Acceleration stimulation

A 6-degree-of-freedom ship motion simulator (SMS) was adopted to induce MS in humans. The acceleration of the SMS was 0.27 g with a sine function frequency of 0.26 Hz. Graybiel’s score was used to measure human MS severity.

All participants underwent acceleration for 15 min in SMS. After acceleration, the subjects were scored according to Graybiel’s score of MS. A score of 0 points indicated no MS, a score of 1 to 4 points indicated slight MS, 5 to 7 points indicated moderate MS, 8 to 15 points indicated severe MS and 16 points or more indicated serious MS. Graybiel’s measurement of MS is based on MS symptoms and signs such as nausea, skin color, cold sweats, drooling, drowsiness, pains, and central nervous system symptoms. The above symptoms were scored with values of 0, 1, 2, 4, 8, or 16 according to their severity. All scores add up to generate a total score.

Human blood samples were obtained before and after exposure to acceleration. Serum samples were used to detect metabolites, hormones (epinephrine, glucocorticoid, insulin, glucagon, and AVP), blood glucose-regulating factors, and cytokines.

2.3. Animal acceleration simulation

Model of rats MS was simulated using an acceleration simulation device. The acceleration device consisted of an electric motor, hob, and plastic box. SD rats were placed in individual plastic boxes. The device was rotated in a clockwise direction at a constant angular acceleration of 16 °/s² until the angular velocity reached 120 °/s² then decelerated to 48 °/ s². After a 1 s pause, this procedure was repeated in a counter-clockwise direction which lasted for 2 h. After rotation MS symptoms were recorded to calculate the motion sickness index (MSI) (Yu et al. 2007). The dejection amounts, urination, tremor, and piloerection of rats were observed immediately after acceleration stimulation. Each fecal particle counts 1 point. Urination and tremor each count 1.2 points. Slight or severe piloerection count 0.6 or 1.2 points. The sum of all scores is MSI.

2.4. Behavioral Testing

Open Field Test (OFT): The OFT is widely used to assess the anxiety in rodents. During OFT, an animal behavior test system (RD1112-IFO-R-4, Mobiledatum, Shanghai, China) was used with a dark cuboid chamber (length: 40cm, width: 40cm, height: 90cm). Each rat was gently placed in the corner of the chamber. After one minute of adaptation, all spontaneous activities were recorded for 5 minutes using a video computer tracking system. The chambers were cleaned between experiments for each rat. The total distance traveled (body center-point) were measured with commercially available software (EthoVision XT 8.5, Noldus, Netherlands).

T-maze Test: rats were fully touched for 1-2min every day for 5-7d so that they show no stress reaction to the experimenter. After a day of equipment adaptation, rats were subjected to acceleration stimulation. After the stimulation, the rats were put into the trunk arm of the T-maze. The experimental method refers to the study of Yang et al. (2020).

2.5. Sample preparation and spectral acquisition

Gas chromatography coupled to time-of-flight mass spectrometry (GC-TOF/MS) was applied to detect the peak metabolite intensity in blood samples. After the plasma was centrifuged at 3,000 g for 20 min, the supernatant was collected. Internal standards including 10 µL of L-2-chlorophenylalanine (0.3 mg/mL) in water and 10 µL of heptadecanoic acid in methanol (1 mg/mL) were introduced to each 100 µL serum sample. The serum samples were shaken, and 300 µL of a methanol and ethyl chloroform mixture (methanol: ethyl chloroform, 1:3 v/v) was added to precipitate the protein. After shaking and storage at -20°C for 10 min, all samples were centrifuged at 10,000 g for 10 min. 300 µL of supernatant was extracted and evaporated. A total of 80 µL of methoxyamine (15 mg/mL in pyridine) was added to the residue and the solution was stored at 37°C for 90 min. A total of 80 µL of BSTFA (1% TMCS) was added to the solution which was maintained at 70°C for 60 min for analysis. 1 µL of the sample was injected into an Agilent 6890N gas chromatograph coupled to a Pegasus HT time-of-flight mass spectrometer (GC-TOF/MS) (Leco Corporation, St. Joseph, MI, USA).

2.6. Data reduction and pattern recognition

The raw data were converted to the NETCDF format using Data Bridge (Perkin-Elmer Inc., U.S.A.) and processed using MATLAB (MathWorks, Inc.) to perform the baseline corrections, peak discrimination and alignment, internal standard exclusion, and normalization to the total sum of the chromatogram. Metabolites with variable influence on projection values of greater than 1.0 and P-values of less than 0.05 were deemed statistically significant.

2.7. The measurement of cytokines

The serum TNF-a, IL-1β, IL-4, IL-5, IL-10, interferon IFN-g, and vascular endothelial growth factor (VEGF) were measured using the magnetic bead Bio-Plex Pro™ Human and Rat Cytokines assay test kit (Bio-Rad Laboratories, Shanghai, China). The kit was used according to the manufacturer’s instructions (Bio-Plex 200 system, Bio-Rad).

2.8. Diabetes immunoassays

The serum ghrelin, leptin, and resistin were determined using the magnetic bead-based Bio-Plex Pro™ human and rat diabetes immunoassays test kit (Bio-Rad Laboratories, Shanghai, China). The kit was used according to the manufacturer’s instructions, and the samples were analyzed (Bio-Plex 200 system, Bio-Rad).

2.9. The measurement of stress hormones

The serum insulin, glucagon, and cortisol levels were measured by radioimmunoassay kits for each hormone (Beijing, North Institute of Biological Technology Co). AVP was analyzed using radioimmunoassay kits (Shanghai, Naval Medical University). Epinephrine was analyzed using an immunoenzyme assay (epinephrine ELISA, ZYMO RESEARCH, China). The insulin, glucagon, corticosterone, and epinephrine measurement methods of animals were the same as humans.

2.10. Western blot analysis.

Rats were anesthetized (3% pentobarbital sodium, 40mg/kg, i.p.) and transcardially perused with 50 ml of PBS (0.01 M, pH7.4) before brains collection to ensure that there were no blood contaminants. The hippocampus were instantly collected and snap frozen in liquid nitrogen, then placed in -80℃ for storage until use. Hippocampus samples were homogenized in RIPA lysis buffer (Strong, Beyotime, China) and further centrifuged at 10,000g at 4℃ for 5 min. Equal amounts of protein (30 µg) were loaded onto 6% or 7.5% SDS-PAGE gel, then electrophoresed and transferred to 0.45µm nitrocellulose filter membranes (Merck Millipore, Germany) using eBlot™ L1 Fast Wet Transfer System (GenScript USA Inc.). Then the NC membranes were blocked with QuickBlock Blocking buffer and incubated overnight at 4 ℃ with the primary antibodies: IRS-1(1:1000, Cell Signaling Technology); Phospho-IRS-1 (Ser307) (1:1000, Cell Signaling Technology); GluA1 (AMPA subtype) (1:1000, abcam); GluA1 (AMPA subtype) (phospho S845) (1:1000, abcam); PSD95(1:500, abcam); GAPDH(1:1000, Beyotime);β-Actin(1:1000, Beyotime). On the following day, the membranes were incubated with the appropriate secondary antibody (1:20000, LI-COR, USA) at room temperature for 1.5 h. Immunoblots were then visualized using Odyssey two-color infrared laser imaging system (LI-COR, USA) and quantified with optical methods using the ImageJ software (ImageJ 1.5, NIH, USA). The results were normalized using GAPDH or β-actin as an internal control.

2.11. Surgery

The head of the rat was fixed on the stereotaxic brain localizer after anesthesia (3% pentobarbital sodium, 40mg/kg, i.p.). Two cannulas implanted into the center of the bilateral hippocampus (anteroposterior, AP: 3.8mm. mediolateral, ML: 2.3mm. in relation to bregma. dorsoventral, DV: 3.0mm from skull surface). After the 7-day recovery period, a total of 5mU/50µl insulin or saline per rat was administrated through the cannulas 30 minutes before acceleration.

2.12. Statistics

All data are expressed as mean ± SEM. The Independent Samples T-test was used for two-group comparison. One-way ANOVA, followed by Turkey test as a post hoc, was performed to analyze the difference between the three or more groups. Pearson correlation is used to test the degree of linear relationship between two variables. Calculations were made using GraphPad Prism 9.0.

3.1. Human blood test and Graybiel’s score

33 out of 50 subjects had seasickness symptoms such as nausea, vomiting, pallor, and sweating after being exposed to acceleration. Human blood glucose (p < 0.01, Table 1) and n-dodecanoic (p < 0.05, Table 1) increased after acceleration which is positively correlated with Graybiel’s score. The L-serine and L-threonine decreased after acceleration which were negatively correlated with Graybiel’s score (p < 0.05, Table 1). Insulin decreased while glucocorticoid, epinephrine and AVP increased after acceleration (p < 0.001, Table 2). Leptin and resistin decreased, while ghrelin increased after acceleration (Table 3). VEGF (p < 0.001) and TNFα (p < 0.05) increased after acceleration (Table 4). Other cytokines showed no significant difference after acceleration.

3.2. Animal blood test and MSI

To determine whether rats after rotation exposure were analogous to human, we simulated MS in rats with device acceleration simulation and investigated correlations between the above-mentioned hormones and MSI after acceleration. The results showed that glucagon, glucocorticoids, epinephrine and AVP were not significantly correlated with MSI after acceleration, while insulin was negatively correlated with MSI (p < 0.05, r = 0.328) (Table 5). After acceleration, ghrelin (r = 0.514, p < 0.05) and resistin (r = 0.630, p < 0.01) were positively correlated with the severity of MS (Table 6). There was no significant difference between cytokine and MSI after acceleration (Table 7).

3.3. MSI after drug administration

In order to further explore, we took drug administration to prove the effect of insulin level regulators on MS in rats. Rotation group was exposed to acceleration for 2 hours (ROT), control group (CTRL) was placed in the same stimulation device without acceleration for 2 hours, streptozotocin group (STZ) was intraperitoneal injected of streptozotocin (50 mg/kg) to lower insulin secretion before acceleration, Insulin group (INS) was intraperitoneal injected of insulin (1unit/kg) 30 min prior to acceleration), streptozotocin & insulin group (SINS) was intraperitoneal injected of streptozotocin (50 mg/kg) to lower the insulin secretion, followed by insulin injection (1unit/kg) prior to acceleration, scopolamine group (SCOP) was intraperitoneal injected of scopolamine (3 mg/kg) prior to acceleration. All drugs are injected 30 minutes before acceleration. The rotation method was the same as protocol.

The results showed MSI of ROT was significantly lower than STZ (p < 0.05) but higher than CTRL (p < 0.001), INS (p < 0.001), SINS (p < 0.05) and SCOP (p < 0.001). And MSI of INS was significantly lower than STZ (p < 0.001) and SINS (p < 0.01). There was no significantly different between the MSI of INS and SCOP (Fig. 1A).

In the previous experiment, we observed the effect of insulin on MS through direct exogenous administration and endogenous reduction of insulin, but streptozotocin may have other side effects on rats. In order to further explore mechanisms, we administered ghrelin (insulin endogenous inhibit) and (D-LYS3)-GHRP-6 (ghrelin antagonist) 30 minutes before acceleration respectively. It turned out that rats injected with ghrelin had higher MSI than control group (CTRL) (p < 0.05) and (D-LYS3)-GHRP-6 (p < 0.05) group. But MSI between CTRL and (D-LYS3)-GHRP-6 group showed no statistically different (Fig. 1B).

3.4 Changes of protein expression in the hippocampus after acceleration stimulation

We investigated the effect of motion sickness on insulin signaling by determining the level and activation of insulin receptor substrate 1 (IRS1), which can be activated by insulin receptors and are capable of activating downstream effector molecules, after acceleration stimulation. The activation of IRS1 was assessed by measuring their phosphorylation levels at the activity-dependent sites. We found that insulin signaling was somewhat disturbed in rat after acceleration stimulation, as evidenced by significant reduction the levels of p-IRS1/IRS1 in the exposure group as compared to the control group (p < 0.01, Fig. 2).

Synapses are the structural basis of memory and cognition, and their alterations usually underlie functional changes of the brain. To learn whether acceleration stimulation can alter synaptic activity, we determined the levels of the synaptic marker proteins, including α-Amino-3-hydroxy-5-methyl-4-isoxazoleprotonic acid (AMPA) receptor subunit, glutamate A1 (GluA1) and the postsynaptic marker postsynaptic density 95 (PSD-95) in the rat hippocampus after acceleration stimulation using Western blots. We found that the level of PSD-95 in the rat hippocampus after acceleration stimulation was higher than that in the stationary control group (p < 0.05, Fig. 2).

3.5. Effect of insulin microinjection into hippocampus on MS symptoms and cognitive ability

We found that both endogenous and exogenous insulin changes will affect the symptoms of MS. To determine the central effect and mechanism of insulin on MS, we gave insulin microinject into hippocampus of MS rats to observe cognitive ability and related signal molecules. It was found that acceleration stimulation could increase MSI, decrease spatial memory and physical strength. In OFT, total distance of exposure group was significantly lower than control group (p < 0.001) and exposure ± insulin group (p < 0.01, microinjection of insulin before acceleration) (Fig. 3A). And the correct choice in T-maze of exposure group was also significantly lower than control group (p < 0.001) and exposure ± insulin group (p < 0.005, Fig. 3B). Insulin can significantly decrease MSI and impairment of spatial memory of rats after acceleration stimulation (Fig. 3C).

Symptoms of MS which caused by acceleration (a stress response), include abnormal gastrointestinal and central nervous system such as dizziness, headache, abdominal discomfort, nausea, vomiting, salivation, cold sweat, and pale skin (Lackner and Graybiel 1983). Threonine is an essential amino acid that helps maintain the protein balance, immune response (Farrar et al. 1989). It performs an anti-fatty liver function in combination with aspartic acid and methionine. Research has indicated that chronic stress can reduce threonine (Wang et al. 2009). As MS is a stress response (Choukèr et al. 2010), we found that MSI was negatively correlated with threonine which may relieve stress and prevent MS.

Serine is a non-essential amino acid that plays a role in the metabolism of fats and fatty acids. Serine also contributes to muscle growth and assists immunoglobulins and antibodies, thus, serine plays an important role in a healthy immune system (Li et al. 2007). Serine is also involved in the manufacturing process of the cell membrane and the synthesis of neurons and muscle tissue (Wolosker et al. 1999). Studies have shown that L-serine plays an important role in the development of the central nervous system and neuronal survival (Tom et al. 2003) and that the oral administration of L-serine can be used to control seizures, eliminate vomiting, improve body weight and inhibit the development of psychiatric symptoms (Hashimoto et al. 2005). In this study, we found that the severity of MS is positively correlated with a decrease in L-serine after exposure to acceleration. During acceleration, supplemental L-serine may help prevent the nausea and vomiting caused by MS.

N-dodecanoic acid is a dicarboxylic acid containing twelve carbon atoms. Studies have shown that free fatty acids can assist in gastric emptying (Little et al. 2007). In this study, the increase of free fatty acids, including n-dodecanoic acid, was positively correlated with the severity of MS. During acceleration, an increase in free fatty acids may help to gastrointestinal emptying in MS.

Increased glucose was positively correlated with the severity of MS. Many studies have demonstrated that blood glucose can stimulate the central and peripheral vagal afferent neurons and their nerve endings and affect gastrointestinal motility and especially gastric motility (Wan and Browning 2008). Recent studies also confirmed that postprandial hyperglycemia within a normal range affects gastrointestinal motility, significantly accelerating esophageal peristalsis and slowing gastric emptying (Kuo et al. 2010). Human after acceleration were previously found to physical and cognitive abilities decreased and serum pyruvate increased. And pyruvate accumulation may contribute to acceleration-induced impairment of physical and cognitive abilities (Mo et al. 2021). Acupuncture was demonstrated that significantly alleviates MS through the IRβ-ERK1/2-dependent IR signal pathway in the dorsal motor nucleus of the vagus nerve (Tian et al. 2018). In this study, we found that insulin is negatively correlated with the Graybiel’s score in humans. The animal experiments demonstrated after exposure to acceleration, insulin is negatively correlated with MSI. In this study, administration of streptozotocin can aggravate MS, while exogenous and endogenous insulin can relieve it. Ghrelin can inhibit the expression of insulin endogenously, so we used ghrelin antagonist (D-LYS3)-GHRP-6 to figure out the pathway. Rats with ghrelin intraperitoneal injection showed more severe MS symptoms, such as defecation, urination, and vomiting than control group. But there was no statistical difference of MSI between (D-LYS3)-GHRP-6 and control group. It showed that ghrelin may affect the secretion of insulin, and exacerbate MS symptoms, but GHRP-6 may not be involved in this pathway. As a metabolism-regulated hormone, insulin plays an important role in the central nervous system. As 20 years ago, Le et al. (1983) found that the average concentration of insulin was 24-fold higher in the brain than in serum (and even 100-fold higher in some brain regions). A high central nervous system insulin concentration may be accumulated partially through peripheral circulation into the central nervous system or may be secreted by the central nervous system itself (Krowicki et al. 1998). Insulin receptor (IR) can act on glutamate and GABA receptors, modulate neuronal synaptic plasticity and protect neurons from oxidative stress. Blake and Smith (2012) have shown that insulin can stimulate the gastric motor by acting directly on the vagus nerve complex rather than through the regulation of blood glucose. Peripheral insulin can cross the blood-brain barrier, and as neuromodulators involved in the nerve conduction process (Muller et al. 2013; Gupta and Dey 2012; Hill et al. 2010). Additionally, some studies have shown that patients with diabetes present with significant nausea, vomiting and other gastrointestinal symptoms (O'Donovan et al. 2003). Mima et al. (2011) have shown that IR expression and signaling pathway activity were significantly decreased in diabetic rat brains. Russo et al. (2005) have shown that insulin can accelerate intestinal motility. Hulse and Patrick (1977) found that insulin could eliminate radiation-induced gastrointestinal emptying. Gastroparesis, a disorder of delayed gastric emptying in the stomach, is usually observed in diabetes mellitus. Continuous subcutaneous insulin infusion (CSII) therapy can manage diabetic gastroparesis (Sharma et al. 2011). Vestibular system is the main central system for sensing external acceleration stimuli. Hippocampus is an important brain region related to memory. There is a wide connection between vestibule and hippocampus. Vestibular information can affect hippocampus through cerebellum, hypothalamus, pedunculopontine tegmental nucleus (Hitier et al. 2014). Vestibular injury can cause impairment of learning and memory function in hippocampus. Wang et al. (2017b) showed that microgravity can cause the loss of spatial memory in the hippocampus. In rodents, peripheral vestibular injury can completely eliminate the discharge of pyramidal cells related to spatial position perception and memory in hippocampal CA1 region, thereby damaging spatial memory capacity (Baek et al. 2010). Previous studies have pointed out that the hippocampus is related to the signal mismatch caused by acceleration stimulation (mechanism of MS). The damage of hippocampal CA1 region may cause disappearance of MS adaptation and changes in behavior (spontaneous activities, etc.) (Wang et al. 2017a). The research indicates that the acceleration stimulus may affect the function of hippocampus, and then cause cognitive abilities such as spatial learning and memory to decline, but its mechanism has not been specifically clarified. We suggested that the results indicating that the symptoms of MS and spatial learning and memory impairment of rats after acceleration stimulation may be related to IR and memory protein, (postsynaptic marker protein postsynaptic density 95, PSD95). Microinjection of insulin into hippocampus can improve spatial learning and memory impairment of motion sickness by activating insulin receptor and its downstream signal pathway (IRS-1/PI3K/AKT/mTOR), and up regulating the activity of memory related protein, PSD95. We confirmed that acceleration stimulation can cause significant decline in learning and memory ability of rats, and the decline is reversed after microinjection of insulin into the hippocampus.

As the center of learning and memory in the brain, the expression of IR in hippocampus is critical to cognitive function. Morphological experiments confirmed that there are many IR expressed in the hippocampus. IR signaling pathway plays an important role in brain synapses and learning ability. Brain IR specific knockout mice showed anxiety and depression like behaviors. The loss of IRβ can also damage the memory function of rats(Gralle 2017).

Neuron synapse is the basis of memory formation which affects memory function. Synaptic marker proteins mainly include presynaptic protein synaptophysin (Syp), synapsin-1, PSD95, α-Amino-3-hydroxy-5-methyl-4-isoxazoleprotonic acid (AMPA) receptors such as GluRs (GluA1, GluA2, etc.), NMDA (N-methyl-D-aspartate) receptors (NR1, NR2α, NR2β) (Zhang et al. 2016). NMDA receptor mediated intracellular signal transduction cascade and expression of new genes are the main molecular mechanisms of learning and memory. Insulin can activate IRS1/PI3K/AKT (IRS2 negatively regulates the formation of memory) through IR, positively regulate CREB activity in hippocampus (Jia et al. 2016), and promote the synthesis of PSD95 through PI3K/AKT/mTOR (main target of rapamycin) pathway. PSD95 can integrate glutamate receptor (including NMDA and AMPA receptor) signal transduction through synaptic. Our study also found that the activity of IRS1 and PSD95 in the hippocampus was reduced after acceleration stimulation. These studies indicate that IR and related pathway in hippocampus play an important role in learning and memory function. In conclusion, insulin level regulators may affect hippocampus through IR, and PSD95 signaling pathway to induce cognitive ability in MS rats.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Conflict of interest

The authors declared no potential conﬂicts of interest with respect to the research, authorship, and publication of this article.

Author Statement

Mengyu Zhong, Hui Shen, Shuang Nie, and Fengfeng Mo are contributed to the design of the work. Mengyu Zhong, Jian Zhu, Bohan Zhang, and Shuang Nie contributed to acquisition of the animal experiment data and original drafting the manuscript. Hongxia Li and Yuxiao Tang are contributed to acquisition of the human data. Mengyu Zhong, Shuang Nie, Hui Shen and Fengfeng Mo are contributed to analysis and interpretation of the data. All authors approved the final version of the manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.

Funding

This work was supported by the Young scientists fond of the National Natural Science Foundation of China (grant number: 81901913), Naval Medical University basic medical research project (grant number: 2022QN015), Naval Medical University project (grant number: 2023MS008), and Major project of Brain Science and Brain-like Research (grant number: 2022ZD0208100).

Acknowledgments

We would like to express our thanks to the Naval Medical University volunteers who participated in this study.

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Table 1 primary antibodies used in this study

Antibody	Specificity	Type	Phosphorylation sites	Source/Reference
IRS-1	IRS-1	Mono-	-	Cell Signaling Technology
Phospho-IRS-1 (Ser307)	P-IRS-1	Mono-	Ser307	Cell Signaling Technology
Anti-GluA1 (AMPA subtype)	GluA1	Mono-	-	abcam
Anti-GluA1 (AMPA subtype) (phospho S845)	P-GluA1	Mono-	Ser845	abcam
Anti-PSD95	PSD95	Mono-	-	abcam
anti-GAPDH	GAPDH	Mono-	-	Beyotime
β-Actin	β-Actin	Mono-	-	Beyotime

Table 2 Association between metabolites and Graybiel’s score in humans

RT (min)	Compound	Change after acceleration	P-value	Pearson Correlation
18.15	glucose	UP	**	0.397
9.93	L-serine	DOWN	*	-0.269
10.26	L-threonine	DOWN	*	-0.295
13.73	n-dodecanoic acid	UP	*	0.302

Pearson Correlation was calculated between metabolites and Graybiel’s score. *p<0.05, **p<0.01.

Table 3 Comparison of hormones before and after acceleration

	Insulin (μIU/ml)	Glucagon (pg/ml)	Glucocorticoid (ng/ml)	Epinephrine (pg/ml)	AVP (pg/ml)
Pre	18.88 ±6.61	163.49±33.36	202.56±40.23	79.52 ±32.88	8.06 ±2.38
Post	13.83±2.47	164.74±43.46	258.88±44.77	97.11 ±43.84	39.14 ±7.96
P-value	***	N.S	***	***	***

Compare hormones before and after acceleration with two-tailed student's t-test. *P<0.05, **P<0.01, ***P<0.001, N.S: no significant difference.

Table 4 Comparison of human serum insulin level regulators before and after exposure

(pg/ml)	Ghrelin	Leptin	Resistin
Pre	141.54±79.62	845.47±688.91	7099.44±4187.05
Post	217.08±121.76	399.82±273.09	2861.09±1545.57
P-value	*	**	**

Compare hormones before and after acceleration with two-tailed student's t-test. *P<0.05, **P<0.01.

Table 5 Comparison of human serum cytokines before and after exposure

(pg/ml)	IL-1β	IL-4	IL-5	IL-10	IFN-g	TNF-a	VEGF
Pre	5.08±3.03	8.65±4.85	11.09±8.29	9.78±10.63	7.70±4.39	0.72±0.40	3.17±1.41
Post	7.18±4.91	9.49±4.45	17.0±5.30	25.32±10.06	7.72±4.36	1.20.36±0.77	8.41.94±3.45
P-value	N.S	N.S	N.S	N.S	N.S	*	***

Compare cytokines before and after acceleration with two-tailed student's t-test. *P<0.05, ***P<0.001. N.S: no significant difference.

Table 6 Association between metabolites and MS in rats

Insulin

(μIU/ml)

Glucagon

(pg/ml)

Glucocorticoid

(ng/ml)

Epinephrine

(pg/ml)

AVP

(pg/ml)

Post

93.55±53.09

248.13±37.71

37.53±9.33

79.96±55.26

175.48±33.71

P-value

N.S

Pearson Correlation was calculated between the change in the level and MSI. *P<0.05, N.S: no significant difference.

Table 7 Association between insulin level regulators and MS in rats

(pg/ml)	Ghrelin	Leptin	Resistin
Post	9798.75±3352.82	852.03±316.03	1534.44±467.94
P-value	*	N.S	**

Pearson Correlation was calculated between insulin level regulators and MSI. *P<0.05, **P<0.01, N.S: no significant difference.

Table 8 Association between serum cytokines and MS in rats

(pg/ml)	IL-1β	IL-4	IL-5	IL-10	IFN-g	TNF-a	VEGF
Post	21.67 ±15.80	76.05±23.98	444.53 ± 73.52	721.60±176.18	189.81 ±52.00	22.05±8.41	45.18±10.88
P-value	N.S	N.S	N.S	N.S	N.S	N.S	N.S

Pearson Correlation was calculated between serum cytokines and MSI. N.S: no significant difference.

No competing interests reported.

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Insulin level regulators may affect cognitive ability caused by motion sickness: an experimental study

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Abstract

Figures

1. Introduction

2. Materials and Methods

2.1. Animals and Human Subjects

2.2. Human Acceleration stimulation

2.3. Animal acceleration simulation

2.4. Behavioral Testing

2.5. Sample preparation and spectral acquisition

2.6. Data reduction and pattern recognition

2.7. The measurement of cytokines

2.8. Diabetes immunoassays

2.9. The measurement of stress hormones

2.10. Western blot analysis.

2.11. Surgery

2.12. Statistics

3. Results

3.1. Human blood test and Graybiel’s score

3.2. Animal blood test and MSI

3.3. MSI after drug administration

3.4 Changes of protein expression in the hippocampus after acceleration stimulation

3.5. Effect of insulin microinjection into hippocampus on MS symptoms and cognitive ability

Discussion

Declarations

References

Tables

Additional Declarations

Supplementary Files

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