The Role Of Hippocampal Insulin Resistance In Cognitive Impairment Induced By Simulated Weightlessness In Rats

BackgroundThe study on astronauts’brain health is an important issue in aerospace medical research.Recent studies have reported a decline in astronauts’cognition,sensation,visual and motor coordination during the spaceflight,which results in the decline of astronauts’working capacity,and further affecting the completion of space missions.Ground-based research also reported that simulated weightlessness can lead to learning and memory impairment,neurotransmitter secretion reduction and neuronal apoptosis aggravation in rodents.However,the specific mechanism of cognitive dysfunction caused by weightlessness is still unclear.Therefore,it is necessary to clarify the effects and causes of the weightlessness on brain structure and function,especially on cognition,and put forward effective countermeasures to future astronauts’brain protection as well.Insulin,an important neuroprotective molecule,acting on promoting synapses formation and maintaining alive of neurons.The endocytosis and efflux is the major way of blood-brain barrier（BBB）transporting insulin into the brain,and several studies also shows that a few neurons and glial cells can also synthesize insulin.It has demonstrated that hippocampal insulin resistance can damage the synaptic plasticity,promote Aβdeposition,inducing Tau protein hyperphosphorylation and aggravating the formation of neurofibrillary tangles,and plays an important role in the pathological process of chronic cognitive dysfunction such as neurodegenerative diseases,aging,diabetes,dementia and so on.It is worth noting that there is a close relationship between hippocampal insulin sensitivity and peripheral insulin sensitivity.More importantly,on orbit flight research,obvious peripheral insulin resistance was observed in astronauts,including islet dysfunction,increased serum insulin and HOMA-IR,reduced glucose tolerance as well as protein and amino acids metabolism disorders.In addition,studies on orbit flight and ground-based have also reported the potential risks of insulin resistance in the peripheral and hippocampus induced by weightlessness,for instance,increased molecules related to inflammation and oxidative stress.Therefore,it is necessary to explore whether weightlessness can lead to hippocampal insulin resistance and thus,inducing neuronal damage and cognitive deficit.Given that,we established the tail-suspended rodent model and explored the mechanism and countermeasures of the hippocampal insulin resistance under the simulated weightlessness.AimsTo investigate whether hippocampus insulin resistance is involved in simulated weightlessness-induced cognitive impairment,and further explore the mechanism and countermeasures.MethodsThe tail-suspended rodent model was used for simulated weightlessness.Rats were grouped according to the random number table.Monitoring the food intake and body weight once a week during modeling.Intraperitoneal glucose tolerance test（IPGTT）was performed to test the rats’glucose tolerance.Fasting blood glucose and insulin were analyzed respectively.Rats’cognitive function was measured by the open field test（OFT）and novel object exploration test（NOR）.Furthermore,Nissl staining,HE staining,immunofluorescence and transmission electron microscopy were used to test the damage of neurons in hippocampus.The change of hippocampal insulin sensitivity was judged by testing the activation of PI3K-Akt pathway under the unstimulated and insulin stimulated condition（0.75 U/kg,BW,30 min,i.p.）,and the negative regulators of Akt phosphorylation including PHLPP-1,PP2A-C and PTEN were tested by Western blotting（WB）to further study the mechanism behind the hippocampal insulin resistance induced by weightlessness.Finally,berberine treatment（200 mg/kg/d,BW,gavage）for 4 weeks was applied to intervene the cognitive impairment induced by simulated weightlessness for further clarify the mechanism.Besides,Evans blue extravasation was performed to study the function of BBB in hippocampus,and the expression of tight junction proteins were detected by WB to explore the impact of simulated weightlessness on insulin transport in hippocampus.And the rat model of intermittent artificial gravity（rats were untied to the standing position for 1 hour every day during the tail suspension）was established to explore the relationship between the gravity and the insulin resistance as well as the structure and function of the BBB.The data are presented as the mean±SD.Student’s t-test was used for comparison between two groups.One-way and two-way ANOVA following Tukey’s Post-Test were used for multiple comparisons.When P<0.05,the difference was considered statistically significant.Results1.Compared with the CON group,the fasting blood glucose and HOMA-IR were increased in the SUS group（n=6,P<0.05,P<0.01）.IPGTT showed the rats’glucose tolerance was obviously decreased after 4-week simulated weightlessness（n=6,P<0.05）.There was no significant difference in the level of fasting serum insulin between the two groups.Besides,simulated weightlessness had no effect on rats’food intake,body weight and lipid metabolism.These results indicated that 4-week simulated weightlessness can lead to obvious peripheral insulin resistance in rats.2.The results of OFT showed that compared with the CON group,the time and distance and in center area,the ratio of time in center area to total time in move and the distance in center area to overall distance traveled were declined significantly in the SUS group（n=15-17,P<0.01）,which suggested an obvious anxiety-like behavior and impaired exploration capacity of rats after 4-week tail-suspension.According to the NOR,both groups of rats exhibited similar total object exploration times.Compared with the CON group,times and duration of visit to the novel object and the discrimination index were significantly decreased in the SUS group（n=13-17,P<0.01）,which indicated that rats’learning and memory capacity was impaired after four-week tail suspension.3.HE staining and Nissl staining showed that the neurons in SUS group were shrunk and loosely arranged with deeply stained nucleus,scattered over the hippocampus.TUNEL staining showed an obvious increase in neuronal apoptosis in the hippocampus of the SUS rats,and the expression of Bax and Cleaved-Caspase-3 were up-regulated with a remarkably down-regulation of Bcl-2 and Caspase-3 meanwhile（n=5-6,P<0.05,P<0.01）.Additionally,according to the transmission electron microscopy,the mitochondria in the hippocampal neurons was slightly swollen,and even some vacuolation can be seen in the SUS rats’hippocampal mitochondria.Besides,the rough endoplasmic reticulum was observed slightly dilated and partly degranulated in the SUS rats’hippocampus neurons Furthermore,it was showed that 4-week simulated weightlessness significantly increased degenerative neurons in hippocampus as evidenced by increased FJB positive neurons and Tau hyperphosphorylation in Ser404,Thr231 and Ser396（n=5-6,P<0.05,P<0.01）.In a addition,the aging-related protein including p-m TOR,P53 and P21 were observed up-regulated remarkably,and Sirt-1 was down-regulated in the SUS rats’hippocampus（n=5,P<0.05,P<0.01）.These results indicated that simulated weightlessness promoted neurodegeneration in hippocampus.4.After fasting for 8 hours,rats were injected with insulin（0.75 U/kg,30 min,i.p.）.The insulin concentration in hippocampus before and after insulin injection were detected by ELISA.Our results showed that in the SUS group,the insulin level in hippocampus decreased significantly after fasting for 8 h,but increased significantly after peripheral injection of insulin for 30 min（n=6,P<0.05）,suggesting that simulated weightlessness reduced insulin in rats’hippocampus but may promote the insulin transport or increase the permeability of BBB in hippocampus.The activation of PI3K-Akt signaling pathway was detected by WB with or without insulin stimulation.It was found that the phosphorylation of Akt in Ser473 site was significantly inhibited after 4-week simulated weightlessness（n=5,P<0.05）,while there was no change in other upstream molecules such as IR and IRS-1.These results indicated that simulated weightlessness induced insulin resistance in hippocampus.PTEN,the negative regulator of Akt phosphorylation was significantly increased in the SUS group（n=5,P<0.05）,which indicated that the up-regulation of PTEN was attributed to the hippocampal insulin resistance caused by simulated weightlessness.5.Berberine（200mg/kg/d,gavage）was given to treat hippocampal insulin resistance induced by simulated weightlessness.Rats were randomly divided into the control group（CON）,the CON rats treated with berberine group（CON+BBR）,the tail-suspended group（SUS）and the SUS rats treated with berberine group（SUS+BBR）.The results showed that the SUS+BBR rats had a significantly lower fasting blood glucose and abnormal glucose tolerance compared with the SUS group（n=6,P<0.05）.The results of OFT and NOR showed that compared with the SUS group,the ratio of time in center area to total time in move and the distance in center area to overall distance traveled as well as the discrimination index of the SUS+BBR group were significantly increased（n=12,P<0.05）,which indicated that berberine can partially alleviate the cognition impairment induced by simulated weightlessness.Besides,berberine selectively reduced the simulated weightlessness-induced overexpression of p-Tau ^S396,down-regulated PTEN and up-regulated the p-Akt ^S473（n=3,P<0.05）.These results indicated that berberine partially reversed the cognitive impairment,Tau hyperphosphorylation and insulin resistance both in hippocampus and peripheral tissues induced by simulated weightlessness.6.According to the double immunofluorescence staining for GLUT1 and CD31,the vascular density and diameter in the SUS rats’hippocampus was decreased significantly compared with the CON group（n=3,P<0.01）.Evans blue extravasation test was used to detect the BBB permeability.However,fluorescence angiograms showed that compared with the control group,the extravasation of Evans blue around the blood vessels was remarkably increased in the SUS rats’hippocampus,with the signs of Evans blue uptaken by nearby neurons,which suggested that simulated weightlessness increased the permeability of BBB in rats’hippocampus.And this conclusion was also supported by a significant increase of albumin in the hippocampus of the SUS group（n=3,P<0.05）.Furthermore,the detection of tight junction proteins showed that occludin was decreased significantly in the hippocampus after 4-week simulated weightlessness（n=3,P<0.05）.These results suggested that 4-week simulated weightlessness increased the permeability of BBB,which may be attributed to the decrease of occludin.7.To explore the impact of gravity on insulin sensitivity and BBB,rats were untied and allowed to return to a normal standing position 1 hour per day during the tail suspension to simulate the intermittent artificial gravity（IAG）.The results showed that IAG rats had a better performance compared with the SUS rats in behavioral tests as evidenced by significantly increased the ratio of time in center area to total time in move and the distance in center area to overall distance traveled as well as the prolonged duration of novel object exploration（n=12,P<0.05,P<0.01）,which suggested that IAG partially improved the cognitive impairment induced by simulated weightlessness in rats.What’s more,the IAG rats showed a lower fasting blood glucose and higher glucose tolerance in comparation with the SUS group（n=6,P<0.05,P<0.01）.However,it showed no difference in Akt phosphorylation and the expression of PTEN between the SUS group and IAG group,which means IAG was only effective on peripheral insulin resistance induced by simulated weightlessness,but has no significant effect on hippocampal insulin resistance.In addition,compared with the SUS group,the vascular diameter in hippocampus was increased in the IAG group（n=3,P<0.05）.And the increased permeability of BBB and down-regulated expression of occludin induced by simulated weightlessness were partially reversed by IAG（n=3,P<0.05,P<0.01）.In summary,IAG improved the cognition impairment induced by the 4-week simulated weightlessness,which may be owed to its antagonism for vascular injury in hippocampus.Conclusions1.Four-week simulated weightlessness impaires cognitive function and induced apoptosis and degeneration of hippocampal neurons,which is attributed to the up regulation of PTEN-induced hippocampal insulin resistance.Berberine partly reverses the above injury.2.Four-week simulated weightlessness significantly reduces the vascular density and diameter,and increases the permeability of blood-brain barrier as well as down-regulates the expression of occludin.Intermittent artificial gravity shows positive signs of improvement on the vascular injury,but has no significant effect on hippocampal insulin resistance caused by the simulated weightlessness.