Effect Of Iron Diets Supplement On Glucose In Psychological Stress Rats And Its Mechanism

In recent years, iron overload is a major concern problem. Lots of factors cause body iron overload, resulting in oxidative stress, and play an important role in neurodegenerative diseases, coronary heart disease and other common disease development. Some studies also found that body iron overload is related to insulin resistance, and play a important role in development of diabetes.Population survey found that the increasing of serum ferritin (SF) were positively correlated with insulin and blood glucose levels, which suggesting that iron overload may be associated with the increasing risk of diabetes; animal experiments also confirmed that inject iron intraperitoneal can lead to iron deposition in isletβcells and liver cell. Iron deposition may cause pancreatic isletβcell apoptosis, then affect insulin secretion. Liver iron deposition can lead to insulin resistance in hepatocyte. The impact will lead hyperglycemia.However, in recent years some researchers believe that the body iron status and diabetes was no necessary connection. And some studies showed that only the excessive intake of heme iron increased the risk of diabetes. Research in this regard is unclear. With the development of technology in modern society and the accelerated pace of life, people are always in a stressed station. For example, military have to defending for our homeland, often have to face unexpected events, such as war broke , perform special tasks. These exceptional circumstances and conditions of special operations soldiers are likely to cause stress. As we all know, the body under stress, hormones such as glucocorticoids, catecholamine hormones, glucagon levels increased substantially and insulin secretion reduced, will cause hyperglycemia. Stress induced hyperglycemia may damage the health of populations. Therefore, prevent excessive hyperglycemia on the protection of diabetes is important.In daily life, in order to prevent anemia, low birth weight, people often supplement iron. Clarify the impact of dietary iron in the mechanism of stress on blood glucose, so that it can provide evidence for the supply of dietary iron for stress groups (especially the military), positive to abnormal glucose metabolism diseases and prevention for the pathogenesis of disease. This article is to clarify this problem. ObjectiveClarify the relation of iron and hyperglycemia, especially in stress station, so that provide experimental basis for the formulation of dietary iron supply to stress people (such as soldiers, athletes, etc.); Clarify the effects of supplement of dietary iron intake on blood glucose especially in stress, clear the mechanism of blood glucose changes, were important for the explanation or provide clues to some diseases, such as abnormal glucose metabolism and pathogenesis, even its prevention.Methods1. The copy of psychological stress modelWith Communication box system, psychological stress rat model. Communication box system is constituted by the transparent acrylic plate which composed of a small ventricular septal A and B. A room insulated hose, B room power. In B, the rats were subjected to 30min / d of the foot shock (voltage 80V), jumping and screaming; A Room of rats received visual and auditory, exposed to psychological stress. (Communication box system below)2. Effect of dietary iron supplement on blood glucose in psychological stress rats(A) Setting of dietary iron contentIron content 80mg/kg was used as normal iron diet group; double it, and take 160mg/kg as the middle iron group; at its basis, double it as high iron content of iron group (320mg/kg).(B) Animals and GroupingChange of blood glucose : male SD rats (purchased from Bikai company), weighting (120±5) g. Feeding diets containing 80 mg/kg iron 7 days adaptively, then the rats were randomly divided into 6 groups, normal iron group (NC), normal Fe + psychological stress group (NP), the iron control group (MC), iron + psychological stress group (MP), high iron control group (HC), high iron + psychological stress group (HP), n = 8 respectively. Animals were caged in clean animal room, the ambient temperature is 24℃±1℃, humidity of 40% to 60%. All single-caged, free to diet and deionized water. 12h/12h light natural circadian rhythm. Extend the time of supplement with iron diets to two weeks: 32 male SD rats (purchased from Bikai company), weight (120±5) g. With 80mg/kg iron containing diets, rats were fed for 7 days, then were randomly divided into four groups, the normal iron group (NC), normal iron stress group (PS), high-speed rail control group (HC) and high-iron stress group ( HP) respectively.(C) DisposalOne week groups: normal iron control, control of iron, high iron control group were fed three dietary iron content of 80, 160, 320 mg / kg diet; To normal iron stress, middle iron stress and high-iron stress, beside corresponding dietary iron diet feeding, they also receive psychological stress exposure. The time of continuous disposal is 7 days. Two weeks groups: normal iron control group fed iron containing diet with 80mg/kg iron, high iron control group fed dirt with 320mg/kg iron. The method of copy psychological stress model is as above. The time of continuous disposal is 14 days.(D) Body weight and Feeds consumptionBody weight and feeds consumption of rats were weighted by electronic balance(E) Change of blood glucose in ratsThe day before stress exposure, after 6 hours of fasting, determine the change of blood glucose in rats; even in the days of psychological stress exposure, rats were fasted for 6 hours. Then with Accu -Chek Active test trips, test blood glucose from rat tail vein within 1hour after stress exposure.3. Effect of dietary iron supplement on iron deposition in serum, liver, pancreas and oxidative stress injury in liver(A) Disposal and Grouping 60 male SD rats were purchased from Bikai company, weighting (120±5) g. Then they were randomly divided into 6 groups, normal iron group (NC), normal Fe + psychological stress group (NP), the iron control group (MC), iron + psychological stress group (MP), high-speed rail the control group (HC), high-speed rail + psychological stress group (HP), n = 8, respectively. The other 12 rats were take as the shock group. Animals were caged in clean animal room, temperature is 24℃±1℃, relative humidity of 40% to 60%. Free to diet and deionized water. Adaptive feeding 7 days to adapt to dietary iron content of 80mg/kg of feeds.(B) Copy psychological stress model in rats Method is same to mention above. Normal, medium and high iron control group were fed with dietary iron content of 80, 160, 320 mg/kg diet; at basis of corresponding iron diets, normal iron stress, medium iron stress and high iron stress groups received psychological stress exposure. Continuous experimental treatment time is 7 days.(C) Body weight and Feeds consumptionThe method is same to mention above.(D) Effect of dietary iron supplementation on iron content of serum, liver and pancreas in stress rats After treatment, anesthetized rats immediately, then received heart perfusion, and take liver, pancreas and other tissues. Atomic absorption spectrophotometer was used to detect iron concentration of rat serum, liver and pancreas.(E) Effect of dietary iron supplementation on oxidative stress injury in rat liverMDA was used as degradation products of lipid peroxidation. MDA and thiobarbituric acid(TBA) could condensation to form a red product which absorb 532nm visible light. We can use it to detect the liver MDA contentWith corresponding kits, we can detect the levels of antioxidant enzymes activity, such as SOD, GSH, GPx, so that we can evaluate oxidative stress injury in liver.4. Effect of dietary iron supplement on stress hormones and phosphorylation of IRS-1 and PKB/Akt in insulin signaling(A) Disposal and Grouping:108 male SD rats, weighting (120±5) g, were divided into 3 groups randomly, they were two-day group, seven-day group and two-week group respectively. Each group was subdivided into four groups, control group (NC), normal stress group (NP), high iron control group (HC) and high-iron stress group (HP). N=8. The other 12 rats as the shock group to receive foot shock. The room condition is same to above. 7 days adaptation, they were fed iron containing diet 80mg/kg.(B) Model of Psychological StressSpecific method is same to the way mentioned above. Normal and high iron control groups were fed iron containing diet, 80 and 320 mg/kg respectively; at basis of feeding corresponding iron containing diet, normal and high iron stress group also expose to psychological stress. 2 day, 7 day and 2 weeks groups receive two days, seven days and two weeks psychological stress exposure respectively. (C) Effect of supplement of dietary iron on stress-related hormones (such as cortisol, epinephrine, glucagon, insulin) in stress ratsWith RIA kits, we test serum cortisol and epinephrine concentrations; using ELISA kits, to detect serum epinephrine, insulin concentration, so that we can know the effect of supplement of dietary iron on stress-related hormones in stress rats.(D) Effect of dietary iron supplementation on insulin sensitivity in psychological stress rats hepatocyteWith Western-blotting, we can detect pIRS-1, IRS-1, pAkt and Akt, so that we can know the effect of iron supplement on the transduction of insulin signal pathway.(E) Effect of dietary iron supplementation on glycogen content in rat liverWith corresponding kit, test glycogen content in liver, so that we can know the effect of dietary iron supplementation on glycogen content in stress rats.4. Statistical MethodsUsing SPSS16.0, using one-way ANOVA (including LSD, SNK to compare every two groups), repeated measures analysis and t test to dispose our data.Results1. Effect of supplement dietary iron on blood glucose in psychological stress rats(A) Effect of supplement dietary iron on feeds consumption in psychological stress ratsDuring the experiment, before and after stress exposure, feeds consumption have no significant differences (P> 0.05); normal, medium and high iron groups were not significantly different (P> 0.05); and three stress groups were not significantly different (P> 0.05).(B) Effect of supplement dietary iron on average body weight gain consumption in psychological stress ratsDuring the experiment, compared with their corresponding control group, the average body weight gain in normal, middle and high iron stress group decreased 13.51%, 13.55% and 16.79% respectively.(C) Effect of supplement dietary iron on blood glucose in psychological stress rats 7-day group: After stress exposure, compared with the corresponding control group, blood glucose in stress rats were elevated in first 4 days of our test (P <0.05), but on the sixth day, only in the high-iron stress group, blood glucose levels were significantly increased (P <0.05).HP group increased 6.41%, 6.51%, 8.07% on 2d, 4d, 6d of stress exposure respectively. Compared with NP group, the gap between HP and NP group becomes larger and lasting time of hyperglycemia becomes longer, and exists statistically significance between two groups during whole experimental session.Dietary iron supplement may cause significant stress and sustained blood glucose elevation: high blood glucose levels in HP, high levels of blood glucose persist in our whole study; Compared with NP, blood glucose increased 6.41%, 6.51%, 8.07% on 2, 4, 6 day, (P <0.01).Two weeks group: In order to understand the stress of dietary iron supplementation on blood glucose levels of dietary supplement, we extend psychological stress exposure time to two weeks. Throughout the experimental period, HP had a higher blood glucose level, compared to NC and NP, there were significant differences (P <0.01).2. Effect of dietary iron supplement on iron deposition in serum, liver, pancreas and oxidative stress injury in liver1. Effect of dietary iron supplement on iron contents in stress rats Using atomic absorption spectrophotometry, we detect of iron contents in rats serum, liver and pancreas. After stress exposure, iron in liver and pancreas ware significantly increased (P <0.05), serum iron decreased significantly (P <0.05) ; With the increasing supplement of dietary iron, iron contents in serum, liver and pancreas were also significantly elevated (P <0.05); compared with NC, after stress exposure, dietary iron supplement could further significantly increase iron contents in liver and pancreas (P <0.01), and improved the state of decreasing serum iron induced by psychological stress exposure.2. Effect of supplement iron on oxidative stress injury in stress rats Corresponding kits were used to detect MDA, SOD, GSH and GPx contents. High dietary iron supplement alone will cause oxidative stress in the liver tissue damage index changes: MDA content was significantly increased, while the activity of other antioxidant enzymes was significantly decreased (P <0.05); and after exposure to psychological stress liver oxidative stress also significantly increased; and the high iron diet supplement, will further increased oxidative stress injury significantly in rats (P <0.01).3. Effect of dietary iron supplement on stress hormones and phosphorylation of IRS-1 and PKB/Akt in insulin signaling1. Effect of supplement dietary iron content on corticosterone, epinephrine, glucagon, insulin and other glucose-related stress hormones in rats of 2 d, 7d and 2 weeks groups2 d group: After psychological stress exposure, compared with NC, stress-related hormones (such as cortisol, epinephrine and glucagon) were significantly elevated while insulin levels significantly decreased (P <0.05); supplement high-iron diet alone, compared with NC, corticosterone, epinephrine, insulin levels were significantly increased (P <0.05); and in HP, compared with NP, levels of serum insulin would significantly increased (P <0.05).7 d group: After psychological stress exposure, compared with NC, corticosterone, epinephrine levels were significantly elevated and insulin levels decreased significantly (P <0.05), while glucagon showed no significant change; high iron diet supplement will cause serum insulin significantly increased (P <0.01), and compared with NC, the other levels of stress hormones was no significant change (P> 0.05); High levels of dietary iron supplement will further significantly increased serum corticosterone, epinephrine, insulin levels (P <0.05).2 weeks group: After psychological stress exposure, compared with corresponding control group, serum epinephrine levels were significantly increased (P <0.05), serum insulin levels decreased significantly (P <0.05); compared with the corresponding control groups, serum corticosterone in stress groups had significantly higher levels; for glucagon, psychological stress exposure and high dietary iron supplement, both can caused no significant change (P> 0.05). Supplement high levels of dietary iron alone, can not cause significantly changes in stress-related hormones (such as cortisol, epinephrine and glucagon) (P> 0.05).2. Effect of supplement dietary iron on insulin signal pathway in psychological stress ratsCompared with their corresponding control groups, after psychological stress exposure, pIRS-1/IRS-1 and pAkt/Akt did not changed significantly (P> 0.05). Compared to the control group, high iron stress group insulin sensitivity decreased significantly (P <0.05). And the high iron and High-iron diet alone did not cause additional changes in insulin sensitivity (P> 0.05).3. Effect of supplement dietary iron on liver glycogen content in psychological stress ratsChange of liver glycogen content is also a factor in the pathway of insulin. Its content reflects that some changes appear in this pathway. Our study showed that a simple dietary supplement with high iron a significantly decreased in liver glycogen content was (P <0.05); after psychological stress exposure, liver glycogen content in HP was significantly decreased (P <0.01), but not the other two groups (P> 0.05).ConclusionUsing Communication box system to copy psychological stress rat model, and set dietary iron content, observed the effect of dietary iron on blood glucose in psychological stress rats, and our study showed that supplement dietary iron can cause hyperglycemia in psychological stress rats. The main conclusions are as follows:1. Supplement high-iron diet alone can cause a transient increase in blood glucose levels; with continued dietary supplement with high iron diet, glucose levels will gradually decreased to normal levels; and to rats exposed to psychological stress, high dietary iron supplement could elevated glucose levels significantly and persist a long time; extend our experiment time to two weeks, blood glucose still stay at a higher level in stress rats.2. Iron content: atomic absorption was used to detect iron disposition state in liver, serum and pancreas. Content of iron in rat liver and pancreatic was significantly increased after psychological stress exposure (P <0.05) and serum iron decreased significantly; and supplement high-iron diet to stress rats, further increased iron deposition in liver significantly, but improved the decreased levels of serum iron induced by stress expoure. Oxidative stress conditions: after stress exposure, oxidative stress indexes in liver was significantly increased; and high dietary iron supplement will further aggravate this injury in stress rats.3. After stress exposure on 2d and 7 d, serum corticosterone, epinephrine, glucagon and other blood-related stress hormones increased significantly, while insulin levels decreased significantly, which also proved that psychological stress modeling copied successfully. Compared with NC, high dietary iron supplement alone only lead to serum insulin increased significantly; and rats exposed to psychological stress, high-iron diet supplement lead to the concentration of serum corticosterone maintained at a higher level.4. Compared with NC, high iron supplement on stress rats were significantly decreased transduction of insulin signaling pathway and the concrete possible reason is continuing to exploring.