The Level Of Risk Factors For Cognitive Impairment In Insulin Resistance And Diabetic Rat Brain

Objective: Recent epidemiological evidence indicates that patients who suffer from T2DM have a two to three fold increased relative risk for Alzheimer's disease (AD). Oxidative stress,β-amyloid protein (β-amyloid, Aβ) deposition and accumulation of fatty acids in the brain are the possible factors which cause cognitive dysfunction.But it is unknown whether those factors are related to cognitive impairment in the state of T2DM and insulin resistance which exist before diabetes.Oxidative stress is recognized as a major key factor of cognitive dysfunction.The brain is especially sensitive to oxidative damage because of the abundance of unsaturated fatty acids. The increasing of the product of lipid peroxidation malondialdehyde (MDA) is the hallmark of oxidative damage.Whether the content of MDA, total antioxidant capacity (T-AOC) and the activity of major antioxidant enzyme such as superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), catalase (CAT) changed in the brain of insulin resistance and T2DM? Beta-amyloid peptides (Aβ) is considered a primary fator of cognitive impairment and AD. Aβis a minor product of beta-amyloid precursor protein (APP) processing that is released after cleavage by so-calledβ-secretase andγ-secretase. A third APP-cleaving enzyme, so-calledα-secretase, cleaves within the Aβsequence and thereby decreases Aβformation, which is the main way of APP proteolytic processing. However, whether the production of Aβand the mRNA level of APP,β-secretase BACE1 andα-secretase ADAM10 are influenced in the condition of insulin resistance and T2DM ,has not been well understood.Accummulation of fatty acids is related to many cognitive dysfunction. The decomposition is mainly through fatty acidsβ-oxidation in mitochondria and peroxisome. Short, medial and long chain fatty acids can be catabolized in mitochondria byβ-oxidation, carnitine acyltransferaseⅠ(CPT-Ⅰ) is the rate-limiting enzyme. Very long chain fatty acids must be handled in peroxisomal byβ-oxidation. There are two pathway in this condition. Acyl-CoA oxidase 1(Acox1), L-bifunctional protein(LBP), Thiolase A (THLA) and Thiolase B(THLB) are mainly oxidized straight-chain fatty acids. Acyl-CoA oxidase3(Acox3), D-bifunctional protein(DBP) and sterol carrier protein x(SCPx) are involved in the oxidation of branched-chain fatty acid. Peroxisome Proliferator Activated Receptorα(PPARα) regulates the metabolism of fatty acids in cells.It is unclear that whether fatty acids accummulate in the brain of insulin resistance and T2DM,and the mRNA expression of the main enzymes involved in mitochondria and peroxisomeβ-oxidation will change or not.In order to study these issues, we developed insulin resistance in male Sprague-Dawley rats by high-fat diet, and further formation of diabetes by peritoneal injection of streptozotocin (STZ). To assay oxidative stress, Aβproduction and fatty acid metabolism in brain of insulin resistance rats and diabetes rats.In order to provide references to researches of diabetic cognitive impairment and prevention of AD.Methods:1 Animals and materialsAccording to Wang XL, we used male SD rats (weight 220-240g) copy the model of insulin resistance. At the basis of insulin resistance,using STZ injection to set up the model of type2 diabetes.Blood were collected for blood index assay.Brain tissues were removed and immediately submerged in liquid Nitrogen, then stored at -70℃for the extraction of total RNA ,the detection of Aβand the level of oxidative stress.2 Assay of blood indexBlood glucose and triglyceride concentration were detected by Olympus HITACHI 7170A automatic biochemistry analysator.3 Detection of the level of oxidative stress in brainThe content of MDA and the activity of SOD, GSH-PX, CAT, T-AOC were determined using colorimetry.4 Brain AβassayAβwas determined by radioimmunoassay method using the Aβassay kit.5 Brain total FFA assay Total fatty acids in the brain were determined using colorimetry.6 Assay of mRNA expressionBrain total RNA was extracted by Promega SV Total RNA Isolation System. The relative mRNA content was measured by quantitative real time RT-PCR , with 18S rRNA as inner standard.7 Statistical analysisData were expressed as mean±SD. SPSS 13.0 soft ware was used. Statistical comparisons were made by Independent-Sample T Test (2-tail). A value of P < 0.05 was considered significant.Results:1 Oral glucose tolerance test (OGTT)The AUC of HF group(31.1383±1.797 mmol/L*h) is significantly higher than that of Con group(27.8217±1.2713 mmol/L*h, P < 0.01). It indicated that the model of insulin resistance is setted up.2 Changes of fasting blood glucose (FBG)Compared with Con group (5.1417±0.5665 mmol/L ),the FBG in DM group (12.7167±1.8622 mmol/L) increased (P < 0.01)., but the changes between IR(5.9533±1.7565 mmol/L) and Con groups had no statistical significance(P > 0.05). It indicated that the model of type 2 diabetes is setted up.3 Changes of serum cholesterolSerum cholesterol in Con, IR and DM groups were respectively 1.6208±0.3274 mmol/L, 1.8750±0.8560 mmol/L 1.4633±0.3734mmol/L. Compared with Con group, IR and DM groups had no statistical significance (P > 0.05, P > 0.05).4 The level of oxidative stress in brain4.1 The content of MDA in brainThe content of MDA in Con, IR and DM groups were respectively 2.5817±0.4977 nmol/mgprot, 3.2199±0.5641 nmol/mgprot, 3.6422±0.7434 nmol/mgprot. The content of MDA in IR and DM groups was higher than that in Con group (P < 0.01, P < 0.05).4.2 The level of T-AOC in brainThe level of T-AOC in Con, IR and DM groups were respectively 4.589±0.4064 U/mgprot, 2.7492±0.2170 U/mgprot, 2.6994±0.2811 U/mgprot. The level of T-AOC in IR and DM groups were significantly lower than that in Con group(P < 0.01, P < 0.01).4.3 The activity of SOD in brainThe activity of SOD in Con, IR and DM groups were respectively 189.1895±19.8206 U/mgprot, 168.5015±14.3083 U/mgprot, 157.6365±16.7645 U/mgprot. The activity of SOD in IR and DM groups were lower than that in Con group (P < 0.05,P < 0.01).4.4 The activity of GSH-PX in brainThe activity of GSH-PX in Con, IR and DM groups were respectively 70.9901±15.1047 U/mgprot, 50.6992±8.7335 U/mgprot, 51.8568±13.2662 U/mgprot. The activity of GSH-PX in IR and DM groups were lower than that in Con group (P < 0.01, P < 0.05).4.6 The activity of CAT in brainThe activity of CAT in Con, IR and DM groups were respectively 8.6647±0.6612 U/mgprot, 7.3753±0.4180 U/mgprot, 7.7356±0.6930 U/mgprot. The activity of CAT in IR and DM groups were lower than that in Con group (P < 0.01,P < 0.05).5 The content of Aβin brainThe content of Aβin Con, IR and DM groups were respectively 0.6675±0.1249 ng/mgprot, 1.1024±0.1767 ng/mgprot, 1.1613±0.0990 ng/mgprot. The content of Aβin IR and DM groups were significantly higher than that in Con group (P < 0.01, P < 0.01).6 The content of total FFA in brainThe content of total FFA in Con, IR and DM groups were respectively 0.6256±0.1174 mmol/gprot, 0.9705±0.1973 mmol/gprot, 0.8387±0.1146 mmol/gprot. The content of total FFA in IR and DM groups were significantly higher than that in Con group (P < 0.01, P < 0.01).7 The expression of brain genes7.1 The relative expression of APP mRNA in brainThe relative expression of APP mRNA in Con, IR and DM groups were respectively 1.7853±0.2472, 2.9749±0.1577, 2.6451±0.2418. The relative expression of APP mRNA in IR and DM group were significantly higher than that in Con group (P < 0.01, P < 0.01).7.2 The relative expression of BACE1 mRNA in brainThe relative expression of BACE1 mRNA in Con, IR and DM groups were respectively 3.2275±0.1456, 5.2675±0.9458,5.4507±0.8602. The relative expression of BACE1 mRNA in IR and DM group were significantly higher than that in Con group (P < 0.01, P < 0.01).7.3 The relative expression of ADAM10 mRNA in brainThe relative expression of ADAM10 mRNA in Con, IR and DM groups were respectively 5.3933±0.5424, 5.8163±0.9998, 5.9193±0.9344. Compared with Con group , IR and DM groups had no statistical significance (P> 0.05, P > 0.05).7.4 The relative expression of PPARαmRNA in brainThe relative expression of PPARαmRNA in Con, IR and DM groups were respectively 2.9033±0.5425, 3.7475±0.3225, 4.4925±0.6161. The relative expression of PPARαmRNA in IR and DM group were significantly higher than that in Con group (P < 0.01, P < 0.01).7.5 The relative expression of CPTⅠmRNA in brainThe relative expression of CPTⅠmRNA in Con, IR and DM groups were respectively 4.3467±0.4359, 7.1600±0.7399, 7.5525±0.8917. The relative expression of CPTⅠmRNA in IR and DM group were significantly higher than that in Con group (P < 0.01, P < 0.01).7.6 The relative expression of ACOX1 mRNA in brainThe relative expression of ACOX1 mRNA in Con, IR and DM groups were respectively 20.8267±2.0094, 28.5100±3.5548, 28.0925±4.6149. The relative expression of ACOX1 mRNA in IR and DM group were significantly higher than that in Con group (P < 0.01, P < 0.01).7.7 The relative expression of LBP mRNA in brainThe relative expression of LBP mRNA in Con, IR and DM groups were respectively 2.4683±0.3750, 3.8850±0.4841, 5.2943±0.8613. The relative expression of LBP mRNA in IR and DM group were significantly higher than that in Con group (P < 0.01, P < 0.01).7.8 The relative expression of THLA mRNA in brainThe relative expression of THLA mRNA in Con, IR and DM groups were respectively 1.0850±0.2748, 1.5733±0.2375, 1.2723±0.2521. The relative expression of THLA mRNA in IR group was significantly higher than that in Con group (P < 0.01),but the changes between DM and Con groups had no statistical significance (P > 0.05).7.9 The relative expression of THLB mRNA in brainThe relative expression of THLB mRNA in Con, IR and DM groups were respectively 1.7267±0.2225, 2.0200±0.3703, 1.4867±0.0954. The relative expression of THLB mRNA in DM group was lower than that in Con group (P < 0.05),but the changes between IR and Con groups had no statistical significance (P > 0.05).7.10 The relative expression of ACOX3 mRNA in brainThe relative expression of ACOX3 mRNA in Con, IR and DM groups were respectively 8.7500±0.4312, 11.1775±2.1998, 12.1800±1.1681. The relative expression of ACOX3 mRNA in IR and DM group were significantly higher than that in Con group (P < 0.01, P < 0.01).7.11 The relative expression of DBP mRNA in brainThe relative expression of DBP mRNA in Con, IR and DM groups were respectively 14.1367±1.4650, 19.0575±2.2587, 18.3650±3.5966. The relative expression of DBP mRNA in IR and DM group were significantly higher than that in Con group (P < 0.01, P < 0.01).7.12 The relative expression of SCPx mRNA in brainThe relative expression of SCPx mRNA in Con, IR and DM groups were respectively 9.4433±1.0632, 10.6500±1.1963, 7.0000±1.0379. The relative expression of SCPx mRNA in DM group was significantly lower than that in Con group (P < 0.01),and the relative expression of SCPx mRNA in IR group was higher than that in Con group (P < 0.05).Conclusion:1 In insulin resistance and type 2 diabetic brain,there are three risk factors causing cognitive impairment :oxidative stress, Aβdeposition and the accumulation of fatty acids.2 In insulin resistance and type 2 diabetic brain, the high level of oxidative stress is related to the enhanced fatty acidβ-oxidation and the decreased activity of major antioxidant enzymes; enhanced mitochondrial and peroxisomal fatty acidβ-oxidation can not decompose so many fatty acids; the increase of Aβis related to the high mRNA expression of APP and BACE1.