| Type2diabetes mellitus (DM) is a chronic metabolic disorder disease,due to the relative insulin deficiency or insulin resistance (IR), which can leadto multiple organs dysfunctions. In recent years, with the improvement ofliving standards and the change of life style, type2diabetes mellitus hasbecome a global disease threatening people’s health. Therefore, to study itspathogenesis has an important significance for prevention and treatment oftype2diabetes mellitus.Disorder in fatty acid metabolism has been recognized as animportant characteristic of type2diabetes mellitus. However, it is scarce thathow profile of fatty acids changed in type2diabetic patients, and whether thechanges are associated with type2diabetes.Single nucleotide polymorphisms (SNPs) of associated genes areimportant factor affecting the metabolism of fatty acid in the gene level, and itis closely related to the occurrence and development of type2diabetes.Researchs have shown that the SNPs in fatty acid desaturase1(FADS1)and2(FADS2) genes, which are coded for delta5desaturase (△5D) and delta6desaturase (△6D), the key enzymes in polyunsaturated fatty acid synthesis,are related to the profile of fatty acids.Then, it is worth to further studywhether the SNPs in FADS gene are related to the disorders of fatty acidmetabolism in type2diabetes?In addition, except the effects in gene level, the expressions and activitiesof key enzymes in the fatty acids synthesis are also important factors whichinfluence the fatty acid metabolism. Liver is the main organ of fatty acidsynthesis in the body, which also can provide fatty acids to the extrahepatictissues. Then, how dose the fatty acids synthesis in the liver change in type2 diabetes? Is this change one of the reasons for fatty acid metabolism disorderin type2diabetes mellitus?In order to explain the above questions, firstly we detect serum profile offatty acids in type2diabetic patients, and analyze the changes of fatty acids tofind the target for further research. Then, we deeply research the relationshipbetween the fatty acids metabolism and type2diabetes in the gene level andthe fatty acids synthesis in the liver to provide strong experimental evidencesfor the prevention of type2diabetes.Part One Profile of serum fatty acids in type2diabetic patientsObjective: To study the profile of serum fatty acids in type2diabeticpatients, and find the target for further research in fatty acid metabolism oftype2diabetes.Methods: A case-control study was employed in this study.752subjectswere collected and divided into2groups: controls group (Con,421cases) andtype2diabetic patients group (DM,331cases). Fasting serum insulin, fastingblood glucose, triglyceride, total cholesterol, and free fatty acid weredetermined. Body mass index (BMI), insulin sensitive index (ISI) and insulinresistance index (HOMA-IR) were calculated. Fasting serum fatty acids withcarbon atoms16-24were determinated by gas chromatography and analyzedaccording to the category.Results：1The general biochemical indexesCompared with Con group, there was no difference in sex ratio. The agesof DM group was significantly higher64(10) vs.51.38(18), P<0.001; BMI ofDM group also significantly increased (25.568±3.316vs.23.177±2.434)P<0.001.Compared with Con group, fasting blood glucose, fasting insulin, totalcholesterol, triglyceride and total free fatty acids in DM group weresignificantly increased, P<0.001; ISI in DM group decreased significantly, andHOMA-IR were significantly increased, P<0.001. 2Profiles of serum fatty acidsFatty acid profile shows: compared with Con group, C16:0, C16:1n-7,C18:1n-9, C18:3n-3(α-linolenic acid, ALA), C20:5n-3(eicosapentaenoic acid,EPA), C22:5n-3(docosapentaenoic acid, DPA) and C20:4n-6(arachidonicacid, AA) increased in DM group, P <0.01, but C18:0, C24:0, C22:6n-3(docosahexenoic acid, DHA) and C18:2n-6(linoleic acid, LA) decreased, P<0.01. C20:0, C22:0and C22:4n-6had no obvious change.To analysis the different types of fatty acids, we found that, comparedwith Con group, total monounsaturated unsaturated fatty acid (Total MUFAs)increased significantly in DM group, P <0.01, while the total saturated fattyacid (Total SFAs), total n-3polyunsaturated fatty acids (Total n-3PUFAs) andtotal n-6polyunsaturated fatty acids (Total n-6PUFAs) had no obviouschange.Summary：1Total freely fatty acids in type2diabetic patients was increasedsignificantly.2The relative content of AA in serum increased and the relative content ofDHA decreased in type2diabetic patients.Part Two Study of correlation between SNP in FADS1-FADS2genecluster and serum PUFAs, desaturase activity and the riskof type2diabetesObjective: To investigate the correlation between the SNPs inFADS1-FADS2gene cluster and the risk of type2diabetes, the PUFAs anddesaturase activity in serum of type2diabetic patients.Methods: A case-control study was employed in this study.752subjectswere collected and divided into2groups, controls (421cases) and the patientswith type2diabetes (331cases)(the inclusion and exclusion criteria as sameas part one of this study). Peripheral blood was collected to extract DNA forgenotyping by Matrix Assisted Laser Desorption/ionization Time of FlightMass Spectrometry (MALDI-TOF MS). Logistic regression was applied to case-control comparisons for the different phenotypic outcomes forco-dominant, dominant and recessive genetic models, under unadjustedcondition, adjusted for sex and age and adjusted for sex, age and BMI toanalyze the association between SNPs in rs174545, rs2072114, rs174602andrs174616and serum PUFAs, desaturase activity and the risk of type2diabetes.Results：1To analyze the content of serum PUFAs and desaturase activity according tothe genotypeIn order to investigate the SNPs in FADS1-FADS2gene cluster andserum PUFAs metabolism in type2diabetic patients, the serum PUFAscontent and the ratio of AA/LA and EPA/ALA were analyzed in healthysubjects and patients with type2diabetes, according to the genotype.1.1The effect of SNP in rs174545on PUFAs and desaturase activityIn healthy people, the content of serum LA increased in subjects carryingG in rs174545(C>G), AA decreased; in patients with type2diabetes, not onlythe content of serum LA increased and AA decreased, but also the contentEPA and DPA declined in subjects carrying G in rs174545(C>G)(P<0.05). Inaddition, the ratio AA/LA and EPA/ALA in serum also decreased in subjectsboth healthy people and type2diabetic patients carrying G in rs174545(C>G)(P<0.05).1.2The effect of SNP in rs2072114on PUFAs and desaturase activityIn healthy people, the content of serum AA decreased in subjectscarrying G in rs2072114(A>G); in type2diabetic patients, not only thecontent of serum LA increased and AA decreased, but also the content EPAand DPA declined in subjects carrying G in rs2072114(A>G)(P<0.05). Inaddition, the ratio AA/LA and EPA/ALA in serum also decreased in subjectsboth healthy people and type2diabetic patients carrying G in rs2072114(A>G)(P<0.05). 1.3The effect of SNP in rs174602on PUFAs and desaturase activityIn healthy people, the content of serum LA increased in subjects carryingG in rs174602(A>G), AA and EPA decreased; in type2diabetic patients notonly the content of serum LA increased and AA decreased, but also thecontent DPA declined in subjects carrying G in rs174602(A>G)(P<0.05). Inaddition, the ratio AA/LA and EPA/ALA in serum also decreased in subjectshealthy people, and the ratio EPA/ALA in serum in type2diabetic patientscarrying G in rs174602(A>G)(P<0.05).1.4The effect of SNP in rs174616on PUFAs and desaturase activityIn healthy people, the content of serum AA decreased in subjectscarrying T in rs174616(C>T); in type2diabetic patients, LA increased insubjects carrying T in rs174616(C>T)(P<0.05); no effect on other PUFAs. Inaddition, the ratio AA/LA in serum also decreased in subjects both healthypeople and type2diabetic patients carrying T in rs174616(C>T)(P<0.05).The above results showed that the SNP mutation in rs174545, rs2072114,rs174602and rs174616can influence PUFAs content and ratio of AA/LA and(or) EPA/ALA in both health people and type2diabetic patients, but the samemutation appears different changes in healthy people and type2diabeticpatients. Among them, SNP in rs174616only effects on metabolism ofn-6PUFAs.2Association SNPs in FADS1-FADS2gene cluster with the risk of type2diabetes2.1Single factor analysis (not excluding the confounding factors)The results showed that rs174616C>T and the risk of type2diabetes hasnegative correlation. In codominant model, individuals carrying rs174616CTand rs174616TT were not susceptible of type2diabetes, the OR values were0.708(95%CI=0.508,0.987, P=0.041) and0.720(95%CI=0.521,0.985,P=0.043); the same results as well as dominant models (P＜0.001), the ORvalue was0.717(95%CI=0.518,0.992, P=0.042). In the recessive model,were not susceptible of type2diabetes, while in the model, there are norelation between rs174616C>T with risk of type2diabetes. In the3models above,174545, rs2072114and rs174602were not relatedwith the risk of type2diabetes.2.2Adjust the gender and age (Adjustment1).Rs174616C>T was related to the risk of type2diabetes in codominantand dominant model after adjusting the gender and age(P＜0.001). In addition,174545, rs2072114and rs174602were not related with the risk of type2diabetes after adjusting the gender and age.2.3Adjust the gender, age and BMI (Adjustment2).Rs174616C>T was related to the risk of type2diabetes in codominantand dominant model after adjusting the gender, age and BMI (P＜0.001). Inaddition,174545, rs2072114and rs174602were not related with the risk oftype2diabetes after adjusting the gender, age and BMI.The above results showed that after adjusting gender, age and BMI, theindividuals carrying rs174616(CT+TT) have a low risk of type2diabetesmellitus, and mutation of this site is a protective factor for type2diabetes.While, rs174545, rs2072114and rs174602mutations have no relation withtype2diabetes.3To analyze the effect of SNP in rs174616on serum PUFAs according to thedominant modelThe results showed that in healthy people carrying rs174616(CT+TT),the ratio AA/LA decreased; in type2diabetic patients carryingrs174616(CT+TT) ratio of AA/LA decreased and LA increased. No changesfound in n-3PUFAs.In summary, after adjusting the gender, age and BMI on interference,individuals carrying rs174616(CT+TT) have a low risk of type2diabetesmellitus, maybe related to the reduce of AA synthesis.Summary：1In the Han population in northern China, the SNPs of FADS1-FADS2genecluster in rs174545, rs2072114, rs174602and rs174616can reduce△5Dand△6D activity both in health people and in type2diabetic patients,and further affect the content of LA, AA and EPA in serum. These SNPs in FADS1-FADS2gene cluster have no effect on the amount of ALA andDHA.2In the Han population in northern China, there is a negativelycorrelationship between the SNP mutation of FADS1-FADS2gene clusterin rs174616and the incidence of type2diabetes, may be related to thereduce of AA synthesis.Part Three Enhanced activity of peroxisomes beta oxidation wasrelated to the increased DHA in the liver of type2diabeticrat liverObjective: To study the mechanism of changed DHA from its synthesisin the liver in type2diabetesMethods: DHA biosynthesis was investigated in a diabetic model bystudying liver fatty acid composition,△5-desaturase (△5D) and△6-desaturase (△6D) gene expression levels and peroxisomal β-oxidation activity.Moreover, the expression levels of genes involved in oxidation were evaluated,as were the relationships between changes in DHA biosynthesis andperoxisomal β-oxidation.Results：1Fatty acid profiles in the liver of ratsBecause of the high-fat diets, C18:0and C18:1n-9increased, resulting inan increase in total fatty acids and a higher ratio of SFAs/PUFAs in the liversof diabetic rats. Among the SFAs, another more obvious change was adecrease in C24:0. For the PUFAs in the livers of diabetic rats, most of thedetected PUFAs decreased, but DHA and the product/precursor ratio,DHA/ALA, increased by almost2-fold and by more than5-fold respectively.These results suggest that the increased DHA might be due to the increase inbiosynthesis in the livers of diabetic rats.2The expression of key enzymes in the DHA synthesis in the livers of ratsIn the livers of diabetic rats, the mRNA and protein levels of△5D and△6D in the ER were decreased, compared to the control animals. However, the mRNA and protein levels of all of the enzymes: straight-chain acyl-CoAoxidase (SCOX), D-bifunctional protein (DBP) and3-Ketoacyl-CoA thiolase(THL), except for sterol carrier protein X (SCPx), in the peroxisome ofdiabetic livers were increased compared to the control animals. Thus, theincreased DHA in the livers of high-fat and low-dose STZ-induced diabeticrats was caused by the β-oxidation enzymes in peroxisome but not by those inthe endoplasmic reticulum (ER).3The activity of DHA biosynthesis in the peroxisome of the livers of ratsThe oxidation activity of peroxisomal β-oxidation was enhanced in thelivers of diabetic rats, in agreement with both increased expressions of theperoxisomal β-oxidation enzymes in DHA biosynthesis and the decreasedlevels of C24:0(very long-chain fatty acid, oxidized only through peroxisomalβ-oxidation) described above. In addition, after a4-h incubation of diabeticliver homogenate with C24:6n-3, substantially lower levels of C24:6n-3andhigher levels of DHA were observed, suggesting that the diabetic liver hasgreater biosynthesis capacity of DHA from C24:6n-3than normal controllivers. These changes were in agreement with the increase in DHA in diabeticlivers.Summary：1In the liver of type2diabetic rats induced by high-fat diet and low-dosestreptozotocin (STZ), the content of DHA increased, but other mostPUFAs decreased significantly.2The increased activity of peroxisomal β-oxidation was related to increasedDHA in the liver of type2diabetic rat.Conclusions：1Not only the total free fatty acids, but the profile of serum fatty acids intype2diabetic patients was changed significantly. In type2diabeticpatients, the relative content of AA in serum increased and the relativecontent of DHA decreased. 2In the Han population in northern China, the SNP of FADS1-FADS2genecluster in rs174616is negatively associated to type2diabetes, which maybe related to the reduce of AA synthesis.3The synthesis of DHA was increased in the liver of type2diabetic ratsinduced by high-fat diet and low-dose streptozotocin (STZ), which wasrelated to increased activity of peroxisomal β-oxidation. |
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