2型糖尿病PUFAs代谢异常与相关基因多态性及过氧化物酶体β-氧化关系的研究
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摘要
2型糖尿病（type2diabetes mellitus，DM）是由于胰岛素相对缺乏或胰岛素抵抗（insulin resistance，IR）而引起的代谢紊乱性疾病，可导致多器官功能损害。近年来，其发病率逐年升高，已成为严重威胁人们健康的全球性疾病。因此，深入了解其发病机制，对于2型糖尿病的预防及治疗有着重要意义。
脂肪酸代谢紊乱已被公认为是2型糖尿病的一个重要特征，2型糖尿病时，各种脂肪酸，尤其是多不饱和脂肪酸，如何改变？与2型糖尿病的关系如何？却研究甚少。
相关基因的单核苷酸多态性（single nucleotide polymorphism，SNP）是从基因水平影响体内脂肪酸代谢的重要因素，并且与2型糖尿病的发生发展密切相关。有研究表明，为多不饱和脂肪酸合成过程中关键酶，△5去饱和酶（△5desaturase，△5D）和△6去饱和酶（△6desaturase，△6D）编码的基因---脂肪酸去饱和酶1（fatty acid desaturase1，FADS1）和2（FADS2）基因的SNPs突变与体内脂肪酸关系密切，那么FADS基因簇的SNP突变是否与2型糖尿病时脂肪酸代谢紊乱有关？
而且，除了基因多态性能够影响体内脂肪酸代谢以外，体内参与脂肪酸代谢的相关酶的表达及活性也是影响脂肪酸代谢的重要因素。肝脏是体内脂肪酸合成的主要器官，也是为肝外组织提供脂肪酸的主要器官。2型糖尿病时，肝脏脂肪酸的合成又会发生哪些改变？与2型糖尿病时的脂肪酸代谢紊乱关系如何？值得进一步研究。
因此，本研究首先对2型糖尿病患者血清中脂肪酸谱进行检测及分析，以了解2型糖尿病患者体内脂肪酸变化情况，选出与2型糖尿病关系密切的脂肪酸作为研究的靶点，从基因水平以及肝脏对脂肪酸合成影响角度深入探讨2型糖尿病时脂肪酸代谢紊乱的机制，为2型糖尿病的预防和治疗提供有力的实验证据。
第一部分2型糖尿病患者血清脂肪酸谱分析
目的：了解2型糖尿病患者体内脂肪酸代谢改变情况，为研究2型糖尿病时脂肪酸代谢紊乱机制寻找靶点。
方法：选择符合条件的研究对象共752例，按照病例对照研究分组（健康人421例，2型糖尿病患者331例），收集空腹血清，测定空腹血糖（fastingblood glucose, FBG）、胰岛素（fasting insulin, FINs）、甘油三酯（triglycerides,TGs）、胆固醇（total cholesterol, TC）及总游离脂肪酸（free fatty acids,FFAs），计算体重指数（body mass index, BMI）；用高效气相色谱法测定所有受试者空腹血清中主要的14种脂肪酸，并进行分类分析，以了解2型糖尿病患者体内脂肪酸代谢情况。
结果：
1一般情况及生化指标
2型糖尿病（DM组）与健康非糖尿病组（Con组）的性别比例一致，没有差异。与Con组相比，DM组的年龄64(10) vs.51.38(18)，明显高于Con组，P<0.001；DM组的BMI（25.568±3.316vs.23.177±2.434）同样明显增加，P<0.001。
与Con组相比，DM组FBG、FINs、TC及TGs均明显增高，P<0.001；通过公式计算得DM组的胰岛素敏感指数（insulin sensitive index, ISI）明显下降，而胰岛素抵抗指数（HOMA-IR）则明显增加，P<0.001。血清总FFAs明显增加，（403.39±82.54μmol/L vs.658.62±125.75μmol/L），P<0.001。
2血清脂肪酸谱
脂肪酸谱显示：与Con相比，DM组血清中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）及C20:4n-6（arachidonic acid, AA）百分含量明显增加，P<0.01；C18:0、C24:0、C22:6n-3（docosahexenoic acid,DHA）和C18:2n-6（linoleic acid, LA）的百分含量明显下降，P<0.01；只有C20:0、C22:0和C22:4n-6变化不明显。
对各种脂肪酸进行归类分析，发现：与Con组相比，DM组的总单不饱和脂肪酸（monounsaturated fatty acids，MUFAs）所占比例明显升高，P<0.01；而总饱和脂肪酸（saturated fatty acid, SFAs），总n-3系多不饱和脂肪酸（polyunsaturated fatty acids，n-3PUFAs）和总n-6系多不饱和脂肪酸（n-6PUFAs）均没有明显变化。
小结：
12型糖尿病患者体内总游离脂肪酸增加。
22型糖尿病患者血清脂肪酸谱发生明显改变，AA含量明显增加，DHA
的含量明显下降。
第二部分FADS1-FADS2基因簇单核苷酸多态性与血清PUFAs、去饱和酶活性及2型糖尿病的关联性研究
目的：探讨FADS1-FADS2基因簇的单核苷酸多态性（single nucleotidepolymorphism, SNP）与2型糖尿病患者血清PUFAs及2型糖尿病之间的相关性。
方法：选择符合条件的研究对象752例（入选和排除标准同第一部分研究），收集外周血，提取DNA，用基质辅助激光解吸电离飞行时间质谱（Matrix Assisted Laser Desorption/ionization Time of Flight MassSpectrometry，MALDI-TOF MS）实验平台进行DNA的SNPs分型。用非条件Logistic回归逐一分别在未校正混杂因素；校正性别和年龄；校正性别、年龄和BMI时，分析rs174545、rs2072114、rs174602和rs174616四个SNP位点与2型糖尿病患者血清PUFAs、去饱和酶活性以及2型糖尿病发病风险的相关性。
结果：
1按照基因型分析血清PUFAs的含量及去饱和酶活性
1.1rs174545位点突变对血清PUFAs及去饱和酶活性的影响
在健康人中，rs174545（C>G）位点携带G基因型的个体血清LA的相对含量增加，AA的含量相对降低；在2型糖尿病患者中，rs17454（5C>G）位点携带G基因型的个体不仅血清LA相对含量增加，AA相对含量降低，还出现了EPA与DPA相对含量的下降（P<0.05）。另外，rs174545（C>G）位点携带G基因型还可以使健康人和2型糖尿病患者血清中AA/LA和EPA/ALA的比值下降（P<0.05）。
1.2rs2072114位点突变对血清PUFAs及去饱和酶活性的影响
在健康人中，rs2072114（A>G）位点携带G基因型的个体血清AA的含量相对降低；在2型糖尿病患者中，rs2072114（A>G）位点携带G基因型的个体不仅血清LA相对含量增加，AA相对含量降低，还出现了EPA与DPA相对含量的下降（P<0.05）。另外，rs2072114（A>G）位点携带G基因型还可以使健康人和2型糖尿病患者血清中AA/LA和EPA/ALA的比值下降（P<0.05）。
1.3rs174602位点突变对血清PUFAs及去饱和酶活性的影响
在健康人中，rs174602（A>G）位点携带G基因型的个体血清LA的含量相对增加，AA和EPA的含量相对降低；在2型糖尿病患者中，rs174602（A>G）位点携带G基因型的个体不仅血清LA相对含量增加，AA相对含量降低，还出现了DPA相对含量的下降（P<0.05）。另外，rs174602（A>G）位点携带G基因型还可以使健康人血清中AA/LA和EPA/ALA，及2型糖尿病患者组血清中EPA/ALA比值下降（P<0.05）。
1.4rs174616位点突变对血清PUFAs及去饱和酶活性的影响
在健康人中，rs174616（C>T）位点携带T基因型的个体血清AA含量相对降低，对其他PUFAs没有影响；在2型糖尿病患者中，rs174616（C>T）位点携带T基因型的个体血清LA相对含量增加（P<0.05），对其他PUFAs也没有影响。另外，rs174616（C>T）位点携带T基因型还可以使健康人和2型糖尿病患者血清中AA/LA的比值下降（P<0.05）。
以上结果说明：rs174545、rs2072114、rs174602和rs174616均可以影响健康人和2型糖尿病患者体内的PUFAs含量与AA/LA和（或）EPA/ALA的比值，其中rs174616只对n-6系PUFAs的代谢有影响。另外，同一种突变因素在健康人和2型糖尿病体内引起的表现不同。
2FADS1-FADS2基因簇的SNP突变与2型糖尿病发病风险的相关性研究
2.1Logistic回归分析（未剔除混杂因素）
结果显示：rs174616C>T与2型糖尿病的发病风险存在着负相关。在共显性模型下，与携带rs174616CC基因型的个体比较，携带rs174616CT和rs174616TT基因型的个体发生2型糖尿病的风险降低，OR值分别为0.708（95%CI=0.508,0.987, P=0.041）和0.720（95%CI=0.521,0.985，P=0.043）；在显性模型下，与携带rs174616CC基因型的个体比较，携带rs174616（CT+TT）基因型的个体发生2型糖尿病的风险降低，OR值为0.717（95%CI=0.518,0.992，P=0.042）；而在隐性模型下，与携带rs174616（CC+CT）基因型的个体比较，携带rs174616TT基因型的个体发生2型糖尿病的风险没有变化。
rs174545，rs2072114和rs174602，与2型糖尿病的发病风险无关。以健康者为对照组，以2型糖尿病为病例组，在3种模型下差异均无统计学意义。
2.2调整性别与年龄（调整1）
结果显示：在调整性别与年龄之后，rs174616C>T与2型糖尿病的发生率仍成负相关。在共显性模型下，携带rs174616CT和rs174616TT基因型的个体比携带rs174616CC基因型的个体，发生2型糖尿病的风险降低，OR值为0.628（95%CI=0.437,0.901，P=0.011）和0.619（95%CI=0.441,0.889，P=0.010）；在显性模型下，携带rs174616（CT+TT）基因型的个体比携带rs174616CC基因型的个体发生2型糖尿病的风险降低，OR值为0.630（95%CI=0.442,0.897，P=0.01）；在隐性模型下，rs174616的基因型与2型糖尿病发病仍然无关。
调整性别与年龄后，其他3个位点：rs174545，rs2072114和rs174602仍与2型糖尿病的发病风险无关。
2.3调整性别、年龄和BMI（调整2）
结果显示：在调整性别、年龄与BMI之后，rs174616C>T与2型糖尿病的发生率仍成负相关。在共显性模型下，携带rs174616CT和rs174616TT基因型的个体比携带rs174616CC基因型的个体，发生2型糖尿病的风险降低，OR值为0.656（95%CI=0.447,0.964，P=0.032）和0.652（95%CI=0.440,0.931，P=0.020）；在显性模型下，携带rs174616（CT+TT）基因型的个体比携带rs174616CC基因型的个体发生2型糖尿病的风险降低，OR值为0.646（95%CI=0.443,0.943，P=0.023）；在隐性模型下，rs174616的基因型与2型糖尿病发病仍然无关。
调整性别、年龄和BMI后，其他3个位点： rs174545，rs2072114和rs174602仍与2型糖尿病的发病风险无关。
以上结果说明，在排除了性别、年龄和BMI的干扰后，携带突变型基因rs174616（CT+TT）的个体比携带野生型基因rs174616CC的个体，发生2型糖尿病的风险下降，即该位点的突变是2型糖尿病发病的保护因素。而rs174545，rs2072114和rs174602三个位点的突变则与2型糖尿病的发病风险无关。
3按照显性模型分析rs174616位点SNP突变对血清PUFAs及去饱和酶活性的影响
为了进一步探讨rs174616与2型糖尿病患者血清PUFAs代谢的关系，接下来我们按照rs174616CC和rs174616（CT+TT）基因型分别对健康非糖尿病患者和2型糖尿病患者血清PUFAs含量，以及AA/LA和EPA/ALA的比值进行了分析。结果显示：在健康人，携带rs174616（CT+TT）基因型的个体比携带rs174616CC基因型的个体AA/LA的比值下降；在2型糖尿病患者中。携带rs174616（CT+TT）基因型的个体比携带rs174616CC基因型的个体LA含量增加，AA/LA比值下降。另外，rs174616位点突变对n-3系PUFAs及EPA/ALA均没有影响。
小结：
1在中国北方汉族人群中，FADS1-FADS2基因簇中rs174545、rs2072114、rs174602和rs174616四个突变位点都能降低健康人和2型糖尿病患者△5D和△6D的活性，从而影响血清中LA、AA和EPA的含量，而对ALA和DHA的含量没有影响。
2在中国北方汉族人群中，FADS1-FADS2基因簇中rs174616位点突变与2型糖尿病的发生呈负相关，即携带rs174616（CT+TT）基因型的个体2型糖尿病的发病风险明显下降，可能与rs174616（CT+TT）基因型能够降低体内AA的合成有关。
第三部分过氧化物酶体β-氧化活性增强与2型糖尿病大鼠肝脏DHA合成增加有关
目的：从肝脏合成脂肪酸角度探讨2型糖尿病时DHA变化的机制
方法：用高脂饮食加小剂量链脲左菌素（streptozotocin, STZ）的方法诱导2型糖尿病大鼠模型，检测2型糖尿病大鼠肝脏脂肪酸谱；△5去饱和酶（△5-desaturase,△5D）和△6去饱和酶（△6D）表达水平；过氧化物酶体β氧化活性和相关酶的表达及DHA合成活性等方面去研究肝脏DHA合成情况，为寻找2型糖尿病时体内DHA改变的原因提供实验依据。
结果：
12型糖尿病大鼠肝脏脂肪酸谱
由于食用的是高脂饮食，与Con组大鼠相比，2型糖尿病大鼠肝脏中C18:0和C18:1n-9（高脂饮食中主要的脂肪酸）明显增加；SFAs/PUFAs的比值增高，而长链饱和脂肪酸C24:0则明显降低。另外，与Con组大鼠相比，2型糖尿病大鼠肝脏中绝大多数的PUFAs（ALA、LA、AA、EPA和DPA）和总PUFAs都明显下降，但是我们却意外的发现DHA和C22:4n-6明显增加，几乎是Con组的2倍；而且2型糖尿病大鼠肝脏DHA/ALA的比值也增加了，大约是Con组的5倍。因此，以上结果提示，
2型糖尿病大鼠肝脏DHA的含量增加，增加的DHA可能是由于在合成增加引起的。
22型糖尿病大鼠肝脏DHA合成过程中关键酶的表达
为了进一步研究2型糖尿病大鼠肝脏DHA增多与DHA合成代谢的关系，我们检测了DHA合成过程中关键酶的表达，结果显示：与Con组相比，2型糖尿病大鼠肝脏△5D和△6D的mRNA和蛋白质表达水平均下降，而过氧化物酶体β氧化过程中的酶直链脂酰CoA氧化酶（straight-chainacyl-CoA oxidase, SCOX）、D双功能蛋白（D-bifunctional protein, DBP）和3-酮脂酰CoA硫解酶（3-Ketoacyl-CoAthiolase, THL）的表达增加。由此推断，2型糖尿病大鼠肝脏DHA增多是由于过氧化物酶体阶段β氧化增强引起的，而与内质网阶段的去饱和、延长反应无关。
32型糖尿病大鼠肝脏过氧化物酶体β氧化阶段DHA合成活性
为了证实过氧化物酶体β氧化阶段与2型糖尿病时肝脏DHA合成增多有关，首先，我们对2型糖尿病大鼠肝脏过氧化物酶体β氧化活性进行了测定。结果显示过氧化物酶体β氧化活性增加。这一结果与此过程中的酶表达增加相符，也与前面的结果中提到2型糖尿病大鼠肝脏C24:0含量下降一致，因为极长链脂肪酸C24:0在体内只能够通过过氧化物酶体被氧化代谢。
接下来，我们用C24:6n-3作为底物，与肝组织匀浆共孵育，直接检测过氧化物酶体β氧化阶段DHA的合成。结果显示：C24:6n-3与肝组织匀浆共孵育4小时后，2型糖尿病大鼠肝脏增加的DHA和减少的C24:6n-3均比对照组变化明显。由此可见，2型糖尿病大鼠肝脏过氧化物酶体β氧化活性增加与DHA合成增强有关。
小结：
1高脂饮食加小剂量STZ诱导的2型糖尿病大鼠肝脏绝大多数PUFAs明显减少，而只有DHA含量增加。
22型糖尿病大鼠肝脏DHA增加，增多的DHA与过氧化物酶体β氧化活性增强有关，而与内质网△5和△6去饱和酶的表达下降无关。
结论：
12型糖尿病患者不仅存在血清总游离脂肪酸的增多，而且血清脂肪酸谱也发生了明显变化，n-3系PUFAs中DHA的含量明显下降；n-6系PUFAs中AA的含量明显增加，LA的含量则明显降低。
2在中国北方汉族人群中，FADS1-FADS2基因簇的rs174616（C>T）位点SNP突变与2型糖尿病的发病风险呈负相关，是2型糖尿病发生的保护因素。这种保护作用可能与rs174616突变能够抑制体内AA合成有关。
3肝脏过氧化物酶体β氧化活性增强与2型糖尿病大鼠肝脏DHA合成增加有关。
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 patients
Objective: 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 indexes
Compared 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 acids
Fatty 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 type2diabetes
Objective: 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 genotype
In 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 activity
In 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 activity
In 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 activity
In 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 activity
In 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 type2diabetes
2.1Single 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 model
The 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 liver
Objective: To study the mechanism of changed DHA from its synthesisin the liver in type2diabetes
Methods: 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 rats
Because 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 rats
In 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 rats
The 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.
脂肪酸代谢紊乱已被公认为是2型糖尿病的一个重要特征，2型糖尿病时，各种脂肪酸，尤其是多不饱和脂肪酸，如何改变？与2型糖尿病的关系如何？却研究甚少。
相关基因的单核苷酸多态性（single nucleotide polymorphism，SNP）是从基因水平影响体内脂肪酸代谢的重要因素，并且与2型糖尿病的发生发展密切相关。有研究表明，为多不饱和脂肪酸合成过程中关键酶，△5去饱和酶（△5desaturase，△5D）和△6去饱和酶（△6desaturase，△6D）编码的基因---脂肪酸去饱和酶1（fatty acid desaturase1，FADS1）和2（FADS2）基因的SNPs突变与体内脂肪酸关系密切，那么FADS基因簇的SNP突变是否与2型糖尿病时脂肪酸代谢紊乱有关？
而且，除了基因多态性能够影响体内脂肪酸代谢以外，体内参与脂肪酸代谢的相关酶的表达及活性也是影响脂肪酸代谢的重要因素。肝脏是体内脂肪酸合成的主要器官，也是为肝外组织提供脂肪酸的主要器官。2型糖尿病时，肝脏脂肪酸的合成又会发生哪些改变？与2型糖尿病时的脂肪酸代谢紊乱关系如何？值得进一步研究。
因此，本研究首先对2型糖尿病患者血清中脂肪酸谱进行检测及分析，以了解2型糖尿病患者体内脂肪酸变化情况，选出与2型糖尿病关系密切的脂肪酸作为研究的靶点，从基因水平以及肝脏对脂肪酸合成影响角度深入探讨2型糖尿病时脂肪酸代谢紊乱的机制，为2型糖尿病的预防和治疗提供有力的实验证据。
第一部分2型糖尿病患者血清脂肪酸谱分析
目的：了解2型糖尿病患者体内脂肪酸代谢改变情况，为研究2型糖尿病时脂肪酸代谢紊乱机制寻找靶点。
方法：选择符合条件的研究对象共752例，按照病例对照研究分组（健康人421例，2型糖尿病患者331例），收集空腹血清，测定空腹血糖（fastingblood glucose, FBG）、胰岛素（fasting insulin, FINs）、甘油三酯（triglycerides,TGs）、胆固醇（total cholesterol, TC）及总游离脂肪酸（free fatty acids,FFAs），计算体重指数（body mass index, BMI）；用高效气相色谱法测定所有受试者空腹血清中主要的14种脂肪酸，并进行分类分析，以了解2型糖尿病患者体内脂肪酸代谢情况。
结果：
1一般情况及生化指标
2型糖尿病（DM组）与健康非糖尿病组（Con组）的性别比例一致，没有差异。与Con组相比，DM组的年龄64(10) vs.51.38(18)，明显高于Con组，P<0.001；DM组的BMI（25.568±3.316vs.23.177±2.434）同样明显增加，P<0.001。
与Con组相比，DM组FBG、FINs、TC及TGs均明显增高，P<0.001；通过公式计算得DM组的胰岛素敏感指数（insulin sensitive index, ISI）明显下降，而胰岛素抵抗指数（HOMA-IR）则明显增加，P<0.001。血清总FFAs明显增加，（403.39±82.54μmol/L vs.658.62±125.75μmol/L），P<0.001。
2血清脂肪酸谱
脂肪酸谱显示：与Con相比，DM组血清中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）及C20:4n-6（arachidonic acid, AA）百分含量明显增加，P<0.01；C18:0、C24:0、C22:6n-3（docosahexenoic acid,DHA）和C18:2n-6（linoleic acid, LA）的百分含量明显下降，P<0.01；只有C20:0、C22:0和C22:4n-6变化不明显。
对各种脂肪酸进行归类分析，发现：与Con组相比，DM组的总单不饱和脂肪酸（monounsaturated fatty acids，MUFAs）所占比例明显升高，P<0.01；而总饱和脂肪酸（saturated fatty acid, SFAs），总n-3系多不饱和脂肪酸（polyunsaturated fatty acids，n-3PUFAs）和总n-6系多不饱和脂肪酸（n-6PUFAs）均没有明显变化。
小结：
12型糖尿病患者体内总游离脂肪酸增加。
22型糖尿病患者血清脂肪酸谱发生明显改变，AA含量明显增加，DHA
的含量明显下降。
第二部分FADS1-FADS2基因簇单核苷酸多态性与血清PUFAs、去饱和酶活性及2型糖尿病的关联性研究
目的：探讨FADS1-FADS2基因簇的单核苷酸多态性（single nucleotidepolymorphism, SNP）与2型糖尿病患者血清PUFAs及2型糖尿病之间的相关性。
方法：选择符合条件的研究对象752例（入选和排除标准同第一部分研究），收集外周血，提取DNA，用基质辅助激光解吸电离飞行时间质谱（Matrix Assisted Laser Desorption/ionization Time of Flight MassSpectrometry，MALDI-TOF MS）实验平台进行DNA的SNPs分型。用非条件Logistic回归逐一分别在未校正混杂因素；校正性别和年龄；校正性别、年龄和BMI时，分析rs174545、rs2072114、rs174602和rs174616四个SNP位点与2型糖尿病患者血清PUFAs、去饱和酶活性以及2型糖尿病发病风险的相关性。
结果：
1按照基因型分析血清PUFAs的含量及去饱和酶活性
1.1rs174545位点突变对血清PUFAs及去饱和酶活性的影响
在健康人中，rs174545（C>G）位点携带G基因型的个体血清LA的相对含量增加，AA的含量相对降低；在2型糖尿病患者中，rs17454（5C>G）位点携带G基因型的个体不仅血清LA相对含量增加，AA相对含量降低，还出现了EPA与DPA相对含量的下降（P<0.05）。另外，rs174545（C>G）位点携带G基因型还可以使健康人和2型糖尿病患者血清中AA/LA和EPA/ALA的比值下降（P<0.05）。
1.2rs2072114位点突变对血清PUFAs及去饱和酶活性的影响
在健康人中，rs2072114（A>G）位点携带G基因型的个体血清AA的含量相对降低；在2型糖尿病患者中，rs2072114（A>G）位点携带G基因型的个体不仅血清LA相对含量增加，AA相对含量降低，还出现了EPA与DPA相对含量的下降（P<0.05）。另外，rs2072114（A>G）位点携带G基因型还可以使健康人和2型糖尿病患者血清中AA/LA和EPA/ALA的比值下降（P<0.05）。
1.3rs174602位点突变对血清PUFAs及去饱和酶活性的影响
在健康人中，rs174602（A>G）位点携带G基因型的个体血清LA的含量相对增加，AA和EPA的含量相对降低；在2型糖尿病患者中，rs174602（A>G）位点携带G基因型的个体不仅血清LA相对含量增加，AA相对含量降低，还出现了DPA相对含量的下降（P<0.05）。另外，rs174602（A>G）位点携带G基因型还可以使健康人血清中AA/LA和EPA/ALA，及2型糖尿病患者组血清中EPA/ALA比值下降（P<0.05）。
1.4rs174616位点突变对血清PUFAs及去饱和酶活性的影响
在健康人中，rs174616（C>T）位点携带T基因型的个体血清AA含量相对降低，对其他PUFAs没有影响；在2型糖尿病患者中，rs174616（C>T）位点携带T基因型的个体血清LA相对含量增加（P<0.05），对其他PUFAs也没有影响。另外，rs174616（C>T）位点携带T基因型还可以使健康人和2型糖尿病患者血清中AA/LA的比值下降（P<0.05）。
以上结果说明：rs174545、rs2072114、rs174602和rs174616均可以影响健康人和2型糖尿病患者体内的PUFAs含量与AA/LA和（或）EPA/ALA的比值，其中rs174616只对n-6系PUFAs的代谢有影响。另外，同一种突变因素在健康人和2型糖尿病体内引起的表现不同。
2FADS1-FADS2基因簇的SNP突变与2型糖尿病发病风险的相关性研究
2.1Logistic回归分析（未剔除混杂因素）
结果显示：rs174616C>T与2型糖尿病的发病风险存在着负相关。在共显性模型下，与携带rs174616CC基因型的个体比较，携带rs174616CT和rs174616TT基因型的个体发生2型糖尿病的风险降低，OR值分别为0.708（95%CI=0.508,0.987, P=0.041）和0.720（95%CI=0.521,0.985，P=0.043）；在显性模型下，与携带rs174616CC基因型的个体比较，携带rs174616（CT+TT）基因型的个体发生2型糖尿病的风险降低，OR值为0.717（95%CI=0.518,0.992，P=0.042）；而在隐性模型下，与携带rs174616（CC+CT）基因型的个体比较，携带rs174616TT基因型的个体发生2型糖尿病的风险没有变化。
rs174545，rs2072114和rs174602，与2型糖尿病的发病风险无关。以健康者为对照组，以2型糖尿病为病例组，在3种模型下差异均无统计学意义。
2.2调整性别与年龄（调整1）
结果显示：在调整性别与年龄之后，rs174616C>T与2型糖尿病的发生率仍成负相关。在共显性模型下，携带rs174616CT和rs174616TT基因型的个体比携带rs174616CC基因型的个体，发生2型糖尿病的风险降低，OR值为0.628（95%CI=0.437,0.901，P=0.011）和0.619（95%CI=0.441,0.889，P=0.010）；在显性模型下，携带rs174616（CT+TT）基因型的个体比携带rs174616CC基因型的个体发生2型糖尿病的风险降低，OR值为0.630（95%CI=0.442,0.897，P=0.01）；在隐性模型下，rs174616的基因型与2型糖尿病发病仍然无关。
调整性别与年龄后，其他3个位点：rs174545，rs2072114和rs174602仍与2型糖尿病的发病风险无关。
2.3调整性别、年龄和BMI（调整2）
结果显示：在调整性别、年龄与BMI之后，rs174616C>T与2型糖尿病的发生率仍成负相关。在共显性模型下，携带rs174616CT和rs174616TT基因型的个体比携带rs174616CC基因型的个体，发生2型糖尿病的风险降低，OR值为0.656（95%CI=0.447,0.964，P=0.032）和0.652（95%CI=0.440,0.931，P=0.020）；在显性模型下，携带rs174616（CT+TT）基因型的个体比携带rs174616CC基因型的个体发生2型糖尿病的风险降低，OR值为0.646（95%CI=0.443,0.943，P=0.023）；在隐性模型下，rs174616的基因型与2型糖尿病发病仍然无关。
调整性别、年龄和BMI后，其他3个位点： rs174545，rs2072114和rs174602仍与2型糖尿病的发病风险无关。
以上结果说明，在排除了性别、年龄和BMI的干扰后，携带突变型基因rs174616（CT+TT）的个体比携带野生型基因rs174616CC的个体，发生2型糖尿病的风险下降，即该位点的突变是2型糖尿病发病的保护因素。而rs174545，rs2072114和rs174602三个位点的突变则与2型糖尿病的发病风险无关。
3按照显性模型分析rs174616位点SNP突变对血清PUFAs及去饱和酶活性的影响
为了进一步探讨rs174616与2型糖尿病患者血清PUFAs代谢的关系，接下来我们按照rs174616CC和rs174616（CT+TT）基因型分别对健康非糖尿病患者和2型糖尿病患者血清PUFAs含量，以及AA/LA和EPA/ALA的比值进行了分析。结果显示：在健康人，携带rs174616（CT+TT）基因型的个体比携带rs174616CC基因型的个体AA/LA的比值下降；在2型糖尿病患者中。携带rs174616（CT+TT）基因型的个体比携带rs174616CC基因型的个体LA含量增加，AA/LA比值下降。另外，rs174616位点突变对n-3系PUFAs及EPA/ALA均没有影响。
小结：
1在中国北方汉族人群中，FADS1-FADS2基因簇中rs174545、rs2072114、rs174602和rs174616四个突变位点都能降低健康人和2型糖尿病患者△5D和△6D的活性，从而影响血清中LA、AA和EPA的含量，而对ALA和DHA的含量没有影响。
2在中国北方汉族人群中，FADS1-FADS2基因簇中rs174616位点突变与2型糖尿病的发生呈负相关，即携带rs174616（CT+TT）基因型的个体2型糖尿病的发病风险明显下降，可能与rs174616（CT+TT）基因型能够降低体内AA的合成有关。
第三部分过氧化物酶体β-氧化活性增强与2型糖尿病大鼠肝脏DHA合成增加有关
目的：从肝脏合成脂肪酸角度探讨2型糖尿病时DHA变化的机制
方法：用高脂饮食加小剂量链脲左菌素（streptozotocin, STZ）的方法诱导2型糖尿病大鼠模型，检测2型糖尿病大鼠肝脏脂肪酸谱；△5去饱和酶（△5-desaturase,△5D）和△6去饱和酶（△6D）表达水平；过氧化物酶体β氧化活性和相关酶的表达及DHA合成活性等方面去研究肝脏DHA合成情况，为寻找2型糖尿病时体内DHA改变的原因提供实验依据。
结果：
12型糖尿病大鼠肝脏脂肪酸谱
由于食用的是高脂饮食，与Con组大鼠相比，2型糖尿病大鼠肝脏中C18:0和C18:1n-9（高脂饮食中主要的脂肪酸）明显增加；SFAs/PUFAs的比值增高，而长链饱和脂肪酸C24:0则明显降低。另外，与Con组大鼠相比，2型糖尿病大鼠肝脏中绝大多数的PUFAs（ALA、LA、AA、EPA和DPA）和总PUFAs都明显下降，但是我们却意外的发现DHA和C22:4n-6明显增加，几乎是Con组的2倍；而且2型糖尿病大鼠肝脏DHA/ALA的比值也增加了，大约是Con组的5倍。因此，以上结果提示，
2型糖尿病大鼠肝脏DHA的含量增加，增加的DHA可能是由于在合成增加引起的。
22型糖尿病大鼠肝脏DHA合成过程中关键酶的表达
为了进一步研究2型糖尿病大鼠肝脏DHA增多与DHA合成代谢的关系，我们检测了DHA合成过程中关键酶的表达，结果显示：与Con组相比，2型糖尿病大鼠肝脏△5D和△6D的mRNA和蛋白质表达水平均下降，而过氧化物酶体β氧化过程中的酶直链脂酰CoA氧化酶（straight-chainacyl-CoA oxidase, SCOX）、D双功能蛋白（D-bifunctional protein, DBP）和3-酮脂酰CoA硫解酶（3-Ketoacyl-CoAthiolase, THL）的表达增加。由此推断，2型糖尿病大鼠肝脏DHA增多是由于过氧化物酶体阶段β氧化增强引起的，而与内质网阶段的去饱和、延长反应无关。
32型糖尿病大鼠肝脏过氧化物酶体β氧化阶段DHA合成活性
为了证实过氧化物酶体β氧化阶段与2型糖尿病时肝脏DHA合成增多有关，首先，我们对2型糖尿病大鼠肝脏过氧化物酶体β氧化活性进行了测定。结果显示过氧化物酶体β氧化活性增加。这一结果与此过程中的酶表达增加相符，也与前面的结果中提到2型糖尿病大鼠肝脏C24:0含量下降一致，因为极长链脂肪酸C24:0在体内只能够通过过氧化物酶体被氧化代谢。
接下来，我们用C24:6n-3作为底物，与肝组织匀浆共孵育，直接检测过氧化物酶体β氧化阶段DHA的合成。结果显示：C24:6n-3与肝组织匀浆共孵育4小时后，2型糖尿病大鼠肝脏增加的DHA和减少的C24:6n-3均比对照组变化明显。由此可见，2型糖尿病大鼠肝脏过氧化物酶体β氧化活性增加与DHA合成增强有关。
小结：
1高脂饮食加小剂量STZ诱导的2型糖尿病大鼠肝脏绝大多数PUFAs明显减少，而只有DHA含量增加。
22型糖尿病大鼠肝脏DHA增加，增多的DHA与过氧化物酶体β氧化活性增强有关，而与内质网△5和△6去饱和酶的表达下降无关。
结论：
12型糖尿病患者不仅存在血清总游离脂肪酸的增多，而且血清脂肪酸谱也发生了明显变化，n-3系PUFAs中DHA的含量明显下降；n-6系PUFAs中AA的含量明显增加，LA的含量则明显降低。
2在中国北方汉族人群中，FADS1-FADS2基因簇的rs174616（C>T）位点SNP突变与2型糖尿病的发病风险呈负相关，是2型糖尿病发生的保护因素。这种保护作用可能与rs174616突变能够抑制体内AA合成有关。
3肝脏过氧化物酶体β氧化活性增强与2型糖尿病大鼠肝脏DHA合成增加有关。
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 patients
Objective: 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 indexes
Compared 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 acids
Fatty 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 type2diabetes
Objective: 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 genotype
In 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 activity
In 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 activity
In 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 activity
In 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 activity
In 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 type2diabetes
2.1Single 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 model
The 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 liver
Objective: To study the mechanism of changed DHA from its synthesisin the liver in type2diabetes
Methods: 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 rats
Because 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 rats
In 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 rats
The 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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