高血糖后代父系和母系糖脂代謝障礙的差異研究

陳文秀 叢 林 姚 潔 袁 靜 陳 薇 方慧琴

高血糖后代父系和母系糖脂代謝障礙的差異研究

陳文秀 叢 林 姚 潔 袁 靜 陳 薇 方慧琴

目的 探討糖尿病合并妊娠和妊娠期糖尿病(GDM)的SD大鼠后代中父系和母系糖脂代謝障礙的差異。方法分別把SD大鼠用STZ小劑量腹腔注射和STZ加高糖高脂飲食誘導(dǎo)成GDM和糖尿病合并妊娠模型，得到F1代大鼠后，分別與正常異性8周大鼠雜交產(chǎn)生F2代，分為對(duì)照組、GDM母系組、GDM父系組、糖尿病合并妊娠母系組與糖尿病合并妊娠父系組。測(cè)定F2代SD大鼠體質(zhì)量、血糖、胰島素、三酰甘油及瘦素水平。 結(jié)果 <1周時(shí)，GDM父系組和GDM母系組體質(zhì)量差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；8周時(shí)，糖尿病合并妊娠父系組和糖尿病合并妊娠母系組體質(zhì)量差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；<1周時(shí)的糖尿病合并妊娠組和8周時(shí)GDM組，父系組和母系組體質(zhì)量差異均無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)。8周時(shí)，糖尿病合并妊娠父系組和糖尿病合并妊娠母系組的胰島素水平、三酰甘油水平差異有統(tǒng)計(jì)學(xué)意義(P<0.05)，空腹血糖水平、瘦素水平差異均無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)。GDM父系組與GDM母系組的空腹血糖水平、胰島素水平和三酰甘油水平差異有統(tǒng)計(jì)學(xué)意義(P<0.05)，瘦素水平差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)。 結(jié)論 糖尿病大鼠的糖脂代謝障礙存在父系和母系差異，糖尿病合并妊娠性成熟期和GDM組出生時(shí)體質(zhì)量差異父系組顯著高于母系組。糖尿病合并妊娠母系組和GDM母系組TG水平的代謝障礙可能更明顯。子代代謝障礙可能與宮內(nèi)高血糖環(huán)境有關(guān)。

妊娠期糖尿病；糖尿病合并妊娠；父系；母系；糖脂代謝

妊娠期糖尿病(gestational diabetes mellitus,GDM)可能引起機(jī)體代謝異常的機(jī)制被稱(chēng)為“胎兒編程”或代謝記憶[1]。宮內(nèi)高血糖影響后代基因甲基化，從而影響胎兒的生長(zhǎng)發(fā)育[2]。在蛋白質(zhì)水平方面， PGC-1α基因甲基化的表達(dá)可能導(dǎo)致代謝異常[3]。在基因方面，胰腺組織中表達(dá)量最多的是過(guò)氧化物酶體增殖激活受體γ 輔激活因子1A (peroxisome proliferatoractivated receptor-γ coactivator-1A,PPARGC1A) 基因，PPARGC1A是調(diào)控線粒體新陳代謝的關(guān)鍵轉(zhuǎn)錄因子，外界生理刺激可以通過(guò)它來(lái)調(diào)控線粒體的生物合成[4]。GDM 和糖尿病合并妊娠可能通過(guò)誘導(dǎo)子代PPARGC1A 基因發(fā)生高甲基化，使子代成年期發(fā)生糖、脂代謝紊亂的風(fēng)險(xiǎn)升高[5]。表觀遺傳在性別傳代方面研究少見(jiàn)，Ding等[6]研究發(fā)現(xiàn)糖耐量減低(impaired glucose tolerance,IGT)在父系后代進(jìn)展比母系更顯著。為探討是否存在性別傳代差異，本研究檢測(cè)了SD大鼠父系和母系的糖脂類(lèi)代謝水平。

1 材料與方法

1.1 大鼠動(dòng)物模型的建立及繁殖 實(shí)驗(yàn)動(dòng)物的選擇和糖尿病動(dòng)物模型制備詳見(jiàn)我們的前期研究[5]。除前期研究中每組隨機(jī)選取12只處死進(jìn)行F1代研究外，現(xiàn)從大量的F1代每組隨機(jī)選取4只雌鼠和4只雄鼠進(jìn)入下一代與正常異性大鼠繁殖，并從F2代中每組隨機(jī)選取8只進(jìn)行研究。統(tǒng)計(jì)學(xué)理論上可以和處死的F1代進(jìn)行遺傳學(xué)比較。

1.2 F2代的分組方法 當(dāng)F1代大鼠達(dá)到8周性成熟時(shí)，將雌性F1代大鼠與正常雄鼠交配，產(chǎn)生F2代母系SD鼠，將雄性F1代大鼠與正常雌鼠交配產(chǎn)生F2代父系SD鼠。具體方式：糖尿病合并妊娠組分為父系(糖尿病合并妊娠♂- C♀)和母系(糖尿病合并妊娠♀-C♂);GDM組分為父系(GDM♂- C♀)和母系(GDM♀-C♂);以及對(duì)照組(C♀ - C♂)。同F(xiàn)1代喂養(yǎng)方法至8周，每組隨機(jī)選取8只子鼠進(jìn)行研究。由此，F(xiàn)2代分為以下5組：糖尿病合并妊娠父系組、糖尿病合并妊娠母系組、GDM父系組、GDM母系組、對(duì)照組。

1.3 取材 F2代飼養(yǎng)至8 周齡，水合氯醛(天津福晨化學(xué)試劑廠) 腹腔注射0.004 mL/kg 麻醉， 分離并采集胰腺組織，置于無(wú)菌EP管中，-80 ℃冰箱保存。注射器抽取心臟血3 mL，室溫靜置20 min，2 000 r/min離心15 min。分離上層血清置于無(wú)菌EP管中，保存于-80℃冰箱。

1.4 體質(zhì)量及血糖檢測(cè)方法 體質(zhì)量采用上海力衡儀器儀表有限公司的電子天平測(cè)量，精確到0.01 g。血糖檢測(cè)：距大鼠尾尖0.2 cm 處剪斷，從尾根向尾尖按摩，待斷端出現(xiàn)完整血滴時(shí)，用血糖儀(瑞士羅氏診斷產(chǎn)品有限公司)測(cè)量。

1.5 血清生化指標(biāo)的檢測(cè) 采用雙抗體夾心酶聯(lián)免疫吸附(ELISA)法檢測(cè)子鼠血清胰島素(insulin,INS)、三酰甘油(triglyceride,TG)、瘦素(leptin,LEP)的含量，按試劑盒(美國(guó)RB公司)說(shuō)明操作。

2 結(jié)果

2.1 糖尿病合并妊娠組和GDM組體質(zhì)量比較 8周時(shí)，糖尿病合并妊娠父系組和糖尿病合并妊娠母系組體質(zhì)量差異有統(tǒng)計(jì)學(xué)意義(F=25.6，P<0.05)；<1周時(shí)，GDM父系組和GDM母系組體質(zhì)量差異有統(tǒng)計(jì)學(xué)意義(F=4.26，P<0.05)；<1周時(shí)的糖尿病合并妊娠組和8周時(shí)GDM組，父系組和母系組體質(zhì)量差異均無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)。見(jiàn)表1。

表1 糖尿病合并妊娠組、GDM組與對(duì)照組不同時(shí)期體質(zhì)量比較

2.2 糖尿病合并妊娠組和GDM組糖脂類(lèi)指標(biāo)比較 8周時(shí)，糖尿病合并妊娠父系組與母系組的胰島素水平、三酰甘油水平差異有統(tǒng)計(jì)學(xué)意義(P<0.05)，空腹血糖水平、瘦素水平差異均無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)。GDM父系組與母系組的空腹血糖水平、胰島素水平和三酰甘油水平差異有統(tǒng)計(jì)學(xué)意義(P<0.05)，瘦素水平差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)。見(jiàn)表2。

表2 糖尿病合并妊娠組、GDM組與對(duì)照組糖脂類(lèi)指標(biāo)比較

3 討論

GDM對(duì)母親和胎兒均有長(zhǎng)遠(yuǎn)的影響，包括圍產(chǎn)期不良結(jié)果、后代的肥胖及葡萄糖耐受不良的長(zhǎng)期風(fēng)險(xiǎn)等[7]。這些遺傳基因在胚胎期，胎兒期，嬰兒期和青春期對(duì)于組織發(fā)育和生長(zhǎng)的調(diào)節(jié)異常是最敏感的。

Ding 等[6]報(bào)道糖尿病大鼠子代血糖可能受到親代性別差異的產(chǎn)生不同代謝影響，在F2代糖尿病合并妊娠組母系發(fā)育早期，存在體質(zhì)量增加和血糖升高或IGT，這也本研究結(jié)果相似。對(duì)人類(lèi)糖尿病性別傳代的研究較少，國(guó)外有建立在胎兒臍帶血與產(chǎn)婦血清的研究[9]，同類(lèi)研究顯示新生兒GDM組脂肪量顯著高于正常組[10]，但均無(wú)性別差異研究。本實(shí)驗(yàn)中GDM組SD大鼠糖、脂代謝均存在性別傳代差異；糖尿病合并妊娠組存在脂代謝性別傳代差異，母系高于父系。這種可遺傳的糖脂代謝的性別傳代特異性，其機(jī)制可能與脫離宮內(nèi)高血糖環(huán)境有關(guān)，或與胰島β細(xì)胞的功能受損，引發(fā)糖耐量受損和胰島素抵抗有關(guān)[5]。也可能與懷孕期間母體或胎兒胰島素抵抗有關(guān)，宮內(nèi)高糖環(huán)境致胎盤(pán)分泌各種脂肪因子和激素[11]。Boney等[12]的研究闡述了三酰甘油水平存在跨代遺傳，本研究也證實(shí)三酰甘油存在跨代遺傳和性別傳代差異，提示在糖尿病孕婦的后代存在脂代謝紊亂的可能，后代心血管相關(guān)疾病的風(fēng)險(xiǎn)增加[13]。

表觀遺傳機(jī)制可能是參與代謝性疾病的發(fā)病機(jī)理的一個(gè)早期事件，對(duì)表觀遺傳引起的疾病起到預(yù)防和治療作用。本課題組的前期研究顯示，F(xiàn)1代糖尿病合并妊娠組和GDM組大鼠的子代PPARGC1A基因可能發(fā)生了高甲基化狀態(tài)，抑制了其mRNA 的表達(dá)，課題組將在F2代后期研究中繼續(xù)開(kāi)展表觀遺傳的研究。GDM和2型糖尿病都是多基因、多因素疾病，PPARGC1A位于染色體4 p15.1,它在各種組織包括脂肪組織,骨骼肌和胰腺，PPARGC1A與2型糖尿病風(fēng)險(xiǎn)增加有關(guān)[14]。除了DNA甲基化可能會(huì)引起糖脂代謝紊亂，糖尿病后代DNA多態(tài)性產(chǎn)生的后果的相關(guān)描述包括體質(zhì)量、巨舌、眼間距過(guò)遠(yuǎn)和畸形足等[15]。另外，線粒體DNA突變也可以讓部分患者發(fā)展為GDM[16]。研究數(shù)據(jù)證實(shí)高風(fēng)險(xiǎn)多態(tài)性對(duì)2型糖尿病患者的影響，同時(shí)證明配合經(jīng)典遺傳學(xué)方法進(jìn)行前瞻性的研究是有價(jià)值的[17]。為預(yù)防GDM的后代風(fēng)險(xiǎn)增加，需要針對(duì)不同地區(qū)人群的差異施行后代長(zhǎng)期隨訪[18]。

綜上所述，糖尿病大鼠的糖脂代謝障礙存在父系和母系差異，可能糖尿病合并妊娠母系組和GDM母系組TG水平的代謝障礙可能更明顯。

[1] Yessoufou A,Moutairou K.Maternal diabetes in pregnancy: Early and long-term outcomes on the offspring and the concept of “metabolic memory” [J]. Exp Diabetes Res, 2011,218598.

[2] 尤子善.妊娠期糖尿病對(duì)母嬰的影響[J].安徽醫(yī)學(xué),2007,28(2):122-124.

[3] Ma JM,Zeng CJ,Zhang L,et al.Increased hepatic peroxisome proliferator-activated receptor coactivator-1α expression precedes thedevelopment of insulin resistance in offspring of rats from severe hyperglycemic mothers[J]. Chin Med J(Engl), 2012, 125(7):1224-1229.

[4] Koo SH,Satoh H,Herzig S,et al.PGC-1 promotes insulin resistance in liver through PPAR-alpha-dependent induction of TRB-3[J]. Nat Med, 2004, 10(5):530-534.

[5] 王婷,叢林,姚潔,等.過(guò)氧化物酶體增殖物激活受體γ 輔激活因子1A 基因及其表觀遺傳修飾對(duì)孕期糖尿病大鼠糖脂代謝的影響[J].中華圍產(chǎn)醫(yī)學(xué)雜志,2014,17(2):119-122.

[6] Ding GL,Wang FF,Shu J,et al.Transgenerational glucose intolerance with Igf2/H19,Epigenetic alterations in mouse islet induced by intrauterine hyperglycemia[J].Diabetes,2012,61(5)：1133-1142.

[7] Buchanan TA,Xiang AH,Page KA.Gestational diabetes mellitus: risks and management during and after pregnancy[J]. Nat Rev Endocrinol, 2012, 8(11):639-49.

[10] Uebel K,Pusch K,Gedrich K, et al.Effect of maternal obesity with and without gestational diabetes on offspring subcutaneous and preperitoneal adipose tissue development from birth up to year-1[J]. BMC Pregnancy Childbirth, 2014,14(138):1-13.

[11] Lacroix M,Kina E,Hivert MF.Maternal/fetal determinants of insulin resistance in women during pregnancy and in offspring over life[J].Curr Diab Rep,2013,13(2):238-244.

[12] Boney CM,Verma A,Tucker R,et al.Metabolic syndrome in childhood: association with birth weight, maternal obesity, and gestational diabetes mellitus[J].Pediatrics,2005,115(3):e290-296.

[13] Retnakaran R,Ye C,Hanley A,et al.Effect of maternal gestational diabetes on the cardiovascular risk factor profile of infants at 1 year of age[J].Nutr Metab Cardiovasc Dis,2013, 23(12):1175-1181.

[14] Shaat N,Lernmark A,Karlsson E,et al.A variant in the transcription factor 7-like 2 (TCF7L2) gene is associated with an increased risk of gestational diabetes mellitus[J].Diabetologia,2007, 50(5):972-979.

[15] Chen Y,Liao WY,Roy AC,et al.Mitochondrial gene mutations in gestational diabetes mellitus[J].Diabetes Res Clin Pract,2000, 48(1):29-35.

[16] Christian SL,Rich BH,Loebl C,et al.Significance of genetic testing for paternal uniparental disomy of chromosome 6 in neonatal diabetes mellitus[J].J Pediatr,1999,134(1):42-46.

[17] Vaxillaire M,Veslot J,Dina C,et al.Impact of common type 2 diabetes risk polymorphisms in the DESIR prospective study[J]. Diabetes,2008,57(1):244-524.

[18] 叢林.妊娠合并糖尿病臨床診斷與治療[J].安徽醫(yī)學(xué),2009,30(4):364-365.

(2014-09-15收稿 2014-12-10修回)

Investigation on paternal and maternal differences of glycolipid metabolism disorder in hyperglycemia SD rat offsprings

ChenWenxiu,CongLin,YaoJie,etal

DepartmentofObstetricsandGynecology,FirstAffiliatedHospitalofAnhuiMedicalUniversity,Hefei230022,China

Objective To investigate the paternal and maternal differences of glycolipid metabolism disorder in the offsprings of SD rats with diabetic pregnancy and gestational diabetes mellitus (GDM).MethodsThe GDM or diabetic pregnancy models of SD rats were induced by intraperitoneal injection of a small dose of STZ or STZ combined with high-sugar and high-fat diet, and then their F1 generation rats were hybridized with normal heterosexual rats of eight weeks to produce the F2 generation hybrid rats, which were divided into five groups: control group, GDM maternal group, GDM paternal group, diabetic pregnancy maternal group, and diabetic pregnancy paternal group. The body mass, levels of blood glucose, insulin, triglyceride and leptin of the F2 generation SD rats in the five groups were detected and compared.ResultsAt <1 week after grouping, the difference in the body mass between the GDM paternal group and the GDM maternal group was statistically significant (P<0.05), while at 8 weeks, the difference in the body mass between the diabetic pregnancy paternal group and the diabetic pregnancy maternal group also became statistically significant (P<0.05). But in the diabetic pregnancy groups at <1 week and the GDM groups at 8 weeks, comparison with body mass between paternal and maternal groups showed no significant difference (P>0.05). At 8 weeks, there had significant differences in the insulin level and triglyceride level between the diabetic pregnancy paternal group and the diabetic pregnancy maternal group (P<0.05), with no significant differences in the fasting blood glucose level and leptin level between the two groups (P>0.05). The differences in the fasting blood glucose level, insulin level, and triglyceride level between the GDM paternal group and the GDM maternal group were significant (P<0.05), but not in the leptin level (P>0.05).ConclusionThere is paternal and maternal differences of glycolipid metabolism disorder in diabetic rats. The paternal and maternal differences of body mass in the diabetic pregnancy groups within sexual maturity period and the GDM groups within newborn period were significant. The metabolic disorder of triglyceride level in the diabetic pregnancy maternal group and the GDM maternal group may be more obvious. The offspring metabolic disorders may be associated with intrauterine environment of hyperglycemia.

Gestational diabetes mellitus; Diabetic pregnancy; Paternal; Maternal; Glycolipid metabolism

2012年度安徽省自然科學(xué)基金項(xiàng)目(項(xiàng)目編號(hào)：1208085MH172 )

230022 合肥 安徽醫(yī)科大學(xué)第一附屬醫(yī)院婦產(chǎn)科產(chǎn)前診斷中心

叢林，conglin1957@163.com

10.3969/j.issn.1000-0399.2015.02.003