DNA array analysis in OLETF, a type 2 diabetic rats, and LETO, a non-diabetic control rats

AMP deaminase (AMPD) plays a crucial role in adenine nucleotide metabolism, and its upregulated activity contributes to diastolic dysfunction of the heart caused by type 2 diabetes mellitus (T2DM) via reduction of the adenine nucleotide pool. Here we examined the mechanism of AMPD upregulation by T2DM. The protein level of 90-kDa full-length AMPD3 was increased in whole myocardial lysates and in the sarcoplasmic reticulum (SR) fraction by 55% and 123%, respectively, in OLETF, a rat model of T2DM, compared to those in LETO, a non-diabetic control, though AMPD mRNA level was unchanged in OLETF. MicroRNA array analysis revealed downregulation (>50%) of 57 microRNAs in OLETF compared to those in LETO, among which miR-301b was predicted to interact with the 3’UTR of AMPD3 mRNA. The 90-kDa AMPD3 level was significantly increased by miR-301b inhibitor, but decreased by a miR-301b mimetic in H9c2 cells. A luciferase reporter assay indicated binding of miR-301b to the 3’UTR of AMPD3 mRNA. The results indicate that translational regulation by miR-301b mediates AMPD3 upregulation in the diabetic heart. Since ATP generated by SR-associated glycolytic enzymes contributes to Ca2+ uptake by SERCA2a, upregulated AMPD3 in the vicinity of the SR may be a therapeutic target for diabetic cardiomyopathy.腺苷酸脱氨酶（AMP deaminase，AMPD）在腺苷酸代谢中发挥关键作用，其活性上调可通过降低腺苷酸库，参与2型糖尿病（T2DM）诱导的心脏舒张功能障碍。本研究探究了T2DM诱导AMPD表达上调的分子机制。与非糖尿病对照LETO大鼠相比，2型糖尿病大鼠模型OLETF的全心肌裂解物及肌浆网（SR）组分中，90 kDa全长AMPD3的蛋白水平分别升高55%和123%，但OLETF大鼠体内AMPD的mRNA水平未发生显著变化。微RNA芯片（microRNA array）分析显示，OLETF大鼠相较于LETO大鼠，有57种微RNA的表达量下调超过50%，其中预测miR-301b可与AMPD3 mRNA的3'非翻译区（3’UTR）结合。在H9c2细胞中，miR-301b抑制剂可显著升高90 kDa AMPD3的蛋白水平，而miR-301b模拟物则使其水平显著降低。荧光素酶报告基因实验证实，miR-301b可结合AMPD3 mRNA的3’UTR。上述结果表明，miR-301b介导的翻译调控是糖尿病心脏中AMPD3表达上调的关键机制。由于肌浆网关联的糖酵解酶产生的ATP可参与SERCA2a介导的钙离子摄取，肌浆网附近上调的AMPD3或可成为糖尿病心肌病的潜在治疗靶点。