LMBD1 蛋白質於心肌之胰島素訊息調節

Abstract

細胞內的能量恆定調節是維繫細胞正常生理的重要因素，而這能量的恆定調 節的失衡則是導致許多疾病發生的開端。lmbrd1 基因所表達出的LMBD1 蛋白質 是一包含九個預測穿膜結構的膜蛋白質。最近的研究顯示細胞內溶酶體中維他命 B12 的細胞質輸出作用與 lmbrd1 基因的對位雙變異有密切關係，會造成病人的 維生素B12 利用缺乏症，導致新生兒許多早期病理症狀。在本研究中，發現只 要有一 lmbrd1 基因對位的變異，便會導致非維生素B12 利用缺乏相關的心臟疾 病。利用 lmbrd1 基因對位單一變異(lmbrd +/-)的小鼠模式系統，發現 18FDG 在 心臟組織有大量累積，並對外加胰島素的再誘發作用，無明顯反映在額外的 18FDG 的增加累積。並發現在此 lmbrd1+/- 小鼠心臟的胰島素接受體是處於持續 活化的狀態，且心博與心肌收縮也異常增加。導致代償性的心肌肥大與局部受損 相關的纖維化病變。隨著lmbrd1+/-小鼠的老化，其心臟的左心室血液射出分率降 低，伴隨著心室功能的惡化。進一步利用初繼代培養大鼠心室細胞，進行 lmbrd1 基因的抑低表現調控，發現其胰島素受體的自身活化提升，並造成下游Akt 激 酶的活化也提升。以共軛焦螢光顯微鏡與活細胞全反射螢光顯微鏡的觀察結果， 發現 LMBD1 蛋白會與胰島素受體與clathrin 聚集在細胞膜同一位置，並會因胰 島素誘發胰島素受體的內吞作用時，同步一起進入細胞內。利用定點突變置換 LMBD1 蛋白質上可能的AP-2 結合位的胺基酸，結果發現 LMBD1 包含兩個與 胰島素受體細胞內吞作用調節有關的區段。綜合本研究的結果，此 lmbrd1 基因 所表達出的 LMBD1 蛋白會參與調節心臟中胰島素受體的細胞內吞作用，並與 其下游相關活性訊息傳遞調控相關，且從 lmbrd1+/- 小鼠模式系統發現，其對位 單一變異會導致心臟左心室心肌肥大及相關病變可能是與其調控胰島素受體細 胞內吞作用與其活性有密切關係。

Energy homeostasis is crucial in maintaining normal biological functions of cells. Disturbances in such balance often lead to various diseases. Limb region 1 (LMBR1) domain containing 1 gene (lmbrd1) encodes a nine-transmembrane LMBD1 protein. Previous study demonstrated that double allele frameshift mutation of lmbrd1 is associated with lysosomal cobalamin export deficiency, suggesting the participation of LMBD1 in the export of cobalamin from lysosome to the cyotsol. In this study, we have distinguished that heterozygous deletion of lmbrd1 is sufficient for causing cardiac diseases through a pathway independent of the vitamin B12 metabolic defect. lmbrd1 ubiquitous heterozygous knockout lmbrd1+/- mice exhibited increase in myocardial glucose uptake and insulin receptor signaling that were insensitive to the administration of additional insulin. Consistent with the constitutively activated insulin receptor signaling, lmbrd1+/- mice exhibited an increase in heart rate and cardiac muscle contractility, leading to the development of compensated pathological hypertrophy and fibrosis. As lmbrd1+/- mice aged, the decrease in ejection fraction and fraction shortening showed signs of ventricular function deterioration. Additional studies using primary ventricular cells demonstrated that knockdown of lmbrd1 resulted in an elevated signaling of insulin receptor (IR) and its downstream molecule Akt. Confocal and live total internal 3 reflection fluorescence microscopy showed that LMBD1 colocalized and co-internalized with clathrin and IR upon insulin induction. Mutagenesis and phenotypic rescue studies further identified the motifs responsible for assisting the endocytosis of IR. Altogether, LMBD1 plays a regulatory role in the plasma membrane as an adaptor protein for insulin receptor endocytosis and modulates the IR metabolic signaling pathway.