Xu G G, Rothenberg P L
Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, USA.
Diabetes. 1998 Aug;47(8):1243-52. doi: 10.2337/diab.47.8.1243.
The insulin receptor (IR) is expressed by insulin-secreting beta-cells, but its cellular function is unknown. We transfected betaTC6-F7 beta-cells with cDNAs encoding either wild-type or mutant kinase-inactive (A/K1018) IRs, and by fluorescence-activated cell sorting generated polyclonal beta-cell lines that overexpressed each receptor type at levels two- to fourfold higher than the parental cells. Beta-cells overexpressing wild-type IRs had a proportional increase in insulin-stimulated tyrosine kinase activity; no change occurred in beta-cells expressing the mutant IR. We observed a threefold increase in cellular insulin content in beta-cells that overexpressed the wild-type IR, as determined by radioimmunoassay. This increase occurred despite a fivefold elevated rate of both basal and 10 mmol/l glucose-induced insulin secretion. Fractional insulin secretion (percentage of total cell insulin releasable at 10 mmol/l glucose) was unchanged in beta-cells overexpressing the wild-type IR compared with the parental beta-cell line. Insulin content and insulin secretion were unaffected by overexpression of kinase-inactive IRs. Steady-state insulin mRNA levels were elevated twofold in the beta-cells overexpressing the wild-type IR and unchanged in the beta-cells expressing the kinase-inactive receptor, as determined by Northern blot analysis. The rate of insulin mRNA degradation measured in the presence of 5 microg/ml actinomycin D was not significantly affected in either cell line. In the absence of glucose, the basal level of (pro)insulin biosynthesis in the beta-cells overexpressing the wild-type IR increased significantly (61%) compared with the beta-cells transfected with the kinase-inactive IR. These data indicate that IR signaling can regulate insulin gene transcription and can modulate the steady-state insulin content of beta-cells.
胰岛素受体（IR）由分泌胰岛素的β细胞表达，但其细胞功能尚不清楚。我们用编码野生型或激酶失活型（A/K1018）IR的cDNA转染βTC6-F7β细胞，并通过荧光激活细胞分选产生多克隆β细胞系，这些细胞系中每种受体类型的过表达水平比亲代细胞高两到四倍。过表达野生型IR的β细胞中，胰岛素刺激的酪氨酸激酶活性成比例增加；表达突变型IR的β细胞则无变化。通过放射免疫测定法测定，我们观察到过表达野生型IR的β细胞中细胞胰岛素含量增加了三倍。尽管基础胰岛素分泌率和10 mmol/l葡萄糖诱导的胰岛素分泌率均升高了五倍，但仍出现了这种增加。与亲代β细胞系相比，过表达野生型IR的β细胞中胰岛素分泌分数（10 mmol/l葡萄糖时可释放的总细胞胰岛素的百分比）没有变化。激酶失活型IR的过表达不影响胰岛素含量和胰岛素分泌。通过Northern印迹分析测定，过表达野生型IR的β细胞中稳态胰岛素mRNA水平升高了两倍，而表达激酶失活型受体的β细胞中则没有变化。在5 μg/ml放线菌素D存在的情况下测量的胰岛素mRNA降解率在两种细胞系中均未受到显著影响。在无葡萄糖的情况下，过表达野生型IR的β细胞中（前）胰岛素生物合成的基础水平与转染激酶失活型IR的β细胞相比显著增加（61%）。这些数据表明，IR信号传导可以调节胰岛素基因转录，并可以调节β细胞的稳态胰岛素含量。
