Kriauciunas K M, Kahn C R, Muller-Wieland D, Reddy S S, Taub R
Research Division, Joslin Diabetes Center, Boston, Massachusetts 02215.
J Clin Endocrinol Metab. 1988 Dec;67(6):1284-93. doi: 10.1210/jcem-67-6-1284.
To determine the role of genetic defects in the insulin receptor in the insulin resistance of lipoatrophic diabetes mellitus, we studied insulin binding, insulin receptor autophosphorylation, and insulin receptor mRNA levels and performed Southern blot analysis of genomic DNA in four siblings, all of whom have some degree of insulin resistance and three of whom have lipoatrophy. The insulin receptor concentration in Epstein-Barr virus-transformed lymphocytes was about 30% of normal in all three lipoatrophic siblings (LA1, LA2, and LA3) and was 55% of normal in the nonlipoatrophic sibling (LAS). Insulin receptor mRNA concentrations in the lymphocytes paralleled insulin binding and ranged from 15-67% of the mean normal level. Insulin binding to fibroblasts was also reduced about 50% in the lipoatrophic siblings. In addition, insulin binding to fibroblasts of LAS and LA2 exhibited a rightward shift of the competition curve, suggesting reduced receptor affinity [ED50, 35 and 50 ng/mL (5845 and 8350 pmol/L); normal, 1-3 ng/mL (167-501 pmol/L)]. Receptor autophosphorylation determined using Triton X-100 extracts of the fibroblasts was decreased in LA1 and LA3, but normal in LA2 and LAS. Using restriction enzyme digests of genomic DNA and probes spanning the entire cDNA of the insulin receptor, no gross alterations in receptor gene structure were detected in any members of this family. In 2 of the lipoatrophic siblings (LA1 and LA3) and in the sibling with insulin resistance but no lipoatrophy (LAS), a unique variant BamHI site was detected using a probe to the alpha-subunit region. This site was not found in 200 normal or diabetic insulin receptor alleles. By use of probes 5' and 3' to the alpha-subunit probe and by genomic cloning analysis, this variant BamHI site was localized to an intron in the insulin receptor gene downstream of exon 3 which encodes amino acids 191-296 of the alpha-subunit of the receptor. These data indicate the complex nature of familial lipoatrophic diabetes mellitus, with alterations in insulin receptor expression and/or function in both clinically affected and non-affected siblings. Both the reduced insulin binding and reduced levels of insulin receptor mRNA in the lipoatrophic siblings suggest that an insulin receptor gene defect contributes to this syndrome. Several members of this family also carry a unique variant insulin receptor gene, which, however, could not be linked to a specific alteration in receptor expression or the presence of lipoatrophy.(ABSTRACT TRUNCATED AT 250 WORDS)
为了确定胰岛素受体基因缺陷在脂肪萎缩性糖尿病胰岛素抵抗中的作用，我们研究了4名兄弟姐妹的胰岛素结合、胰岛素受体自身磷酸化及胰岛素受体mRNA水平，并对其基因组DNA进行了Southern印迹分析。这4名兄弟姐妹均有一定程度的胰岛素抵抗，其中3人有脂肪萎缩。在所有3名脂肪萎缩的兄弟姐妹（LA1、LA2和LA3）中，爱泼斯坦-巴尔病毒转化淋巴细胞中的胰岛素受体浓度约为正常水平的30%，而在无脂肪萎缩的兄弟姐妹（LAS）中为正常水平的55%。淋巴细胞中的胰岛素受体mRNA浓度与胰岛素结合情况相似，为正常平均水平的15% - 67%。脂肪萎缩的兄弟姐妹中，胰岛素与成纤维细胞的结合也减少了约50%。此外，LAS和LA2的成纤维细胞与胰岛素的结合竞争曲线向右移动，提示受体亲和力降低[半数有效剂量（ED50）分别为35和50 ng/mL（5845和8350 pmol/L）；正常为1 - 3 ng/mL（167 - 501 pmol/L）]。用成纤维细胞的Triton X - 100提取物测定的受体自身磷酸化在LA1和LA3中降低，但在LA2和LAS中正常。使用基因组DNA的限制性酶切及跨越胰岛素受体整个cDNA的探针，未在该家族任何成员中检测到受体基因结构的明显改变。在2名脂肪萎缩的兄弟姐妹（LA1和LA3）以及有胰岛素抵抗但无脂肪萎缩的兄弟姐妹（LAS）中，用α亚基区域的探针检测到一个独特的BamHI位点变异。在200个正常或糖尿病胰岛素受体等位基因中未发现该位点。通过使用α亚基探针两侧的5'和3'探针以及基因组克隆分析，该BamHI位点变异定位于胰岛素受体基因外显子3下游的一个内含子中，外显子3编码受体α亚基的第191 - 296位氨基酸。这些数据表明家族性脂肪萎缩性糖尿病具有复杂性，临床受累和未受累的兄弟姐妹均存在胰岛素受体表达和/或功能改变。脂肪萎缩的兄弟姐妹中胰岛素结合减少及胰岛素受体mRNA水平降低均提示胰岛素受体基因缺陷与该综合征有关。该家族的几名成员还携带一个独特的胰岛素受体基因变异，但这与受体表达的特定改变或脂肪萎缩的存在并无关联。（摘要截短于250词）
