Epithelial Function and Development Group, Department of Functional and Comparative Genomics, Institute of Integrative Biology, Faculty of Health and Life Sciences, University of Liverpool, Liverpool L69 7ZJ, UK.
Domest Anim Endocrinol. 2013 Apr;44(3):139-44. doi: 10.1016/j.domaniend.2012.12.001. Epub 2013 Jan 24.
Glucose transport across the enterocyte brush border membrane by sodium/glucose cotransporter-1 (SGLT1, coded by Slc5a1) is the rate-limiting step for intestinal glucose transport. The relevance of SGLT1 expression in predisposition to diabetes mellitus and to obesity was investigated in dogs. Cultured Caco-2/TC7 cells were shown to express SGLT1 in vitro. A 2-kbp fragment of the Slc5a1 5' flanking region was cloned from canine genomic DNA, ligated into reporter gene plasmids, and shown to drive reporter gene expression in these cells above control (P < 0.001). To determine the effect of the 3 known SNPs in this region on promoter function, new promoter/reporter constructs (all permutations of these 3 SNPs) were created by site-directed mutagenesis. No significant differences in promoter function were seen, suggesting that these SNPs do not have a significant effect on the constitutive transcription of SGLT1 mRNA in dogs. A search for novel SNPs in this region in dogs was made in 2 breeds predisposed to diabetes mellitus (Samoyed and cairn terrier), 2 breeds that rarely develop diabetes (boxer and German shepherd), and 2 breeds predisposed to obesity (Labrador retriever and cocker spaniel). The Slc5a1 5' flanking region was amplified from 10 healthy individuals of each of these breeds by high-fidelity PCR with the use of breed-labeled primers and sequenced by pyrosequencing. The sequence of the Slc5a1 5' flanking region in all individuals of all breeds tested was identical. On this evidence, variations in Slc5a1 promoter sequence between dogs do not influence the pathogenesis of diabetes mellitus or obesity in these breeds.
葡萄糖通过肠上皮刷状缘膜的转运是由钠/葡萄糖协同转运蛋白-1（SGLT1，由 Slc5a1 编码）来完成的，它是肠道葡萄糖转运的限速步骤。SGLT1 的表达与糖尿病和肥胖的易感性的相关性在犬中进行了研究。体外培养的 Caco-2/TC7 细胞表达 SGLT1。从犬基因组 DNA 中克隆出 Slc5a1 5'侧翼区的 2kbp 片段，将其连接到报告基因质粒中，并在这些细胞中驱动报告基因表达高于对照（P<0.001）。为了确定该区域内的 3 个已知 SNP 对启动子功能的影响，通过定点诱变创建了新的启动子/报告基因构建体（这些 SNP 的所有排列组合）。未观察到启动子功能的显著差异，这表明这些 SNP 对犬 SGLT1 mRNA 的组成型转录没有显著影响。在易患糖尿病的 2 个犬种（萨摩耶犬和凯恩梗犬）、2 个很少发生糖尿病的犬种（拳击犬和德国牧羊犬）和 2 个易患肥胖的犬种（拉布拉多猎犬和可卡犬）中，在该区域内搜索新的 SNP。使用带有品种标记的引物，通过高保真 PCR 从每个品种的 10 个健康个体中扩增 Slc5a1 5'侧翼区，并通过焦磷酸测序进行测序。所有测试品种的所有个体的 Slc5a1 5'侧翼区序列均相同。根据这一证据，犬中 Slc5a1 启动子序列的变异并不影响这些品种中糖尿病或肥胖的发病机制。
