How much insulin is enough? A quantitative assessment of the transdifferentiaton potential of liver.
How much insulin is enough? A quantitative assessment of the transdifferentiaton potential of liver.
复制标题
多少胰岛素才足够？
DOI：
10.1007/s00125-006-0549-0
复制
发表时间：
2007
期刊：
影响因子：
8.2
通讯作者：
Tisdale,JF
中科院分区：
文献类型：
--
作者：
Perl,S;Hirshberg,B;Harlan,DM;Tisdale,JF
To the Editor: Adenoviral vector-mediated expression of the pancreatic and duodenal homeobox 1 gene (Pdx1; current designation in mice insulin promotor factor-1, Ipf1) in mice has previously been reported to result in hepatic, glucose-regulated insulin secretion sufficient to ameliorate experimentally induced diabetes [1]. In studies designed to assess the clinical applicability of this approach, we developed a protocol employing non-human primates rendered insulin-deficient (and therefore diabetic) through selective intra-arterial streptozotocin (STZ) infusion into the pancreas [2]. We obtained and amplified the adenoviral vector in which the CMV promoter drives rat Pdx1 expression (Ad-CMV-Pdx1 referred to hereafter as Ad-Pdx1), kindly provided by S. Ferber, Endocrine Institute, Sheba Medical Centre, Tel-Hashomer, Israel. In order to document vector activity prior to proceeding to the large animal model, we tested Ad-Pdx1 lots for activity in 8-to 10-week-old C57Bl6 mice (Jackson Laboratories, Bar Harbor, ME, USA), which were housed and handled in accordance with the guidelines set forth by the Committee on the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources in a protocol approved by the Animal Care and Use Committee of the National Institute of Diabetes, Digestive, and KidneyDiseases. Mice were rendered diabetic with STZ (200 mg/kg ip). We treated 40 mice with the Ad-Pdx1 vector, but compared with control mice, observed no improvement in glycaemia. All the mice lost significant weight and subsequently had to be killed (Fig. 1 a), further supporting the absence of an insulin anabolic effect. To test whether our negative results could be explained by the vector’s failure to promote Ipf1 expression, we employed quantitative real-time PCR (qPCR) to assay for both Ipf1 and insulin, and western blotting to assess IPF1 protein expression. These assays clearly indicated vectordriven Ipf1 expression in the liver of Ad-Pdx1-treated mice; RNA was detected at a low PCR cycle number, and western blotting revealed IPF1 protein levels comparable to that from a rat insulinoma cell line (Fig. 1 b, c). In order to further quantitate insulin gene expression, we removed approximately 25% of a control mouse liver, divided it into four quarters, and extracted RNA; one quarter was used as a negative control, and to the other three we added the RNA extracted from either 100, 10 or 1 mouse islets. While insulin RNA from one islet was easily detectable (Table 1), that from Ad-Pdx1-treated mouse livers was at (shown) or below the limit of detection using 40 PCR cycles, similar to the negative control liver (final amplification plot in black). These results demonstrate that the hepatic insulin message induced by Ad-Pdx1 is less than that of one islet’s RNA mixed with RNA from one-sixteenth of a liver. As four more PCR cycles were required to detect insulin message from the Ad-Pdx1-treated liver (assuming the signal represents specific amplification) compared with the cycle number when one islet was mixed with one-sixteenth of a control mouse liver, the Ad-Pdx1-induced insulin message was 24 or 16-fold less than the insulin message resulting from one islet in one-sixteenth of a liver control. Thus, the overall liver insulin RNA content induced by Ad-Pdx1 is less than that of 1 islet equivalent.
