How much insulin is enough? A quantitative assessment of the transdifferentiaton potential of liver

To the Editor: Adenoviral vector-mediated expression ofthe 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 ameliorateexperimentally induced diabetes [1]. In studies designed toassess the clinical applicability of this approach, wedeveloped a protocol employing non-human primatesrendered insulin-deficient (and therefore diabetic) throughselective intra-arterial streptozotocin (STZ) infusion intothe pancreas [2]. We obtained and amplified the adenoviralvector in which the CMV promoter drives rat Pdx1expression (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 proceedingto the large animal model, we tested Ad-Pdx1 lots foractivity in 8- to 10-week-old C57Bl6 mice (JacksonLaboratories, Bar Harbor, ME, USA), which were housedand handled in accordance with the guidelines set forth bythe Committee on the Care and Use of Laboratory Animalsof the Institute of Laboratory Animal Resources in aprotocol approved by the Animal Care and Use Committeeof the National Institute of Diabetes, Digestive, and KidneyDiseases. Mice were rendered diabetic with STZ (200 mg/kg i.p.). We treated 40 mice with the Ad-Pdx1 vector, butcompared with control mice, observed no improvement inglycaemia. All the mice lost significant weight andsubsequently had to be killed (Fig. 1a), further supportingthe absence of an insulin anabolic effect.To test whether our negative results could be explainedby the vector’s failure to promote Ipf1 expression, weemployed quantitative real-time PCR (qPCR) to assay forboth Ipf1 and insulin, and western blotting to assess IPF1protein expression. These assays clearly indicated vector-driven Ipf1 expression in the liver of Ad-Pdx1-treated mice;RNAwas detected at a low PCR cycle number, and westernblotting revealed IPF1 protein levels comparable to thatfrom a rat insulinoma cell line (Fig. 1b,c). In order tofurther quantitate insulin gene expression, we removedapproximately 25% of a control mouse liver, divided it intofour quarters, and extracted RNA; one quarter was used as anegative control, and to the other three we added the RNAextracted from either 100, 10 or 1 mouse islets. Whileinsulin RNA from one islet was easily detectable (Table 1),that from Ad-Pdx1-treated mouse livers was at (shown) orbelow the limit of detection using 40 PCR cycles, similar tothe negative control liver (final amplification plot in black).These results demonstrate that the hepatic insulin messageinduced by Ad-Pdx1 is less than that of one islet’s RNAmixed with RNA from one-sixteenth of a liver. As fourmore PCR cycles were required to detect insulin messagefrom the Ad-Pdx1-treated liver (assuming the signalrepresents specific amplification) compared with the cyclenumber when one islet was mixed with one-sixteenth of acontrol mouse liver, the Ad-Pdx1-induced insulin messagewas 2