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      IGF-I Transcript Levels in Whole-Liver Tissue, in Freshly Isolated Hepatocytes, and in Cultured Hepatocytes from Lean and Obese Zucker Rats

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          Abstract

          Background: The mechanisms underlying the maintenance of normal to high rates of linear growth and plasma insulin-like growth factor I (IGF-I) levels in spite of a low growth hormone secretion in obese children remain unknown. Among the animal models of early-onset obesity, obese Zucker (fa/fa) rats (which are homozygous for an inactivating missense mutation in the leptin receptor) are particularly appropriate, because their linear growth shows this growth hormone independence. Methods: To study the regulation of IGF-I synthesis in this model, we have established primary cultures of hepatocytes derived from 12-week-old Zucker male obese and lean rats. The rat IGF-I gene contains six exons, and alternative splicing generates different mRNAs, one of which (called IGF-1B) has been shown to be decreased by fasting. We report steady state mRNA levels for IGF-I (all transcripts) and for IGF-IB in hepatocytes after 3 days in culture, in freshly isolated hepatocytes, and in whole-liver tissue. RT-PCRs using primers specific for IGF-I or IGF-IB were performed with two different internal competitors for quantification. Results: In primary cultures of hepatocytes, the IGF-IB mRNA was increased by >50-fold (p = 0.01) in cells derived from obese animals as compared with cells from lean animals. However, these transcript levels were not significantly different when measured in freshly isolated hepatocytes or in whole-liver tissue. Conclusions: Increased IGF-IB transcription could be an intrinsic characteristic of cultured hepatocytes harbouring leptin receptors that bear the fa mutation. However, the modulation of this characteristic by cell-cell interactions and by in vivo hormone and metabolic status remains to be studied.

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          Most cited references 4

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          Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.

          A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.
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            Identification and expression cloning of a leptin receptor, OB-R.

            The ob gene product, leptin, is an important circulating signal for the regulation of body weight. To identify high affinity leptin-binding sites, we generated a series of leptin-alkaline phosphatase (AP) fusion proteins as well as [125I]leptin. After a binding survey of cell lines and tissues, we identified leptin-binding sites in the mouse choroid plexus. A cDNA expression library was prepared from mouse choroid plexus and screened with a leptin-AP fusion protein to identify a leptin receptor (OB-R). OB-R is a single membrane-spanning receptor most related to the gp130 signal-transducing component of the IL-6 receptor, the G-CSF receptor, and the LIF receptor. OB-R mRNA is expressed not only in choroid plexus, but also in several other tissues, including hypothalamus. Genetic mapping of the gene encoding OB-R shows that it is within the 5.1 cM interval of mouse chromosome 4 that contains the db locus.
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              Modulation of insulin activities by leptin.

              Leptin mediates its effects on food intake through the hypothalamic form of its receptor OB-R. Variants of OB-R are found in other tissues, but their function is unknown. Here, an OB-R variant was found in human hepatic cells. Exposure of these cells to leptin, at concentrations comparable with those present in obese individuals, caused attenuation of several insulin-induced activities, including tyrosine phosphorylation of the insulin receptor substrate-1 (IRS-1), association of the adapter molecule growth factor receptor-bound protein 2 with IRS-1, and down-regulation of gluconeogenesis. In contrast, leptin increased the activity of IRS-1-associated phosphatidylinositol 3-kinase. These in vitro studies raise the possibility that leptin modulates insulin activities in obese individuals.
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                Author and article information

                Journal
                HRE
                Horm Res Paediatr
                10.1159/issn.1663-2818
                Hormone Research in Paediatrics
                S. Karger AG
                1663-2818
                1663-2826
                2003
                2003
                19 March 2003
                : 59
                : 3
                : 135-141
                Affiliations
                aResearch Unit on the Biology of Reproduction and Development, Research Center, Ste-Justine Hospital, and bDepartment of Pediatrics, University of Montreal, Montreal, Que., Canada
                Article
                69066 Horm Res 2003;59:135–141
                10.1159/000069066
                12637793
                © 2003 S. Karger AG, Basel

                Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

                Page count
                Figures: 2, Tables: 3, References: 39, Pages: 7
                Categories
                Original Paper

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