Rat insulin turnover in vivo

Zucker lean and obese rats were injected under pentobarbital anesthesia with 125I-labeled insulin; at timed intervals from 30 to 120 sec, blood samples were extracted and used for the estimation of insulin levels by RIA. A group of rats from each series was maintained under a constant infusion of noradrenaline. For each insulin determination, a duplicate blood sample containing the same amount of insulin as that used in the RIA, but without the radioactive label, was used as a blank for insulin measurement. The radioactivity in these tubes was then used for the measurement of insulin label per ml blood. From plasma label decay curves and insulin concentrations, the insulin pool size, half-life, and rate of degradation were calculated. Obese rats had higher insulin levels (2.43 nM) and showed less effect of noradrenaline than their lean counterparts, in which insulin distribution volume shrank with noradrenaline treatment. The half-life of plasma insulin was similar in all groups (range, 226-314 sec). Pool size and overall degradation rates were higher in obese (198 femtokatals) than in lean rats (28 femtokatals). It is postulated that obese rats synthesize and cleave much more insulin than lean controls despite their higher circulating levels of insulin.

Different modulation of adipocytokine expression in four main white adipose tissue sites in the rat: mesenteric, perigonadal, retroperitoneal and subcutaneous

White adipose tissue (WAT) is a disperse organ acting as energy storage depot and endocrine/paracrine controlling factor in the management of energy availability and inflammation. WAT sites response under energy-related stress is not uniform. In the present study we have analyzed how different WAT sites respond to limited food restriction as a way to better understand the role of WAT in the pathogenesis of the metabolic syndrome.

Semiquantitative RT-PCR measurement of gene expression in rat tissues including a correction for varying cell size and number

Background: Current methodology of gene expression analysis limits the possibilities of comparison between cells/tissues of organs in which cell size and/or number changes as a consequence of the study (e.g. starvation). A method relating the abundance of specific mRNA copies per cell may allow direct comparison or different organs and/or changing physiological conditions. Methods: With a number of selected genes, we analysed the relationship of the number of bases and the fluorescence recorded at a present level using cDNA standards. A lineal relationship was found between the final number of bases and the length of the transcript. The constants of this equation and those of the relationship between fluorescence and number of bases in cDNA were determined and a general equation linking the length of the transcript and the initial number of copies of mRNA was deduced for a given pre-established fluorescence setting. This allowed the calculation of the concentration of the corresponding mRNAs per g of tissue. The inclusion of tissue RNA and the DNA content per cell, allowed the calculation of the mRNA copies per cell. Results: The application of this procedure to six genes: Arbp, cyclophilin, ChREBP, T4 deiodinase 2, acetyl-CoA carboxylase 1 and IRS-1, in liver and retroperitoneal adipose tissue of food-restricted rats allowed precise measures of their changes irrespective of the shrinking of the tissue, the loss of cells or changes in cell size, factors that deeply complicate the comparison between changing tissue conditions. The percentage results obtained with the present methods were essentially the same obtained with the delta-delta procedure and with individual cDNA standard curve quantitative RT-PCR estimation. Conclusion: The method presented allows the comparison (i.e. as copies of mRNA per cell) between different genes and tissues, establishing the degree of abundance of the different molecular species tested.