RESUMEN
The aim of the present study was to investigate the influence of dietary protein level on the protein anabolic effects of growth hormone (GH) and insulin-like growth factor-I (IGF-I). Female growing rats were fed on either a high- or a low-protein diet with crude protein contents of 222 and 83 g/kg respectively. The diets contained the same amount of metabolizable energy (15.1 MJ/kg) and were given during a 14 d period. During the same time, three groups of rats (n 8) on each diet received subcutaneous infusions of either saline, recombinant human GH (rhGH) or recombinant human IGF-I (rhIGF-I). rhGH and rhIGF-I were given in doses of 360 and 500 micrograms/d respectively. The low-protein diet alone reduced significantly (P < 0.05) IGF-I concentrations in serum and in tissue taken from the gastrocnemius muscle as well as IGF-I mRNA from the same muscle. The responses to rhGH and rhIGF-I in terms of muscle IGF-I and its mRNA were variable. However, when rhIGF-I was infused into rats on the high-protein diet, significantly elevated levels of IGF-I in muscle tissues could be observed. This was associated with a significantly (P < 0.05) increased N balance, whereas rhGH significantly (P < 0.05) enhanced the N balance in rats on the low-protein diet. Thus, it can be concluded that the level of dietary protein ingested regulates not only the effect of IGF-I on whole-body N economy but also the regulation of IGF-I gene expression in muscles. The exact mechanism by which GH exerts its protein anabolic effect, however, remains to be elucidated.
Asunto(s)
Proteínas en la Dieta/administración & dosificación , Hormona del Crecimiento/sangre , Factor I del Crecimiento Similar a la Insulina/metabolismo , Nitrógeno/metabolismo , Animales , Metabolismo Energético , Femenino , Hormona del Crecimiento/administración & dosificación , Humanos , Factor I del Crecimiento Similar a la Insulina/administración & dosificación , Factor I del Crecimiento Similar a la Insulina/genética , Músculos/efectos de los fármacos , Músculos/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/metabolismoRESUMEN
It is well established that nutrition is an important regulator of both serum insulin-like growth factor-I (IGF-1) and its binding proteins (IGFBPs). The Western ligand blot method (WLB) for simultaneous determinations of IGFBPs in serum or plasma samples was evaluated and validated with emphasis on its reproducible capabilities. After electrophoretic separation and transfer, the membranes were incubated with a mixture of recombinant labeled human (GF-I/IGF-II(rhIGF-I/rhlGF-II) and band intensities measured by autoradiography. The typical electrophoretic profile for pig serum, as determined with molecular weight markers, showed four mainbands of approximately 42-39, 32, 30-28 and 24 kDa which seemed to correspond to IGFBP-3, IGFBP-2, IGFBP-1 and IGFBP-4 respectively. Likewise, a triplet of approximately 42-39 kDa (IGFBP-3), a broad area called IGFBP-30 region (most probably IGFBP-1, -2 and -3 variants) and a third band of approximately 24 kDa (IGFBP-4) were seen in rat samples. Determination of IGFBP-2 and -1 in rat serum samples, as two separate bands on 12% gels was difficult due to their close electrophoretic migration and possibly to the reported lower levels of IGFBP-2 in adult rat serum. Dilutions tested on 0.2 micron nitrocellulose membranes with samples volumes between 0.25 to 1.5 microliters (1:10-1:60 dilutions), showed IGFBPs curves with good linearity which suggest first, that there exist a quantitative relation between the amount of each protein and the densitometric response and second, that the transfer of the proteins was linear across the range of 0.25 to 1.5 microliters (1:10-1:60 dilutions). Moreover, the results also suggest that losses were equally spread and that the proteins retained their binding properties after the transfer process. Reproducibility showed intraassay coefficients of variation (CVs) of 15% or lower using either a transfer device without cooling system or a combination of a transfer device with cooling system and manually defined band boundaries. In summary, it was shown that the optimized experimental conditions here described for the WLB method, allow reliable simultaneous measurements of the main pig and rat serum IGFBPs and therefore, could be utilised to detect changes in the serum profile after dietary manipulations.