Non-suppressible insulin-like activity

Non-suppressible insulin-like activity (NSILA-S) was determined in dogs after acute changes in the blood sugar and after injection of growth hormone. Serum was chromatographed over Sephadex G-50 columns equilibrated in 1 m acetic acid and NSILA-S was determined in the fractions between 55 and 85 % column volume using a protein binding assay and the conventional fat pad assay. The results obtained with these two methods correlated rather well (r = 0.74). Hyperglycaemia induced by an intravenous glucose load, by intravenous administration of mannoheptulose or both was not followed by an increase in NSILA-S levels. The injection of insulin and human growth hormone did not lead to alterations of the NSILA-S levels. It is concluded that total NSILA-S levels in the dog do not change acutely following manipulations of the blood sugar and that, in all likelihood, NSILA-S plays no role in the regulation of blood glucose. This work was supported by grant No. 3.595.-0.75 from the Schweizerische National¬ fonds. Address reprint requests to Dr. E. R. Froesch, Stoffwechsellabor, Kantonsspital, 8091 Zurich. Downloaded from Bioscientifica.com at 11/29/2018 01:08:16AM via free access The insulin-like effects of non-suppressible insulin-like activity (NSILA-S) have been extensively studied in vivo and in vitro (for review see Oelz et al. 1972). In more recent studies it has been demonstrated that NSILA-S exerts not only insulin-like effects in the classical sense but also marked growth pro¬ moting effects (Morell 8c Froesch 1973) and that NSILA-S is a potent sulphation factor (Zingg 8c Froesch 1973; Froesch et al. 1976). However, the phy¬ siological significance of NSILA-S remains to be elucidated. In 1975 (Zapf et al. 19756) we demonstrated the presence in serum of a highly specific carrier protein for NSILA-S and Kaufmann et al. (1977) found that the halflife of NSILA-S in the rat is of the order of magnitude of 4 h. Since more than 95 % of NSILA-S in the circulation is present in the bound form, which has this rather long half-life the finding of Megyesi et al. (1975) who de¬ scribed acute changes in total NSILA-S in the serum of human subjects came rather as a surprise. The experiments, the results of which are described in this study were car¬ ried out in order to verify whether or not acute changes in the NSILA-S levels can be obtained by acute changes in blood sugar and by a single injection of growth hormone. MATERIALS AND METHODS Mannoheptulose (Sigma) was infused as a solution of 20 "la. Human growth hormone (Serono) containing 2 U/mg was dissolved in saline (pH 9) in a concentration of 0.2 mg/ml. Crystalline pork insulin (Actrapid®, Novo) was diluted with sterile saline to a final concentration of 1 U/ml before injection. Glucagon-free pork insulin (Hoechst) was used as a standard in the rat fat pad assay (Froesch et al. 1963). Highly purified NSILA-S was labelled with 12äI and a preparation containing 4.5 mU/mg was used as standard in the protein binding assay (Zapf et al. 1977). Both were a generous gift from E. Rinderknecht and R. E. Humbel, Department of Biochemistry, University of Zurich. NSILA-S was extracted from individual sera by the method of Schlumpf et al. (1976), with the exception that Sephadex G-50 was found to give a better sepa¬ ration of NSILA-S from its binding protein than Sephadex G-75. Using this method, NSILA-S appears between 55 and 85 % of total bed volume. The fractions in this region were pooled, lyophilized, rinsed with 10 ml of 0.1 M NHjHC03, re-lyophilized and their NSILA-S content was then determined. For the fat pad assay we used male ZBZ Cara rats weighing 120-130 g (Froesch et al. 1963). In addition, NSILA-S was also determined in the protein binding assay (Zapf et al. 1977). All experiments were carried out using 5 adult, pure bred female Labrador dogs weighing 23.4 + 2.1 kg (mean + standard error). The dogs were a generous gift from Dr. W. Rossbach, Hoffmann-LaRoche. All experiments on these dogs were carried out after an overnight fast. Injections and blood collections were performed by puncture of the antebranchial cephalic vein. Blood samples were al¬ lowed to clot for 2 h at 4°C, centrifuged at 4000 r. p. m. and the serum was stored at -20°C. For statistical analysis Student's i-test and linear regression were employed. Downloaded from Bioscientifica.com at 11/29/2018 01:08:16AM via free access