E. R. Froesch

Binding of nonsuppressible insulinlike activity to human serum: Evidence for a carrier protein☆

Abstract When 125 I-labeled nonsuppressible insulinlike activity—soluble in acid/ethanol (NSILA-S) is incubated with human serum between 10 and 20% of the radioactivity are bound to serum proteins and can be displaced specifically by cold NSILA-S. Chromatography of the incubation mixture on Sephadex G-200 at pH 7.5 reveals three peaks of radioactivity in the large molecular weight region and a fourth one corresponding to low molecular unbound labeled NSILA-S. An excess of cold NSILA-S during preincubation leads to the disappearance of the two major large molecular weight peaks and to a concomitant increase of the peak eluting in the low molecular weight range. Binding of 125 I-labeled NSILA-S is highly sensitive to small concentrations of cold NSILA-S, whereas insulin, ACTH and human growth hormone are completely ineffective in displacing bound 125 I-labeled NSILA-S. NSILA-S preparations of different purity show displacement according to their specific biological activities. Furthermore, binding of 125 I-labeled NSILA-S to serum pH- and time-dependent and displays saturation characteristics. Chromatography of serum on Sephadex G-200 with 0.15 m acetic acid/0.15 m NaCl localizes the binding fraction in the 50,000–70,000 molecular weight range. Boiling of serum for 5 min abolishes binding completely. These studies help explain why the molecular weight of NSILA varied considerably from one group of investigators to the other.

Fibroblasts as an experimental tool in metabolic and hormone studies. II. Effects of insulin and nonsuppressible insulin-like activity (NSILA-S) on fibroblasts in culture.

Abstract. The effects of insulin and purified nonsuppressible insulin‐like activity from serum (NSILA‐S) on glucose, DNA‐metabolism and growth of chicken embryo fibroblasts are compared. Insulin stimulates growth, glucose consumption, lactate production and thymidine uptake only in very high concentrations (1–100 mU/ml), whereas NSILA‐S is effective at concentrations which may be present under physiological conditions. On a molar basis NSILA‐S is approximately 20 times as active as insulin. Although a potent stimulator of all these metabolic indices NSILA‐S can only partially replace serum. NSILA‐S may be one of the growth factors present in serum but it does not seem to be the only one.

Specific binding of nonsuppressible insulinlike activity to chicken embryo fibroblasts and to a solubilized fibroblast receptor

Abstract 125I-labeled nonsuppressible insulinlike activity—soluble in acid/ethanol (NSILA-S) binds to cultured chicken embryo fibroblasts and to an extract obtained by treating the fibroblasts with Triton X-100. Binding to intact cells and to the cell extract is timetemperature, and pH-dependent and shows saturation kinetics. The apparent intrinsic association constant for binding of NSILA-S to intact cells is 109 M−1, the number of binding sites per cell approximately 6000. Cold NSILA-S preparations of different purity displace bound 125I-labeled NSILA-S according to their biological potency. Moreover, cold NSILA-S displaces bound 125I-labeled NSILA-S in concentrations in which it also stimulates thymidine incorporation into fibroblast DNA. Insulin displaces bound 125I-labeled NSILA-S only at concentrations above 1 mU/ml. Glucagon, ACTH, human growth hormone and inactivated NSILA-S are ineffective. The displacement curves obtained with human serum are similar to those obtained in the presence of cold NSILA-S.

Effects of insulin and of NSILA-S on the perfused rat heart: glucose uptake, lactate production and efflux of 3-0-methyl glucose.

Abstract. 200 μU of insulin per ml perfusion medium stimulated glucose uptake and lactate production by the isolated perfused rat heart about 5 and 10 fold, respectively. Nonsuppressible insulin‐like activity (NSILA‐S) extracted from human serum had almost the same maximal effects. Mol per mol the two substances were equally effective and, correspondingly, the dose‐response curves were very similar. NSILA‐S accelerated the efflux of 3–0‐methyl glucose from the heart cells to the same extent as insulin. It can be concluded that NSILA‐S acts on heart muscle in a very similar way to that of insulin.

Inhibition of Phosphorylase-a by Fructose-1-Phosphate, α-Glycerophosphate and Fructose-1,6-Diphosphate: Explanation for Fructose-Induced Hypoglycaemia in Hereditary Fructose Intolerance and Fructose-1,6-Diphosphatase Deficiency*

Abstract. The effect of fructose‐1‐phosphate, α‐glycerophosphate and fruetose‐1,6‐diphosphate on the activity of liver phosphorylase‐a has been investigated at different concentrations of inorganic phosphorus.—Inhibition of phosphorylase‐a by fructose‐1‐phosphate is competitive, by α‐glycerophosphate it is noncompetitive and by fructose‐1,6‐diphosphate uncompetitive. The Ki is 4 × 10‐3 M for fructose‐1‐phosphate and in the range of 5 to 6 × 10‐2 M for fructose‐1,6‐diphosphate and α‐glycerophosphate. The activity of phosphorylase‐a and its inhibition by fructose‐1‐phosphate depends very much on the concentration of inorganic phosphorus.—In fructose‐1,6‐diphosphatase deficiency glucose production from gluconeogenic precursors is impossible, whereas in hereditary fructose intolerance accumulated fructose‐1‐phosphate blocks the residual abnormal hepatic aldolase. Our findings explain fructose induced hypoglycaemia in hereditary fructose intolerance and fructose‐1,6‐diphosphatase deficiency by a block in glycogenolysis at the level of phosphorylase‐a.

Nonsuppressible insulin-like activity (NSILA) of human serum. V. Hypoglycaemia and preferential metabolic stimulation of muscle by NSILA-S.

Abstract 10 mU of NSILA‐S, as determined by the fat pad assay, lowered blood glucose and free fatty acids of adrenalectomized rate for a much longer period than 10 mU of crystalline insulin. NSILA‐S was not measurably inactivated by the liver during a 2 h cyclical perfusion, whereas insulin rapidly lost activity with a half‐life of 42 min. 6 mU of NSILA‐S injected intravenously stimulated the incorporation of [6‐14C] glucose into the diaphragm of streptozotocin‐diabetic rats to a much greater extent than 6 mU of insulin, whereas their effects on the incorporation of carbon 14 into the fat pad were equal.

Biological properties of NSILA-S.

Publisher Summary This chapter discusses the biological properties of NSILA-S. The effects of NSILA-S and of insulin on adipose tissue in vitro are indistinguishable. Pooled epididymal adipose tissue of the rat serves as the routine assay for NSILA-S preparations in laboratory. Pure NSILA-S has an activity on adipose tissue equivalent to 400 mU/per milligram of protein. Considering the molecular weight of 7500, it is mole per mole, approximately 50 times less active on adipose tissue than insulin. Whereas serum ILA seems to influence the conversion of labeled glucose to glycogen in adipose tissue in vitro to a lesser extent than insulin, this is not true for extracted NSILA-S. The chapter describes an experiment which was carried out with adipose tissue from fasted-refed rats. The results demonstrate that 250 μU of NSILA-S per milliliter of incubation medium exert the same maximal effects as maximal concentration of insulin. The conversion of 14 C-labeled glucose to total lipids, fatty acids, and glycogen is the same, and so is the oxidation of the 14 C of glucose to 14 CO 2 . Furthermore, both hormones block glycerol release completely. The decrease of glycogen content which normally occurs during in vitro incubations of adipose tissue from fasted-refed rats is entirely abolished by both hormones.

Comparative Study of the Metabolism of U‐14C‐Fructose, U‐14C‐Sorbitol and U‐14C‐Xylitol in the Normal and in the Streptozotocin‐Diabetic Rat*

Abstract. Young male rats metabolize xylitol, sorbitol and fructose with a half‐life of 165 seconds. Within 10 min. after the intravenous injection of a load of these substrates more than 70% of the carbon‐14 in plasma is present as 14C‐glucose. The labelling of plasma glucose from these substrates appears to be insulin‐independent. A small percentage of labelled xylitol, sorbitol and fructose is retained by the liver as glycogen and total lipids. In the normal rat, this process appears to be insulin‐independent. In the chronic streptozotocin‐diabetic rat, insulin acutely enhances glycogen synthesis from all three substrates and reduces their incorporation into total lipids. Once these substrates have been converted to glucose the further storage as muscle glycogen and adipose tissue total lipids is entirely dependent on insulin, both in the normal and in the streptozotocin‐diabetic rat. Fructose makes an exception since it is metabolized by adipose tissue independently of insulin. In the light of these results in the rat a re‐evaluation of the use of various carbohydrates and polyols in parenteral nutrition of normal and diabetic subjects appears desirable.

Lipase Activity in the Fat Cake and Aqueous Phase of Adipose Tissue Homogenate of Fed, Fasted and Fasted-Refed Rats

Abstract. 1. Free fatty acid production by the fat cake of adipose tissue was found to be much greater in fasted‐refed than in fasted and fed rats, respectively. Preincubation with adrenalin and dibutyryl cyclic AMP stimulated lipase activities in fasted and fed rats, but not in fasted‐refed rats. Basal lipase activity in the fat cake of fasted‐refed rats was higher than adrenalin or dibutyryl cyclic AMP activated lipase activity of fasted or fed rats. Activation of lipase activity by refeeding lasted for several days.—2. Lipase activity in the aqueous phase of adipose tissue homogenate of all rats was in the same range.—3. Free fatty acid release during the assay of lipase in the fat cake proceeded for a period of up to 120 minutes although not in an entirely linear manner. The ratio of free fatty acids to glycerol release was 2.9. The pH optimum of the reaction was 7.4. NaCl, NaF, protamine and heparin markedly inhibited the lipase reaction. Heparin in the preincubation medium did not reduce the lipase activity in the fat cake.— 4. The very active triglyceride lipase activity of adipose tissue of fasted‐refed rats explains earlier findings showing that intact adipose tissue of fasted‐refed rats exhibits a very high glycerol release. It appears that tissue levels of triglyceride lipase increase during refeeding under the influence of insulin, i.e. at a time when the level of cyclic AMP in the tissue supposedly is low.