Ida K. Mariz

Insulin-like growth factors I and II of nonmammalian sera ☆

We have compared the insulin-like growth factors (IGFs) of a number of mammalian and nonmammalian vertebrate sera. Sera were subjected to acid gel filtration through G-75 Sephadex and separate binding protein and free IGF fractions were pooled. The IGF pools were subjected to flatbed isoelectric focusing and the concentration of IGF I in the fractions was detected with an immunoassay using 125I-hIGF I and the somatomedin C antibody of Underwood and Van Wyk. IGF II in the fractions was detected with either the 125I-IGF rat placental membrane RRA or the 125I-IGF II serum binding protein assay. One or more basic peaks of IGF I immunoactivity were detected in all the mammalian sera studied (human, guinea pig, rat, bovine, kangaroo, and opossum) and all the nonmammalian species studied (chicken, turtle, toad, and trout). Neutral and slightly acidic peaks of IGF II receptor reactivity were observed in human, cow, and guinea pig serum. No distinct peaks of IGF II were observed in adult rat, opossum, or kangaroo serum. Distinct peaks of IGF II activity in the neutral to slightly acidic range were clearly recognized in chicken and turtle serum, no such peaks were recognized in toad and trout serum. All mammalian sera studied contained acid stable, binding proteins with high affinity, saturable binding of 125I-IGF II. None of the nonmammalian sera studied contained demonstrable specific 125I-IGF II binding. These observations document the presence of separate IGF I and IGF II peptides in chickens and turtles but only IGF I in amphibians and fish. These observations indicate that the gene duplication giving rise to two separate IGFs arose before reptilian evolution. The acquisition of a specific IGF binding protein is limited to mammals.

Heterogeneity of insulin receptors on fat cell membranes

Abstract A highly purified and well characterized fat cell membrane preparation has been shown to retain the insulin binding characteristics of intact fat cells. Two major binding sites were identified, one a high affinity, low capacity site (K D = 5 × 10 −10 M) and the other a lower affinity, high capacity site (K D = 3 × 10 −9 M). Insulin binding to the membranes was prevented by insulin analogues (desoctapeptide and desalanine insulin) and proinsulin in direct relationship to their biological activities.

Effects of Cortisol and Diethylstilbestrol on Growth Hormone Release by Rat Pituitary in vitro.

The secretion of growth hormone in response to hypoglycemia and the administration of arginine in man has been shown to be greatly influenced by certain non-pituitary hormones. Corticosteroids decrease growth hormone response to these stimuli in man (1,2) while estrogens and the synthetic estrogenic compound diethylstilbestrol facilitate the release of growth hormone(3,4). The mechanism of action of these modifying effects has not been established. Corticosteroids have little effect on the concentration of growth hormone in the pituitary of the normal rat(5). If corticosteroids are administered during the induction of hypoglycemia, the expected drop in growth hormone content is inhibited(5). Estrogens on the other hand decrease the concentration of growth hormone even when compared to pair fed control rats(6). The modifying effects of estrogens and corticosteroids on growth hormone secretion could be exerted either at the level of the hypothalamus by affecting the secretion of the somatotropin releasing factor or these hormones could affect the secretion of growth hormone by acting directly on the somatotropic cells themselves. The observations of Pecile and Müeller (5) have suggested that cortisol may deplete the hypothalamus of somatotropin releasing factor. In their experiments hypothalamic extracts from cortisol treated animals had a much reduced capacity to cause a depletion of bioassayable pituitary growth hormone in recipient animals when compared to hypothalamic extracts from normal rats. Cortisol-treated recipient rats retained their ability to respond with a depletion of bioassayable growth hormone when injected with hypothalamic extracts from normal rats. Although one can not have complete confidence in the specificity of bioassay of crude pituitary extracts, these observations suggest that cortisol affects growth hormone release at the level of the hypothalamus. Despite these observations we felt that it was important to study possible influences of corticosteroids and estrogens directly on the pituitary in the absence of hypothalamic tissue.

Characterization of the binding of a rat somatomedin to receptors in human placental cell membranes.

Abstract The somatomedins presumably initiate their growth promoting effects by first binding to specific cell surface receptors in responsive tissues. The specific and high affinity binding of [125I]-rat somatomedin to human placental membranes was saturable and reversible with a dissociation constant of 4.5 × 10−9 M calculated from Scatchard analysis of competitive binding experiments. Competition for [125I]-rat somatomedin binding to placental receptors by other somatomedins and growth factors suggest a close structural relationship between rat somatomedin and the human somatomedin, insulin-like growth factor I.

Insulin Storage and Release in Rats Bearing Growth Hormone Secreting Tumors

The effects of chronic hypersomatotropism on pancreatic islet function were studied in rats bearing the growth hormone secreting tumor MtTW15. These rats did not become hyperglycemic and minimally hyperinsulinemic in the fed state following prolonged massive elevations of serum growth hormone. Islets from tumor-bearing rats were grossly larger and beta cells more granulated than in control rats by light microscopy. Electron microscopy confirmed the increase in cell cytoplasm and increased number of beta granules per cell. Isolated islet preparations from tumor-bearing rats contained four times the insulin found in control rats. Fed control and tumor-bearing rats had comparable serum glucose and immunoreactive insulin concentrations after glucose intragastrically and tolbutamide intravenously. After a forty-eight-hour fast the tumor-bearing rats had higher serum immunoreactive insulin than controls and their insulin levels were higher after glucose and tolbutamide than controls. Some of the tumor-bearing rats became hypoglycemic during the forty-eight-hour fast. The increased stored insulin in the tumor-bearing rats was mobilized by a combination of the individually potent insulinogenie stimuli theophylline and glucagon.

[59] Isolation of rat somatomedin

Publisher Summary This chapter discusses that although many litres of human serum or kilograms of human serum of Cohn Fraction IV have been the starting material for isolation of human insulin-like growth factors (hIGF), it is possible to obtain sufficient insulin-like growth factor I (rIGF I) from 1 to 2 litres of rat serum for iodination and standards for radioimmunoassay (RIA) and for characterization with highly sensitive methods. As measured in the human hIGF I/Sm C RIA, normal rat serum is from 2 to 4 times as potent as human serum. Moreover, rIGF I is only about one-third as potent as hIGF in this assay. This suggests that the true level of rIGF I in rat serum is about 600 to 900 ng/ml. Workers have isolated rIGF II—multiplication stimulating activity (MSA)—from conditioned medium of Buffalo rat hepatocytes. A method that led to the isolation and characterization of a peptide very similar in amino acid sequence to hIGF II has also been described in the chapter. The heterogeneity of IGF II related peptides in the conditioned medium is a problem. In addition to the rat IGF II isolated, there are at least six additional peptides that probably represent partially processed precursor molecules.