Characterization of an insulin-like growth factor (IGF) receptor and the insulin-like effects of IGF-1 in the bony fish, Lates calcarifer.

The present work is part of a broad phylogenetic study of the insulin superfamily of peptides in lower vertebrates. In the bony fish barramundi (Lates calcarifer), the presence of IGF receptors were investigated in the liver by means of competitive binding studies. The results suggested the presence of a type 1-like but no type 2-like IGF receptor. We also demonstrated insulin-like effects of intraperitoneally injected recombinant human (rh)-IGF-1 in barramundi with rh-IGF-1 and rh-insulin showing similar effects with respect to induction of hypoglycemia and stimulation of incorporation of [14C]-glucose into muscle glycogen.

IGF-2-like peptides are present in insulin cells of the elasmobranchian endocrine pancreas: an immunohistochemical and chromatographic study.

Evidence for the presence of peptides, related to insulin-like growth factor 2 (IGF-2), has been obtained in the endocrine pancreas of the elasmobranchian species Raja clavata, the sting ray. By radioimmunoassay, IGF-2-like immunoreactivity was detected in Raja pancreas extract. Further characterization of this activity by acid gel chromatography revealed two distinct peaks of IGF-2-like immunoreactivity with apparent molecular weights of approximately 8.2 kDa and 4.5 kDa. Using the same IGF-2 antibody as well as antisera specific for mammalian IGF-1, insulin, glucagon, somatostatin and pancreatic polypeptide in double immunofluorescence studies, IGF-2-like immunoreactivity was located exclusively in insulin-immunoreactive cells. In contrast, IGF-1-like immunoreactivity was mainly observed in somatostatin- and glucagon-immunoreactive cells. A varying proportion (0-70%) of insulin-immunoreactive cells, however, displayed both IGF-1- and IGF-2-like immunoreactivity. Absorption studies indicated that the IGF-2-like peptides in Raja are different from mammalian and submammalian insulin and mammalian IGF-1, but similar to mammalian IGF-2. Thus, IGF-2-like peptides seem to occur during evolution as early as the phylogenetic development of the elasmobranchians. Furthermore, the results indicate a particularly conservative evolution of the islet IGF-2 system.

Occurrence of members of the insulin superfamily in central nervous system and digestive tract of protochordates.

Antisera specific for mammalian insulin-like growth factor 1 (IGF-1) and mammalian insulin and the double immunofluorescence technique were used for this study. IGF-1-like-immunoreactivity was localized in entero-endocrine cells in the gastro-intestinal tract of the protochordates Ciona intestinalis and Branchiostoma lanceolatum. Some of the specimens also showed IGF-1-like-immunoreactive (-IR) perikarya and fibers in the central nervous system. Whilst in rat endocrine pancreas, IGF-1-IR and insulin-IR occurred in different cell populations, in Ciona and Branchiostoma the vast majority of entero-endocrine cells and central neurons were IGF-1-like- +insulin-IR. A minor portion exhibited IGF-1-like-IR alone. For further characterization of the IGF-1-like-IR material, in Ciona intestinalis, peptides related to IGF-1 were identified by radioimmunoassay and gel chromatography. In accordance with the immunohistochemical results, IGF-I-like-IR was detected both in cerebral ganglion and in gastro-intestinal tract. Using acid gel chromatography, in Ciona gastro-intestinal tract the IGF-1-like-IR was found to occur in two peaks, with apparent molecular weights of approximately 16 kDa and 3 kDa. Absorption studies with insulin- and IGF-related peptides, with crude extracts and the peak material obtained after gel chromatography, indicated that the IGF-1-like peptides in Ciona are different from mammalian insulin and IGF-1. The findings are in accordance with the presence of a common insulin/IGF precursor molecule in protochordates.

Identification of IGF-1 receptors in primitive vertebrates.

This is the first report of the existence of insulin-like growth factor (IGF-1) receptors in three representatives of lower vertebrates: the osteichtyes, chondrichtyes and cyclostomi. Competitive binding studies and affinity labelling of brain membranes from Cottus scorpius (sea scorpion), Raja clavata (ray) and Myxine glutinosa (atlantic hagfish) identified a mammalian type 1 or IGF-1 receptor by its binding specificity and the molecular size of its alpha-subunit. IGF-1 and IGF-2 are almost equally potent in displacing receptor-bound 125I-IGF-1 or 125I-IGF-2, and the proteins labeled with both tracers have a molecular size of 100,000-120,000 under reducing conditions. There was no evidence for the presence of a mammalian type 2 or IGF-2/mannose 6-phosphate receptor in brains of Cottus, Raja or Myxine. In all three species the binding of 125I-IGF-1 and 125I-IGF-2 was significantly higher in brain compared with liver and gastrointestinal tract, and the IGF-1 receptor could only be identified with certainty in Raja liver. It is concluded that the brain of three lower vertebrates express mammalian IGF-1 receptors, whereas IGF-2-mannose 6-phosphate receptors could not be detected.

Peptides related to insulin-like growth factor 1 in the gastro-entero-pancreatic system of bony and cartilaginous fish.

Evidence for the presence of peptides, related to insulin-like growth factor 1 (IGF-1) has been obtained in serum and various organs of representatives of osteichthyes and chondrichthyes, i.e., the bony fish Myoxocephalus (Cottus) scorpius and the cartilaginous fish Raja clavata. The peptides were identified by means of gel chromatography and an IGF-1 radioimmunoassay. IGF-1-like immunoreactivity was detected in three different apparent molecular mass forms, i.e., 17 kDa, 6 kDa and 4 kDa, the occurrence of which seemed to depend on the species. When the same antiserum was used immunohistochemically, IGF-1-like immunoreactivity was observed in endocrine cells of the open type in the intestinal mucosal epithelium. These cells exhibited distinct and species-specific distribution patterns. Endocrine cells of the pancreas as well as epithelial cells of the pancreatic duct also showed IGF-1-like immunoreactivity. Occasionally, IGF-1-like immunoreactivity was observed also in interstitial cells. The distribution patterns and densities of the IGF-like immunoreactive cells correlated with the results obtained by radioimmunoassay of the crude extracts. Absorption studies indicated that the IGF-1-like peptides observed differ from mammalian and submammalian insulins as well as from mammalian IGF-1.

Presence of IGF-1-like peptides in the neuroendocrine system of the Atlantic hagfish, Myxine glutinosa (Cyclostomata): evidence derived by chromatography, radioimmunoassay and immunohistochemistry.

By the use of radioimmunoassay and chromatography peptides related to insulin-like growth factor 1 (IGF-1) have been identified in the cylostomian species Myxine glutinosa. IGF-1-like-immunoreactivity was detected in serum as well as in brain, intestine, pancreas and liver. After acid gel chromatography, the IGF-1-like immunoreactivity eluted as one major peak, with an apparent molecular weight of between 2-4 kDa. When the same antiserum was applied immunohistochemically, IGF-1-like-immunoreactivity was observed in endocrine cells of the mucosal epithelium throughout the primitive intestinal tube. These cells were of the open type and occurred in small clusters. In addition, the majority of the endocrine cells of the pancreas of Myxine displayed IGF-1-like-immunoreactivity. In some of the specimens investigated IGF-1-like-immunoreactive perikarya and fibers were observed on all levels of the brain. Distribution patterns and densities of the IGF-1-like-immunoreactive structures in Myxine correlated with the measurements obtained by radioimmunoassay. Absorption studies with insulin- and IGF-related peptides as well as with crude extracts and the peak material obtained after gel chromatography indicated that the IGF-1-like peptides in Myxine are different from mammalian and non-mammalian insulins as well as from mammalian IGF-1. Generally, the results suggest a long phylogenetic history of IGF-1-like peptides and indicate their fundamental functional impact in all vertebrates.

Circulating forms and biological activity of intact and truncated insulin-like growth factor I in adult and neonatal rats.

A variant of IGF-I with a truncated aminoterminal region has been isolated and shown to display increased biological activity in vitro, but weak affinity of binding to the IGF binding proteins compared with intact IGF-I. In the present study, the circulating molecular forms and biological activity of intact and truncated IGF-I were compared after in vivo administration. Adult and 10-day-old rats were given 125I-truncated or 125I-intact IGF-I iv. In both adult and 10-day-old rats 125I-truncated IGF-I showed weaker affinity of binding to the IGF binding proteins and greater degradation than 125I-intact IGF-I. Serum half-life was 2 h for 125I-truncated IGF-I and 3 h for 125I-intact IGF-I in adult rats. The half-life in 10-day-old rats was 20.5 min for 125I-truncated IGF-I and 27 min for 125I-intact IGF-I. The uptake of 125I-truncated IGF-I into the kidney, liver and brain of 10-day-old rats was significantly higher than for 125I-intact IGF-I 15 min after iv administration. The insulin-like effects of the IGF-I peptides were examined in vitro and in vivo. Truncated IGF-I stimulated [3-3H]glucose incorporation into free fatty acids in adipocytes in vitro to a greater extent than did intact IGF-I. In vivo administration of both intact and truncated IGF-I to adult rats significantly decreased serum glucose levels and significantly increased the incorporation of [U-14C]glucose into glycogen.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of altered nutritional status upon insulin-like growth factors and their binding proteins in neonatal rats.

To investigate the role of nutrition in the regulation of IGFs during the perinatal period, 10-d-old rats were infused intravenously with various concentrations of nutrients for 24 h. Breast-fed litter mates served as controls. The effect of caloric intake on concentrations of IGF-I and IGF-II as well as IGF-binding proteins in serum, liver, and brain of neonatal rats was studied. A total of 45 rats from 10 litters was infused with solutions ranging from a caloric intake of 0 (saline) to 75% (glucose, amino acids, and lipids) of the estimated intake of control rats. In serum, both IGF-I and -II concentrations fell markedly in response to fasting. Serum IGF-II levels were linearly related to caloric intake in the pooled data from all groups. Concentrations of IGF-II, but not IGF-I, in liver and brain were depressed by caloric restriction. In contrast to the fall in IGF concentrations, activity of IGF-associated binding proteins rose in serum and in liver cytosol 2- to 4-fold in response to decreased nutrient intake. In serum, but not liver, the rise in binding protein activity was inversely related to to caloric intake. In liver, cytosol, but not serum, the rise in binding protein activity was inversely related to total serum amino acid concentration. Thus, IGF concentrations in preweanling rats change in response to alterations of nutrient intake. The fasting induced decrements in IGF levels, as well as the elevations in IGF-associated binding protein activity, may serve as a protective mechanism to depress growth in times of caloric restriction.

The study of insulin-like growth factors in Tilapia, Oreochromus mossambicus.

Whole and acid-separated serum samples from fed, starved, and refed Tilapia were analyzed for insulin-like growth factors 1 (IGF-1) and 2 (IGF-2) using human fetal brain radioreceptorassay (RRA-IGF-1), rat liver membrane radioreceptorassay (RRA-IGF-2), and radioimmunoassay (RIA-IGF-1). Triidothyronine (T3) and thyroxine (T4) levels were measured by commercial kits for RIA. For serum separation, acid Sephadex G-50 and G-100 and neutral Sephadex G-200 columns were used. Whole serum and separated serum cross-reacted in RRA-IGF-1, but only slightly in RRA-IGF-2. IGF activity eluted in two peaks after acid G-50 chromatography. Peak I eluted at the void volume, and peak II eluted with an apparent molecular weight of approximately 7 kDa. The 7 kDa activity did not cross-react in RIA-IGF-1 excluding identity with human intact or truncated IGF-1, but did suggest the presence of an IGF-1 variant form. Whole serum was separated over a neutral G-200 column, and all activity eluted at the void volume indicated an apparent molecular weight equal to or greater than 250 kDa. No IGF-binding activity was displayed by either whole serum or peak I after acid G-50 chromatography. Despite significant changes in body weight, an influence of starvation and refeeding on serum IGF activity could not be established. No correlation was seen between serum IGF and T3 and T4 levels.