Comparative studies on fish and bird insulin receptors

1987

Insulin receptor assays using canine liver membranes and erythrocytes were validated for use In evaluating Insulin receptor binding parameters within a healthy population of dogs. Association, dissociation, saturabllity and specificity were demonstrated for Insulin receptors of canine liver membranes and erythrocytes. Assay precision was evaluated by measuring Interassay coefficient of variation (liver membrane " 25.OX; erythrocytes ” 9.3%). Because the number of hlgh-affinity receptor sites and highaffinlty K^ were determined using physiological concentrations of insulin, they were considered to be more physiologically important than the total number of receptor sites, low-affinlty Kp and Ke, which were derived using supra-physlologlcal concentrations of insulin. The X maximum binding correlated with the number of high-affinity receptor sites and Ke, but negatively correlated with high-affinity Kp. In addition to correlating binding parameters within and between tissues, the e...

Molecular and Cellular Endocrinology, 1993

Insulin receptor and insulin sensitivity in a chicken hepatoma cell line Kev wordx Chicken hepatoma (LMH) cell line; Chicken hepatocyte; insulin receptor: Phosphotyrosine protein: Tyrosine kinase; Amino acid transport

General and Comparative Endocrinology, 1971

This paper describes an adaptation of the radioimmunoassay of Hales and Randle for the determination of insulin levels in fish serum and in media used for the incubation of fish islets. Using codfish insulin as a reference standard, guinea pig anticod insulin serum, and "'I-labeled cod insulin, instead of the standard mammalian components, the average fasting serum insulin level (*SEM) was found to be 36 + 9 pU/ml in goldfish (Carassius auratus) and 19 * 6 pU/ml in toadfish (Opsanus tau). Serum insulin levels of fed goldfish were nearly double that of fasted fish. Glucose (40 or 100 mg/lOO ml) or leucine (1 mg/ml), stimulated the release of insulin by incubated islets of 0. tau. The leucine effect appeared to be enhanced b> the simultaneous addition of glucose (40 mg/lOO ml) to the incubation medium, Arginine (1 mg/ml) had no effect upon the release of insulin in vitro. The significance of the results is discussed. Marked chemical, biologic, and immunologic differences have been demonstrated between insulins of different animal species (Smith, 1966; Falkmer and Wilson, 1967). These differences result in poor affinity of fish insulin for antibody to mammalian insulin and make the measurement of insulin levels in fish, with commonly available antimammalian insulin sera and mammalian insulin standards difficult, if not impossible (Tashima and Cahill, 1968; Watkins et al., 1968; Dixit et al., 1969). This paper describes an adaptation of the insulin immunoassay of Hales and Randle (1963) in which cod fish insulin and guinea pig anticodfish-insulin serum (GPACIS) were substituted for the equivalent mammalian components. This sys-'Aided by Grants Nos. 5 ROl AM06034 and 5 SO1 FR05641.

General and Comparative Endocrinology, 1992

Amphibia undergo regular annual cycles of metabolic activity that are influenced by both exogenous factors and hormones. Insulin binding to crude frog hepatic membranes was studied throughout the year. The general character of insulin binding was similar to that in other vertebrates; the maximum specific binding was achieved after 4 hr at 4", the optimum pH was 7.8, half-maximal displacement of bound insulin was from 9 X lo-"' to 1 X lo-?W, and insulin analogs competed for the insulin receptor in line with their relative biological potencies. A biphasic Scatchard plot and negative cooperativity of the receptor were also observed in frog liver membranes. Affinity constants from Scatchard plots revealed high and low affinity binding sites which were unchanged during the year. The seasonal cycle, however, markedly affected the binding capacity for both sites. Maximum binding occurred in May-June and the minimum in November-December for both classes of receptors. Binding capacities ranged from 1.71 to 11.33 fmol/mg protein for the high affinity sites and from 432 to 3171 fmol/mg protein for the low affinity sites. It is concluded that annual cycles of insulin binding reflect modulation of receptor number rather than receptor affinity. Q

Comparative biochemistry and physiology. A, Comparative physiology, 1982

The effect of insulin was studied on blood glucose, and on the glycogen level of liver, muscles and heart in fed and in starved catfish (Ictalurus melas). Fish received intraperitoneally 60 iu/kg body weight of bovine insulin, or physiological saline and were sacrificed after 2, 4, 8, 24, 72 hr from injection. Insulin caused a decrease of blood glucose level, both in fed and in fasted animals, and the effect is more evident in fed animals. After insulin treatment, liver glycogen shows a decrease which is significant at the 8th and 24th hr in fasted and in fed animals respectively; after 72 hr the glycogen level in livers of fed and fasted animals is still very low. Insulin increases the glycogen level both in white and in dark muscle, both in fed and in fasted fish, although with different characteristics, but at the 72nd hr in all animals, the increases are significant. Hormone treatment does not change heart glycogen levels in fed catfish till the 24th hr, then there is a net decr...

Fish Physiol Biochem, 2001

Two insulins were isolated from an extract of the Brockmann bodies of channel catfish (Ictalurus punctatus). The primary structure of insulin I is: A-Chain: GIVELCCHKP 10 CSLHDLQNYC 20 N; B-Chain: GAPQHLCGSH 10 LVDALYLVCG 20 PNGFFYNPK. Insulin II has three amino-acid substitutions compared with insulin I: A14His → Gln, B1Gly → Val, and B13Asp → Glu. Despite some unusual amino acid substitutions in the catfish insulins compared with human insulin, such as A5Gln → Leu, B21Glu → Pro and B22Arg → Asn, those residues believed to constitute the receptor-binding domain are conserved. Consistent with this, catfish insulins I and II were equipotent in inhibiting the binding of [3-[ 125 I] iodotyrosine-A14] human insulin to the soluble human insulin receptor and were only 3-fold less potent than human insulin in the same assays. An analysis of mRNA expression in Brockmann bodies by reverse-transcriptase PCR identified two proinsulin sequences for the channel catfish containing a single, highly conserved C-peptide whose deduced amino acid sequence is REVDPLLGFL 10 PPKSAPEGEL 20 AEYPYKEYSE 30 LMVKR. PCR of genomic DNA with specific proinsulin primers spanning the intron II interrupting the C-peptide of all vertebrate insulins, produced two introns of 105 and 104 bp, respectively. The nucleotide sequences of the introns differ in 13 positions. Because of the high degree of conservation in insulin and C-peptide and the large variability in the small intron, we conclude that the two insulins isolated are the products of different genes and do not simply represent different alleles. The channel catfish is a diploid species that may have undergone gene or chromosome duplication and therefore we propose that multiple insulin genes may not be restricted to polyploid species such as salmonids or sturgeons.

Diabetes Care, 1981

Biosynthetic human insulin (BHI) and pancreatic human insulin were compared with respect to receptor binding in a heterologous assay system: displacement of pork A14-125I-monoiodoinsulin from receptors on pig hepatocytes. The concentrations of human insulin giving half-maximal displacement were identical for both preparations, i.e., 0.5 nM. Their relative potency was 1.01 ± 0.14 (SD, N = 5), suggesting that biosynthetic and pancreatic human insulin exert the same biologic activity.

Diabetes Care, 1981

Biosynthetic human insulin (BHI) was compared with highly purified human pancreatic and pork insulin with regard to its ability to bind to erythrocytes of normal and insulin-dependent diabetic subjects (type I diabetes). The binding affinity or capacity of erythrocyte from both normal and diabetic subjects were comparable for biosynthetic and pancreatic human insulin. In contrast, binding of pork insulin to erythrocytes was significantly decreased at low insulin concentrations in normal as well as in diabetic subjects due to a reduced receptor affinity. The affinity of the “empty sites” was 5.25 × 10−8 M−1 with pork insulin in normal subjects and 6.1 × 10−8 M−1 in diabetic subjects; with both human insulins, the affinities were 6.9 × 10−8 M−1 in normal subjects and 8.6 × 10−8 M−1 in diabetic subjects. The number of insulin receptors per erythrocyte was calculated as being 30 in normal subjects and 35 in diabetic patients.

European Journal of Biochemistry, 1983

Insulin from an elasmobranch, the spiny dogfish (Squalus acantlzias) has been purified to near homogeneity by means of acid-ethanol extraction and salt precipitation. The amino acid sequences of the performic-acid-oxidised A and B chains have been determined and exhibit some unusual features. The A chain contains a total of 22 amino acids; only the insulin from coypu (a member of the Rodentia suborder, Hystricomorpha), has previously been reported to contain an extension past the A,, asparagine. The B,, histidine, which is involved in the formation of the insulin hexamers in higher vertebrates through the co-ordination of zinc, is present in this elasmobranch insulin. Several substitutions relative to bovine insulin occur in the proposed receptor binding region (A,Gln+ His, B, Glu +Pro, B,,Arg+Lys, B,,Phe+Tyr). In spite of these substitutions, the maximal response in the rat epididymal fat cell assay is the same for bovine and dogfish insulins; the concentration required to produce the half-maximal response is, however, approximately threefold greater for dogfish insulin than that of bovine insulin.

Journal of Biological Chemistry, 1973