High affinity binding sites for proinsulin in human IM-9 lymphoblasts

Summary Proinsulin and insulin binding in IM-9 lymphoblasts show curvilinear Scatchard plots, which may be explained by two binding sites, negative cooperativity of receptors, or both. Using flow-cytometric analysis of insulin binding, we were able to distinguish and separate two different IM-9 cell fractions. In both fractions, Scatchard plots for specific binding of insulin and proinsulin were linear, suggesting the presence of two distinct populations of receptors. Type 1 cells showed low capacity but high affinity of insulin binding (16,300±3,000 sites/cell; K_d 0.4±0.1 nmol/l). Proinsulin and insulin-like growth factor 1 (IGF-1) were significantly less potent in competition. MA-20, a specific antibody against human insulin receptors, inhibited insulin binding by 80%, while the specific antibody against human IGF-1 receptors, IR-3, had no effect. Pretreatment with insulin decreased insulin binding by 90%. ¹²⁵I-insulin displayed stepwise dissociation with the rate markedly enhanced by cold insulin. Type 2 cells exhibited significantly different binding characteristics with higher capacity but lower affinity of ¹²⁵I-insulin binding (430,000±25,000 sites/cell, _p<0.001 vs type 1; K_d 2±0.4 nmol/l, p<0.02 vs type 1). Proinsulin competed with similar potency for insulin binding, while IGF-1 was still less potent. ¹²⁵I-proinsulin showed a significantly higher binding affinity than ¹²⁵I-insulin (K_d 0.5±0.3 nmol/l, p<0.05) with 50,000±10,000 binding sites/cell. C-peptide was able to compete for ¹²⁵I-proinsulin, but not for ¹²⁵I-insulin binding. MA-20 did not influence ¹²⁵I-proinsulin binding, but inhibited ¹²⁵I-insulin binding by 50%, whereas IR-3 increased proinsulin binding 1.5-fold with no effect on insulin binding. Preincubation with insulin decreased insulin binding by 50% and proinsulin binding by 10%. The dissociation of ¹²⁵I-proinsulin showed linear first-order kinetics and was not significantly accelerated by cold proinsulin. Furthermore, the tyrosine phosphorylation of a 65 kDa protein was stimulated to a significantly greater extent by proinsulin than by insulin, indicating activation of different signalling cascades. DNA analysis revealed that type 1 cells were predominantly in the G₁ phase, whereas type 2 cells were in the S and G₂ + M phases of the cell cycle. We conclude, that IM-9 lymphoblasts were separated by flow-cytometry into one fraction with typical insulin receptors and a second fraction with high affinity binding sites for proinsulin. High affinity proinsulin binding sites were distinguished from typical insulin receptors by: 1) higher affinity for proinsulin than insulin, 2) inhibition of proinsulin binding by C-peptide but not by the insulin receptor antibody MA-20, 3) non-co-operative first order dissociation kinetics of proinsulin binding, 4) resistance to down-regulation by insulin, and 5) differences in signal transduction.Abbreviations IR-3 Monoclonal anti-IGF-1 receptor antibody - BSA bovine serum albumin - FACS fluorescence-activated cell sorting - FCS fetal calf serum - FITC fluorescein isothiocyanate - G₁, S, G₂ + M cell cycle phases - HEPES N-[2-hydroxyethyl]piperazine-N-[2-ethanesulphonic acid] - IGF-1/2 insulin-like growth factor-1/2 - MA-20 monoclonal anti-insulin receptor antibody - NIDDM non-insulin-dependent diabetes mellitus - PBS phosphate buffered saline - TCA trichloroacetic acid - type 1 IM-9 cell fraction with low insulin binding capacity - type 2 IM-9 cell fraction with high insulin binding capacity     

Binding and biological effects of insulin,insulin analogues and insulin-like growth factors in rat aortic smooth muscle cells. Comparison of maximal growth promoting activities

Summary Binding and growth promoting effects of insulin, insulin analogues modified in the B chain, proinsulin, insulin-like growth factor-I and -II were studied in cultured rat aortic smooth muscle cells. Specific binding of¹²⁵I-insulin was 0.9±0.2% of total ¹²⁵I-insulin added, and the IC₅₀-value was estimated to 8.9 pmol/1. The insulin analogue B10 Asp tended to be more potent than insulin in displacing ¹²⁵I-insulin, B28 Asp was equipotent, B9 Asp/B27 Glu was approximately 100 times less potent and insulin-like growth factor-I more than 1000 times less potent than insulin. Specific binding of ¹²⁵I-insulin-like growth factor-I after 4 h incubation at 10 °C was five times higher than the specific binding of insulin (4.4±0.4% of total ¹²⁵I-insulin-like growth factor-I added), and the IC₅₀-value was 0.3 nmol/l. Insulin was approximately 500 times less potent than insulin-like growth factor-I in displacing ¹²⁵I-insulin-like growth factor-I. The insulin analogue B10 Asp was slightly more potent and analogue B28 Asp was equipotent with insulin. Analogue B9 Asp/B27 Glu was ten times less potent and proinsulin was more than ten times less potent than insulin. The order of potency was similar for ³H-thymidine incorporation into DNA: insulin-like growth factor-I > B10 Asp > insulin-like growth factor-II > insulin > B28 Asp > B9 Asp/B27 Glu > proinsulin. The maximal effect of insulin-like growth factor-I on ³H-thymidine incorporation was 71±16% higher than the maximal effect of insulin. The maximal effect of insulin-like growth factor-II was at least as high as the effect of insulin-like growth factor-I. Furthermore, the maximal effect of B10 Asp was 62±10% higher than the maximal effect of insulin. Insulin-like growth factor-I and B10 Asp tended to increase cell number more than insulin. In conclusion, this study shows that insulin analogues interact with different potencies with receptors for insulin and insulin-like growth factor-I in vascular smooth muscle cells and that insulin-like growth factors and the insulin analogue B10 Asp have more pronounced growth effects than insulin. Substitution of the amino acid Asp for His at position B10 in insulin makes the molecule more similar to insulin-like growth factor-I, chemically and probably also biologically.     

Receptors for insulin and insulin-like growth factor in cultured arterial smooth muscle cells depend on their growth state

The binding of 125I-labelled insulin and 125I-labelled insulin-like growth factor (IGF) to cultured arterial smooth muscle cells from rats was studied during various growth states of the cells. The level of binding of 125I-labelled insulin to the cells was low in growing cells and high in stationary cells. The level of 125I-labelled IGF binding to the cells was high in growing cells and low in stationary cells. In addition, the effect of unlabelled IGF and insulin on the binding of both 125I-labelled hormones to the cells was examined during various growth states. In growing cells insulin displaced 125I-labelled insulin from its binding sites; IGF competed weakly with 125I-labelled insulin for the binding sites. In parallel, IGF displaced 125I-labelled IGF binding whereas insulin competed weakly with 125I-labelled IGF for the binding sites. In stationary cells both hormones displaced 125I-labelled IGF binding. Insulin-like growth factor also displaced 125I-labelled insulin binding whereas insulin could not significantly displace 125I-labelled insulin from the binding sites. Insulin only competed with 125I-labelled insulin for the binding sites after removal of the fetal calf serum from the culture medium.     

Measurement of human proinsulin by an indirect two-site immunoradiometric assay

Summary An indirect two-site immunoradiometric assay is described for the measurement of human proinsulin in plasma. Polyethylene tubes coated with purified guinea-pig antibodies to insulin were used to extract proinsulin and insulin from plasma. Rabbit antibody to human C peptide was then added to react with the C-peptide moiety of the bound proinsulin. The uptake of this antibody was measured by the subsequent binding of¹²⁵I-sheep antibody to rabbit IgG. The binding of radioactivity to the tubes was a function of the proinsulin concentration in the sample. The sensitivity of the assay was 0.006 pmol/ml. Only 200 l of plasma was required in the assay and the¹²⁵I-labelled antibody was produced from readily available reagents. The polyethylene tubes remained stable for at least 5 months after coating. The mean fasting proinsulin level was 0.009 pmol/ml in sixteen normal subjects and 0.025 pmol/ml in twelve maturity onset diabetics. Oral glucose produced an 8 fold increase in proinsulin concentration but a decline in the plasma proinsulin/insulin molar ratio. Four patients with insulinoma had extremely elevated proinsulin levels and proinsulin/insulin ratios.     

The insulin receptor of embryonic chicken cartilage.

Highly purified plasma membranes have been obtained from embryonic chicken cartilage by physical means rather than enzymatic digestion. Rapid and reversible binding of [125I]iodoinsulin to these membranes is demonstrated. Similar to the insulin-binding properties of rat liver and adipocytes and human mononuclear cells, optimal specific binding of insulin to chondrocyte plasma membranes has a sharp pH optimum at 8.0, and maximal binding occurs at 2--4 C. Analysis of equilibrium binding reveals a curvilinear Scatchard plot, whose high affinity segment generates a maximum affinity of 1.0 X 10(9) M-1, and a receptor concentration of 0.4 pmol/mg membrane protein. This affinity constant is similar to those generated for insulin binding to membranes prepared from embryonic chicken liver (2.5 X 10(9) M-1), rat liver (1.4 X 10(9) M-1), and mouse liver (0.6 X 10(9) M-1), whereas the receptor concentration is less than that of embryonic chicken liver membranes (1.1 pmol/mg), which in turn was less than those of rat liver membranes (2.8 pmol/mg) and mouse liver membranes (3.5 pmol/mg). Kinetic studies show augmentation of insulin-receptor dissociation by excess insulin when initial receptor occupancy, is low, suggesting that negative cooperativity is present. There is little or no interaction of other hormones with the chondrocyte insulin receptor, with the exception of proinsulin and the insulin-like growth factors. Porcine proinsulin, bovine proinsulin, somatomedin C, and nonsuppressible insulin-like protein prevent [125I]iodoinsulin binding to chondrocyte plasma membranes with dose-response curves which are parallel to that of unlabeled porcine insulin itself, but with molar potencies relative to porcine insulin of 15%, 9%, 2.5%, and 1.4%, respectively. Porcine insulin and proinsulin both prevent binding of [125I]iodosomatomedin C to chondrocyte plasma membranes but with molar potencies less than 1% that of unlabeled somatomedin C. These observations are consistent with the presence of a specific independent insulin receptor in embryonic chicken cartilage which is similar in its characteristics to the insulin receptor in previously described tissues. Insulin has a weak interaction with the chondrocyte receptor for somatomedin C. Interaction with the somatomedin receptor may be the mechanism by which insulin exerts anabolic effects on cartilage when used in pharmacological amounts.     

NSILA-carrier protein abolishes the action of nonsuppressible insulin-like activity (NSILA-S) on perfused rat heart

Summary Human serum in a concentration of 10% in the perfusion medium failed to increase glucose uptake by the isolated perfused rat heart, indicating that nonsuppressible insulin-like activity (NSILA) in whole serum was inactive in this system. When NSILA-carrier protein was added to partially purified NSILA-S, its biological activity on the rat heart disappeared. In contrast, the action of insulin was not affected by the presence of NSILA-carrier protein. Binding of¹²⁵I-labelled NSILA-S to rat heart was inhibited by NSILA-carrier protein.¹²⁵I-labelled insulin binding was not inhibited. These results support the hypothesis that NSILA-S bound to serum carrier protein is a large molecular compound which does not readily diffuse out of the capillary bed and therefore does not exert insulin-like effects in vivo.     

A rapid, sensitive and versatile two-site immunoradiometric assay for insulin

A two-site immunoradiometric assay for insulin is described which is both rapid (processing time 60 min) and highly sensitive (lower detection limit 2 pM). Insulin is bound by a 125I-labelled mouse monoclonal antibody raised against human proinsulin and binding assessed by immunoprecipitation with an immunoadsorbent prepared from guinea pig polyclonal antisera raised against bovine insulin. Human, rat, bovine and porcine insulins (10-600 pM) showed similar reactivities in the assay. The human insulin-like peptides, proinsulin, des-31,32-proinsulin and des-64,65-proinsulin (25 pM) had reactivities which were 44.7%, 63.2% and 73.4% of that of insulin, respectively. The assay was highly reproducible with a coefficient of variation of 2.3% for the highest human insulin standard (1000 pM) and 5.5% for the lowest (2 pM). The assay was suitable for determining the concentration of insulin in plasma of fasting human subjects, in normal and tumour-bearing rats and for in vitro studies of insulin secretion from rat pancreatic islets.     

Autocrine tumour growth regulation by somatomedin C: an in-vitro model

A human primary haemangiosarcoma was derived from a patient with severe hypoglycaemia. Cell line established from that tumour secreted somatomedin C in serum-free culture media. Immunoreactive somatomedin from the media eluted from Sephacryl S-200 in two peaks of 160 000 and 8000 molecular weights. Similar results were obtained when medium was acidified and chromatographed on Sephadex G-50. Binding of tracer concentrations of 125I-labelled somatomedin C to human haemangiosarcoma cells was much higher than that of 125I-labelled insulin. Half-maximal displacement of 125I-labelled somatomedin C binding occurred at an unlabelled somatomedin C concentration of 0.7 nmol/l. Insulin competed with 125I-labelled somatomedin for binding to this receptor, but 150-fold more insulin was required for half-maximal displacement. Somatomedin secreted by human haemangiosarcoma cells and purified from serum-free media strongly stimulated [methyl-3H]thymidine incorporation into the DNA of these cells. Inhibition of somatomedin C secretion by cortisol resulted in the inhibition of tumour cell proliferation but stimulation of somatomedin secretion by human GH stimulated the cell proliferation rate. It appears that production of somatomedin C in human haemangiosarcoma cells plays a part in the regulation of tumour growth by an autocrine mechanism.     

Growth hormone activates phospholipase C in proximal tubular basolateral membranes from canine kidney.

To delineate pathways for "signal" transduction by growth hormone (GH) in proximal tubule, we incubated basolateral membranes isolated from canine kidney with human growth hormone (hGH) or human prolactin (hPrl) and measured levels of inositol trisphosphate (InsP3) in suspensions and of diacylglycerol extractable from the membranes. Incubation with hGH, but not hPrl, increased levels of InsP3 and diacylglycerol in a concentration-dependent manner. Half-maximal effects occurred between 0.1 and 1 nM hGH. Increased levels of InsP3 were measured after as little as 5 sec of incubation with 1 nM hGH, and increase was maximal after 15 sec. Increases were no longer detectable after 60 sec because of dephosphorylation of InsP3 in membrane suspensions. hGH did not affect rates of dephosphorylation. hGH-stimulated increases in InsP3 were detectable in membranes suspended in 0, 0.1, and 0.2 mM calcium but not in 0.3 or 1.0 microMs calcium. 125I-labeled hGH-receptor complexes with Mr values of 66,000 and 140,000 were identified in isolated basolateral membranes. Our findings establish that GH activates phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) in isolated canine renal proximal tubular basolateral membranes, potentially after binding to a specific receptor. This process could mediate "signal" transmission by GH across the plasma membrane of the proximal tubular cell and elsewhere.     

Proinsulin autoantibodies are more closely associated with Type 1 (insulin-dependent) diabetes mellitus than insulin autoantibodies

Summary The disease association of autoantibodies to proinsulin and insulin was compared in patients with Type 1 (insulin-dependent) diabetes mellitus and first-degree relatives. Following the recommendation of the Fourth International Workshop on the Standardization of insulin autoantibodies, autoantibodies were determined by fluid-phase radioimmunoassay using equimolar concentrations of mono¹²⁵I-A14-insulin or -proinsulin to detect insulin or proinsulin autoantibodies, respectively. A higher prevalence of proinsulin autoantibodies vs insulin autoantibodies was found in 97 patients with Type 1 diabetes prior to insulin treatment (34.0 % vs 22.7 %, p< 0.05) and in 16 islet cell antibody-positive relatives (43.8% vs 31.3%, NS). There was only one serum positive for insulin and proinsulin autoantibodies in 110 islet cell antibody-negative first degree relatives (0.9 %). None of 88 normal sera contained proinsulin autoantibodies or insulin autoantibodies. There was a close correlation of proinsulin autoantibody and insulin autoantibody titres in individual sera (r=0.95, p< 0.01) due to crossreaction of all insulin autoantibodies with proinsulin. However, some proinsulin autoantibodies did not crossreact with insulin. Background binding in normal sera was lower for proinsulin autoantibodies. We conclude that proinsulin autoantibodies have a higher association to acute Type 1 diabetes than insulin autoantibodies.Part of this work was presented at the 26th Annual Meeting of the EASD in Copenhagen, 10^th–13^th September 1990     

Specific and direct radioimmunoassay for human proinsulin in serum

Summary A routine radioimmunoassay for human proinsulin in serum has been developed. The reagents used were: antibodies against the C-peptide part of the proinsulin molecule, human proinsulin as the standard and¹²⁵I-labelled synthetic human Tyr-C-peptide as the tracer. The first step in this assay comprises the binding of proinsulin to insulin antibodies covalently coupled to Sepharose (S-AIS). Although bound to the solid-phase S-AIS, the proinsulin retains its second immunogenic site, viz., the C-peptide part of the molecule, accessible. Hence a surplus of C-peptide antibodies is added to the S-AIS-bound proinsulin, and the residual amount of C-peptide antibody is determined by addition of¹²⁵I-Tyr-C-peptide. The detection limit is approximately 0.01 pmol/ml. The advantages of this method are: (1) its high specificity (proinsulin is determined as a molecule having both an insulin and a C-peptide moiety), (2) its simplicity and rapid performance, and (3) the low detection limit of the assay. Fasting sera from 24 nondiabetics, 9 maturityonset diabetics and 10 newly diagnosed insulin requiring diabetics showed the following concentrations of proinsulin: 0.009±0.005, 0.022±0.23 and 0.010±0.009 pmol/ml (mean±SD). One hour after 1.75 g/kg oral glucose, the values increased to 0.052±0.023, 0.046±0.022 and 0.032±0.022 pmol/ml. The fasting proinsulin constituted 19, 23 and 17% of the IRI, respectively, whereas 1 h post glucose these values changed to 8, 9 and 31% of IRI. Serum from 10 insulin-treated diabetics containing insulin antibodies contained from 0–1.80 pmol/ml, whereas the C-peptide levels in the same patients were 0–0.35 pmol/ml. It is suggested that insulin requiring diabetics hypersecrete proinsulin due to the inability of their B-cell to arrange proinsulin in secretory granules for adequate proinsulin/insulin conversion.     

Angiotensin II-binding sites in rat and primate isolated renal tubular basolateral membranes

In the kidney, angiotensin II influences reabsorptive processes by a direct tubular effect(s). The receptors mediating the response may be located on either the luminal (brush border) and/or the contraluminal (basolateral) membranes of the tubular epithelial cells. In the studies reported here, we identify specific [125I]angiotensin II-binding sites in rat and baboon tubular basolateral membranes. Specific binding was saturable, largely reversible, and proportional to membrane protein concentration. Structural specificity was confirmed by the use of angiotensin analogs and structurally unrelated polypeptides. The latter did not compete with radioligand for binding. Scatchard analyses of binding inhibition data indicated a single class of high affinity sites in rat (Kd = 2.2 +/- 0.2 nM; n = 12) and two classes of sites in baboon [Kd = 1.32 (n = 1) and 0.6 +/- 0.1 nM; n = 2) basolateral membranes. The binding site concentrations were 929 +/- 138 fmol/mg protein (rat) and 463 and 439 +/- 120 fmol/mg protein (baboon). Rat binding sites were affected by the addition of cations, as chloride salts, to the incubation medium. Na+ (100-200 mM) decreased the Kd from 4.2 +/- 0.4 nM in the absence of cations to 2.7 +/- 0.3 nM (n = 4). Mg2+ (4 mM) had no effect on Kd, but increased the binding site concentration from 556 +/- 84 to 915 +/- 166 fmol/mg protein. In contrast, 2 mM Ca2+ increased the Kd to 5.3 +/- 0.6 nM, and Mg2+ and Ca2+ added together affected neither Kd nor number of binding sites. Bound (eluted from membranes) and free (in medium) radioactivity, after incubation with membranes to steady state, were 54-68% (n = 3) and 85% (n = 2) intact [125I] angiotensin II, respectively, as determined by rebinding to fresh membranes. These data are inconsistent with binding to a degradative enzyme and indicate the presence of specific [125I] angiotensin II-binding sites in renal tubular basolateral membranes.     

Receptor binding and biological effect of insulin in human adipocytes

Summary The binding of¹²⁵I-labelled insulin to human adipocytes was studied at 37° C. The precipitability of the¹²⁵I-labelled insulin preparation (0.03 nmol/l) in trichloroacetic acid and the concentration of biologically active insulin (7.5 nmol/l) remained constant in buffer incubated with human adipocytes (100 l cells/ml suspension) for 30–60 minutes at 37° C, whereas more than half of the insulin was inactivated by rat fat cells under the same conditions. A constant level of binding of¹²⁵I-labelled insulin (0.03 nmol/l) to human adipocytes was obtained after 45 minutes. The apparent dissociation constant of receptor binding was about 0.2 nmol/l as compared to about 2 nmol/l for rat adipocytes. Conversion of [U-¹⁴C]glucose to lipids was stimulated half-maximally by about 0.05 nmol/l of insulin (similar to rat adipocytes). Thus, half-maximal stimulation of human adipocytes was obtained with a receptor occupancy of about 20–30 per cent.     

Structural determinants of ligand recognition by type I insulin-like growth factor receptors: use of semisynthetic insulin analog probes

We undertook a systematic analysis of the structural determinants necessary for ligand recognition by the type I insulin-like growth factor (IGF) receptor by investigating the binding of semisynthetic insulin analogs to IGF receptors from human placental cell membrane fragments. Analogs were prepared by synthetic and semisynthetic methods. Three groups of insulin analogs were synthesized: the first group contained insulin analogs modified at the amino-terminal position of the insulin A chain and included acetyl-insulin and human proinsulin; the second group included analogs in which B chain residues B26-B30 [despentapeptide insulin (DPI)], B25-B30 (deshexapeptide insulin), and B24-B30 (desheptapeptide insulin) were removed; the third group contained insulin analogs in which B chain residues B26-B30 were removed (DPI) and phenylalanine(B25) substituted with other amino acids, including alanine, serine, leucine, and tyrosine. Half-maximal inhibition of binding of radiolabeled IGF-I to placental cell membrane fragments was used as an index of relative binding affinity (K1/2). To determine further if semisynthetic insulin analogs bound to the type I IGF receptor, placental membrane fragments were affinity labeled with radiolabeled IGF-I in the presence and absence of submaximal concentrations of unlabeled hormone, insulin, or semisynthetic analogs, and the labeled proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Insulin had a 500 times lower affinity for the type I IGF receptor than IGF-I [K1/2 = 140 +/- 69 nM (mean +/- SD)] whereas proinsulin and acetyl insulin had a more than 100 times lower affinity than insulin for this receptor type. Removal of insulin B chain amino acid residues 26-30 (DPI) did not negatively affect the binding of the insulin-derived peptide and actually increased the apparent affinity of ligand-receptor association approximately 2-fold. However, further removal of phenylalanine(B25) (deshexapeptide insulin) and phenylalanine(B24) (desheptapeptide insulin) decreased the binding of ligand to the type I IGF receptor progressively by several orders of magnitude. Substitution of phenylalanine(B25) of DPI with tyrosine, a substitution that actually increased the homology of this analog to IGF-I, resulted in a 4- to 5-fold increase in the relative apparent affinity of the analog for the type I IGF receptor (K1/2 = 31 +/- 4 nM). On the other hand, substitution of phenylalanine(B25) with alanine, serine, and leucine decreased the relative apparent binding affinity approximately 2- to 8-fold.(ABSTRACT TRUNCATED AT 400 WORDS)     

Production of anti-idiotypic antisera to rat GH antibodies capable of binding to GH receptors and increasing body weight gain in hypophysectomized rats

Anti-idiotypic antibodies to rat GH antibodies were produced in both sheep and mice and shown to be capable of mimicking GH by inhibiting 125I-labelled ovine GH (oGH) binding to sheep liver membranes. The sheep anti-idiotypes were characterized further and shown to (1) inhibit 125I-labelled oGH binding to oGH antibodies, (2) inhibit 125I-labelled oGH binding to rat adipocytes and (3) be incapable of inhibiting the binding of either 125I-labelled ovine prolactin or 125I-labelled bovine insulin to sheep liver membranes. This indicated that the antibodies were not limited to certain species or tissues, but were hormone specific. Finally, these anti-idiotypic antibodies were also capable of stimulating an increase in body weight gain in hypophysectomized rats, suggesting that they may be functional as well as structural mimics of GH, although the increased body weight gain was not accompanied by any increase in circulating concentrations of insulin-like growth factor-I.     

Binding and degradation of 125I-insulin by renal glomeruli and tubules isolated from rats

Summary Isolated rat renal glomeruli and tubules were shown to exhibit specific binding of ¹²⁵I-insulin and enzymatic degradation of the hormone. Binding to both renal fractions reached a plateau by 1h at 22 °C and increased linearly with increasing protein concentrations. Binding was inhibited in both preparations by insulin and its analogues in the order of relative potency: insulin > despentapeptide insulin > proinsulin, but insulin was ten times more potent in inhibiting ¹²⁵I-insulin binding to glomeruli than that to tubules, indicating a different affinity of receptors for the hormone in the two renal fractions (about 17 versus 210 g unlabelled insulin/l inhibiting 50% of the ¹²⁵I-insulin binding to glomeruli and tubules, respectively). Bound ¹²⁵I-insulin dissociated at a faster rate from tubules than from glomeruli; this release was accelerated by unlabelled insulin in both renal fractions, but to a greater extent in glomeruli than in tubules. Two-thirds of the total bound material released from glomeruli was found to be intact insulin as measured by trichloroacetic acid precipitation, whereas only one-third of the material released from tubules was intact. No direct relationship between binding and degradation of ¹²⁵I-insulin in these renal fractions could be demonstrated, however, because of the release of proteolytic enzymes into the incubation medium resulting in almost all degradation being extracellular. Although differing in their affinity for ¹²⁵I-insulin the high affinity glomerular insulin receptor and the lower affinity tubular insulin receptor have characteristics similar to those of insulin receptors in insulin responsive tissues.     

Specific non-saturable binding of insulin by human platelets

Studies on the nature and characterization of the specific binding of 125I-insulin to intact human platelets have been undertaken. Although under conditions of physiological buffer osmolality, a binding equilibrium was attained in 4-6 h at 4 degrees C, at higher temperatures (17 degrees C or 24 degrees C) equilibrium was reached only in the presence of very high buffer osmolality or 25 mM NaF. Under conditions of normal osmolality and in the absence of NaF, binding at 17 degrees C was not saturable. This phenomenon was specific for insulin or insulin-like hormones (the insulin-like growth factors) and did not occur with 125I-labelled growth hormone, ACTH or beta-endorphin. The non-saturable uptake of insulin appeared due to an energy-dependent specific sequestration or internalization of insulin by mechanisms probably involving the microtubule system. This study emphasizes the need to restrict this non-saturable uptake of insulin if one wishes to adequately study the platelet plasma membrane receptors for insulin. These data also indicate that there is a major interaction of insulin and insulin-like hormones with normal human platelets and support previous demonstrations of major insulin effects on platelet physiology in both normal and diabetic states.     

IGF-I stimulation of luteinizing hormone secretion, IGF-binding proteins (IGFBPs) and expression of mRNAs for IGFs, IGF receptors and IGFBPs in the ovine pituitary gland

Circulating concentrations of insulin-like growth factor-I (IGF-I) are reduced in juvenile sheep during nutritional growth restriction and the associated delay in puberty. Since exogenous IGF-I has been shown to stimulate luteinizing hormone (LH) secretion, it is postulated that endogenous IGF-I may act as a stimulatory metabolic signal to the pubertal ovine hypothalamo-pituitary axis, yet its site of action is unknown. Using coronal hypothalamic and pituitary sections from pubertal ewe lambs, in vitro autoradiography was used to localise 125I-labelled IGF-I binding, and gene expression for components of the IGF system was localised by in situ hybridisation using oligonucleotide probes. High concentrations of 125I-IGF-I binding were seen in the pars tuberalis (PT) and pars distalis (PD) of the pituitary, and relatively little in the hypothalamus; binding in the PT but not the PD was displaced by excess unlabelled IGF-I. Large amounts of mRNA were detected for the type-1 receptor (IGF-1R) and for IGF-binding protein (IGFBP)-5, localised to the PT and PD, and less intense specific hybridisation signals were obtained with mRNAs for IGF-II, type-2 receptor (IGF-2R) and IGFBP-3. There was some evidence for specific hybridisation to IGFBP-4 mRNA in the PT. IGF-I, IGFBP-1 and IGFBP-2 mRNAs were not detected in PT and PD. None of the genes were expressed in hypothalamic tissue. Western-ligand binding on PD extracts from male castrates revealed by their molecular weights the likely presence of IGFBPs-2, -3, and -5. Finally, cultured PD cells from abattoir-killed sheep were challenged with IGF-I (0.1, 1, 10 or 30 nM) or luteinizing hormone-releasing hormone (LHRH, 10 nM) alone, or both together. Basal LH output was stimulated by 10 nM IGF-I (120+/-11.2%, P>0.05), 30 nM IGF-I (148+/-12.8%, P<0.01), and LHRH alone (200+/-16.1%, P<0.001); there was no additive or subtractive effect of LHRH and IGF-I given together. Thus, an intrapituitary IGF system exists in sheep and the present results are consistent with an endocrine role for IGF-I in nutritional modulation of LH secretion at the level of the pituitary gland.     

Lipogenic and mitogenic effects of insulin during conversion of Ob17 cells to adipose-like cells

Chronic exposure to insulin of confluent cells of a preadipocyte clonal line (Ob17) leads to an acceleration of their development into adipose cells. The short-term effects of insulin have been examined by the stimulation of [14C] alpha-aminoisobutyrate uptake and the long-term effects by the increase in the activity levels of several lipogenic enzymes and in the intracellular triacylglycerol content. These metabolic effects of insulin occur within a physiological range of concentrations (EC50 congruent to 1 nM). As compared to insulin, dose-response curves obtained with proinsulin on these parameters show at least a 10-fold decrease in sensitivity of the cells. In contrast, the growth-promoting effects of both insulin and proinsulin occur at supraphysiological concentrations (EC50 greater than 300 nM). This mitogenic response is likely mediated through binding to receptors of insulin-like growth factors. Our data demonstrate that long-term effects of insulin on lipid synthesis can be dissociated from its effect on cell growth. Therefore the Ob17 cell line should be a useful model to study the role of insulin in the regulation of lipid synthesis in adipose cells.     

Binding specificity and intramolecular signal transmission of uncleaved insulin proreceptor in transformed lymphocytes from a patient with extreme insulin resistance

Summary An alteration of an amino acid sequence in the processing site of the insulin proreceptor by a point mutation of the insulin receptor gene produced extreme insulin resistance. We characterized functional properties of the unprocessed insulin receptor in transformed lymphocytes from a patient. Insulin binding to intact cells and to a partially purified insulin receptor preparation was radically decreased to 20% and 18% of the control values, respectively. In competitive insulin binding to intact cells, [Leu^A3]-, [Leu^B24]-, [Ser^B24-insulin, and mini-proinsulin ([B(1–29)-Ala-Ala-Lys-A(1–21)]-insulin) had the same relative binding activity in both the patient's and the control cells, but proinsulin and IGF-I were markedly less able to displace ¹²⁵I-insulin in the patient's cells. In contrast to the study in intact cells, proinsulin and IGF-I as well as other insulin analogues had the same relative binding activity to bind to the partially lectin-purified insulin receptor preparations from both the patient's and the control cells. As regards the signal transduction after receptor binding, insulin-stimulated autophosphorylation of the unprocessed insulin proreceptor occurred proportionally to the amount of decreased insulin binding. With 0.025% trypsin treatment, the abnormal binding characteristics and autophosphorylation were normalized through conversion to functionally normal receptors. In spite of the abnormal processing, self-association of receptors into oligomeric structures was observed in the proreceptor. These results suggest that the unprocessed insulin proreceptor in the plasma membranes has an altered conformation which affects its binding characteristics but not its intramolecular signal transmission.