Isolation and partial characterization of insulin of the honeybee (Apis mellifica).

In the honeybee (Apis mellifica), insulin-like material was partially purified with acid ethanol extractions by a classic method for recovering insulin and following gel filtration on a Sephadex G-50 column. The preparations were characterized by their ability to cross-react with porcine insulin antibodies. Insulin-like biological activity was demonstrated using the insulin bioassay. Stimulation of glucose oxidation or lipogenesis was measured by isolated rat adipocytes. Insulin seems to be more widespread in invertebrates than was previously assumed.

Insulin as a growth regulator of arterial smooth muscle cells: effect of insulin of I.G.F.I.

The proliferation of smooth muscle cells in the vessel wall plays an important role in the development of an atherosclerosis. The insulin-like growth factor I (I.G.F.I) is an important growth factor for cultivated arterial smooth muscle cells. The plasma I.G.F.I level is controlled by growth hormone and insulin--both factors seem to be important for the development of a diabetic macroangiopathy. In order to evaluate whether I.G.F.I is associated with the development of a macroangiopathy we measured the serum I.G.F.I level in type 2 diabetics with and without macroangiopathy and in non diabetic patients with atherosclerosis and in healthy controls. The range of the individual I.G.F.I levels was wide and the I.G.F.I level between the various groups was not statistically significant. However, our studies with cultured cells show that modulation of the cellular responsiveness to I.G.F.I is as important in the growth control as are changes in the plasma I.G.F.I level. We observed that cultured smooth muscle cells were able to produce an I.G.F.I-like factor. The synthesis and secretion of this factor was dependent on insulin. These findings explain one mechanism by which insulin may influence the growth of smooth muscle cells in the vessel wall.

Interaction of receptors for insulin-like growth factor I, platelet-derived growth factor, and fibroblast growth factor in rat aortic cells.

Serum contains various growth factors which regulate the proliferation of cells. We investigated the growth of cultured arterial smooth muscle cells under the influence of insulin-like growth factor I (IGF I), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF), and examined the effect of these growth factors on the binding of [125I] IGF I and on the binding of [125I]PDGF to these cells. IGF I, FGF, and PDGF stimulated [6-3H]thymidine incorporation into DNA of confluent cultures of cells which were incubated in modified Dulbecco's modified Eagle medium. However, the effect of these growth factors on DNA synthesis was much more potent in Dulbecco's modified Eagle medium with 1% fetal calf serum. FGF and PDGF potentiated the growth-promoting effect of IGF I. The binding of [125I]IGF I to the cells was increased after a preincubation with FGF and PDGF. The binding was potently increased by FGF (100 ng/ml) after a preincubation time of 30 min. There was an increase in binding during the first 3 h of preincubation followed by a decrease after 4-5 h. PDGF (10-1000 ng/ml) stimulated [125I]IGF I binding only after 2 h of preincubation. The stimulation was dose dependent. Maximal stimulation of the binding was observed after 3 h of preincubation followed by a decrease after 4-5 h of preincubation. Specific binding sites for PDGF on smooth muscle cells could be demonstrated too. A preincubation of confluent cells with IGF I caused a dose-dependent increase in [125I]PDGF binding. These results support the hypothesis that the regulation of the binding of a specific growth factor by a second growth factor is important for the control of cell growth.

Radioimmunoassay for the measurement of insulin-like growth factor I in patients with pituitary disease in comparison with commercially available somatomedin-C radioimmunoassays.

A highly sensitive and specific radioimmunoassay for the measurement of insulin-like growth factor I (IGF I) has been developed. The IGF I concentrations were measured in sera of normal subjects, patients with acromegaly, hypophysectomized patients and patients with hyperprolactinaemia. The results were compared with the results obtained after measurement of IGF I in the sera of the same patients with two commercially available radioimmunoassays for somatomedin-C. IGF I was separated from its carrier protein using Sep-Pak C18 cartridges. The total recovery of IGF I by this method was about 100%. The in-house assay shows a high specificity for IGF I and a high sensitivity. As little as 0.1 microgram/l of IGF I can be detected. Only a small amount of serum (25 microliter) is necessary for the IGF I determination and a great number of serum samples (more than 100 per day) can be processed. The mean immunoreactive IGF I concentration was 379 +/- 159 micrograms/l in normal adult subjects, 3340 +/- 1094 micrograms/l in acromegalic patients, and 52 +/- 11 micrograms/l in growth-hormone deficient patients. Patients with hyperprolactinaemia had an IGF I concentration of 433 +/- 112 micrograms/l.

Evidence for a potent angiotensin I degrading enzyme different from angiotensin I converting enzyme in rat vascular tissues.

There are indications for the existence of an intrinsic renin angiotensin system in vascular walls, which is assumed to participate in blood pressure regulation and in pathogenesis of arterial hypertension. It was evaluated if and to what extent the decapeptide angiotensin (A) I, one of the natural substrates of A I converting enzyme (ACE), is degraded by other peptidases than ACE in rat vascular tissues. A I and A II degradation was studied in arterial and venous vascular wall extracts. The activities ranged between 0.068 +/- 0.025 U and 0.044 +/- 0.025 U. The enzymes involved were biochemically characterized by determination of isoelectric points (pI), pH optima, molecular weights and by investigation of their inhibition behavior in vitro. One potent A I degrading enzyme (AIDE) was identified with pI between 3.6 and 3.9, and pH optimum at 7.75. In vitro studies revealed that AIDE activity was not blocked by the specific ACE inhibitors MK 421 or MK 422 (both 11 nMol/ml). The molecular weight of AIDE ranged between 440,000 and 457,000. The results indicate that AIDE is not identical to ACE (pI 4.2-5.0; pH optimum 8.3). AIDE was also observed in aortic smooth muscle cells cultured in vitro. AIDE decreased following bilateral nephrectomy or administration of aldosterone combined with sodium chloride loading, whereas it was elevated in spontaneously hypertensive rats (Okamoto strain). Since AIDE metabolizes A I, one of the substrates of ACE, it may indirectly affect A II formation and bradykinin inactivation as well.

Insulin-like growth factor I/somatomedin-C: a rapid isolation procedure with FPLC.

Insulin-like growth factor I (IGF I)/somatomedin-C (SM-C) was purified from lyophilized human serum by acid-ethanol extraction. The extract was precipitated with acetone-ethanol. The precipitate was purified by Sephadex G-50 chromatography. The protein peak within a molecular weight range of 5000-10 000 was further purified with FPLC-reversed phase chromatography using a Pep RPC HR 5/5 column (Pharmacia) with a solvent system of acetonitrile (CH3CN) and 0.1% trifluoroacetic acid (TFA) in water. The purification of IGF I was monitored by radioimmunoassay for SM-C. Purity was established by analytical isoelectric focusing and by SDS polyacrylamide gel electrophoresis. Analytical isoelectric focusing showed one single protein band with an apparent pI of 8.3 +/- 0.1. SDS polyacrylamide gel electrophoresis showed also one single protein band with an apparent molecular weight of 7000. Biological activity was demonstrated by measuring the (3H)thymidine incorporation into DNA of cultured arterial smooth muscle cells.

Effect of thyroid hormone on insulin action in cultured arterial smooth muscle cells.

The effect of triiodothyronine (T3) on insulin action in cultured smooth muscle cells of the rat aorta were studied. Insulin binding to smooth muscle cells was increased by T3. The Scatchard analysis indicated an increase of insulin binding sites and an elevation of the affinity of insulin to its binding sites. T3 alone had no significant effect on DNA, RNA and protein synthesis in smooth muscle cells and did not increase the transport of 3-O-methylglucose into the cells. However, the insulin-induced 3-O-methylglucose transport into the cells was stimulated. The insulin-stimulated protein synthesis was not significantly altered by T3 treatment, whereas the effect of insulin on DNA and RNA synthesis was even reduced in the presence of T3. The study suggests, that T3 treatment produces predominantly a stimulation of processes which are necessary for the production of energy. The anabolic effect of insulin is decreased and must by inferior to the credit of energy production.

Internalization of colloidal gold-labelled insulin complexes into cultivated human smooth muscle cells.

The binding and internalization of colloidal gold-labelled insulin complexes into cultivated human arterial smooth muscle cells was studied. We could demonstrate that the colloidal gold-labelled insulin complex was bound to specific receptors on the cell surface of smooth muscle cells and internalized after 30 min at 20 degrees C. The gold-labelled insulin complexes entered the cell via vesicles and escaped from them in the cytoplasm. The biological activity of the complexes was determined by measuring their effect on DNA synthesis in smooth muscle cells and incorporation of (14C)-glucose into isolated fat cells. The binding characteristics of the complexes were examined by competitive inhibition of 125I-insulin binding to isolated fat cells in the presence of increasing concentrations of gold-labelled insulin complexes or unlabelled insulin. The gold-labelled insulin complex showed a growth promoting and metabolic effect and was able to compete with 125I-insulin for its binding sites.

Investigations of components of the renin-angiotensin system in rat vascular tissue.

Investigations were performed on components of the renin-angiotensin system (RAS) in homogenate extracts of vascular tissue and aortic smooth muscle cells cultivated in vitro. Determinations of isoelectric points and pH optima indicated the existence in aortic homogenate extracts of two local angiotensin I (AI)-forming enzymes (AIFE) that were different from those of plasma, renal cortex, veins, and aortic smooth muscle cells. The pH optima for AI-converting enzyme (ACE) from vascular tissues, aortic smooth muscle cells, and plasma were in the same range (pH 8.0-8.5), and in agreement with those measured previously in other tissues. In contrast, in vitro studies with the ACE inhibitors MK-421 and MK-422 and measurement of isoelectric points suggested that aortic ACE was different from the plasma enzyme. AIFE and ACE activities were found to be elevated in spontaneously hypertensive rats (SHR). The biochemical characteristics of the enzymes investigated in the vascular tissue of SHR were not different from those of the normotensive controls. AI- and AII-degrading enzymes were found both in aortic tissue and in aortic smooth muscle cells. One potent AI-degrading enzyme different from ACE was observed in aortic tissue. A high ratio of AI/AII immunoreactivities in arterial walls suggests the availability of renin substrate, and that AI-degrading enzymes are the rate-limiting enzymes for AII formation. The results further support the concept of an intrinsic vascular RAS.

Regulation of receptors for insulin-like growth factors in cultured arterial smooth muscle cells by thyroid hormone.

The effect of tri-iodothyronine (T3) on the binding of 125I-labelled insulin-like growth factors (IGF) to cultured arterial smooth muscle cells was investigated. When cells which were grown to confluency were incubated with 125I-labelled IGF and different concentrations of T3, low concentrations of T3 (0.1-10 nmol/l) increased the binding of 125I-labelled IGF. High concentrations of T3 (1 mumol/l) could not induce this effect. Scatchard analysis of the binding of 125I-labelled IGF in the presence of different concentrations of unlabelled IGF showed a high-affinity, low-capacity binding system and a second lower affinity, high-capacity binding system. In the presence of T3 (1 nmol/l) Scatchard analysis indicated that the affinity of 125I-labelled IGF to its binding sites was altered. After a 2-h preincubation of the cell layers with Dulbecco's Modified Eagle's Medium containing T3 (1-100 nmol/l), the 125I-labelled IGF binding was enhanced in a dose-dependent manner by T3. Scatchard analysis showed a significant increase in the number of IGF binding sites.

Two separate receptors for insulin and insulinlike growth factors on arterial smooth muscle cells.

Insulin and insulinlike growth factors (IGF) are related polypeptides that have similar biological activities. Both factors produce metabolic effects as well as growth effects. Most cells have separate receptors for IGF and insulin. In the present study we have demonstrated specific IGF receptors in cultured smooth muscle cells of rat aorta. The properties and specificity of these receptors were compared with those of the insulin receptor in the same cell system. The specific binding of both 125I-IGF and 125I-insulin binding in the order of potency: insulin proinsulin IGF. The potency of IGF in displacing 125I-insulin was rapid and reversible. Maximal 125I-IGF binding occurred at 20 degrees C with a specific binding of 10%. At this temperature specific 125I-insulin binding was 1.3% and occurred in biphase. The pH optimum for 125I-IGF binding was between pH 7 and 8. Both receptors show a high degree of specificity. IGF, insulin and proinsulin competed for 125I-IGF binding in the order of potency: IGF proinsulin insulin. The potency of insulin in displacing 125I-IGF was about 2000 times lower than that of IGF itself. In addition, IGF, insulin and proinsulin competed for 125I-insulin was about 450 times lower than that of insulin. These results indicate two separate binding sites for insulin and IGF on arterial smooth muscle cells.

Characterization of insulin binding sites in cultured smooth muscle cells of rat aorta.

Insulin receptors could be demonstrated in cultured smooth muscle cells of rat aorta. The specific binding of 125I-insulin was time-, temperature- and pH-dependent. The optimal temperature for our studies was 12 degrees C. At this temperature maximal specific binding was 0.5% of total counts at 120 min incubation. The pH-optimum for the binding process was between 7.5 and 8. Degradation of 125I-insulin at 12 degrees C was 14%, no degradation of binding sites could be measured at this temperature. Dissociation of 125I-insulin was rapid. 50% of the labeled hormone remained associated with the cells. Half-maximal inhibition of 125I-insulin binding was produced by insulin at 4 X 10(-11) mol/l. Scatchard-analysis gave curvilinear plots, that may suggest negative cooperativity. Specificity of binding was studied in competition experiments between 125I-insulin, insulin, proinsulin, insulin-like growth factors and human growth hormone. Half-maximal inhibition of 125I-insulin binding was produced by proinsulin at 2 X 10(-9) mol/l and by insulin-like growth factors at 9 X 10(-9) mol/l. Human growth hormone had no significant effect on the insulin binding.

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.

Binding and biological actions of insulin-like growth factors on human arterial smooth muscle cells.

Insulin-like growth factors (IGF) were isolated from human serum and compared with some biological actions of IGF supplied by Dr. J. Hapf, Zürich. Both factors were potent mitogens. They stimulated DNA-, RNA- and protein synthesis in cultivated human arterial smooth muscle cells. Furthermore, they enhanced the aminoacid transport. Our protein fraction (IGF Ulm) had a more potent biological activity than IGF (Zürich). Specific binding receptors for IGF (Zürich) on human arterial smooth muscle cells could be demonstrated. Specific binding of 125I-IGF (Zürich) was 10%. Half-maximal displacement was achieved by 250 ng/ml of unlabeled IGF (Zürich), by 1.2 micrograms/ml of IGF (Ulm), by 6.3 micrograms/ml of pro-insulin and by 17.8 micrograms/ml of insulin. In separate studies we could demonstrate that sera of normal adults, diabetic, acromegalic and hypophysectomized patients showed different growth-promoting activity in human arterial smooth muscle cells.

Thyroid hormone effects on beta-adrenergic receptors in isolated fat cells in rats.

The effect of thyroid hormones (thyroxine and triiodothyronine) on catecholamine receptors in isolated rat fat cells was investigated. Binding of (3H)isoproterenol and (3H)norepinephrine were increased by thyroid hormones. (3H)isoproterenol binding was more enhanced than (3H)norepinephrine binding. Triiodothyronine had a more potent effect than thyroxine. (3H)isoproterenol was used to estimate the number or affinity of beta-adrenergic receptors in rat fat cells treated with thyroid hormones. The binding sites for (3H)isoproterenol were the same in untreated and with triiodothyronine treated fat cells. The equilibrium dissociation constants (KD) for the interaction of receptors with (3H)isoproterenol were altered in thyroid hormone treated cells. There was a significant difference between the untreated and triiodothyronine treated fat cells in the affinity of beta-adrenergic receptor binding sites for (3H)isoproterenol. Thyroid hormone could alter negative cooperative site-to-site interaction among the binding sites for (3H)isoproterenol.

Characterization of beta-adrenergic receptors by (3H) isoproterenol in adipocytes of humans and rats.

The specific binding of (3H) isoproterenol to isolated fat cells of human and rat was characterized. Binding of (3H) isoproterenol to isolated fat cells of rat was saturable with 420 pmol of (3H) isoproterenol bound/100 mg of lipid. Half-maximal saturation occurred at 5 microM providing an estimate of the equilibrium dissociation constant, KD, for the interaction of (3H) isoproterenol with its binding sites. Kinetic analysis of (3H) isoproterenol binding provided a value of 2.01 x 10(4) min-1. M-1 for the forward bimolecular rate constant, k1. Dissociation of (3H) isoproterenol was a first order reaction with a rate constant, k2, of 0.62 x 10(-1) min-1. The ratio k2/k1 = 3.07 microM provides an independent measurement of the KD for the interaction of (3H) isoproterenol with its binding sites which is in agreement with the values obtained by steady state analysis (3 to 5 microM). The apparent equilibrium dissociation constant, KD, for the interaction of (3H) isoproterenol with its receptor in human fat cells obtained by steady state analysis was 1 to 0.9 microM. Scatchard- and Hill-analysis suggest the possibility of different negatively cooperative interactions among the binding sites in human and rat. beta-Adrenergic agonists competed for the binding sites. The order of potency was isoproterenol greater than epinephrine greater than norepinephrine. Compounds such as DOPA, dopamine and (m-Hydroxyphenyl)2-methyl-aminoethanol which are structurally related to catecholamines had little or no affinity for (3H) isoproterenol binding sites.

Effect of insulin on growth of cultured human arterial smooth muscle cells.

The effect of insulin (10--10 000 mU/1) on the proliferation of cultured human arterial smooth muscle cells was studied. Smooth muscle cells were cultivated by explanation. Cells from the third to the fifth subculture were used. Proliferation was studied by growth curve experiments. Insulin stimulated cell proliferation in all concentrations (p less than 0.001). Growth was however stimulated more by a medium containing 10% fetal calf serum. The highest concentration of insulin produced only 35% of the effect of 10% fetal calf serum. Our results support the hypothesis that insulin may play a role in atherosclerosis.

Thyroid hormone stimulation of lipolysis and cyclic adenosine 3', 5'-monophosphate accumulation in human adipose tissue.

The role of thyroid hormones on lipolysis in human subcutaneous adipose tissue was investigated. Incubation of subcutaneous fat pads with thyroxine (0.1--10 000 nM) augmented the subsequent isoproterenol stimulation of lipolysis, measured by glycerol release. The basal lipolysis could not by stimulated by thyroxine. The theophylline- and dibutyryl-cyclic AMP stimulated lipolysis also could not be increased by thyroxine at these concentrations. In separate studies, the effect of thyroxine (0.01 pM--1 microM) and triiodothyronine (0.01 pM--1 microM) on cyclic AMP accumulation was examined. No effect of thyroid hormones on cyclic AMP accumulation was seen in non-isoproterenol stimulated tissue. Fat pads stimulated by isoproterenol and then treated with thyroid hormones showed marked increases in accumulation of cyclic AMP as compared to control tissue in the presence of isoproterenol alone.

Insulin as a cellular growth regulator of rat arterial smooth muscle cells in vitro.

Smooth muscle cells were grown from thoracic aortas of rats. The effect of insulin on the proliferation of these cells was studied by comparing the growth of cells in culture medium containing insulin and 1% fetal calf serum with growth of cells in culture medium containing only 1% serum and in culture medium containing 10% serum. Insulin in concentrations of 10, 50, 100, 1000 and 10 000 microunits/ml induced smooth muscle cells to stationary growth more rapidly than basal medium. This could be demonstrated as well in the logarithmic growth as in confluent cells grown in medium with 1% serum. However, the highest concentration of insulin did not stimulate growth to the same degree as medium containing 10% serum. Cells that were older in culture life (11th passage) did not show a growth response to insulin.

The effect of insulin and insulin-like growth factors on cell proliferation of human smooth muscle cells.

The effect of insulin and insulin like growth factors on the proliferation of cultured human arterial smooth muscle cells was investigated. Smooth muscle cells were cultivated by explanation. The effect of insulin and of an isolated serum protein fraction containing insulin like growth factors on the proliferation of human arterial smooth muscle cells was studied by incorporation of 3H-thymidine into DNA of the cells. Insulin stimulated in all concentrations the proliferation of the cells more than culture medium with 1% fetal calf serum. Insulin could not stimulate growth to the same degree as medium with 10% serum. On the other hand the isolated proteins had a more potent growth-promoting effect than medium with 10% serum.