PubMed Health⌕ Search

Biomedical subjects

C de Haën

Publications and source records attributed to C de Haën.

At least 55 records · Page 3Linked to original sources
PubMed1982

The lack of insulin mimetic or antagonistic effects of methyl-alpha-D-Mannoside in iso-osmolar solutions.

Transduction of insulin binding into metabolic control in isolated rat adipocytes apparently requires intact cell surface carbohydrate. The ability of certain lectins and some glycosides to mimic and/or inhibit the actions of insulin had been cited as evidence supporting the hypothesis that a concanavalin A-like binding site on fat cells is crucial to this function. Such a binding site could explain the stimulation by methyl-alpha-D-mannoside of glucose oxidation or its ability to antagonize the effect of insulin on lipolysis. The present study corroborated these effects of methyl-alpha-D-mannoside in hyperosmolar medium, but shows that the effects vanish when osmolarity is maintained within physiological limits. Osmolarity alone could not explain all of the complex effect observed, but it can be concluded that earlier data suggesting methyl-alpha-D-mannoside mimics or antagonizes the actions of insulin cannot be used to support the above hypothesis.

Adipose Tissue↗
PubMed1982

On the mechanisms of inhibition of insulin action by small-molecular-weight trypsin inhibitors.

Evidence from a number of laboratories has suggested that the mechanism of insulin action involves the release of an intracellular mediator polypeptide from the plasma membrane. It has been proposed that activation of a protease with trypsin-like specificity is involved in release of the putative mediator. In an effort to assess the potential role of such a protease in intact cells, the present study tested the effects of a variety of low-mol-wt protease inhibitors on insulin's metabolic action in isolated rat epididymal fat cells. The protease inhibitors studied included p-aminobenzamidine, benzamidine, phenylguanidine, diisopropylfluorophosphate, leupeptin, and the competitive substrate N-alpha-tosyl-L-arginine methylester. Leupeptin was devoid of activity. Most of the other inhibitors used were able to interfere with insulin-stimulated metabolism if used in sufficiently high concentrations, concentrations considerably higher than those required for inhibition of known proteases or inhibition of intracellular processes in a previously described system which involves a trypsin-like enzyme. Moreover, they displayed various activities unrelated to protease inhibition that could explain their effects on insulin action better than protease inhibition. While none of the data on individual inhibitors were by themselves convincing enough to either confirm or reject the hypothesis concerning the involvement of a protease with trypsin-like specificity in insulin action, taken together our results do weaken the hypothesis considerably and in particular render the involvement of an extracellular trypsin-like enzyme improbable.

Adipose Tissue↗
PubMed1980

Effects of hydrogen peroxide on basal and hormone-stimulated lipolysis in perifused rat fat cells in relation to the mechanism of action of insulin.

Since insulin is known to stimulate intracellular hydrogen peroxide production in rat epididymal fat cells, the effects of exogenous hydrogen peroxide on rates of basal and hormone-stimulated lipolysis were investigated in a perifusion system. H2O2 (60 microM) caused a weak and transient stimulation of basal lipolysis that did not interfere with subsequent activation of lipolysis by hormones. More importantly, lipolysis stimulated submaximally with ritodrine (10(-7) M) or glucagon (10(-9) M) was inhibited by H2O2 in a manner similar to insulin, although slight differences in time course were noted. Ritodrine served as a beta-adrenergic agonist resistant to oxidative destruction by H2O2. The inhibition of lipolysis was reversible upon cessation of perifusion with H2O2. These findings ruled out oxidative destruction of the hormone or cell death as explanations for the antilipolytic effect of H2O2. Like insulin, H2O2 also inhibited 1-methyl-3-isobutylxanthine (4 X 10(-6) M)-stimulated lipolysis, but whereas insulin inhibited lipolysis stimulated by dibutyryl-cAMP (4 X 10(-4) M), H2O2 further enhanced it. These findings add another case to the growing list of insulin effects on adipocytes that can be mimicked by exogenous H2O2, and they hint at a site where the mechanisms of action of the two agents may differ.

1-Methyl-3-isobutylxanthine↗
PubMed1979

The insulin-like effect of hydrogen peroxide on pathways of lipid synthesis in rat adipocytes.

In addition to the well known insulin-like effects of certain concentrations of H2O2 on glucose transport and oxidation in isolated rat adipocytes, the present work demonstrates that lipid synthesis from glucose is also enhanced over a narrow range of H2O2 concentrations (0.15 to 0.5 mM) added to the incubation medium. As in the case of insulin, H2O2 was found to stimulate greater glucose incorporation into glyceride-fatty acid than incorporation into glyceride-glycerol. As part of a multifaceted regulation of lipogenesis, H2O2, like insulin, increased the amount of pyruvate dehydrogenase in the active form without increasing the total amount of pyruvate dehydrogenase. Pyruvate dehydrogenase activity increased within 5 min of H2O2 incubation, reached a maximum at 15 min and declined thereafter as the H2O2 disappeared from the incubation medium. While medium glucose per se was found to activate the enzyme, it is unlikely that the effect of H2O2 was mediated by the known enhancement of glucose transport since the effects on the enzyme were maximal in the absence of glucose in the incubation medium. These findings add to the growing list of insulin effects that are reproduced by H2O2, and strengthen the hypothesis that assigns H2O2 the role of "second messenger" of insulin.

Adipose Tissue↗
PubMed1979

Insulin-stimulated intracellular hydrogen peroxide production in rat epididymal fat cells.

Insulin stimulation of hydrogen peroxide production by rat epididymal fat cells was investigated by studying the oxidation of formate to CO2 by endogenous catalase. Under optimal concentrations of formate (0.1 to 1 mM) and glucose (0.275 mM), insulin stimulated formate oxidation 1.5- to 2.0-fold. Inhibitors of catalase activity, including nitrite and azide, inhibited both basal and insulin-stimulated formate oxidation at concentrations that did not interfere with insulin effects on glucose C-1 oxidation or glucose H-3 incorporation into lipids. The addition of exogenous catalase increased formate oxidation only slightly, while exogenous H2O2 (0.5 mM) stimulated formate oxidation by endogenous catalase strongly. These data indicate that the insulin-stimulated H2O2 production was intracellular. Insulin dose-response curves for formate oxidation were identical with those for glucose H-3 incorporation into lipids. The dependence of relative insulin effects on the logarithm of the glucose concentration was bell-shaped for formate oxidation and correlated highly with the coresponding dependences of glucose C-1 oxidation and glucose H-3 incorporation into lipids. This suggests that insulin stimulation of intracellular H2O2 production is linked to glucose metabolism. Since it is known that extracellular H2O2 can mimic insulin in several respects, these observations suggest that H2O2 may act as a "second messenger" for the observed effects of insulin.

Adipose Tissue↗
PubMed1979

[12-Homoarginine]glucagon: synthesis and observations on conformation, biological activity, and copper-mediated peptide cleavage.

Specific modification of the single lysine residue (Lys-12) in glucagon with O-methylisourea has been effected by blocking the reactivity of the amino terminal histidine with copper, providing a method for obtaining [12-homoarginine]glucagon. It was found that as a side reaction, under the conditions of the modification reaction, Cu(II) catalyzed cleavage of the polypeptide chain between Asp-9 and Tyr-10, and between Lys-12 and Tyr-13. This observation may be of value for development of a sequence-specific peptide cleavage procedure. The dilute solution conformations of glucagon and [12-homoarginine]-glucagon were compared by circular dichroism, fluorescence, phosphorescence, energy transfer, and optical detection of magnetic resonance. The results indicate that conversion of Lys-12 to homoarginine does not alter the helix content the side chain conformation in the vicinity of the tyrosine and tryptophan residues, or the relative distances and orientations between these residues. However, the modification reduces the hormone potency towards activation of lipolysis in isolated rat epididymal fat cells by a factor of seven. We attribute the loss of potency to an interference with a specific interaction between the lysine residue and the fat cell hormone receptor, and not to a change in the solution conformation of the hormone.

Adipose Tissue↗
PubMed1978

N epsilonB29-(+)-biotinylinsulin and its complexes with avidin. Synthesis and biological activity.

Insulin was modified with d-biotin-N-hydroxysuccinimide ester in dimethylformamide. Mono-, di-, and triacylated insulins were separated by preparative isoelectric focusing. Monoacylated derivatives (isoelectric point 5.1) were fractionated twice on DEAE-cellulose to yield pure N epsilonB29-biotinylinsulin. The structure of the product was established by amino acid analysis before and after deamination. N epsilonB29-biotinylinsulin had biological activity indistinguishable from insulin on glucose oxidation and lipid synthesis assays using isolated rat epididymal fat cells. Complexes of N epsilonB29-biotinylinsulin with avidin, having essentially all but one binding site filled with biotin, were prepared in order to obtain a 1:1 insulin:avidin ration. The elicited identical maximal biological responses, but showed a potency decreased to 5% of that of insulin. Such complexes conjugated with ferritin will provide a useful tool in the development of electron microscopic stains of insulin receptors.

Adipose Tissue↗
PubMed1978

Characterization of proinsulin-insulin intermediates in human plasma.

This work addressed the problem of heterogeneity of immunoreactive insulin (IRI) in human plasma. Subjects with normal glucose tolerance were given 75g of an oral glucose solution, followed in 30 min by an intravenous infusion of 30g of arginine over 30 min. At the end of the infusion blood was withdrawn for analysis. IRI was extracted from plasma of individual subject by immunosorbent columns and was fractionated by gel filtration, disc gel electrophoresis and isoelectric focusing. Human IRI components were identified by molecular size, immunoreactivity with a human proinsulin antibody, sensitivity to trypsin, and by comparison of electrophoretic mobility and isoelectric point with porcine pancreatic products, after suitable correction for electric charge and molecular weight differences. The pattern of IRI heterogeneity was the same among six healthy subjects. Heterogeneity of proinsulin-size IRI in circulation was more marked than that of insulin-size material. Proinsulin and desdipeptide proinsulin were present in approximately equal amounts accompanied by minor amounts of split proinsulin and monodesamido-desdipeptide proinsulin. Insulin-size IRI contained over 80% insulin. Minor amounts of monodesamidoinsulin and diarginylinsulin were observed in some cases. The types of IRI components observed in plasma are evidence in support of a physiologic role of trypsin-and carboxypeptidase B-like enzymes in the conversion of proinsulin to insulin. Moreover, this study provides a base line for investigation of abnormalities in proinsulin-to-insulin conversion that may be associated with certain pathologic states.

Humans↗
PubMed1977

Isolation and amino-terminal sequence analysis of a new pancreatic trypsinogen of the African lungfish Protopterus aethiopicus.

The purification and characterization of three pancreatic trypsinogens A1, A2, and A3, from the African lungfish, Protopterus aethiopicus, is reported. These zymogens are activated by trypsin, by enterokinase, by an acid protease from Aspergillus oryzae, and by autoactivation. The three trypsinogens contain the same amino-terminal amino acid sequence, beginning with the activation peptide Leu-Pro-Leu-Glu-Asp-Asp-Lys-. Like the activation peptide of the previously characterized trypsinogen B [Reeck, G. R., & Neurath, H. (1972) Biochemistry 11, 503] of the same organism, it lacks the tetraaspartyl sequence characteristic of other vertebrate trypsinogens. Two of the corresponding lungfish trypsins were found to have identical amino-terminal sequences for at least 27 residues. These data suggest that the three enzymes are allelic variants. In contrast, the amino acid sequences differ sufficiently from that of trypsinogen B of the same organism to indicate that trypsinogens A and B are the products of different gene loci.

Amino Acid Sequence↗