Effect of methoxyindole 2-carboxylic acid and 4-pentenoic acid on adipose tissue metabolism.

1. Administration of methoxyindole 2-carboxylic acid to rats caused an increase in circulating free fatty acids which was associated with rapid hypoglycemia in fasted rats and liver glycogenolysis without hypoglycemia in fed rats. 2. The incorporation of labeled glucose, pyruvate and acetate carbons into triacylglycerol-glycerol, triacylglycerol-fatty acids and CO2 was inhibited in epididymal fat pads from methoxyindole 2-carboxylic acid-treated rats and by the addition of methoxyindole 2-carboxylic acid in vitro. In contrast, palmitate esterification and oxidation were enhanced by methoxyindole 2-carboxylic acid. 3. The activity of enzymes associated with fatty acid synthesis was reduced to a varying degree in the presence of methoxyindole 2-carboxylic acid in the reaction mixture in concentrations lower than those used to inhibit glucose and pyruvate metabolism in the intact tissue in vitro. 4. 4-Pentenoic acid, a potent inhibitor of pyruvate and palmitate metabolism in the liver, was considerably less effective in adipose tissue. 5. The effect of the two hypoglycemic substances investigated on adipose tissue metabolism seems to be different.

Extracellular metabolism of cyclic AMP.

Intravenously administered cyclic [8-3H]AMP to rats was quickly eliminated from the circulation. After 2 min 93% of the administered radioactivity disappeared from the plasues was recovered mainly in the form of nucleotides, ATP, ADP, AMP and IMP. In vitro contact of cyclic AMP with perfused liver, isolated liver cells and adipose tissue resulted in a rapid breakdown of the nucleotide, presumably on the outer surface of the cells. The degradation products have been identified mainly as adenosine and inosine. Incubation of adipose tissue and isolated liver cells with [3H] AMP also resulted in the breakdown of the nucleotide in themedium. The rate of AMP degradation by these tissues was faster than that for cyclic AMP degradation. The data suggest that cyclic AMP is readily metabolized on the outer surface of cells to products which may be converted within the cells to nucleotides. These findings seem of importance for the quantitative assessments of cellular cyclic AMP outflow during hormonal stimulation.

Independent modulation of hepatic protein kinase activities.

The effects of hormonal status on protein kinase activity was examined in homogenates of rat liver. Protein kinase activity was evaluated from incorporation of 32P from [gamma-32P]ATP into protamine or histone as receptor substrates. Protamine phosphorylation in the presence or absence of cyclic AMP exceeded histone phosphorylation by at least a factor or two. Hypophysectomy markedly increased protamine phosphorylation in the presence or absence of saturating amounts of cyclic AMP. In contrast, hypophysectomy only slightly increased cyclic AMP independent phosphorylation of histone. These results could not be amounted for by differences in ATPase or protein phosphase activities. Cortisone (2 mg/day x 3) decreased total protein kinase activity in livers of hypophysectomized rats when protamine was substrate, but had no effect on the total activity toward histone. Growth hormone (100 mug/day x 3) significantly increased histone, but not protamine phosphorylation in livers of hypophysectomized rats. Administration of 5 mug of triiodothyonine/day to hypophysectomized rats also markedly increased the phosphorylation of histone, but not protamine when saturating amounts of cyclic AMP were present. These results support the hypothesis that liver may contain more than one type of protein kinase activity and that the different protein kinase activities can be separately affected by hormones. Such control distal to cyclic AMP might allow selective modulation of cyclic AMP-dependent processes in cells which carry out more than one such process.

Turnover of growth hormone receptors in rat adipocytes.

Adipocytes isolated from the epididymal fat pads of normal rats specifically bound [125I]human GH [( 125I]hGH). Preincubation of cells with 20 micrograms/ml cycloheximide, an inhibitor of protein synthesis, produced a progressive loss of ability to bind [125I]hGH specifically. Loss of binding sites with time followed first order kinetics and had a half-time of about 45 min regardless of whether GH was present or absent during treatment with cycloheximide. Nonspecific binding of labeled hormone was unchanged by cycloheximide. Similar results were obtained when adipocytes were incubated with 200 micrograms/ml puromycin, another inhibitor of translation, but incubation with 5 micrograms/ml actinomycin D, an inhibitor of transcription, for 2.5 h had no effect on the binding of [125I]hGH by adipocytes. The findings are not attributable to cell death, since oxidation of [U-14C] glucose to 14CO2 and binding of [125I]insulin were unaffected in replicate cell populations exposed to the same treatments. Diminished binding could not be attributed to an effect of cycloheximide to hasten the degradation of receptor-bound hGH. Treatment of adipocytes with 0.1 mg/ml trypsin for 10 min virtually abolished their ability to bind [125I]hGH specifically, but binding capability gradually returned after removal of trypsin and was nearly restored to pretrypsin levels by 2 h. Addition of cycloheximide to the incubation medium after removal of trypsin completely prevented recovery of binding capability. Covalent binding of [125I]hGH to its receptors with disuccinimidyl suberate followed by sodium dodecyl sulfate-gel electrophoresis and autoradiography of proteins isolated from adipocyte membranes revealed three specifically labeled bands corresponding to mol wt of 250-300, 130, and 56 kilodaltons. Treatment of adipocytes with cycloheximide before cross-linking resulted in a proportional reduction in all three labeled bands, suggesting a similar half-life for all three entities. Similarly, all three labeled entities reappeared in parallel as adipocytes recovered from treatment with trypsin. The data strongly suggest that receptors for GH turn over rapidly on the surface of adipocytes and that ongoing protein synthesis is required to maintain binding capacity. The data do not permit distinction between rapid turnover of the receptor proteins themselves and a short-lived protein(s) which might be required to insert the receptors into the membrane.

Covalent binding of growth hormone to surface receptors on rat adipocytes.

GH specifically binds to receptors on the surface of adipocytes and produces a variety of biological effects in these cells. To gain insight into the nature of the GH receptors, [125I] human GH ([125I]hGH) was cross-linked to surface binding sites on intact rat adipocytes using the bifunctional reagent disuccinimidyl suberate. Plasma membranes were isolated, and after solubilization with sodium dodecyl sulfate (SDS), the proteins were subjected to electrophoresis on 5% or 7.5% polyacrylamide gel. Autoradiography of the 7.5% gels revealed three iodinated bands corresponding to apparent molecular weights of 56, 130, and more than 240 kilodaltons. The more than 240-kilodalton band contained approximately as much 125I as the 130-kilodalton species and about twice as much as the 56-kilodalton species. When run on the more porous 5% gel, the more than 240-kilodalton band resolved into two bands, corresponding to apparent molecular weights of 240 and 310 kilodaltons. Excess unlabeled human or bovine GH, but not ovine PRL, competed with [125I]hGH for binding and prevented the formation of all of the labeled bands. Treatment of the membranes and extracted proteins with dithiothreitol resulted in the generation of additional 130-kilodalton material at the expense of both the 310- and 240-kilodalton species, but failed to alter the amount of 125I that migrated with the 56-kilodalton species. The same pattern of labeling was seen regardless of whether protease inhibitors were present during isolation of membrane proteins or when membrane proteins were isolated under conditions that favored proteolysis, suggesting that the 56-kilodalton species is not a degradative product of the higher molecular weight species. When [125I]hGH was cross-linked to adipocytes in which total binding was decreased by hypophysectomy or starvation of the donor rats or by treatment of the cells with cycloheximide, there was a proportionate diminution in labeling of all species. It thus appears that the GH receptor contains a 130-kilodalton subunit, a portion of which is in disulfide linkage with higher molecular weight complexes and, in addition, contains a 56-kilodalton species. It cannot be determined from these studies if the various labeled protein complexes are components of a single or multiple classes of GH receptors in the adipocyte membrane.

Binding and degradation of [125I]human growth hormone in rat adipocytes.

Iodinated human GH [( 125I]hGH) binds to both specific and nonspecific sites on the surface of adipocytes isolated from the epididymal fat of normal rats. When adipocytes were incubated at 37 C with 1 nM [125I]hGH, specific binding increased for 30-60 min and thereafter remained approximately constant as long as the hormone was present in the medium. When cells that had bound [125I]hGH were removed from the incubation medium and reincubated in hormone-free medium at 37 C, half of the specifically bound 125I was released into the medium about every 30 min, and about half of the nonspecifically bound 125I was released in about 60 min. These rates were seen regardless of whether the time allowed for hormone binding was 15, 30, or 60 min. About 90% of the 125I released was soluble in 5% trichloroacetic acid and was in the form of iodotyrosine. The rate of 125I release from specific binding sites decreased by a factor of 4 when the temperature was lowered from 37 to 17 C. Replacement of some of the sodium chloride in the buffer with 25 mM ammonium chloride had little or no effect on the amount on 125I that bound to cells when [125I]hGH was present in the medium, but virtually completely blocked the release of 125I from cells transferred to hormone-free medium. Ammonium chloride also significantly reduced both the release of 125I from nonspecific binding sites and the amount of 125I recovered in trichloroacetic acid-soluble form. Cloroquine, leupeptin, or colchicine nearly doubled the specific binding of [125I]hGH after 180 min and markedly slowed the release of 125I when cells were transferred to hormone-free medium. All of these agents also significantly reduced the rate of release of 125I from nonspecific binding sites. Incubation of adipose tissue from hypophysectomized rats with ammonium chloride, leupeptin, or colchicine failed to alter the ability of GH to increase glucose oxidation, induce refractoriness, or promote lipolysis in the presence of theophylline. We conclude that GH binds virtually irreversibly to both specific and nonspecific sites on the adipocyte surface and is then internalized and degraded in the lysosomones. These events appear to be independent of the cellular processes that lead to expression to GH responses.

Adenosine 3',5'-monophosphate-dependent loss of growth hormone binding in rat adipocytes.

GH exerts a number of metabolic effects on adipose tissue. Depending on the circumstances, it may increase or decrease glucose metabolism and lipolysis. These effects appear to be mediated by a single class of receptors, which bind GH with high affinity. Incubation of isolated rat adipocytes with a variety of lipolytic agents, including catecholamines, forskolin, or (Bu)2cAMP, decreased the specific binding of [125I]human (h) GH within 10 min. In the presence of 10 microM forskolin, GH binding declined to less than 20% of the control value within 50 min. Cholera and pertussis toxins, which increase cAMP secondary to ADP ribosylation of guanine nucleotide-binding proteins associated with hormone receptors, also decreased the binding of GH. None of these agents affected the rate of loss of cell-associated 125I when added to cells that had previously equilibrated with [125I]hGH. The inhibitory effects of forskolin and (Bu)2cAMP were at least as great when binding was measured in the presence of the protease inhibitor leupeptin, suggesting that increased rates of internalization and processing of bound hormone could not account for the decline in binding. Scatchard plots of data obtained in the presence of forskolin or (Bu)2cAMP were linear and parallel to control plots, indicating that the decline in binding could be accounted for by a decrease in the number of binding sites, with no change in affinity. To determine whether phosphorylation affected binding to receptors already present in the membrane or modified the turnover of receptors, we studied adipocyte ghosts, whose cellular apparatus for receptor turnover is disrupted. Incubation of adipocyte ghosts with cAMP-dependent protein kinase decreased the binding of [125I]hGH by 25%. The data suggest that cAMP-dependent phosphorylation of the GH receptor or a closely associated membrane protein renders the receptor incapable of binding GH.

Evidence for a role of protein kinase C in the stimulation of lipolysis by growth hormone and isoproterenol.

The mechanism that underlies activation of lipolysis by GH is not yet understood. Although cAMP is thought to be involved, the biochemical linkages between GH and cAMP are unknown, and lipolysis produced by GH differs from that produced by such typical activators of adenylate cyclase as the catecholamines with regard to time course, maximum response, and dependence on other factors such as glucocorticoids or theophylline. The present studies were undertaken to evaluate the possibility that activation of protein kinase C by GH may play an important role in the production of the delayed increase in lipolysis. Phorbol myristate acetate (PMA), a known activator of protein kinase C, increased lipolysis in segments of adipose tissue of hypophysectomized rats, and, as with GH, this effect was potentiated by theophylline. The lipolytic effects of PMA were concentration-dependent, required a shorter lag period than those of human GH, increased as the concentration of PMA was raised from 0.1 to 10 microM, and were additive at all concentrations with lipolysis produced by saturating concentrations of GH. The lipolytic actions of GH, but not PMA, were potentiated by dexamethasone in adipose tissue of normal rats. Sphingosine and staurosporine, which are known to inhibit protein kinase C, blocked the lipolytic effects of PMA and severely reduced lipolysis in response to GH in tissues of both normal and hypophysectomized rats. Although higher concentrations of sphingosine interfered with the specific binding of 125I-labeled human GH to isolated adipocytes, the inhibitory effects of sphingosine cannot be attributed to interference with GH binding, since it decreased lipolysis by at least 50% when used at concentrations that were too low to reduce binding significantly. Staurosporine produced little (approximately 20%) or no decrease in binding. At concentrations that severely reduced lipolysis in response to GH and dexamethasone, sphingosine had little or no effect on lipolysis in response to dibutyryl cAMP or forskolin. Staurosporine and sphingosine, however, severely inhibited lipolysis in response to isoproterenol. We conclude that protein kinase C activity plays an important role in hormone-stimulated lipolysis probably by an action exerted on the transduction pathway proximal to cAMP. The present data are equally consistent with the possibilities that GH and/or isoproterenol activate protein kinase C, or that protein kinase C is constitutively active to some extent. It is likely that protein kinase C activity is permissive for, rather than a mediator of, the lipolytic actions of GH and isoproterenol.

Antibodies to cytoplasmic sequences of cloned liver growth hormone (GH) receptors recognize GH receptors associated with tyrosine kinase activity.

GH stimulates tyrosyl phosphorylation of GH receptors in 3T3-F442A fibroblasts, and highly purified GH receptor preparations exhibit tyrosine kinase activity. Paradoxically, however, the GH receptor cloned from liver exhibits no sequence similarity to receptors with known signal transduction mechanisms, including those exhibiting ligand-activated tyrosine kinase activity. These observations raise the possibility that there are two kinds of receptors for GH: the first represented by the cloned liver GH receptor, and the second by a tyrosine kinase-containing GH receptor. To inquire into the possibility of two distinct GH receptors, we determined whether the cloned liver GH receptor shares structural similarities with the tyrosine kinase-associated GH receptor. When the cloned rabbit liver GH receptor is expressed in human kidney 293 cells, it migrates with a mol wt appropriate for the tyrosine kinase-associated GH receptor, despite the calculated mol wt of the cloned GH receptor being 60,000 smaller than that of the tyrosine kinase-associated GH receptor. The recognition of tyrosine kinase-associated GH receptor by antipeptide antibodies to three different epitopes on the cytoplasmic domain of the cloned liver GH receptor was also tested. Tyrosyl phosphorylated [125I]human GH-receptor complexes were prepared by immunoprecipitation with phosphotyrosyl-binding antibody; this subpopulation of GH-receptor complexes was recognized by all three antipeptide antibodies. The antibodies also recognized similarly isolated tyrosyl phosphorylated GH-receptor complexes, which had been further phosphorylated in solution on tyrosyl residues upon addition of [gamma 32P] ATP. Furthermore, highly purified GH receptors prepared by sequential immunoprecipitation using phosphotyrosyl-binding antibody and any one of the three antipeptide antibodies incorporated 32P into tyrosyl residues upon the addition of [gamma 32P] ATP. These results provide evidence that tyrosine kinase-associated GH receptors share sequence similarity in the cytoplasmic domain with the cloned liver GH receptor. The cloned GH receptor and the tyrosine kinase-associated GH receptor, therefore, are likely to be the same receptor or related receptor isoforms.

Cellular effects of growth hormone on adipocytes.

Adipocytes are physiological targets for GH in both growing and nongrowing individuals. In adipocytes that have been deprived of GH for at least 3 h, GH initially produces a response that is characterized by increased metabolism of glucose and inhibition of the lipolytic effects of catecholamines. This insulin-like effect disappears within 2-3 h despite continued stimulation and cannot be elicited again unless cells are deprived of GH for at least 3 h. Despite refractoriness to the insulin-like action of GH, the lipolytic effect of GH is evident at this time. Although termination of the insulin-like response and induction of both refractoriness and lipolysis all depend upon synthesis of RNA and proteins, these 3 effects of GH appear to be neither temporally nor causally related. Scatchard analysis of ligand binding data suggests that these various effects are produced by interaction of GH with a single class of receptors. However, since modification of either the hormone or the carbohydrate moiety of the receptor can selectively attenuate either the insulin-like or the lipolytic response, more than one hormone receptor interaction is likely. Northern analysis indicates the presence of at least 2 alternately spliced mRNA transcripts for the GH receptor, and at least 3 different complexes are seen after GH is covalently crosslinked to intact adipocytes. Refractoriness does not result from changes in either the number or affinity of GH receptors, but may result from increased cytosolic calcium. Although the protein kinase C activator phorbol myristate acetate mimics both the insulin-like and lipolytic actions of GH, increased activity of protein kinase C probably does not mediate either action of GH. The intracellular mediators of the diverse actions of GH are unknown at this time.

[A method of quantitative evaluation of left-ventricular functional reserve in patients with heart aneurysm]. / Metodika kolichestvennoi otsenki funktsional'nogo rezerva levogo zheludochka u bol'nykh s anevrizmoi serdtsa.

Twenty-four patients with postinfarction aneurysms of the heart and 9 patients with chest pains were investigated in order to assess the value of left-ventricular reserve parameters, proposed by the authors, i. e. the ratio between the percentage of change in ejection time and diastolic elasticity in the course of isometric stress and the increment at arterial blood pressure test. These parameters are shown to be more valuable for the assessment of left-ventricular function, as compared to conventional ventriculographic and intraventricular pressure parameters, measured at rest. The changes in ejection fraction and diastolic elasticity at isometric stress are a fairly reliable signal that the resection of heart aneurysm may be contraindicated.

[Classification of the degree of severity of coronary insufficiency in patients with stenocardia at rest and moderate exertion based on isometric load data]. / Klassy tiazhesti koronarnoi nedostatochnosti u bol'nykh so stenokardiei pokoia i malykh napriazhenii po dannym izometricheskoi nagruzki.

A classification of the severity of coronary insufficiency has been developed on the basis of isometric exercise (hand-grip) testing for patients with angina of minor effort and angina at rest. A study of 177 coronary patients has demonstrated that the increment of the pulse-pressure index by the time of ST depression, as compared to the resting values, is the most valuable diagnostic quantitative marker of the severity of coronary insufficiency at isometric testing. Three degrees of the severity of coronary insufficiency (extremely severe, severe and moderately severe) have been identified with respect to this parameter.

[Age-related aspects of intensive therapy of acute poisoning of chemical etiology]. / Vozrastnye aspekty intensivnoi terapii ostrykh otravlenii khimicheskoi etiologii.

Assay of the clinical course of poisoning with acetic acid, dichloroethane, carbophos, chlorophos, phenobarbital and sodium etaminal in 2538 patients made it possible to define, with the use of the probit analysis, the 25, 50, 75 and 95% concentration thresholds of the lethality in acute poisonings with acetic acid, carbophos and dichloroethane. It was established that under exposure to the toxic substances undergoing different chemical changes in the body, the increase of the lethality risk varied in different age groups. The differences in the structure of outcomes in patients of different age groups formed the basis for making up a classification of the toxicity of the chemical compounds. The differences revealed require a differential approach to the determination of the scope of intensive care of poisonings in terms of the age-associated features.

[Expert criteria of the degree of severity of the chemical trauma in acute poisonings by organophosphate insecticides]. / Ekspertnye kriterii stepeni tiazhesti khimicheskoi travmy pri ostrykh otravleniiakh fosfororganicheskimi insektitsidami.

The article deals with quantitative assessment of severity of chemical trauma (according to life-threatening characteristics) in case of acute peroral poisoning with OPI (carbophos and chlorophos). Standard diagrams were plotted which help to assess the life threatening values of the given compounds depending on the initial blood poison level or recorded decrease in enzyme cholinesterase activity. Basic opportunity to assess severity of body lesions in case of poisoning with OPI according to character of specific clinical signs was evidenced.