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M Sonenberg

Publications and source records attributed to M Sonenberg.

At least 37 records · Page 2Linked to original sources
PubMed1989

Expression of growth hormone-independent adipogenesis by a 3T3 cell variant.

We have examined the regulation of adipogenesis of a 3T3-F442A cell variant. The variant, designated 3T3-GH-independent clone 16 (GI-16), was isolated after serum-induced adipogenic commitment. 3T3-GI-16 fibroblasts displayed a lower serum requirement for adipogenesis than the 3T3-F442A parent cell. Insulin-stimulated adipogenesis of 3T3-GI-16 cells in serum-free medium (SFM) was extensive in the absence of GH, as judged by oil red O staining or glycerol-3-phosphate-dehydrogenase activity, a property not associated with the 3T3-F442A cell. In SFM devoid of GH the concentration of insulin required to promote half-maximal adipogenesis of 3T3-GI-16 fibroblasts was 5 nM. The expression of GH-independent adipogenesis by 3T3-GI-16 cells was not due to exposure to adipogenic stimuli during routine passage, as insulin-stimulated differentiation was not a function of the inoculation density in nonadipogenic cat serum. We noted that nine proteins resolved by polyacrylamide gel electrophoresis behaved in a differentiation-dependent manner during adipogenesis of 3T3-GI-16 and 3T3-F442A fibroblasts in SFM. The concentrations of all nine proteins were regulated in a GH-independent manner during insulin-stimulated adipogenesis of 3T3-GI-16 fibroblasts. In contrast, the presence of insulin alone markedly altered the expression of only two of the proteins during differentiation of 3T3-F442A cells. The observed changes in the expression of five presently uncharacterized differentiation-dependent proteins were most likely due to employment of SFM. Our results suggest that expression of GH-independent insulin-induced adipogenesis of 3T3-GI-16 fibroblasts reflects a prior commitment by GH during our selection protocol. These results are discussed in the context of a model in which adipogenesis in vivo is postulated to proceed through the sequential action of GH and insulin on target cells.

Adipose Tissue↗
PubMed1989

Growth hormone-dependent events in the adipose differentiation of 3T3-F442A fibroblasts: modulation of macromolecular synthesis.

GH is necessary but not sufficient to induce adipose differentiation of 3T3-F442A fibroblasts in serum-free medium. Human (h) GH (2 nM) treatment of 3T3-F442A cells in serum-free medium caused a time-dependent (maximal at 48-72 h) and dose-dependent (EC50, approximately 0.2 nM) decrease (40-60%) in de nova protein synthesis. Insulin-like growth factor-I (IGF-I; 17 nM), PRL (2 nM), and glucagon (20 nM) did not decrease de novo protein synthesis, whereas bovine GH was equipotent with hGH. The half-lives of 35S-labeled proteins of 3T3-F442A cells were 21 and 57 h for cells maintained in serum-free medium for 3 days without or with hGH (2 nM), respectively. The total protein content of cells maintained in hGH (2 nM) for 1-4 days was unaffected compared to cells in serum-free medium alone. IGF-I (17 nM) treatment of cells for 4 days doubled the protein content of cells compared to control values in serum-free medium. hGH (2 nM) pretreatment of cells for 1-4 days had no effect on total RNA synthesis. hGH (2 nM) but not IGF-I (17 nM) treatment (3 days) resulted in a 7-fold decrease in cytoplasmic 18S rRNA content (as measured by DNA-RNA hybridization) of cells compared to that of control cells maintained in serum-free medium. When 3T3-F442A cells were transferred to serum-free medium there was a progressive decrease in DNA synthesis. The presence of hGH enhanced the rate at which DNA synthesis decreased for 3T3-F442A cells. IGF-I (17 nM) increased DNA synthesis by 6- and 8-fold after 2 and 3 days of IGF-I exposure. 3T3-F442A cells maintained in serum-free medium for 3 days responded to the addition of platelet-derived growth factor (2 U/ml) and insulin (1.6 microM) with a 56-fold increase in DNA synthesis, assayed 24 h later. 3T3-F442A cells treated with hGH (2 nM) for 3 days before platelet-derived growth factor and insulin addition exhibited a diminished DNA synthetic response, demonstrating that GH-exposed cells were partially refractory to mitogenic stimulation. GH had no effect on any aspect of macromolecular synthesis in 3T3-C2 cells, which have a low frequency of adipogenesis. Based upon these results a cell cycle model for the role of GH in the adipose differentiation of 3T3-F442A cells was proposed.

Adipose Tissue↗
PubMed1989

Antagonism by growth hormone of insulin-sensitive hexose transport in 3T3-F442A adipocytes.

We have studied the effects of GH on basal and insulin-stimulated hexose transport by 3T3-F442A adipocytes in a hormonally defined serum-free medium. Adipocytes preincubated in defined medium exhibit a low level of hexose transport which is acutely (15 min) stimulated (greater than 5-fold) by insulin (EC50, 0.1-0.2 nM). GH has acute (15-45 min) insulin-mimetic (greater than 2-fold) and chronic (4-48 h) diabetogenic (50-80%) effects on basal and insulin-stimulated hexose transport. The insulin-mimetic effect of GH has a higher EC50 (2 nM) than its diabetogenic effect (EC50, 0.2 nM). Chronic GH exposure decreases the maximal responsiveness (50-80%) and the acute sensitivity (approximately 2-fold) of hexose transport to insulin. Insulin-stimulated transport is more (approximately 5-fold) sensitive to the diabetogenic effect of GH than is basal transport. Insulin binding and degradation were not altered by chronic exposure to GH. The diabetogenic effect of GH may occur at a postinsulin binding level.

Adipose Tissue↗
PubMed1988

Anti-fertility and other actions of gossypol analogues.

From a series of gossypol derivatives studied, we conclude that the carbonyl groups of gossypol are needed for inhibition of erythrocyte anion transport and the hydroxy groups affect but are not essential to that inhibition. In an in vitro mouse erythroleukemia cytocidal assay, the most active compounds were gossypol and apogossypol. The latter was not active in the inhibition of erythrocyte anion transport or in a spermicidal assay. Of the more simple structures related to gossypol, those that were active in the cytocidal and spermicidal assays were bi-aromatic, linked by a 1- and not a 4-carbon chain and had free phenolic hydroxyl groups. These results are included in a discussion of the specificity and mechanism of action of gossypol.

Animals↗
PubMed1988

Role of insulin in growth hormone-stimulated 3T3 cell adipogenesis.

The role of insulin during GH-stimulated adipogenesis of 3T3-F442A fibroblasts was investigated. Adipogenesis in defined medium (DM), as quantified by the level of glycerol-3-phosphate dehydrogenase activity, revealed that there existed a strict requirement for both insulin and GH during adipogenesis. The concentration of insulin required to elicit half-maximal adipogenesis was approximately 20 nM. Insulin-like growth factor I was less effective than insulin in promoting adipogenesis, indicating that insulin action during differentiation was most likely mediated through the insulin receptor. Cellular viability was not compromised by the absence of insulin, as judged by colony-forming efficiency or trypan blue exclusion. Deletion of insulin from DM supplemented with 1 nM recombinant human GH reduced glycerol-3-phosphate dehydrogenase activity to uninduced levels. Removal of other individual DM constituents did not have this effect. The growth factors fibroblast growth factor, platelet-derived growth factor, and bombesin did not substitute for insulin during GH-stimulated adipogenesis. The characteristic increase in cell number observed during serum-based differentiation, reflecting clonal expansion of young adipocytes, did not occur in DM supplemented with insulin, and insulin-like growth factor I were necessary for this event. These results suggest that insulin functions in concert with GH as a coinducer of the differentiating signals.

Adipose Tissue↗
PubMed1986

Antagonistic effect of butyrate on hexamethylene bisacetamide induced differentiation of murine erythroleukemia cells.

Butyrate, at concentrations greater than 0.75 mM, induces hemoglobin accumulation in murine erythroleukemia cells (MELC). At concentrations below 0.75 mM, butyrate inhibits hemoglobin accumulation induced by hexamethylene bisacetamide (HMBA) as well as HMBA induced commitment to terminal cell division. The blocking of HMBA induced differentiation does not result from growth inhibition. When cells were exposed to HMBA and butyrate for 4 days and then both inducers were removed, the cells did not terminally divide. On the other hand, cells exposed to HMBA for 4 days, with subsequent removal of HMBA, did go on to terminally divide. Thus, butyrate blocks the ability of HMBA to accumulate the intracellular signals for terminal cell division. A 48-h pretreatment of cells with butyrate did not inhibit the ability of subsequent HMBA treatment, after butyrate removal, to induce terminal cell division. These results might suggest that cells do not generate a memory of exposure to HMBA in the presence of butyrate or a memory of exposure to butyrate when used as a pretreatment under these conditions. HMBA, at concentrations below 1.0 mM, does not induce MELC differentiation, but such concentrations actually enhance dimethyl sulfoxide induced differentiation of MELC. Equimolar concentrations of short chain fatty acids (1 to 7 carbons) were tested for their ability to block HMBA induced differentiation of MELC. Butyrate and valerate (4 and 5 carbons, respectively) had blocking activities similar to each other, whereas the other fatty acids exhibited little or no blocking of HMBA induced differentiation.

Acetamides↗
PubMed1985

Effect of gossypol on erythrocyte membrane function: specific inhibition of inorganic anion exchange and interaction with band 3.

The effects of gossypol on membrane functions of the human erythrocyte were studied. Gossypol (10 microM) had no effect on spontaneous hemolysis, osmotic fragility, cell volume, cholinesterase activity, hexose transport, ouabain-sensitive inorganic cation transport, ouabain-insensitive inorganic cation transport and nucleoside transport. Conversely, 10 microM gossypol inhibited inorganic anion transport by approximately 90% for three different substrates, i.e., phosphate, sulfate and chloride. Inhibition of inorganic anion transport was specific as 10 microM gossypol had no effect on the eight aforementioned membrane-related functions of the human erythrocyte. Inhibition inorganic anion transport was characterized using sulfate as the substrate and had the following features: it was potent, with a Ki of approximately 3 microM; it was rapid, with onset occurring in less than 1 min; it was potently blocked by physiological concentrations of albumin and plasma with 50% blocking achieved at 0.03% (w/v) albumin; it occurred by a noncompetitive kinetic mechanism; it was independent of medium Ca++, Mg++ or pH. Gossypol was bound to human erythrocytes and cell membranes isolated from erythrocytes. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid is a potent inhibitor of anion transport and can be covalently bound to band 3. Covalently bound 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid blocked a fraction of gossypol binding to erythrocyte membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1984

Transport of the folate compound methotrexate decreases during differentiation of murine erythroleukemia cells.

The relationship of folate transport and chemically induced differentiation of murine erythroleukemia cells (MELC) was examined. MELC were found to have a carrier-mediated transport system for reduced folates. Chemically induced differentiation of MELC caused a 10-fold decrease in the rate of influx of the folate analog methotrexate. The loss of methotrexate transport during differentiation resulted from a 15-fold decrease in the Vmax for the influx while the Km remained unchanged. Inducer dose dependencies for hemoglobin accumulation and loss of methotrexate influx were similar. A dimethyl sulfoxide-resistant variant of MELC did not express complete accumulation of hemoglobin or loss of methotrexate transport when treated with dimethyl sulfoxide. The decrease of folate influx during differentiation was not a result of: 1) changes in cell densities or growth phase; 2) growth-related changes of the medium; or 3) a direct inhibition of folate influx by the inducers. Loss of folate compound transport correlated with MELC differentiation.

Acetamides↗
PubMed1984

Cytocidal effect of gossypol on cultured murine erythroleukemia cells is prevented by serum protein.

The interaction of gossypol (G) with cultured murine erythroleukemia cells (MELC) was studied in vitro. G was cytocidal (inhibited growth greater than 90%) to MELC at greater than 10 microM, but not at less than 5 microM in medium supplemented with 10 and 15% fetal calf serum (FCS). Five micromolar of G was cytocidal in 2 and 5% FCS. Serum albumin (2%) also decreased the effective cytocidal dose of G. This inhibition was reversible if extracellular drug (30 microM) was removed after 1 h but not after 24 h. Uptake of [14C]G by MELC (approximately 10(6) cells/ml) was saturable with half-maximal uptake at 8 microM in the absence of FCS. This uptake was concentrative, i.e., 75-fold relative to the total [14C]G concentration. In the presence of both FCS (approximately 2%) and serum albumin (approximately 0.03%), the accumulation of [14C]G (10 microM) by MELC was decreased approximately 50%. Direct binding of G to albumin, assayed by quenching of intrinsic fluorescence, was stoichiometric with respect to micromolar albumin content suggesting an apparent affinity of approximately 10(-7) M. Visible absorption spectra for G in the presence of serum albumin exhibited batho- and hyperchromic shifts of the maxima at approximately 380 nm. These studies demonstrate that: 1) G, at pharmacologically relevant concentrations, is cytocidal for MELC; 2) serum protein, e.g., albumin, can reduce both the cytocidal effects of G and the uptake of [14C]G by MELC; and 3) these effects are probably the result of G binding to serum protein, e.g., albumin, which reduces the free effective concentration of the drug.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Binding and degradation of 125I-labeled insulin by a clonal line of rat pituitary tumor cells.

Receptor sites for insulin on GH3 cells were characterized. Uptake of 125I-labeled insulin by the cells was dependent upon time and temperature, with apparent steady-states reached by 120, 20 and 10 min at 4, 23 and 37 degrees C, respectively. The binding sites were sensitive to trypsin, suggesting that the receptors contain protein. Insulin competed with 125I-labeled insulin for binding sites, with half-maximal competition observed at 5 nM insulin. Neither adrenocorticotropic hormone nor growth hormone competed for 125I-labeled insulin binding sites. 125I-labeled insulin binding was reversible, and saturable with respect to hormone concentration. 125I-labeled insulin was degraded at both 4 and 37 degrees C by GH3 cells, but not by medium conditioned by these cells. After a 5 min incubation at 37 degrees C, products of 125I-labeled insulin degradation could be recovered from the cells but were not detected extracellularly. Extending the time of incubation resulted in the recovery of fragments of 125I-labeled insulin from both cells and the medium. Native insulin inhibited most of the degradation of 125I-labeled insulin suggesting that degradation resulted, in part, from a saturable process. At steady-state, degradation products of 125I-labeled insulin, as well as intact hormone, were recovered from GH3 cells. After 30 min incubation at 37 degrees C, 80% of the cell-bound radioactivity was not extractable from GH3, cells with acetic acid.

Animals↗
PubMed1983

Chlortetracycline as a probe of membrane-associated calcium and magnesium: interaction with red cell membranes, phospholipids, and proteins monitored by fluorescence and circular dichroism.

The fluorescence emission and circular dichroism spectra of chlortetracycline (CTC) have been measured, including the effects of multivalent cations (Ca, Mg, La), of medium polarity, and of interaction with human red cell membranes, lipids, and a variety of proteins. An obligatory role of Ca in the association of CTC with membranes was demonstrated. Binding and kinetic constants for the CTC-Ca chelate interaction with membranes and phospholipids were determined. The results suggest that the CTC-Ca chelate fluorescence is greatly enhanced in the vicinity of membrane phospholipid head groups. The circular dichroism spectra indicate a number of distinct CTC conformations corresponding to chelation of specific cations, to interaction with membranes and phospholipids, and to medium polarity. The high quantum yield CTC-Ca conformation associated with membranes or phospholipids was identified by its characteristic circular dichroism spectrum and is different from the CTC-Ca conformation in nonpolar media (80% methanol).

Calcium↗
PubMed1983

Effects of gossypol on PAH transport in the rabbit kidney slice.

The present work was carried out to investigate if gossypol, a toxic weak organic acid (pK = 7.2) contained in cottonseed, is secreted by the renal proximal tubule through the organic anion transport system in the rabbit. The slice uptake of p-aminohippurate (PAH), a prototypical organic anion, was significantly inhibited (by 30%) only when the medium concentration of gossypol was raised to 10(-4) M. However, gossypol at the latter concentration also induced a 30% inhibition of the slice uptake of tetraethylammonium (TEA), a prototypical organic cation. Moreover, gossypol at 10(-4) M significantly decreased the slice oxygen consumption (congruent to 30%) and Mg-ATPase (70%) and Na-K-ATPase (90%) activities of renal cortical microsomes, while it significantly decreased the intracellular (K+). These results indicate that gossypol inhibits PAH uptake through nonspecific nephrotoxic effects on cell metabolism and Na-K-ATPase activity rather than through its specific interaction with the organic anion transport system.

Adenosine Triphosphatases↗
PubMed1983

Hormones modulate adipocyte membrane potential ATP and lipolysis via free fatty acids.

The hypothesis that lipolytic hormones reduce the mitochondrial electrical potential in rat white adipocytes via free fatty acids (FFA) was examined. Hormonal effects on plasma and mitochondrial membrane potentials were evaluated with [3H]triphenylmethylphosphonium (TPMP+) and 86Rb+. FFA generation was controlled by varying medium albumin concentrations. In 4.0% albumin buffer, adrenocorticotropin or l-epinephrine increased intracellular FFAs, produced cellular TPMP+ efflux, ATP depletion, release of FFAs and glycerol, and no change in 86Rb+ distribution. In 0.5% albumin buffer, greater intracellular FFA accumulation accompanied greater TPMP+ and ATP depletion, significant loss of cell-associated 86Rb+, and a concomitant inhibition of FFA and glycerol release. Exogenous addition of FFAs mimicked the effect of hormones on adipocyte TPMP+ distribution. TPMP+ and 86Rb+ uptake into adipocyte "ghosts" were unaffected by hormones. We suggest that mitochondrial membrane depolarization is a metabolic response to hormones via FFA accumulation by white adipocytes. The additional hormonal effects that were observed in 0.5% albumin buffer may be related to inhibition of lipolysis secondary to intracellular ATP depletion.

Adenosine Triphosphate↗
PubMed1983

The membrane potential of human platelets.

The membrane potential of the human platelet was investigated using the membrane potential probes 3,3'-dipropyl-2,2'-thiadicarbocyanine iodide and tritiated triphenylmethylphosphonium bromide. The membrane potential in physiologic buffer was estimated to be 52-60 mV inside negative. The membrane was depolarized when extracellular potassium or hydrogen ion concentrations were increased. Changes in extracellular sodium, chloride, or calcium ion concentration had no measurable effect on membrane potential. Elevated extracellular potassium has been shown to increase platelet sensitivity to the aggregating agent, adenosine diphosphate. Our results show that changes in extracellular ion concentrations that depolarize platelets increase platelet sensitivity to aggregating agents. These results suggest that membrane potential changes may play a role in modulating the response of platelets to aggregating agents.

Benzothiazoles↗
PubMed1982

Triphenylmethylphosphonium cation distribution as a measure of hormone-induced alterations in white adipocyte membrane potential.

Triphenylmethylphosphonium (TPMP+) partitions into the mitochondrial and cytosolic compartments in the rat white adipocyte in a potential-dependent fashion. The relationship between [3H]TPMP+ distribution, intracellular cAMP generation and lipolysis in response to hormones and cAMP-mimetic compounds was examined. Half-maximal [3H]TPMP+ efflux and glycerol release were produced by 15 and 9 nM adrenocorticotropin, 170 and 110 nM 1-epinephrine, 70 and 27 microM isobutylmethylxanthine and 800 and 750 microM dibutyryl cAMP, respectively. Hormone-stimulated cAMP generation was also correlated with [3H]TPMP+ efflux and lipolysis in terms of concentration dependency. In kinetic experiments, glycerol release and [3H]TPMP+ efflux in response to adrenocorticotropin or cholera toxin proceeded over a similar time course, whereas an earlier rise in cAMP generation was detected. The depolarizing effect of lipolytic compounds was localized to the mitochondrial compartment. When cells were incubated in elevated-[K+]0 buffer, the stimulatory effect of dibutyryl cAMP on [3H]TPMP+ efflux and lipolysis persisted, suggesting that maintenance of the plasma membrane potential is not critical for demonstration of these responses. When the extracellular concentration of serum albumin, which provides binding sites for free fatty acids, was increased from 1 to 3%, an increase in glycerol release and a decrease in [3H]TPMP+ efflux was observed. We suggest that intracellular free fatty acid accumulation in response to lipolytic agents causes dissipation of the mitochondrial membrane potential and efflux of [3H]TPMP+ from the organelle and cell.

Adipose Tissue↗
PubMed1981

Stimulus-secretion coupling in isolated adrenal chromaffin cells: calcium channel activation and possible role of cytoskeletal elements.

The catecholamine secretory function of a preparation of isolated bovine adrenal chromaffin cells has been further characterized under conditions designed to elucidate the mechanism of calcium channel activation and the possible role of cytoskeletal elements in stimulus-secretion coupling. Three related sets of data were obtained: (1) Differences in kinetics, Ca dependence, strength, and additivity of the secretory response to acetylcholine (ACh) versus excess K; (2) the effects on secretion of the Ca channel-blocking agents, Ni, Mg, and verapamil; and (3) the Ca dependence of vinblastine action on ACh- and K-evoked secretion. The results suggest that a major portion of the Ca influx required for catecholamine release enters the cell via voltage-dependent Ca channels with some additional Ca influx via the ACh receptor channel. Comparison of the present secretion data with corresponding known electrophysiological properties of isolated chromaffin cells provides added evidence for a role of chromaffin cell action potentials in regulation of Ca influx and the secretory response. Elevated Ca concentrations enhanced K-evoked secretion to levels comparable to that of Ach but did not induce a vinblastine block of K-evoked release. This provides further evidence against a role of microtubules in the common exocytosis event per se. However, a role of cytoskeletal elements in directing the movement of secretory granules, or an action of vinblastine at cholinergic receptors, remain distinct possibilities.

Acetylcholine↗