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Biomedical subjects

M D Lane

Publications and source records attributed to M D Lane.

At least 145 records · Page 8Linked to original sources
PubMed1982

Effect of differentiation on the adenylate cyclase system of 3T3-C2 and 3T3-L1 cells. Determination of choleragen substrates in differentiating 3T3-L1 and nondifferentiating 3T3-C2 cells.

3T3-L1 preadipocytes, when treated with 3-isobutyl-1-methylxanthine, dexamethasone, and insulin, differentiate into cells with the morphological and biochemical properties of adipocytes; the closely related 3T3-C2 cells, under identical conditions, exhibit a low frequency of adipocyte conversion. During differentiation, 3T3-L1 preadipocytes acquire an increased responsiveness to certain agonists (e.g. isoproterenol and adrenocorticotropic hormone) that influence lipolysis and lipogenesis through activation of adenylate cyclase, whereas 3T3-C2 cells do not. It has been suggested that changes in hormone responsiveness of 3T3-L1 cells during differentiation result from increased amounts of the guanyl nucleotide-binding protein of adenylate cyclase, as demonstrated by choleragen-catalyzed [32P]ADP ribosylation of 42 and 49-50-kilodalton particulate peptides. Particulate fractions from nondifferentiating 3T3-C2 cells, like those from 3T3-L1 cells, contained choleragen substrates of 42 and 46-47 (doublet) kilodaltons. Incubation of intact 3T3-L1 or 3T3-C2 cells with choleragen prior to preparation of particulate fractions prevented the subsequent in vitro choleragen-dependent [32P]ADP ribosylation of only these peptides. Increased incorporation of radioactivity into both the 42 and 46-47-kilodalton peptides was observed during differentiation of 3T3-L1 cells. However, a similar increase was also observed in nondifferentiating 3T3-C2 cells subjected to the differentiation protocol. Therefore, increased hormone responsiveness of 3T3-L1 adipocytes cannot be explained solely on the basis of increased labeling, and perhaps increased amounts, of the guanyl nucleotide-binding protein.

Adenosine Diphosphate Ribose↗
PubMed1982

Synthesis, turnover, and down-regulation of epidermal growth factor receptors in human A431 epidermoid carcinoma cells and skin fibroblasts.

Epidermal growth factor (EGF) receptors extracted with Triton X-100 from human skin fibroblasts and A431 epidermoid carcinoma cells rapidly lose EGF-binding activity precipitable with polyethylene glycol. The presence of concanavalin A which can cross-link and, thereby, aggregate the receptors, allowed quantitative recovery of the lost EGF-binding activity. Scatchard analysis of EGF binding of Triton X-100-solubilized receptors showed that A431 cells and skin fibroblasts possess approximately 1.5 X 10(6) and 7 X 10(4) EGF-binding sites/cell, respectively, which exhibit similar affinities for the ligand. The heavy isotope density-shift method was employed to determine whether differences in rates of receptor synthesis or decay account for the large difference in number of receptors/cell between the two cell types. After shifting cells to medium containing heavy (15N, 13C, and 2H) amino acids, light and heavy receptors, solubilized from total cellular membranes, were resolved by isopycnic banding on density gradients and then quantitated. It was demonstrated that A431 cells synthesize EGF receptors at a rate 12 times faster than skin fibroblasts and that the half-life for receptor decay of A431 cells is somewhat longer (t1/2 = 16 h) than that (t1/2 = 9 h) of fibroblasts. Down-regulation of cell surface and total cellular EGF-binding capacity in A431 cells occurs with a t1/2 of 2-3 h and results in a 70-83% decrease in receptor level in 12 h. Scatchard analysis revealed that these changes in EGF binding were due to an alteration of receptor number and not EGF-binding affinity. Rates of EGF receptor synthesis and inactivation/decay were determined by the heavy isotope density-shift method. No change in the rate of receptor synthesis occurred as a consequence of EGF receptor down-regulation. Down-regulation, however, caused a decrease in receptor half-life from 16 to 4.5 h. These results indicate that EGF-dependent regulation of EGF receptor level in A431 cells involves an alteration of the rate of receptor inactivation.

Carcinoma, Squamous Cell↗
PubMed1982

Association and assembly of triglyceride and phospholipid with glycosylated and unglycosylated apoproteins of very low density lipoprotein in the intact liver cell.

Using estrogen-induced chick liver cells which synthesize and secrete large amounts of very low density lipoprotein (VLDL), we have previously shown (Siuta-Mangano, P., Howard, S., Lennarz, W. J., and Lane, M. D. (1982) J. Biol. Chem. 257, 4292-4300) that the major protein constituent of VLDL, the 350,000 molecular weight apoprotein (apoprotein B), is synthesized as a single polypeptide to which core oligosaccharides are added co-translationally. This system has now been employed to study the assembly of the apoproteins of VLDL with their glycerolipid (triglyceride and phospholipid) components and the secretion of the VLDL glycerolipids. In the presence of cycloheximide such that VLDL apoprotein synthesis is inhibited 98%, the secretion of lipids labeled from a [3H]palmitate pulse by hepatocyte monolayers was halted only after completed apoprotein chains had cleared the cell. Under conditions whereby tunicamycin inhibited [3H]glucosamine incorporation into apoprotein B by 98% and [3H]leucine incorporation into the VLDL apoproteins minimally, the unglycosylated form of apoprotein B assembled with the usual complement of triglyceride and phospholipid as did glycosylated apoprotein B to form a VLDL which was readily secreted by the hepatocyte. Taken together, these findings demonstrate that whereas apoprotein synthesis is necessary for the secretion of the major lipid components of VLDL, glycosylation of apoprotein B is not required for either the assembly of VLDL glycerolipids or for the secretion of the VLDL particle.

Animals↗
PubMed1982

Electric pulse-induced fusion of 3T3 cells in monolayer culture.

Swiss mouse 3T3-C2 fibroblasts, grown to confluence in monolayer culture, are shown to fuse when exposed to electric fields. Exposure to five repetitive electric pulses of about 1 kilovolt per centimeter with a duration of 50 microseconds caused approximately 20 percent of the cells to become fused (multinucleate) when 1 millimolar magnesium was present in the medium. The effects of minimum thresholds of field strength, pulse duration, and number of pulses were determined. Cell disruption was observed when the electric field exceeded 2.0 kilovolts per centimeter or the pulse was of longer duration than 120 microseconds.

Animals↗
PubMed1982

Insulin-induced down-regulation of insulin receptors in 3T3-L1 adipocytes. Altered rate of receptor inactivation.

Fully differentiated 3T3-L1 adipocytes, maintained in the presence of insulin, exhibit up-regulation of insulin-binding capacity when insulin is removed from the culture medium. Both cell surface and total cellular insulin receptors increase by 1.8- to 2.0-fold during the 24-h period following the removal of insulin. When up-regulated 3T3-L1 cells are exposed to 10(-8) M insulin down-regulation of insulin receptors occurs with a t1/2 of 2-3 h. Down-regulation was complete after a 10-h exposure to insulin and resulted in a 50-60% decrease in levels of cell surface and total cellular insulin-binding capacities, respectively. Scatchard analysis revealed that these changes in insulin binding are due to an alteration of receptor number and not insulin-binding affinity. To clarify the mechanism(s) by which the regulation of insulin receptor level occurs, rates of receptor synthesis and degradation were determined by the heavy isotope density-shift method. No change in the rate of receptor synthesis occurred as a consequence of up-regulation or down-regulation. Up-regulation, however, caused an increase in receptor half-life from 8.1 h in the control cells to 14.8 h. Subsequent down-regulation brought about a return of receptor half-life to 6.9 h. These results indicate that insulin-dependent regulation of insulin receptor level in 3T3-L1 adipocytes involves a change in the rate of receptor degradation. Further studies indicated that regulation of insulin receptor level has physiological significance, since up-regulated cells exhibit an increased responsiveness of 2-deoxyglucose uptake to insulin compared to down-regulated cells.

Adipose Tissue↗
PubMed1982

Acetyl coenzyme A carboxylase. Rapid purification of the chick liver enzyme and steady state kinetic analysis of the carboxylase-catalyzed reaction.

Avidin affinity chromatography was used to rapidly purify acetyl-CoA carboxylase to homogeneity in high yield from chicken liver. Dissociation of the purified carboxylase with dodecyl sulfate yielded a single size class of subunit polypeptide of 225,000 daltons. A steady state kinetic analysis of the carboxylase-catalyzed carboxylation of acetyl-CoA gave rise to intersecting line patterns in all double-reciprocal plots of initial velocity with each substrate pair, i.e. ATP . Mg and HCO3(-) and acetyl-CoA. It was concluded that the kinetic mechanism involves a quaternary complex of the enzyme, ADP, Pi, and acetyl-CoA rather than a double displacement as previously believed. The ordered addition of ATP, HCO3(-), and then acetyl-CoA, to the citrate-activated form of the carboxylase is the kinetic mechanism most consistent with the results.

Acetyl-CoA Carboxylase↗
PubMed1982

Lipoprotein lipase suppression in 3T3-L1 cells by an endotoxin-induced mediator from exudate cells.

Conditioned medium from cultures of mouse peritoneal exudate cells incubated wih endotoxin contains a mediator that markedly suppresses (greater than 90%) lipoprotein lipase (triacylglycero-protein acylhydrolase, EC 3.1.1.34) activity in differentiating 3T3-L1 mouse preadipocytes. The effect is dependent upon the amount of mediator and is evident as early as 30 min after the addition of the mediator-containing medium to 3T3-L1 cell cultures. Neither endotoxin nor conditioned medium from cultures of exudate cells not exposed to endotoxin shows the presence of the mediator. Lysates of the exudate cells are also unable to suppress the lipase activity. Increasing the amount of insulin does not reverse this suppression, even at 1000 times the concentration used for standard experiments. The lipoprotein lipase suppression mediator present in the conditioned medium of endotoxin-treated exudate cells is heat labile and has an apparent molecular weight of at least 12,000. The mediator does not inhibit lipoprotein lipase activity directly nor does it affect the half-life of enzyme activity released in the medium. The present study demonstrates that endotoxin promotes the release of a mediator from exudate cells that suppresses the activity of lipoprotein lipase in 3T3-L1 preadipocytes.

Adipose Tissue↗
PubMed1982

Control of insulin receptor level in 3T3 cells: effect of insulin-induced down-regulation and dexamethasone-induced up-regulation on rate of receptor inactivation.

Chronic exposure of 3T3 mouse fibroblasts to insulin or to the glucocorticoid dexamethasone induces down-regulation and up-regulation, respectively, of cell-surface and total cellular insulin binding capacity. Both processes are reversed upon withdrawal of the inducer. Scatchard analysis of insulin binding for receptors in the down- and up-regulated states indicates that the changes in binding capacity result primarily from alterations in insulin receptor level. That these alterations in total receptor level are due to changes in cell-surface receptor level is indicated by the fact that the level of trypsin-insensitive, presumably intracellular, insulin binding sites does not change appreciably upon down- and up-regulation. The effects of insulin-induced down-regulation and dexamethasone-induced up-regulation on the rates of insulin receptor synthesis and decay were assessed by the heavy-isotope density-shift technique. Cells were shifted to medium containing heavy (2H, 13C, 15N) amino acids and, at various times after the shift, light and heavy receptors solubilized from total cellular membranes were resolved by isopycnic banding on density gradients and then quantitated. It was demonstrated that the insulin- and dexamethasone-induced alterations in insulin receptor level were due entirely to changes in the rate constant for receptor inactivation. The decrease in the first-order rate constant for receptor decay caused by dexamethasone is unexpected in view of the known action of steroid hormones in the induction of the synthesis of specific proteins.

Animals↗
PubMed1981

Formation and turnover of triglyceride-rich vesicles in the chick liver cell. Effects of cAMP and carnitine on triglyceride mobilization and conversion to ketones.

Refractile cytoplasmic vesicles are formed in less than 10 h when chick liver cell monolayers are incubated with serum-free medium containing 0.9 mM oleate. These vesicles are identical in microscopic appearance to those formed in monolayers by de novo fatty acid synthesis (Tarlow, D. M., Watkins, P. A., Reed, R. E., Miller, R. S., Zwergel, E. E., and Lane, M. D. (1977) J. Cell Biol. 73, 332-353), but require about one-seventh the incubation time to achieve comparable size. After release from the cells by lysis in hypotonic medium, the vesicles can be isolated by flotation at 27,000 X g. Electron microscopy reveals that the isolated vesicles are rimmed by a membrane. Analysis of vesicles isolated from cells labeled with [14C]oleate or [14C]acetate showed that greater than 95% of their 14C content was in the form of triglyceride and that most cellular [14C]triglyceride was contained in the triglyceride-rich vesicles. Exposure of cells to dibytyryl-cAMP after removal of oleate from the medium caused the disappearance of triglyceride-rich vesicles within 36 h. In the absence of cyclic nucleotide, the vesicles persist. Consistent with this morphological change, dibutyryl-cAMP caused a 5.5-fold activation of the apparent rate of mobilization of cellular [14C]triglyceride from cells previously labeled with [14C]oleate. L-(--)-Carnitine alone had no effect; however, when added with dibutyryl-cAMP, cellular triglyceride mobilization was activated 7.4-fold. Although [14C]triglyceride was the principal 14C-labeled product secreted in the absence of cyclic nucleotide and comprised 90% of the total, [14C]acetoacetate and [14C] beta-hydroxybutyrate became major products when cells were treated with dibutyryl-cAMP. Thus, dibytyryl-cAMP activated ketogenesis from cellular [14C]triglyceride by 200-fold and when added with L-(--)-carnitine, by 400-fold. Cells containing triglyceride-rich vesicles labeled with [2-glyceryl-3H]triglyceride were generated by incubation with medium containing [2-3H]glycerol. A comparison of the rates of loss of cellular [1-oleoyl-14C- and [2-glyceryl-3H]triglyceride revealed that substantial re-esterification, i.e. recycling, of 14C-fatty acid released by lipolysis occurred. Under conditions where recycling of 3H label ws minimal, it was determined that 15% of the cellular [2-glyceryl-3H]triglyceride was secreted "en bloc," i.e. without prior lipolysis. En bloc secretion was not affected by dibutyryl-cAMP. The rate of lipolysis of vesicle-associated [2-glyceryl-3H]triglyceride was increased 2.2-fold in the presence of dibutyryl-cAmP. Chloroquine markedly inhibited the dibutyryl-cAMP-dependent lipolysis suggesting the participation of lysosomes in the mobilization of triglyceride-rich vesicles. Mechanisms are presented which could account for the effects of cAMP and carnitine on the turnover of vesicle triglyceride both at the level of lipolysis and the utilization of the released fatty acids by mitochondria...

Animals↗
PubMed1981

On the mechanism of preadipocyte differentiation. Masking of poly(ADP-ribose) synthetase activity during differentiation of 3T3-L1 preadipocytes.

Differentiation of 3T3-L1 preadipocytes, induced by methylisobutylxanthine (MIX), dexamethasone (DEX), and insulin, produces cells with the morphological and biochemical characteristics of adipocytes. Poly(ADP-ribose) synthetase activity in 3T3-L1 cells treated with MIX, DEX, and insulin underwent an abrupt decrease in activity, remained low for several hours, and then increased; this rise was delayed by readdition of MIX, DEX, and insulin. The drop of synthetase activity represents the earliest alteration of a specific enzyme yet detected during the differentiation of 3T3-L1 cells. Nondifferentiating 3T3-C2 control cells do not exhibit changes in poly(ADP-ribose) synthetase activity when treated with MIX, DEX, and insulin. The transient reduction in poly(ADP-ribose) synthetase activity in 3T3-L1 cells occurred prior to the appearance of the adipocyte phenotype induced by the above agents and was not observed when preparations were assayed in the presence of DNase I. It is evident that poly(ADP-ribose) synthetase activity was masked following treatment with MIX, DEX, and insulin since the synthetase is sensitive to agents that affect the physical properties of DNA. This transient reduction in activity may be an early event in differentiation which reflects changes in chromatin structure.

1-Methyl-3-isobutylxanthine↗
PubMed1981

Post-translational glycosylation-induced activation of aglycoinsulin receptor accumulated during tunicamycin treatment.

Tunicamycin, which inhibits N-linked oligosaccharide chain addition to nascent polypeptides, interrupts glycosylation of the insulin receptor in 3T3-L1 adipocytes giving rise to inactive receptors. Chronic exposure of cells to low levels (100 ng/ml) of high performance liquid chromatography-purified tunicamycin causes a greater than or equal to 90% depletion of insulin binding to cell surface and Triton X-100-extractable receptors and a 93% inhibition of [3H]glucosamine incorporation into protein in alkali-stable form. Under identical conditions, protein synthesis was inhibited less than 10%. Recovery of insulin binding activity after the removal of tunicamycin achieves 70-80% of control activity within 36 h. Concomitant with the withdrawal of tunicamycin, cells were shifted to medium containing heavy (greater than 95% 15N, 13C, 2H) amino acids after which Triton X-100-solubilized "light" and "heavy" insulin receptors were separated isopycnically on CsCl density gradients. A kinetic analysis of the recovery of functional receptors revealed that the initial appearance of previously synthesized light receptor was followed, after a short lag, by newly synthesized heavy receptor. Similar levels of light receptor, but no new heavy receptor, accrue in the presence of cycloheximide. This strongly suggests that inactive aglycoinsulin receptor accumulated post-translationally during chronic treatment with tunicamycin and then re-entered the glycosylation pathway when the inhibitor was removed giving rise to a functional insulin receptor.

Animals↗
PubMed1981

Very low density lipoprotein synthesis and secretion. Extrusion of apoprotein B nascent chains through the membrane of the endoplasmic reticulum without protein synthesis.

The hypothesis was tested that chain elongation/translocation during protein synthesis forces the growing polypeptide chain through the membrane of the endoplasmic reticulum into its luminal compartment for secretion. Estrogen-induced chick liver cells which synthesize and secrete the 350,000-dalton apoprotein B of very low density lipoprotein were employed to address this issue. Treatment of [3H]leucine pulse-labeled cells with cycloheximide arrested apoprotein B nascent chains on membrane-bound polysomes without affecting the secretion of completed chains. Puromycin added immediately following the washout of cycloheximide discharged these nascent chains which were subsequently translocated through the membrane and quantitatively secreted into the medium. The size distribution of the secreted apoprotein B nascent chains approximated that of the polysome-bound apoprotein B nascent chains prior to discharge with puromycin. These findings indicate that the translocation of polysome-associated nascent chains through the membrane into the luminal space of the endoplasmic reticulum requires neither protein synthesis nor the attachment of the polypeptide to the ribosome. Since puromycin-discharged nascent chains are readily secreted by these cells, this approach may be used to study the structural requirements for the co- and post-translational events of apoprotein B processing, e.g. glycosylation and assembly of the lipid components of very low density lipoprotein.

Animals↗