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Biomedical subjects

M D Lane

Publications and source records attributed to M D Lane.

At least 163 records · Page 9Linked to original sources
PubMed1981

Role of glycosylation and protein synthesis in insulin receptor metabolism by 3T3-L1 mouse adipocytes.

The roles of glycosylation and protein synthesis in the maintenance of insulin receptor levels and turnover rates in 3T3-L1 adipocytes were investigated. The heavy isotope density-shift technique was employed to determine the effects of inhibitors of these processes on the rates of synthesis and degradation of cellular insulin receptors. Inhibitors of protein synthesis--i.e., cycloheximide and puromycin--markedly decreased the rate of degradation of the insulin receptor, the half-life for receptor decay increasing from 7.5 hr without to 25 hr with inhibitor. The continued synthesis of a short-lived protein appears to be necessary for normal insulin receptor turnover. Tunicamycin, a potent inhibitor of core oligosaccharide addition in the formation of N-glycosidically linked glycoproteins, caused the depletion of cell-surface and total cellular detergent-extractable insulin receptors. This inhibitor totally prevented the formation of functional newly synthesized insulin receptor, yet receptor degradation was affected minimally. Thus, glycosylation of the receptor appears to be required for its activation after translation.

Adipose Tissue↗
PubMed1981

Insulin receptor synthesis and turnover in differentiating 3T3-L1 preadipocytes.

3T3-L1 "preadipocytes" can be induced to differentiate in culture into cells having the morphological and biochemical characteristics of adipocytes. The binding of 125I-insulin to the cell-surface of differentiated and undifferentiated 3T3-L1 cells and nondifferentiating 3T3-C2 cells was compared. In the absence of agents which induce adipocyte conversion, ie, insulin or insulin plus methylisobutylxanthine (MIX) and dexamethasone (DEX), 3T3-L1 cells fail to express the adipocyte phenotype and maintain a constant number of insulin binding sites. Induction of adipocyte conversion with 3T3-L1 cells in the presence of insulin causes apparent down-regulation of insulin receptors followed by a 12--15-fold increase in receptor number which parallels differentiation. Approximately 170,000 insulin binding sites per cell are expressed when greater than 75% of the cells have differentiated. The rise of insulin receptor level is differentiation-dependent. 3T3-C2 cells, which do not differentiate in the presence of insulin or insulin plus MIX and DEX, exhibit only insulin-induced down-regulation of insulin receptors. The increase of insulin receptor level in 3T3-L1 cells in receptor-specific since the levels of epidermal growth factor receptor or choleragen receptor, respectively, remain constant or decrease substantially. A heavy isotope, density-shift technique was used to analyze insulin receptor synthesis and turnover in cells labeled with "heavy" (2H, 13C, and 15N) amino acids. Solubilized newly-synthesized "heavy" and old "light" receptors were separated by isopycnic banding on CsCl gradients and quantitated. The size of the soluble receptor isolated after isopycnic banding in CsCl gradients is approximately 400,000 daltons. Mixing of "light" and "heavy" membranes prior to extraction of receptor revealed no change in "light" or "heavy" receptor isopycnic banding densities. Thus, no detectable interchange of subunits occurs between receptor molecules during extraction or equilibrium centrifugation. Insulin receptor synthesis and turnover, studied by the density-shift technique showed that the rise of receptor level during differentiation results primarily from an increased rate of receptor synthesis. The rate of insulin receptor degradation was not significantly altered. The t1/2 for degradation of the insulin receptor in differentiated 3T3-L1 cells in culture was 6--7 hours in the presence of insulin. Removal of insulin from the medium did not materially affect the rate of receptor degradation. Inhibition of protein synthesis with cycloheximide causes a lengthening of the t1/2 for insulin receptor degradation to 26 hours. Thus, the synthesis of a short-lived protein appears to be required for a critical step in the pathway of insulin receptor degradation.

1-Methyl-3-isobutylxanthine↗
PubMed1980

Induction of fatty acid synthetase synthesis in differentiating 3T3-L1 preadipocytes.

3T3-L1 preadipocytes, cloned from 3T3 mouse embryo fibroblasts, differentiate in monolayer culture into cells with morphological and biochemical characteristics of adipocytes. Deposition of cytoplasmic triglyceride is associated with an increased lipogenic rate and a coordinate rise in the activities of many lipogenic enzymes (Mackall, J.C., Student, A.K., Polakis, S.E., and Lane, M.D. (1976) J. Biol. Chem. 251, 6462-6464). During differentiation induced by a 48-h treatment of postconfluent cells with methylisobutylxanthine, dexamethasone, and insulin, fatty acid synthetase activity increased to a level 19.5-fold higher than that of undifferentiated 3T3-L1 cells or nondifferentiating 3T3-C2 cells. The rate of [3H]leucine incorporation into immunoadsorbable fatty acid synthetase rose to a maximum and then declined to a new level 12.5-fold higher in differentiated than in undifferentiated 3T3-L1 cells. The kinetics of the changing [3H]leucine incorporation rate was reflected in the kinetics of the rise in fatty acid synthetase activity. The rate of degradation of fatty acid synthetase, determined by pulse-chase experiments, was unaffected by differentiation, the t1/2 remaining constant at 1.4 days. It is concluded that the higher level of fatty acid synthetase activity in differentiated 3T3-L1 cells can be attributed entirely to an increased rate of enzyme synthesis. The rate of total cellular protein synthesis also increases early early in differentiation, lending support to a model in which the synthesis of a large number of "differentiated proteins" is coordinately induced.

Adipose Tissue↗
PubMed1980

Loss of choleragen receptors and ganglioside upon differentiation of 3T3-L1 preadipocytes.

3T3-L1 preadipocytes differentiate in culture into cells having the enzymatic and morphological characteristics of adipocytes. Differentiation is accompanied by a decrease in total cellular ganglioside content; the ganglioside level is 1.8 to 2.5-fold higher in undifferentiated than in differentiated cells. Gangliosides GM3 and GD1a constitute a majority of total cell gangliosides in both cell types, while ganglioside GM1, the putative choleragen receptor, constitutes less than 5%. Differentiation results in a 75 to 85% decrease in ganglioside GM1. An inverse correlation exists between the percentage of adipocytes in the cell population and: 1) total ganglioside and ganglioside GM1 content, and 2) surface ganglioside GM1 as estimated by choleragen binding or fluorescent staining of bound choleragen. Nondifferentiating 3T3-C2 control cells do not exhibit changes in total ganglioside, ganglioside GM1, or choleragen binding that are observed with 3T3-L1 cells.

Adipose Tissue↗
PubMed1980

Insulin receptor synthesis and turnover in differentiating 3T3-L1 preadipocytes.

A density-shift method is described for analyzing insulin receptor synthesis and turnover in cultured cells labeled with "heavy" amino acids (2H, 13C, and 15N). Solubilized newly synthesized heavy and old "light" receptors are separated by isopycnic banding on CsCl gradients and then quantitated. Insulin receptor synthesis and turnover were studied by this technique in 3T3-L1 preadipocytes which undergo an increase in insulin binding capacity during differentiation. The results indicate that the increase in insulin binding capacity is a consequence of new receptor synthesis, that the insulin receptor has a relatively short half-life (6.7 hr), and that an increased rate of receptor synthesis contributes to the increase of insulin receptor level during differentiation.

Adipose Tissue↗
PubMed1980

Molecular structure and intermolecular interactions of N1'-methoxycarbonylbiotin methyl ester: a model for carboxybiotin.

The crystal structure of N1'-methoxycarbonylbiotin methyl ester, a model for N1'-carboxybiotin, has been determined. The ureido carbonyl bond has more double bond (keto) character than does the corresponding bound in free biotin, which has single bond (enolate) character. In addition, there is an interesting intermolecular interaction between the ureido carbonyl oxygen and a methyl group. Comparison of the molecular structure and crystal packing with those of free biotin suggests that the coenzyme may have evolved with the incorporation of the ureido moiety because the electronic configuration of this region of the molecule is sensitive to N1' carboxylation. On decarboxylation, the ureido carbonyl bond becomes more polarized (C-O-), thereby facilitating the deprotonation of N1' and increasing its nucleophilicity. As a result, carboxylation can occur readily. On carboxylation, the carbonyl bond is depolarized (C = O), allowing the carboxylated coenzyme to interact with nonpolar groups and carboxylate them. Thus, the carboxylation and decarboxylation of biotin appear to act as a mechanistic switch, turning off and on the polarization of the ureido carbonyl bond as well as modulating the nucleophilicity of N1'.

Biotin↗
PubMed1979

Hormonal regulation of acetyl-CoA carboxylase activity in the liver cell.

Chick liver cell monolayers synthesize fatty acids at in vivo rates and are responsive to insulin and glucagon. High rates of fatty acid synthesis are maintained with insulin present and lost slowly without insulin. Glucagon or 3',5'-cyclic AMP cause immediate cessation of fatty acid synthesis. The site of inhibition appears to be cytoplasmic acetyl-CoA carboxylase which catalyzes the first committed step of fatty acid synthesis. Liver carboxylase exists either as catalytically inactive protomers or active filamentous polymers. Citrate, an allosteric activator of the enzyme, is required for both catalysis and polymerization. Glucagon and cAMP cause an immediate decrease in the cytoplasmic citrate concentration of chick liver cells apparently by inhibiting the conversion of glucose to citrate at the phosphofructokinase reaction. Since fatty acid synthesis and citrate level are closely correlated, citrate appears to be a feed-forward activator of the carboxylase in vivo. Compelling evidence indicates that carboxylase filaments are present in the intact cell when citrate levels are high and depolymerize when citrate levels fall. Hence, carboxylase activity and fatty acid synthetic rate appear to be determined by cytoplasmic citrate level.

Acetyl-CoA Carboxylase↗
PubMed1979

Acute control of fatty acid synthesis by cyclic AMP in the chick liver cell: possible site of inhibition of citrate formation.

Glucagon and N,(6)O(2)-dibutyryl cyclic adenosine 3',5'-cyclic monophosphate (Bt(2)cAMP) inhibit fatty acid synthesis from acetate by more than 90% and prevent citrate formation in chick hepatocytes metabolizing glucose. With substrates that enter glycolysis at or below triose-phosphates, e.g., fructose, lactate, or pyruvate, Bt(2)cAMP has no effect on the citrate level and its inhibitory effect on fatty acid synthesis is substantially reversed. Because acetyl-CoA carboxylase requires a tricarboxylic acid activator for activity, it is proposed that regulation of fatty acid synthesis by Bt(2)cAMP is due, in part, to changes in the citrate level. Reduced citrate formation appears to result from a cAMP-induced inhibition of glycolysis. Bt(2)cAMP inhibits (14)CO(2) production from [1-(14)C]-, [6-(14)C]-, and [U-(14)C]glucose and has little effect on (14)CO(2) formation from [1-(14)C]- or [2-(14)C]pyruvate or from [1-(14)C]fructose. [(14)C]Lactate formation from glucose is depressed 50% by Bt(2)cAMP. In the presence of an inhibitor of mitochondrial pyruvate transport lactate accumulation is enhanced, but continues to be lowered 50% by Bt(2)cAMP. The activity of phosphofructokinase is greatly decreased in Bt(2)cAMP-treated cells while the activities of pyruvate kinase and acetyl-CoA carboxylase are unaffected. It appears that decreased glycolytic flux and decreased citrate formation result from depressed phosphofructokinase activity. Fatty acid synthesis from [(14)C]acetate is partially inhibited by Bt(2)cAMP in the presence of fructose, lactate, and pyruvate despite a high citrate level. Incorporation of [(14)C]fructose, [(14)C]pyruvate, or [(14)C]lactate into fatty acids is similarly depressed by Bt(2)cAMP. Synthesis of cholesterol from [(14)C]acetate or [2-(14)C]pyruvate is unaffected by Bt(2)cAMP. These results implicate a second site of inhibition of fatty acid synthesis by Bt(2)cAMP that involves the utilization, but not the production, of cytoplasmic acetyl-CoA.-Clarke, S. D., P. A. Watkins, and M. D. Lane. Acute control of fatty acid synthesis by cyclic AMP in the chick liver cell: possible site of inhibition of citrate formation.

Animals↗
PubMed1978

Effect of tunicamycin on the secretion of serum proteins by primary cultures of rat and chick hepatocytes. Studies on transferrin, very low density lipoprotein, and serum albumin.

Using rat or chick hepatocyte monolayers, we have studied the effect of tunicamycin, a specific inhibitor of protein glycosylation, on the synthesis and secretion of serum proteins. Tunicamycin inhibited glucosamine incorporation into rat liver transferrin and the apoprotein B chain of chick liver very low density lipoprotein (VLDL) by 75 to 90%. In contrasts, amino acid incorporation into these two glycoproteins, as well as into the normally unglycosylated proteins, rat serum albumin and apoprotein A of chick liver VLDL, was decreased by only 10 to 25% in the presence of the antibiotic. Despite the inhibitory effect of tunicamycin on glycosylation, secretion of all four proteins was virtually unimpaired. Thus, the carbohydrate moieties of rat liver transferrin or apoprotein B of chick liver VLDL do not appear to play an essential role in the secretion process.

Amino Acids↗
PubMed1978

Multiple phosphorylation of acetyl-CoA carboxylase in chick liver cells. A cyclic AMP-independent process.

When chick liver cells in monolayer culture were incubated with 32Pi in the presence of insulin, acetyl-CoA carboxylase became extensively labeled with 32Pi reaching a stoichiometry of 9 to 10 mol of phosphoryl group per mol of 240,000-dalton enzyme subunit. The covalently bound phosphate was found to be metabolically labile, turning over with a t1/2 of approximately 2 h (enzyme t1/2 approximately equal to 24 h). Addition of Bt2cAMP altered neither the rate nor extent of phosphorylation. Contrary to other reports, the fully phosphorylated acetyl-CoA carboxylase appears to be catalytically active.

Acetyl-CoA Carboxylase↗
PubMed1978

Acetyl-CoA carboxylase. Evidence for polymeric filament to protomer transition in the intact avian liver cell.

Digitonin treatment of chick liver cells in monolayer culture perforates the plasma membrane, causing release of acetyl-CoA carboxylase and other cytosolic enzymes. The rate of carboxylase release is affected by conditions known to alter the position of the protomer-polymer (filament) equilibrium of the enzyme. Citrate, an allosteric activator of the carboxylase, induces polymerization of the protomeric avidin-sensitive form giving rise to the avidin-insensitive polymeric filamentous form. When cells are exposed to N6,O2-dibutyryl cyclic adenosine 3':5'-monophosphate which lowers intracellular citrate levels, the rate of carboxylase release from digitonin-treated cells is greatly accelerated. The presence of avidin, which rapidly enters the cell during digitonin treatment, inactivates carboxylase under conditions that promote depolymerization and rapid release, but not under conditions which promote polymerization and slow release. These findings indicate that carboxylase filaments exist in the intact chick liver cell when the cytoplasmic citrate level is high and undergo depolymerization when citrate levels fall.

Acetyl-CoA Carboxylase↗