Glucose regulation of acetyl-CoA carboxylase in hepatoma and islet cells.

The regulation of acetyl-CoA carboxylase (ACC) by glucose and other fuel molecules has been examined in Fao Reuber hepatoma cells and Syrian hamster insulin tumor (HIT) cells in order to determine whether lipogenic substrates acutely alter ACC activity and to examine the mechanism of such regulation. In Fao cells, preincubated in simple medium without substrates, glucose addition results in a rapid activation of ACC. This effect, mimicked by other fuels such as lactate, is characterized by an increase in enzyme Vmax and a decrease in the activation constant for citrate. Several lines of evidence indicate that this activation of ACC is due to enzyme dephosphorylation, including the kinetic changes observed, the persistence of enzyme activation through ACC isolation, the necessity of inclusion of sodium fluoride/EDTA in the cell lysis buffer for preservation of the glucose-induced change, and the direct demonstration of diminished 32P-labeling of ACC after glucose exposure. Identical effects of glucose are also observed in HIT cells, although the ACC activation is smaller in magnitude and less sensitive than that observed in Fao cells. Other insulin secretagogues such as glutamine, lactate, and isobutylmethylxanthine are also found to activate HIT ACC. Others have suggested that glucose-induced changes in malonyl-CoA in beta-cells may be linked to glucose-induced insulin secretion. However, studies conducted in late passage HIT cells, which fail to secrete insulin in response to glucose stimulation, reveal the same glucose-induced activation seen in early passages, secretion-competent HIT cells, suggesting that glucose-induced ACC activation is not by itself sufficient to provoke insulin secretion. Taken together, these findings indicate that glucose and other fuel molecules can play a major role in the rapid regulation of the fatty acid synthesis pathway. The activation of fatty acid synthesis by substrate-induced ACC dephosphorylation insures ultimate fuel storage of glucose-derived carbon as fatty acid, while substrate-induced increases in the ACC product, malonyl CoA, would serve to simultaneously limit the rate of fatty acid oxidation through its allosteric regulation of carnitine palmitoyltransferase I.     

Acetyl-CoA carboxylase in Reuber hepatoma cells: variation in enzyme activity, insulin regulation, and cellular lipid content.

Reuber hepatoma cells are useful cultured lines for the study of insulin action, lipid and lipoprotein metabolism, and the regulation of acetyl-CoA carboxylase (ACC), the rate-limiting enzyme of fatty acid biosynthesis. During investigations in different clonal lines of these cells, we have uncovered marked intercellular variability in the activity, enzyme content, and insulin regulation of ACC paralleled by differences in cellular neutral lipid (triglyceride) content. Two contrasting clonal lines, Fao and H356A-1, have been studied in detail. Several features distinguish these two lines, including differences in ACC activity and enzyme kinetics, the content of the two major hepatic ACC isozymes (Mr 280,000 and 265,000 Da) and their heteroisozymic complex, the extent of ACC phosphorylation, and the ability of ACC to be activated on stimulation by insulin and insulinomimetic agonists. As studied by Nile Red staining and fluorescence-activated cell sorting, these two lines also display marked differences in neutral lipid content, which correlates with both basal levels of ACC activity and inhibition of ACC by the fatty acid analog, 5-(tetradecyloxy)-2-furoic acid (TOFA). These results emphasize the importance of characterization of any particular clonal line of Reuber cells for studies of enzyme regulation, substrate metabolism, and hormone action. With respect to ACC, studies in contrasting clonal lines of Reuber cells could provide valuable clues to understanding both the complex mechanisms of intracellular ACC regulation in the absence and presence of hormones and its regulatory role(s) in overall hepatic lipid metabolism.     

Both insulin and epidermal growth factor stimulate lipogenesis and acetyl-CoA carboxylase activity in isolated adipocytes. Importance of homogenization procedure in avoiding artefacts in acetyl-CoA carboxylase assay.

Epidermal growth factor (EGF) stimulates lipogenesis by 3-4-fold in isolated adipocytes, with a half-maximal effect at 10 nM-EGF. In the same batches of cells insulin stimulated lipogenesis by 15-fold. Freezing and prolonged homogenization of adipocytes results in release of large quantities of pyruvate carboxylase from broken mitochondria, and sufficient pyruvate can be carried through into assays for this enzyme to cause significant interference with assays of acetyl-CoA carboxylase in crude adipocyte extracts. This may account for the high amount of citrate-independent acetyl-CoA carboxylase activity reported to be present in adipocyte extracts in some previous publications. This problem may be eliminated by homogenizing very briefly without freezing. By using the modified homogenization procedure, EGF treatment of adipocytes was shown to produce an effect on acetyl-CoA carboxylase activity almost identical with that of insulin. Both messengers increase Vmax. without significant effect on the Ka for the allosteric activator, citrate.     

Adaptive synthesis of fatty acid synthetase and acetyl-CoA carboxylase by isolated rat liver cells.

Hepatocytes were isolated at specified times from livers of diabetic and insulin-treated diabetic rats during the course of a 48-h refeeding of a fat-free diet to previously fasted rats. The rates of synthesis of fatty acid synthetase and acetyl-CoA carboxylase in the isolated cells were determined as a function of time of refeeding by a 2-h incubation with l-[U-¹⁴C]leucine. Immunochemical methods were employed to determine the amount of radioactivity in the fatty acid synthetase and acetyl-CoA carboxylase proteins. The amount of radioactivity in the fatty acid synthetase synthesized by the isolated cells was also determined following enzyme purification of the enzyme to homogeneity. Enzyme activities of the fatty acid synthetase and acetyl-CoA carboxylase in the cells were measured by standard procedures. The results show that isolated liver cells obtained from insulintreated diabetic rats retain the capacity to synthesize fatty acid synthetase and acetyl-CoA carboxylase. The rate of synthesis of the fatty acid synthetase in the isolated cells was similar to the rate found in normal refed animals in in vivo experiments [Craig et al. (1972) Arch. Biochem. Biophys. 152, 619–630; Lakshmanan et al. (1972) Proc. Nat. Acad. Sci. USA69, 3516–3519]. In addition the relative rate of synthesis of fatty acid synthetase was stimulated greater than 20-fold in the diabetic animals treated with insulin. Immunochemical assays, when compared with enzyme activities, indicated the presence of an immunologically reactive, but enzymatically inactive, form or “apoenzyme” for both the fatty acid synthetase and acetyl-CoA carboxylase. The synthesis of these immunoreactive and enzymatically inactive species of protein, as well as the synthesis of the “holoenzyme” forms of both enzymes, requires insulin.     

Subcellular location of a soluble factor that stimulates DNA replication in permeable animal cells

The enzymatic isolation of cells with bacterial collagenase has proved to be a powerful technique for the study of a wide variety of tissues. Unfortunately, for some applications such as the isolation of cells from membranous bone, the cellular damage that results from the exposure of the cells to cytotoxic contaminants of bacterial collagenase has limited the usefulness of this approach. The use of chromatographically purified collagenase alone is often ineffective or very slow to release cells from tissue. We have found that two enzymes are necessary and sufficient to isolate cells from neonatal mouse calvaria: purified collagenase and neutral protease. These two enzymes can be chromatographically purified on a preparative scale to yield 100 mg amounts of each enzyme. The purified enzymes can be recombined in amounts which will digest calvaria at the same rate as the crude bacterial collagenase from which they were derived. The cells that are isolated using the purified enzymes are undamaged, as indicated by the measurement of their equilibrium density on gradients of Ficoll and sodium metrizoate. Cells isolated with crude collagenase never reach an equilibrium density upon isopyknic centrifugation, whereas cells isolated with the purified enzymes reach an equilibrium density of 1.074 g/ml in 90 min.     

Recognition site of nuclear factor I, a sequence-specific DNA-binding protein from HeLa cells that stimulates adenovirus DNA replication.

Nuclear factor I is a 47-kd protein, isolated from nuclei of HeLa cells, that binds specifically to the inverted terminal repeat of the adenovirus (Ad) DNA and enhances Ad DNA replication in vitro. We have studied the DNA sequence specificity of nuclear factor I binding using cloned terminal fragments of the Ad2 genome and a set of deletion mutants. Binding of nuclear factor I protects nucleotides 19-42 of Ad2 DNA against DNase I digestion. Filter binding assays show that deletion of the first 23 nucleotides does not impair binding while a deletion of 24 nucleotides reduces binding severely. However, binding studies on Ad12 DNA indicate that nucleotide 24 can be mutated. Fragments containing the first 40 bp are bound normally while the first 38 bp are insufficient to sustain binding. Taken together, these results indicate that the minimal recognition site of nuclear factor I contains 15 or 16 nucleotides, located from nucleotide 25 to nucleotide 39 or 40 of the Ad2 DNA. This site contains two of the four conserved nucleotide sequences in this region. Sequences flanking the minimal recognition site may reduce the binding affinity of nuclear factor I. In accordance with these binding studies, DNA replication of a fragment that carries the sequence of the terminal 40 nucleotides of Ad2 at one molecular end is enhanced by nuclear factor I in an in vitro replication system.     

Hormonal regulation of carbamoyl-phosphate synthetase I synthesis in primary cultured hepatocytes and Reuber hepatoma H-35. Defective regulation in hepatoma cells

Regulation of carbamoyl-phosphate synthetase I (CPS) synthesis by various hormones was compared in primary cultured hepatocytes from adult rat and in Reuber hepatoma H-35 by pulse labeling of the cells with [35S]methionine. CPS synthesis in hepatocytes was stimulated 8-fold and 5-fold by dexamethasone and glucagon respectively. CPS synthesis in hepatocytes was synergically (about 50-fold) stimulated by a combination of dexamethasone and glucagon. Less synergic stimulation was observed by combining dexamethasone with N6, O2'-dibutyryladenosine 3',5'-monophosphate (dibutyryl-cAMP) or with isoproterenol. The basal level of CPS synthesis in hepatoma cells was higher than that in hepatocytes. CPS synthesis in hepatoma cells was stimulated by dexamethasone and dibutyryl-cAMP but the extent was only 3-fold and 1.8-fold respectively. The synergic effect of combination of dexamethasone and dibutyryl-cAMP was not observed in hepatoma cells. Neither glucagon nor isoproterenol exhibited an appreciable effect on CPS synthesis in hepatoma cells. Insulin and epinephrine suppressed CPS synthesis both in hepatocytes and hepatoma cells. The effect of epinephrine was indicated to be through alpha-adrenergic receptors. The effects of insulin and epinephrine were additive on CPS synthesis both in hepatocytes and hepatoma cells.     

Characterization of Staphylococcus aureus cell wall glycan strands, evidence for a new beta-N-acetylglucosaminidase activity

Using sequential digestion with the glycyl-glycine endopeptidase lysostaphin followed by the pneumococcal N-acetylmuramyl-L-alanine amidase (amidase), the glycan strands of the peptidoglycan of Staphylococcus aureus were purified and analyzed by a combination of reverse-phase-high pressure liquid chromatography (HPLC) and mass spectrometry. Reverse-phase-HPLC resolved the glycan strands to a family of major peaks, which represented oligosaccharides composed of repeating disaccharide units (N-acetylglucosamine-[beta-1, 4]-N-acetylmuramic acid) with different degrees of polymerization and terminating with N-acetylmuramic acid residues at the reducing ends. The method allowed separation of strands up to 23-26 disaccharide units with a predominant length between 3 and 10 and an average degree of polymerization of approximately 6. Glycan strands with a higher degree of polymerization (>26 disaccharide units) represented 10-15% of the total UV absorbing glycan material. A unique feature of the staphylococcal glycan strands was the presence of minor satellite peaks that were present throughout the HPLC elution profile eluting either just prior or shortly after the major oligosaccharide peaks. A number of observations including mass spectrometric analysis suggest that the satellites are the products of an N-acetylglucosaminidase activity that differs from the atl gene product and that appears to be involved with modification of the glycan strand structure.     

Nicotine inhibits collagen synthesis and alkaline phosphatase activity, but stimulates DNA synthesis in osteoblast-like cells.

Use of smokeless tobacco is associated with various oral lesions including periodontal damage and alveolar bone loss. This study was performed to test the effects of nicotine on bone-forming cells at concentrations that occur in the saliva of smokeless tobacco users. Confluent cultures of osteoblast-like cells isolated from chick embryo calvariae were incubated for 2 days with nicotine added to the culture medium (25-600 micrograms/ml). Nicotine inhibited alkaline phosphatase in the cell layer and released to the medium, whereas glycolysis (as indexed by lactate production) was unaffected or slightly elevated. The effects on medium and cell layer alkaline phosphatase were concentration dependent with maximal inhibition occurring at 600 micrograms nicotine/ml. Nicotine essentially did not affect the noncollagenous protein content of the cell layer, but did inhibit collagen synthesis (hydroxylation of [3H]proline and collagenase-digestible protein) at 100, 300, and 600 micrograms/ml. Release of [3H]hydroxyproline to the medium was also decreased in a dose-dependent manner, as was the collagenase-digestible protein for both the medium and cell layer. In contrast, DNA synthesis (incorporation of [3H]thymidine) was more than doubled by the alkaloid, whereas total DNA content was slightly inhibited at 600 micrograms/ml, suggesting stimulated cell turnover. Morphologic changes occurred in nicotine-treated cells including rounding up, detachment, and the occurrence of numerous large vacuoles. These results suggest that steps to reduce the salivary concentration of nicotine in smokeless tobacco users might diminish damaging effects of this product on alveolar bone.     

Detection and characterization of a novel factor that stimulates DNA polymerase alpha

A novel factor that stimulates DNA polymerase alpha activity on poly(dA) X oligo(dT) has been identified and partially purified from mouse FM3A cells. The assay system for the factor contained poly(ethylene glycol) 6000. The activities of DNA polymerase alpha on poly(dA) X oligo(dT) in the presence and absence of the stimulating factor were increased greatly by the addition of poly(ethylene glycol). Stimulation by the factor was observed at all the primer to template ratios tested from 0.01 to 0.3. The highest activity was observed at the ratio of 0.05, corresponding to about 3.3 primers on one template in the presence of the factor. The concentration of DNA polymerase alpha used in the assay affected the stimulation by the factor, and the stimulation became more prominent at concentrations of the enzyme lower than 0.04 unit per assay. The stimulating factor lowered the Km value of DNA polymerase alpha for the template-primer, though they had no effect on the Km value for dTTP substrate. The results of product analysis suggested that the stimulation by the factor is mainly due to the increase in the initiation frequency of DNA synthesis from the primers. The stimulating factor specifically stimulated DNA polymerase alpha but not DNA polymerases beta and gamma. Furthermore, the factor formed a complex with DNA polymerase alpha under a certain condition.     

Microinjection of deoxynucleotides into mouse cells. No evidence that precursors for DNA synthesis are channeled

The technique of microinjection was applied for the introduction of radioactive, phosphorylated precursors of DNA synthesis into living mouse cells in culture. Autoradiographs proved that DNA was well labeled by injected dTTP, dCTP, dATP, dGTP, and (to a minor extent) dTMP, but the efficiency was much lower when CDP, ADP, or GDP was used. For practical reasons, injections into nuclei were preferred, but injections into cytoplasm showed no principal difference with the autoradiographed nuclei. The kinetics of the uptake of the injected material agreed neatly with the calculation (from pool sizes and polymerization rate) that the intracellular deoxytriphosphates are sufficient for about 10 min of DNA synthesis. All this evidence strongly argues against the concept that precursors of DNA synthesis are channeled in vivo within a multienzyme complex and suggests a free diffusion of deoxynucleotides within the cell. Injected thymidine was less able to enter the deoxy nucleotide metabolism compared with thymidine from the culture medium. A mutant cell line deficient in thymidine kinase did not accumulate intracellular thymidine. These data indicate that thymidine kinase is a membrane associated enzyme and that uptake and phosphorylation of thymidine are coupled reactions.     

Purification from a human hepatoma cell line of a basic fibroblast growth factor-like molecule that stimulates capillary endothelial cell plasminogen activator production, DNA synthesis, and migration.

A 17,500-dalton protein which stimulates plasminogen activator production in cultured bovine capillary endothelial cells has been purified from a SK-Hep-1 human hepatoma cell lysate by using heparin affinity chromatography and fast protein-liquid ion exchange chromatography. The purified molecule stimulated plasminogen activator production in a dose-dependent manner between 0.01 and 1 ng/ml. It also stimulated collagenase synthesis, DNA synthesis, and motility in capillary endothelial cells in the same concentration range. This molecule was identified as a basic fibroblast growth factor-like molecule on the basis of its biological activity, its affinity for heparin-Sepharose, and its cross-reactivity with a polyclonal antibody raised against the human placental basic fibroblast growth factor.     

The biotinyl domain of Escherichia coli acetyl-CoA carboxylase. Evidence that the "thumb" structure id essential and that the domain functions as a dimer

Biotin carboxyl carrier protein (BCCP) is the small biotinylated subunit of Escherichia coli acetyl-CoA carboxylase (ACC), the enzyme that catalyzes the first committed step of fatty acid synthesis. Similar proteins are found in other bacteria and in chloroplasts. E. coli BCCP is a member of a large family of protein domains modified by covalent attachment of biotin to a specific lysine residue. However, the BCCP biotinyl domain differs from many of these proteins in that an eight-amino acid residue insertion is present upstream of the biotinylated lysine. X-ray crystallographic and multidimensional NMR studies show that these residues constitute a structure that has the appearance of an extended thumb that protrudes from the otherwise highly symmetrical domain structure. I report that expression of two mutant BCCPs lacking the thumb residues fails to restore growth and fatty acid synthesis to a temperature-sensitive E. coli strain that lacks BCCP when grown at nonpermissive temperature. Alignment of BCCPs from various organisms shows that only two of the eight thumb residues are strictly conserved, and amino acid substitution of either residue results in proteins giving only weak growth of the temperature-sensitive E. coli strain. Therefore, the thumb structure is essential for the function of BCCP in the ACC reaction and provides a useful motif for distinguishing the biotinylated proteins of multisubunit ACCs from those of enzymes catalyzing other biotin-dependent reactions. An unexpected result was that expression of a mutant BCCP in which the biotinylated lysine residue was substituted with cysteine was able to partially restore growth and fatty acid synthesis to the temperature-sensitive E. coli strain. This complementation was shown to be specific to BCCPs having native structure (excepting the biotinylated lysine) and is interpreted in terms of dimerization of the BCCP biotinyl domain during the ACC reaction.     

Single-stranded-DNA-dependent ATPase from HeLa cells that stimulates DNA polymerase alpha-primase activity: purification and characterization of the ATPase

A single-stranded DNA-dependent ATPase that cofractionates during the early stages of purification of a multiprotein DNA polymerase alpha complex from HeLa cells has been purified to homogeneity. The ATPase is part of a 16S multienzyme DNA polymerase alpha complex that is fully active in SV40 DNA replication in vitro. The ATPase hydrolyzes ATP to ADP in a reaction that is completely dependent on the presence of DNA. DNA in single-stranded form is strongly preferred as a cofactor, and polydeoxynucleotides with adenine or thymidine residues are highly effective. Glycerol gradient sedimentation showed that the purified ATPase sedimented at an s20,w of 7 S, and polyacrylamide gel electrophoresis under denaturing conditions reveals two polypeptides with relative molecular weights of 83,000 and 68,000. Both of these polypeptides have purine nucleotide binding sites as revealed by photoaffinity cross-linking experiments. ATP binds to the two subunits more efficiently than GTP, and CTP or UTP does not cross-link with the two polypeptides. DNA synthesis catalyzed by purified HeLa cell DNA polymerase alpha-primase is stimulated in the presence of ATPase and ATP at an optimum concentration of 2 mM. Analysis of the DNA product by gel electrophoresis indicates that with poly(dT) but not phage M13 DNA as template the ATPase overcomes a lag and decreases the length of nascent DNA chains synthesized by the DNA polymerase alpha-primase complex.     

A role for ornithine in the regulation of putrescine accumulation and ornithine decarboxylase activity in Reuber H35 hepatoma cells

We investigated the ability of intracellular ornithine to alter both the biosynthesis of putrescine and the activity of ornithine decarboxylase in Reuber H35 hepatoma cells in culture incubated with 12-O-tetradecanoylphorbol 13-acetate (TPA). In confluent cultures of H35 cells, the addition of TPA (1.6 μM) caused the activity of ornithine decarboxylase to increase by more than 100-fold within 4 h. When exogenous ornithine (0.1–1.0 mM) was added to the culture medium with TPA, a marked dose-dependent increase in the production of putrescine was observed. The activity of ornithine decarboxylase in the same cultures incubated with ornithine decreased in a similar dose-dependent manner. The addition of arginine (0.1–1.0 mM) (but not lysine or histidine) to the H35 cells in culture concomitant with TPA also led to a relative increase in putrescine biosynthesis and a decrease in ornithine decarboxylase activity compared to cultures not receiving the amino acids. A similar response to exogenous ornithine and TPA was observed in a series of less confluent rapidly growing cultures which were in culture for a shorter period of time. The confluent cultures possessed a basal level of arginase (55 units/mg protein) which increased approx. 2-fold upon treatment with TPA. The intracellular concentration of ornithine in the unstimulated cells was in the order of 0.02–0.03 mM. Upon incubation of the cells with exogenous ornithine or arginine, the intracellular pools of these amino acids increased 4- to 8-fold.     

Long-term regulation by theophylline of fatty acid synthetase, acetyl-CoA carboxylase and lipid synthesis in cultured glial cells.

The long-term regulation of fatty acid synthetase and acetyl-CoA carboxylase and of fatty acid and sterol synthesis was studied in C-6 glial cells in culture. When theophylline (10(-3) M) was added to the culture medium of these cells, rates of lipid synthesis from acetate and activities of synthetase and carboxylase became distinctly lower than in cells that were untreated. This effect appeared after approximately 12 h, and after 48 h enzymatic activities were reduced approx. 2-fold and rates of lipid synthesis from acetate 3- to 4-fold. The likelihood that the decrease in fatty acid synthesis from acetate was caused by the decrease in activities of fatty acid synthetase and acetyl-CoA carboxylase was established by several observations. These indicated that the locus of the effect probably did not reside at the level of acetate uptake into the cell, alterations in acetate pool sizes or conversion of acetate to acetyl-CoA. Moreover, de novo fatty acid synthesis was found to be the predominant pathway in these glial cells, whether treated with theophylline or not. The mechanism of the effect of theophylline on fatty acid synthetase was shown by immunochemical techniques to involve an alteration in content of enzyme rather than in catalytic efficiency. The change in content of fatty acid synthetase was shown by isotopic-immunochemical experiments to involve a decrease in synthesis of the enzyme. The mechanism whereby theophylline leads to a decrease in lipogenesis and in the synthesis of fatty acid synthetase may not be mediated entirely by inhibition of phosphodiesterase and an increase in cyclic AMP levels, because dibutyryl cyclic AMP (10(-3) M) only partially reproduced the effect.