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A Ichihara

Publications and source records attributed to A Ichihara.

At least 199 records · Page 11Linked to original sources
PubMed1984

Identification of trypsin inhibitor in bovine pituitary extracts as a survival factor for adult rat hepatocytes in primary culture.

Adult rat hepatocytes in primary culture cannot survive more than 2 days in the absence of calf serum. An extract of bovine pituitary gland had a similar effect to calf serum on the survival of hepatocytes, but its specific activity was 70 times that of calf serum. The survival factor was purified from bovine pituitary gland, and obtained in a homogeneous state judging by sodium dodecylsulfate-polyacrylamide gel electrophoresis and high performance liquid chromatography. Its purification was achieved by acid extraction of the pituitary gland, gel filtrations on Sephadex G-75 and Ultrogel AcA 202, ion-exchanger chromatography on CM-cellulose, and then reverse phase high performance liquid chromatography. The purified factor was effective at 10 ng/ml and maximally effective at 100 ng/ml. The overall recovery of its activity was 30%, and the specific activity of the purified factor was 3,100 times that in the acid extract. The molecular weight (Mr 8,000), estimated by Sephadex G-200 filtration, and the strong basic character (pI 10.5) of the purified survival factor suggested that it may be the trypsin inhibitor found in various tissues. Indeed, the amino acid composition of the pure material was identical with that of bovine pancreatic trypsin inhibitor (bPTI). The purified survival factor had the same inhibitory activity on trypsin as commercial bPTI and, conversely, commercial bPTI greatly enhanced survival of rat hepatocytes. Thus, the survival factor in bovine pituitary gland was identified as bPTI.

Amino Acids↗
PubMed1984

Glucagon and glucose as major regulators of glycogen metabolism in primary cultured rat hepatocytes.

The short-term controls of glycogen synthase [EC 2.4.1.11] and glycogen phosphorylase [EC 2.4.1.1] by major regulators, such as insulin, glucose, catecholamine, and glucagon, were compared in a simple, yet organized experimental system, i.e., adult rat hepatocytes in primary culture. Glycogen synthase was activated by glucose markedly and dose-dependently (5-40 mM), but insulin alone (1 X 10(-8) M) activated this enzyme only two-fold. Therefore, activation of the enzyme by the two regulators together was mostly due to activation by glucose. Glucagon at a concentration of 5 X 10(-10) M suppressed this activation almost completely. Glucagon at this concentration activated phosphorylase considerably and this activation was slightly inhibited by insulin. Phenylephrine also activated phosphorylase, and this activation was inhibited by phenoxybenzamine or prazosin, suggesting that activation by catecholamine is through the alpha 1-adrenergic receptor. Similarly a high concentration of glucose diminished the effects of glucagon and phenylephrine. These results suggest that in rat liver, glycogen metabolism is controlled mainly by glucagon, catecholamine, and glucose; the former two activate phosphorylase and inactivate synthase, while glucose activates synthase strongly and inactivates phosphorylase partially. Insulin plays a minor role in both reactions. Thus, the liver is primarily an organ for glucose production, which is regulated by hormones, not for glycogen storage, which is increased only by a high glucose concentration in the portal blood.

Animals↗
PubMed1984

Reciprocal expressions of alpha 1- and beta-adrenergic receptors, but constant expression of glucagon receptor by rat hepatocytes during development and primary culture.

The stimulations of cyclic AMP formation and adenylate cyclase activity by glucagon and isoproterenol were both found to be highest in neonatal rat hepatocytes and to decrease during development. Adult hepatocytes still showed a considerable response to glucagon, but a negligible response to isoproterenol. The decrease in cyclic AMP formation during development can be explained in the case of the response to beta-adrenergic agonist as due to decrease of its receptor number, judging from binding of [125I]iodocyanopindolol to purified plasma membranes. But in the case of the glucagon response, the decrease in the response may be due to change of post-receptor components of the adenylate cyclase system, because the receptor number tended to increase during development, as shown by binding of [125I]iodoglucagon. Similarly, alpha 1-adrenergic receptors increased in number during development, but their IC50 value did not change, as measured by binding of [3H]prazosin to plasma membranes. Previous studies on primary cultures of adult rat hepatocytes showed that the beta-adrenergic response and its receptor number increased markedly during short-term culture (Nakamura, T., Tomomura, A., Noda, C., Shimoji, M., & Ichihara, A. (1983) J. Biol. Chem. 258, 9283-9289). However, in this work the amount of alpha 1-adrenergic receptor of adult rat hepatocytes was found to decrease by one third during 1-2 days culture. Therefore, changes of alpha 1- and beta-adrenergic receptors during development of rat liver and during primary culture of adult rat hepatocytes were reciprocal, although the directions of change in the two conditions were opposite. The additions of various hormones to primary cultures of adult rat hepatocytes did not affect the reciprocal changes of adrenergic receptors, suggesting that these hormones did not regulate the changes of the receptors.

Aging↗
PubMed1983

Reciprocal effects of epidermal growth factor on key lipogenic enzymes in primary cultures of adult rat hepatocytes. Induction of glucose-6-phosphate dehydrogenase and suppression of malic enzyme and lipogenesis.

In primary cultured hepatocytes of adult rats epidermal growth factor (EGF) caused 2- to 3-fold induction of glucose-6-phosphate dehydrogenase (EC 1.1.1.49, G6P dehydrogenase) within 2 days. The effect of EGF was additive with a similar effect of insulin. The half-maximum dose of EGF for the induction was 1 ng/ml. Induction of this enzyme by these hormones was shown by immunotitration to be due to increase of the amount of enzyme. Furthermore, this increase in the amount of enzyme was found to result from increase of syntheses of mRNA and enzyme protein. In contrast, the induction of malic enzyme (EC 1.1.1.40, L-malate:NADP+) oxidoreductase) by insulin plus triiodothyronine was strongly suppressed by the concomitant addition of EGF. Induction of G6P dehydrogenase by EGF, like that by insulin, was not suppressed by either glucagon or dibutyryl cAMP, whereas that of malic enzyme was suppressed additively by EGF and dibutyryl cAMP. EGF also suppressed stimulation of lipogenesis by insulin, measured as incorporation of [1-14C]acetate into triglycerides and phospholipids. Another difference between the inductions of G6P dehydrogenase and malic enzyme was in their dependence on cell density; G6P dehydrogenase induction by insulin and EGF was high at low cell density (3 X 10(4) cells/cm2) and less at higher cell density (13 X 10(4) cells/cm2), whereas induction of malic enzyme was high at higher cell density and less at lower cell density. These results are consistent with the dual role of G6P dehydrogenase in lipogenesis in resting cells and in synthesis of nucleic acid in growing cells. Malic enzyme plays a role only for lipogenesis in mature hepatocytes.

Animals↗
PubMed1983

Hormonal regulation of translatable mRNA of glucose-6-phosphate dehydrogenase in primary cultures of adult rat hepatocytes.

The quantity of translatable mRNA of glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ 1-oxidoreductase, EC 1.1.1.49) in primary cultures of adult rat hepatocytes subjected to different hormonal conditions was determined with a reticulocyte-lysate, cell-free system. The level of glucose-6-phosphate dehydrogenase mRNA was about 5-fold higher in the presence of insulin than in its absence. This increase of glucose-6-phosphate dehydrogenase mRNA reached a maximum 12 h after the addition of insulin. The maximum level of induction of glucose-6-phosphate dehydrogenase mRNA required 10(-8) M insulin. Glucagon and triiodothyronine had no effect on the glucose-6-phosphate dehydrogenase mRNA level. The increase of glucose-6-phosphate dehydrogenase activity correlated with the increase in level of mRNA of this enzyme. This suggests that the changes in glucose-6-phosphate dehydrogenase activity in response to the above hormonal changes are primarily due to changes in the amount of mRNA coding for this enzyme.

Animals↗
PubMed1983

Acquisition of a beta-adrenergic response by adult rat hepatocytes during primary culture.

In freshly isolated parenchymal hepatocytes of adult rats, the beta-adrenergic agonist isoproterenol (Ip) did not stimulate cAMP formation, protein kinase activity, or glycogenolysis, although glucagon markedly stimulated all these activities. However, the beta-adrenergic response appeared when rat hepatocytes were cultured as monolayers. This response had already appeared after 2-h culture and increased during further culture. The appearance of the beta-adrenergic response during culture was blocked by cycloheximide, actinomycin D, or alpha-amanitin. Thus adult rat hepatocytes acquired marked ability to respond to Ip during culture through the syntheses of mRNA and protein. Freshly isolated hepatocytes from postnatal rats showed a high beta-adrenergic response that did not increase further during culture. This response gradually decreased during development and had almost disappeared about 60 days after birth. In plasma membranes prepared from freshly isolated cells of adult rats the basal and NaF-stimulated activities of adenylate cyclase (EC 4.6.1.1) were similar to those of cultured cells and the enzyme activity was also stimulated by guanyl-5'-yl imidodiphosphate. However, in plasma membranes of freshly isolated cells Ip scarcely stimulated adenylate cyclase, but glucagon did. The intact cells, whether they were freshly isolated or cultured, accumulated cAMP when exposed to cholera toxin. Moreover, the two subunits of GTP-binding regulatory protein (also named G/F or Ns site) were detected by [32P]ADP ribosylation with cholera toxin and [32P]NAD+ in freshly isolated cells as well as in cultured cells. These results indicate that freshly isolated and cultured hepatocytes of adult rats contain sufficient levels of all the components of the postreceptor-adenylate cyclase system for activity. However, the number of beta-adrenergic receptors measured by binding of [125I]iodocyanopindolol, a potent beta-adrenergic antagonist, was very low in purified plasma membranes of freshly isolated cells (20 fmol/mg of protein), and the number increased about 6-fold without change in the dissociation constant (Kd = 132 pM) when the cells were cultured for 7 h. This increase in beta-adrenergic receptor sites was completely abolished by cycloheximide and alpha-amanitin. Thus it is concluded that the unresponsiveness of adult rat hepatocytes to Ip was due to a very low amount of beta-adrenergic receptor and that the appearance of a beta-adrenergic response during primary culture was due to new synthesis of beta-adrenergic receptor through synthesis of mRNA.

Adenylyl Cyclases↗
PubMed1983

Specific inhibitors of eukaryotic DNA synthesis and DNA polymerase alpha, 3-deoxyaphidicolin and aphidicolin-17-monoacetate.

Of several phytotoxins isolated from culture filtrates of Phoma betae Frank PS-13, an incitant of leaf spot disease of sugar beet, three have been identified as aphidicolin, 3-deoxyaphidicolin and aphidicolin-17-monoacetate. Aphidicolin is a selective inhibitor of eukaryotic DNA polymerase alpha (Ikegami et al. (1978) Nature 275, 458-460). Consequently, we studied the action mechanism of 3-deoxyaphidicolin and aphidicolin-17-monoacetate. These aphidicolin analogues markedly inhibited the in vivo DNA synthesis of sea urchin embryos and HeLa cells but not RNA and protein syntheses. Only DNA polymerase alpha, not DNA polymerase beta and gamma, was inhibited by these drugs. The mode of action of these analogues on DNA polymerase alpha from the sea urchin was competitive inhibition with respect to dCTP with Ki values of 0.44 micrograms/ml for deoxyaphidicolin and 0.89 micrograms/ml for aphidicolin monoacetate, respectively. None of the other three dNTPs competed with these drugs. A similar inhibitory mode was observed using the enzyme from HeLa cells and toad oocytes. These drugs at a concentration of 2 micrograms/ml caused a delay in the cleavage of fertilized eggs of the sea urchin and decomposition before blastulation, indicating the possibility of achromosomal cleavage because of the absence of DNA synthesis. Based on the above, it is concluded that these analogues can be used as other inhibitors of eukaryotic DNA synthesis and DNA polymerase alpha.

Animals↗
PubMed1983

alpha-Adrenergic regulation of enzymes of amino acid metabolism in primary cultures of adult rat hepatocytes.

In primary cultures of rat hepatocytes, addition of dexamethasone (10 microM) plus glucagon (0.5 microM) caused several-fold increases in the activities of serine dehydratase (EC 4.2.1.13), tryptophan oxygenase (EC 1.13.11.11), and tyrosine aminotransferase (EC 2.6.1.5) in 24 h. These inductions were inhibited by insulin. Addition of epinephrine or phenylephrine at 10 microM blocked these inductions. This suppressive effect of adrenergic compounds was completely abolished by the alpha-adrenergic antagonist phenoxybenzamine at 10 microM. Immunochemical analysis with antiserum to serine dehydratase showed that the changes in enzyme activity were due to changes in the amount of enzyme. Epinephrine was effective even when glucagon was replaced by dibutyryl cAMP (50 microM), indicating that alpha-adrenergic suppression of enzyme inductions was mediated by a cAMP-independent mechanism. Furthermore, the findings that prazosin antagonized this epinephrine effect, but yohimbine did not, indicate that the alpha 1- but not the alpha 2-receptor is involved in this inhibition. However, the alpha-adrenergic effect was different from that of insulin in that, unlike the latter, the inductions of tryptophan oxygenase and tyrosine amino-transferase by dexamethasone alone were not inhibited. The alpha-adrenergic action apparently counteracts the action of glucagon and cAMP. For determination of the beta-adrenergic effect of catecholamines on the inductions of enzymes, beta-adrenergic compounds were tested without glucagon. Isoproterenol or epinephrine plus phenoxybenzamine induced tryptophan oxygenase and tyrosine aminotransferase. Induction of serine dehydratase was shown by isoproterenol only in the presence of 1-methyl-3-isobutylxanthine, an inhibitor of phosphodiesterase. These results indicate that catecholamines play dual roles in regulation of the amount of enzyme through their alpha 1- and beta-adrenergic actions.

Amino Acids↗
PubMed1983

Effects of prostaglandins and divalent cations on cAMP production in isolated rat hepatocytes.

In intact hepatocytes, prostaglandin E2 (PGE2) inhibits up to 60% of the cyclic AMP (cAMP) formation in response to glucagon. PGE2 was found also to inhibit cAMP production in response to Mn2+, and Mn2+ counteracted the effect of PGE2 on the response to glucagon. However, when added to cells with ruptured plasma membranes, PGE2 had no effect on cAMP production, even though the ruptured cells had an equally strong response to glucagon as did intact cells. Only when cells were ruptured in the presence of PGE2 and incubated as very dense suspensions did PGE2 inhibit a response to glucagon up to 20%. These results may be explained by assuming the existence of a cytosolic factor necessary for transmitting the inhibitory effect of PGE2. The role of divalent cations in cAMP formation in intact cells was investigated. After extraction of cations with ethylenediamine-tetraacetic acid (EDTA), cAMP production in response to glucagon was reduced by more than 60%. Addition of Mn2+ restored cAMP formation completely, whereas Mg2+ was somewhat less effective, and Ca2+ could not restore any activity. Even low concentrations of Ca2+ appeared under certain conditions to repress adenylate cyclase activity.

Animals↗
PubMed1983

Reciprocal modulation of growth and differentiated functions of mature rat hepatocytes in primary culture by cell--cell contact and cell membranes.

In primary monolayer cultures of rat mature hepatocytes, many metabolic functions as well as cell growth are regulated by cell density. There are two types of regulatory response of these functions to change of cell density. Growth-related functions, such as DNA synthesis, induction of glucose-6-phosphate dehydrogenase, 2-aminoisobutyric acid transport, synthesis of cellular protein, and cholesterogenesis, are stimulated by low cell density. In contrast, functions related to hepatocyte-specific characters, such as the inductions of tyrosine aminotransferase, serine dehydratase, and malic enzyme and synthesis of triglycerides, are stimulated by high cell density. The reciprocal responses of these cellular activities to cell density were mimicked by addition of plasma membranes purified from adult rat liver to hepatocytes cultured at low cell density. The modulator activity was heat labile and trypsin sensitive. The activity was also found in plasma membranes from kidney, brain, and erythrocytes, although the specific activities of these preparations seemed to be different. These results suggest that the reciprocal regulations of cell growth and hepatocyte-specific functions are mediated by some surface components via cell-cell contact.

Acetates↗
PubMed1983

Density-dependent growth control of adult rat hepatocytes in primary culture.

Adult rat hepatocytes in primary culture, which show various liver functions, did not show any mitosis at confluent cell density, although they entered the S phase and remained in the G2 phase, judging by cytofluorometry, when insulin and epidermal growth factor (EGF) were added to 2-day cultures (Tomita, Y., Nakamura, T., & Ichihara, A. (1981) Exp. Cell Res. 135, 363-371). However, when the cell density was decreased by half or one third, the number of nuclei and cell number increased to 1.5-2.0 times that after culture for 35 h with insulin and EGF. Moreover, at these lower densities, DNA synthesis started much earlier, although at the usual high density DNA synthesis with these two hormones did not start until the hepatocytes had been cultured for over 40 h. These results suggest that proliferation of mature rat hepatocytes is regulated by the cell density. First, cells in G0 enter the G1 phase density-dependently; then cells in the G1 phase seem to be stimulated to enter the S phase by insulin and EGF, and a low cell density may permit cells after DNA synthesis to enter the M phase. DNA synthesis of rat hepatocyte cultures at low cell density was strongly inhibited by co-culture with a dense culture. Therefore, the density-dependent mechanism of hepatocyte proliferation seems to involve regulation by a soluble inhibitor(s) secreted by the hepatocytes into the culture medium.

Animals↗
PubMed1983

Different effect of amino acid deprivation on syntheses of intra- and extracellular proteins in rat hepatocytes in primary culture.

Primary cultured rat hepatocytes synthesize various proteins and secrete half of them into the medium. When the cells were cultured in medium deficient in amino acids for 30 h, they maintained their normal level of intracellular protein synthesis, but their synthesis of extracellular proteins decreased by half. This reduced production of extracellular proteins could be restored by addition of amino acid mixture to the medium. Since secretion of protein was not inhibited and no albumin accumulated in the cells on amino acid deprivation, the reduced secretion of extracellular protein was due to inhibition of its synthesis rather than inhibition of its secretion. Addition of the protease inhibitor leupeptin to cells cultured in medium supplemented with amino acids inhibited protein degradation 60%, but it did not change the rate of protein synthesis. Addition of this inhibitor to cells in amino acid deficient medium strongly inhibited the syntheses of both intra- and extracellular proteins. Electromicroscopic examination of the cells showed that amino acid deprivation markedly decreased the amount of rough endoplasmic reticulum and caused the appearance of autophagic vacuoles. These results show that extracellular amino acids control the syntheses of both intra- and extracellular proteins, but have a more direct effect on the latter, the intracellular proteins being synthesized by reutilization of amino acids liberated on protein degradation. Moreover, protein degradation seems to be accelerated by formation of phagosomes, when the medium is deficient in amino acids. When this protein degradation is inhibited by a protease inhibitor, the synthesis of intracellular proteins decreases. Therefore, there seem to be two compartments of amino acids in hepatocytes, one supplied by lysosomal degradation of proteins and used mainly for synthesis of intracellular protein, and the other supplied from exogenous amino acids and used for syntheses of both intra- and extracellular proteins.

Amino Acids↗
PubMed1983

Long-term polyvinylpyrrolidone storage. Histochemical and ultrastructural observations in two biopsy cases.

Biopsy specimens of lymph nodes or bone marrow taken from two cases, which seemed to have received intravenous infusions of polyvinylpyrrolidone (PVP) more than 15 years ago, were examined histopathologically, histochemically or electron microscopically. In the lymph nodes, proliferation of foam cells, often with multinuclear giant cells, and basophilic vacuolar massive deposition were found markedly in the lymphatic sinuses and sporadically in the parenchyma. In the bone marrow, basophilic foam cells predominated, together with infrequent appearance of multinuclear giant cells and basophilic vacuolar deposits. The basophilic materials stored in the foam cells and multinuclear giant cells, as well as basophilic vacuolar massive deposits, were histochemically proved to stain with the special stains for demonstration of PVP. Electron microscopically, storage inclusions observed in the cytoplasm of PVP-storing macrophages were comprised of electrondense, amorphous substances or of vacuolar, fine reticulogranular, flocculent materials and were bounded by a single delimitating membrane. Most of the inclusions, particularly osmiophilic ones, showed intense acid phosphatase activity, while larger, vacuolar inclusions had a reduced or no enzyme activity. Extracellularly, vacuolar storage inclusions with no enzyme activity were found. The possible mechanism of long-term PVP storage in the tissues was briefly discussed.

Adult↗
PubMed1983

Hormonal regulation of translatable mRNA of tryptophan 2,3-dioxygenase in primary cultures of adult rat hepatocytes.

Tryptophan 2,3-dioxygenase [EC 1.13.11.11] in primary cultures of adult rat hepatocytes was induced 3-4 fold by 1 microM dexamethasone and 6-7 fold by dexamethasone plus glucagon (0.1 microM). Changes of the enzyme activity, amount of enzyme, measured by immunotitration, and rate of enzyme synthesis, assayed by measurement of [3H]leucine incorporation into the enzyme protein, were closely correlated. Furthermore, in a reticulocyte lysate system for cell-free protein synthesis, mRNA of the enzyme was translated to the protein corresponding to the subunit of tryptophan 2,3-dioxygenase, which was identified by SDS-polyacrylamide gel electrophoresis. The activity of translatable mRNA of the enzyme was increased more than 10-fold by dexamethasone and its final content in total mRNA was 0.34%. Glucagon alone did not increase mRNA activity, but dexamethasone plus glucagon increased mRNA activity to twice that with dexamethasone alone, the maximal content of the mRNA being 0.77% of the total mRNA content 12 h after addition of hormones. Insulin (0.1 microM) caused 75% inhibition of the maximum increase of mRNA activity of the enzyme induced by dexamethasone and glucagon. Epinephrine (10 microM) also caused 58% inhibition of the maximum increase. Insulin and epinephrine also suppressed increase of mRNA of tryptophan 2,3-dioxygenase induced by dexamethasone alone. Therefore, dexamethasone alone or together with glucagon stimulated transcription of tryptophan 2,3-dioxygenase increasing its mRNA and enzyme synthesis in hepatocytes. Conversely, insulin and epinephrine suppressed these increases of mRNA synthesis and thus decreased enzyme synthesis.

Animals↗
PubMed1982

Use of hepatocytes in primary culture for biochemical studies on liver functions.

Rat parenchymal hepatocytes isolated with collagenase were cultured as monolayers in Williams medium E supplemented with calf serum. Freshly isolated cells showed very low activities of various liver functions, and they had to be cultured for 6-24 h to allow recovery of these functions. Insulin and dexamethasone greatly increased cell viability in primary. After culture for 24 h, these cells showed various liver functions as seen in vivo and responded well to various added hormones and amino acids. The concentrations of amino acids in the medium regulated synthesis of serum proteins and insulin stimulated lipogenesis, which in turn regulated synthesis of lipoproteins. Insulin also stimulated glycogen synthesis and the stimulation was parallel with the number of insulin receptors. Glucagon stimulated glycogenolysis and its stimulation involved the function of the cytoskeleton. Glucagon and dexamethasone induced various enzymes of amino acid catabolism, such as tryptophan oxygenase, tyrosine aminotransferase and serine dehydratase. These inductions were inhibited by insulin or catecholamine. The effect of catecholamine was due to its alpha-adrenergic action. The beta-action of isoproterenol was low in freshly isolated cells, but increased during culture of the cells. Acquirement of hormonal responses during neonatal development can be studied in this culture system. Mature hepatocytes in culture are usually quiescent, but when insulin and epidermal growth factor were added, DNA synthesis by the cells increased markedly and they showed density-dependent growth. In this culture system, serum could be omitted for 2 days when the dishes were coated with fibronectin without appreciable change of functions, but serum was needed for longer culture of the cells. A factor that increased cell survival was found in serum and in pituitary gland. These results show that hepatocytes in primary culture are a simple and useful system for studies of liver functions in vitro and related works were also reviewed.

Adrenergic beta-Agonists↗
PubMed1982

Hormonal regulations of glucose-6-phosphate dehydrogenase and lipogenesis in primary cultures of rat hepatocytes.

In primary cultured monolayer hepatocytes of adult rats, insulin (1 x 10(-8) M) induced glucose-6-phosphate dehydrogenase [EC 1.1.1.49, G6PDH] several fold in 48 h. It also induced lipogenesis, measured as [1-14C]acetate incorporation in 2 h, in these cells. Of the various lipids, triglycerides and phospholipids were induced markedly, while cholesterol and its esters were not induced. The increase of G6PDH and lipogenesis were parallel. Glucagon, dibutyryl cyclic AMP, triiodothyronine, dexamethasone, epinephrine, isoproterenol, and dibutyryl cyclic GMP were also tested under similar conditions, but none of them caused significant induction of G6PDH or lipogenesis. Use of anti-G6PDH serum showed that induction of G6PDH by insulin was due to increase in the amount of enzyme protein. Insulin was found to increase the rate of synthesis of G6PDH about 3-fold. SDS-polyacrylamide gel electrophoresis of the immunoprecipitable protein revealed that besides G6PDH another radioactive fraction (Mr 37,000) was increased by insulin. This suggests that complete synthesis of G6PDH protein is slowed down in primary cultured hepatocytes and that an apparent nascent peptide of the enzyme accumulates. Although on long-term treatment (48 h), glucagon and dibutyryl cyclic AMP had no effect on lipogenesis, when added with [14C]acetate for 2 h they strongly inhibited lipogenesis. Significant inhibition of lipogenesis by short-term treatment with glucagon was seen even in cells with a high capacity for lipogenesis induced by long-term treatment with insulin. Insulin again stimulated lipogenesis in short-term treatment, but its effect was slight. It is concluded from these results that insulin exerts long-term stimulation of lipogenesis by inducing enzymes related to lipogenesis including G6PDH as well as causing slight stimulation by enhancing supply of substrate for lipogenesis. Glucagon seems to play a minor role in long-term control, but it causes short-term inhibition of lipogenesis.

Animals↗