scholarly journals Increase of l-serine dehydratase activity under gluconeogenic conditions in adult-rat hepatocytes cultured on collagen gel/nylon mesh

1981 ◽  
Vol 198 (3) ◽  
pp. 499-504 ◽  
Author(s):  
W W N Mak ◽  
H C Pitot
Keyword(s):  
Enzyme Activity ◽  
Hormonal Regulation ◽  
Collagen Gel ◽  
Glucose Production ◽  
Rat Hepatocytes ◽  
Cultured Hepatocytes ◽  
Adult Rat ◽  
Nylon Mesh ◽  
Serine Dehydratase ◽  
Dehydratase Activity

Hormonal regulation of L-serine dehydratase [L-serine hydro-lyase (deaminating), EC 4.2.1.13] was studied in primary cultures of adult-rat hepatocytes. The hepatocytes were isolated by collagenase perfusion and maintained in culture on collagen-gel/nylon-mesh substrata. L-Serine dehydratase activity was measured with [14C]threonine as substrate. The enzyme activity in hepatocytes of normal adult rats was low and declined rapidly in culture in L-15 medium containing 0.1 micro M-insulin and even more in the presence of glucose. L-Serine dehydratase activity in hepatocytes of rats with streptozotocin-induced diabetes was initially 20-fold higher than that of normal rats, but fell rapidly to a low value by 4 days in culture. Hormonal regulation of the enzyme activity was manifested by treatment of the cultured hepatocytes with insulin (0.1 micro M), glucagon (0.3 micro M), dexamethasone (10 micro M) and combinations of these hormones. Either glucagon or dexamethasone in the absence of insulin enhanced the activity of L-serine dehydratase, but failed to do so in the presence of insulin. Treatment with both hormones resulted in a 2-3-fold increase in enzyme activity in culture on days 3 and 4. Under conditions in which the enzyme activity was enhanced, glucose production by the cultured hepatocytes was concomitantly increased. Glucose production resulted in part from gluconeogenesis from pyruvate and not entirely from glycogenolysis. The gluconeogenic conditions of culture resulted in a decrease in cellular lipids in the cultured hepatocytes, as evidenced by ultrastructural studies.

scholarly journals Measurement of the metabolism of cytochrome P-450 in cultured hepatocytes by a quantitative and specific immunochemical method

1982 ◽  
Vol 204 (1) ◽  
pp. 281-290 ◽  
Author(s):  
Sammye L. Newman ◽  
Joyce L. Barwick ◽  
Nabil A. Elshourbagy ◽  
Philip S. Guzelian
Keyword(s):  
Cultured Cells ◽  
De Novo ◽  
Internal Standard ◽  
Rat Hepatocytes ◽  
Cellular Protein ◽  
Small Samples ◽  
Cultured Hepatocytes ◽  
Immunochemical Method ◽  
Adult Rat ◽  
Cytochrome P 450

We have defined conditions that permit quantitative and specific measurement of the metabolism of the major phenobarbital-inducible form of cytochrome P-450 protein in primary non-proliferating monolayer cultures of adult rat hepatocytes. Isolated antibodies specifically directed against phenobarbital cytochrome P-450 are used to immunoprecipitate the cytochrome from lysates of cultured hepatocytes pulse-labelled with [3H]leucine. Phenobarbital cytochrome P-450 protein is then isolated from the immunoprecipitate by electrophoresis on polyacrylamide gradient slab gels. Specificity of the assay for phenobarbital cytochrome P-450 was established by competition experiments involving other forms of purified cytochrome P-450 as well as by testing antibodies directed against these other forms of the cytochrome. Using purified phenobarbital cytochrome P-450, radiolabelled in both its haem and apoprotein portions, as an internal standard, we demonstrated that, with this immunoassay, recovery of cytochrome P-450 from microsomal samples is nearly complete. Basal rates of synthesis of phenobarbital cytochrome P-450 representing as little as 0.02–0.05% of total cellular protein synthesis were reliably and reproducibly detected in hepatocyte culture maintained in serum-free medium for 72h. Moreover, inclusion of phenobarbital in the culture medium for 96h stimulated not only synthesis de novo of phenobarbital cytochrome P-450 protein, but also accumulation of spectrally and catalytically active cytochrome P-450. Advantages of this immunoassay are that metabolism (synthesis or degradation) of the haem or protein of this important form of the cytochrome can be measured conveniently in the small samples available from cultured cells without the necessity of preparing subcellular fractions.

Effects of chronic uraemia on the formation of glucose and urea plus ammonia from l-alanine, l-glutamine and l-serine in isolated rat hepatocytes

1986 ◽  
Vol 70 (6) ◽  
pp. 627-634 ◽  
Author(s):  
Rita A. Klim ◽  
Marta Albajar ◽  
Reginald Hems ◽  
Dermot H. Williamson
Keyword(s):  
Glucose Production ◽  
Rat Hepatocytes ◽  
Nitrogen Release ◽  
Isolated Rat Hepatocytes ◽  
Metabolic Intermediates ◽  
Chronic Uraemia ◽  
Serine Dehydratase ◽  
Glucose Synthesis ◽  
Lactate And Pyruvate ◽  
Isolated Rat

1. The effects of chronic uraemia on glucose production and nitrogen release (urea plus ammonia formation) from alanine, glutamine or serine in isolated rat hepatocytes were studied. 2. Uraemia increased the rate of formation of urea plus ammonia from all three amino acids by 38-93% when they were present at a final concentration of 10 mmol/l. At lower concentrations (2 mmol/l) the rate of nitrogen release was not significantly increased. 3. Hepatocytes from normal rats whose food intake had been restricted to the level of that of uraemic rats did not show the increased rates of nitrogen release. 4. The increased rates of nitrogen release with hepatocytes from uraemic rats were not accompanied by increased rates of glucose synthesis. Instead, accumulation of metabolic intermediates occurred: lactate and pyruvate (alanine or serine as substrates) and glutamate (glutamine as substrate). 5. Livers of uraemic rats had increased activities of glutarninase (30%) and serine dehydratase (100%). 6. Hepatocytes from normal rats treated with phlorhizin to increase the plasma glucagon/insulin ratio behaved in a similar manner to hepatocytes from uraemic rats. They had increased serine dehydratase activity, and increased rates of utilization of serine or glutamine. 7. The possible implications of these findings for human uraemia are discussed.

scholarly journals Insulin-mimetic actions of phorbol ester in cultured adult rat hepatocytes. Lack of phorbol-ester-elicited inhibition of the insulin signal

1993 ◽  
Vol 289 (2) ◽  
pp. 549-555 ◽  
Author(s):  
A Quentmeier ◽  
H Daneschmand ◽  
H Klein ◽  
K Unthan-Fechner ◽  
I Probst
Keyword(s):  
Phorbol Ester ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Glycogen Synthesis ◽  
Primary Cultures ◽  
Glucose Production ◽  
Rat Hepatocytes ◽  
Simultaneous Increase ◽  
Additive Effects ◽  
Adult Rat ◽  
Adult Rat Hepatocytes

The actions of the phorbol ester phorbol 12-myristate 13-acetate (PMA) on glucose metabolism, amino acid transport and enzyme inductions were studied in primary cultures of adult-rat hepatocytes and compared with the effects of insulin. PMA and insulin stimulated glycolysis 5- and 7-fold respectively. The half-maximal effective dose of PMA was 60 nM. Stimulation of glycolysis was accompanied by an insulin- or PMA-dependent and okadaic acid-sensitive activation of 6-phosphofructo-2-kinase and pyruvate kinase, as well as by an increase in fructose 2,6-bisphosphate. Glucose production from glycogen was decreased to 50% by PMA and to 15% by insulin, whereas glycogen synthesis was stimulated 2- and 7-fold respectively. PMA also increased aminoisobutyrate uptake, induced ornithine decarboxylase and counteracted the glucagon-dependent induction of phosphoenolpyruvate carboxykinase. PMA strongly antagonized the hormonal activation of glycogen synthesis, but all other insulin actions assayed were not decreased by the phorbol ester. Whereas additive effects of PMA and insulin were not detected, PMA and a simultaneous increase in the glucose concentration had additive effects on glycolysis and glycogen metabolism. Cell exposure to insulin resulted in receptor autophosphorylation and a more than 10-fold activation of the receptor tyrosine kinase. PMA did not alter these effects, and also had no effect on the receptor phosphorylation status in the absence of insulin. Long-term (15 h) pretreatment of the cells with PMA abolished all PMA effects, but not the insulin effects. It is concluded that PMA does not generally antagonize the action of insulin in differentiated adult hepatocytes, and that insulin and PMA may use related signal-transduction pathways.

Hormonal Control of Serine Dehydratase mRNA in Primary Cultures of Adult Rat Hepatocytes 1

1984 ◽  
Vol 95 (1) ◽  
pp. 37-45 ◽  
Author(s):  
Chiseko NODA ◽  
Mineko TOMOMURA ◽  
Toshikazu NAKAMURA ◽  
Akira ICHIHARA
Keyword(s):  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Hormonal Control ◽  
Adult Rat ◽  
Serine Dehydratase ◽  
Adult Rat Hepatocytes

Rapid loss of δ-aminolevulinic acid dehydratase activity in primary cultures of adult rat hepatocytes: A new model of zinc deficiency

Life Sciences ◽  
1982 ◽  
Vol 31 (11) ◽  
pp. 1111-1116 ◽  
Author(s):  
Philip S. Guzelian ◽  
Laurel O'Connor ◽  
Suzanne Fernandez ◽  
Winnie Chan ◽  
Philip Giampietro ◽  
...  
Keyword(s):  
Zinc Deficiency ◽  
Aminolevulinic Acid ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Rapid Loss ◽  
New Model ◽  
Adult Rat ◽  
Acid Dehydratase ◽  
Adult Rat Hepatocytes ◽  
Dehydratase Activity

scholarly journals Fetal phenotypic expression by adult rat hepatocytes on collagen gel/nylon meshes.

1979 ◽  
Vol 76 (1) ◽  
pp. 283-287 ◽  
Author(s):  
A. E. Sirica ◽  
W. Richards ◽  
Y. Tsukada ◽  
C. A. Sattler ◽  
H. C. Pitot
Keyword(s):  
Phenotypic Expression ◽  
Collagen Gel ◽  
Rat Hepatocytes ◽  
Adult Rat ◽  
Adult Rat Hepatocytes
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