The Mechanism of Insulin-Induced Increase of the Rate of De Novo Purine Biosynthesis in Primary Cultured Rat Hepatocytes

1986 ◽  
pp. 623-631
Author(s):  
Masami Tsuchiya ◽  
Mitsuo Itakura ◽  
Kamejiro Yamashita
Keyword(s):  
De Novo ◽  
Rat Hepatocytes ◽  
Purine Biosynthesis ◽  
Primary Cultured Rat Hepatocytes ◽  
De Novo Purine Biosynthesis ◽  
Cultured Rat Hepatocytes

Increased de novo purine synthesis by insulin through selective enzyme induction in primary cultured rat hepatocytes

1990 ◽  
Vol 258 (5) ◽  
pp. C841-C848 ◽  
Author(s):  
M. Tsuchiya ◽  
H. Yoshikawa ◽  
M. Itakura ◽  
K. Yamashita
Keyword(s):  
Enzyme Induction ◽  
De Novo ◽  
Feedback Inhibition ◽  
Rat Hepatocytes ◽  
Purine Metabolism ◽  
Hepatocyte Proliferation ◽  
Adenylate Energy Charge ◽  
Purine Synthesis ◽  
Primary Cultured Rat Hepatocytes ◽  
Cultured Rat Hepatocytes

The proliferative effect of insulin on de novo purine synthesis and on the expression of various enzymes of purine metabolism were studied in primary cultured rat hepatocytes. Insulin greater than 1.5 x 10(-8) M increased DNA and de novo purine synthesis to 260-390 and 270-420%, respectively, 24 and 8 h after the administration. Insulin at 1.5 x 10(-7) M increased the specific activity of amidophosphoribosyltransferase (ATase) to 154-180%, hypoxanthine-guanine phosphoribosyltransferase to 129%, and adenine phosphoribosyltransferase (APRT) to 205%, in contrast to unchanged xanthine dehydrogenase at 80%. Enzyme induction was supported by the results of kinetic analysis and the inhibition of the insulin-induced increase in enzyme activities by protein synthesis inhibitors. Insulin increased ATP to 127% and decreased AMP, ADP, 5'-guanylic acid (GMP), and guanosine 5'-diphosphate (GDP), respectively, to 73, 69, 73, and 69%. Insulin increased adenylate energy charge from 0.83 to 0.90 without changing total feedback inhibitory potential on ATase. No obvious increase of 5-phosphoribosyl-1-pyrophosphate supply was suggested, although its apparent availability for purine ribonucleotide synthesis was increased to 208-245%, reflecting mainly induced APRT activity to 205%. It is concluded that hepatocyte proliferation by insulin, as evidenced by purine metabolism, is mediated by the selective gene activation of anabolic enzymes and increased ATP as the basis to activate multiple metabolic pathways without remarkable changes of substrate availability or feedback inhibition.

scholarly journals Carbocyclic substrates for de novo purine biosynthesis.

1991 ◽  
Vol 266 (25) ◽  
pp. 16699-16702
Author(s):  
D.S. Liu ◽  
C.A. Caperelli
Keyword(s):  
De Novo ◽  
Purine Biosynthesis ◽  
De Novo Purine Biosynthesis

The Use of Primary Cultured Rat Hepatocytes for the Assessment of Xenobiotic Effects on Biotransformation

1991 ◽  
Vol 19 (2) ◽  
pp. 209-213
Author(s):  
Gabi Schepers ◽  
Christiane Aschmann ◽  
Sabine Mörchel
Keyword(s):  
Cell Viability ◽  
Rat Hepatocytes ◽  
Standard Test ◽  
In Vitro Test ◽  
Chlorinated Phenols ◽  
Test Protocol ◽  
Primary Cultured Rat Hepatocytes ◽  
Cultured Rat Hepatocytes ◽  
Different Response

An in vitro test protocol is reported, which, using primary cultured rat hepatocytes, allows for the screening of xenobiotic effects on biotransformation as well as on basal cellular functions. O-Deethylation of 7-ethoxycoumarin (7-EC) and subsequent conjugation of the metabolite 7-hydroxycoumarin (7-HC) with sulphate or glucuronic acid are determined, as representative parameters for the hepatic biotransformation. Cell viability is examined by measuring cellular ATP content and leakage of lactate dehydrogenase. With respect to immediate and delayed effects on biotransformation reactions, the standard test protocol includes exposure to xenobiotics for 1, 24 and 48 hours. Different response patterns could be demonstrated for the solvents dimethylformamide (DMF) and dimethylsulphoxide (DMSO), and the chlorinated phenols, pentachlorophenol (PCP) and hexachlorophene (HCP), which are known to uncouple mitochondrial respiration. Short-term incubation with the solvents resulted in decreased 7-EC- O-deethylation without signs of cytotoxicity. PCP and HCP inhibited 7-EC- O-deethylation and 7-HC-conjugation, affecting sulphate and glucuronide formation differently. 24-hour exposures to PCP and HCP resulted in decreased 7-ethoxycoumarin- O-deethylase activity, which correlated with diminished cell viability, while DMSO and DMF enhanced 7-EC- O-deethylation at sub-cytotoxic concentrations. After exposure for 48 hours to the solvents, enzyme induction was even more pronounced.

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