Carbohydrate reactivation of thyroxine 5'-deiodinase (type II) in cultured mouse neuroblastoma cells is dependent upon new protein synthesis.
The T3 concentration in brain predominantly reflects local production from T4 rather than T3 uptake from the circulating pool. We recently demonstrated that rat brain T3 content is increased by glucose feeding compared to chow feeding. One possible mechanism for this effect is an increase in brain T4 5'-deiodinase (5'-D) activity. Our recent preliminary studies of neuroblastoma (NB) cells demonstrate that renewal of RPMI-1640 medium stimulates T4 5'-D type II (NB T4 5'-D II) activity in these cells. The present studies were performed to determine the mechanism of this response. Studies were performed on NB cells supported in thyroid hormone-depleted (deficient) medium. This approach increased NB T4 5'-DII activity 4-fold compared to that in thyroid hormone-replete medium. Medium renewal further stimulated enzyme activity (7- to 9-fold; maximum at 6 h) in each group. The difference between the hypothyroid group and control was sustained over a 24-h period. Subsequent studies demonstrated that glucose (11 mM) was the specific medium ingredient mediating the medium renewal response. A progressive increase in NB T4 5'-DII activity was noted over 8 h during RPMI-1640 salt plus glucose (11 mM) incubation. This was equivalent to the effect of complete medium containing glucose (11 mM). Coincubation with insulin (10(-7)-10(-9) M) did not modify the enzyme response to glucose. In addition, fructose (10 mM) had a similar effect on enzyme activity. Glycerol and essential and nonessential amino acids also modestly increased NB T4 5'-DII activity compared to that in the control group (P less than 0.01). Actinomycin-D (1 microM), cycloheximide (100 microM), and puromycin (100 microM) significantly (P less than 0.001) decreased the glucose effect on T4 5'-DII by 5-, 9-, and 17-fold, respectively, after 6 h of incubation. In addition, puromycin (10-200 microM) inhibited both NB T4 5'-DII activity and [3H]amino acid incorporation during incubation in glucose. There was a significant correlation between these parameters (r = 0.8; P less than 0.001). The enzyme activity decay curves in the glucose-activated and control groups subsequent to puromycin (100 microM) addition at 8 h were parallel. The fractional turnover rate was 13%/h in the controls and 11%/h in the glucose groups. The calculated enzyme production rate was significantly higher (P less than 0.005) in the glucose group compared to that in the control group (17.4 vs. 6.8 fmol/mg protein.h).(ABSTRACT TRUNCATED AT 400 WORDS)
Gavin
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LA
Division of Endocrinology-Metabolism, Veterans Administration Medical Center, San Francisco, California 94121.
Moller
M
M
McMahon
F
F
Gulli
R
R
Cavalieri
R R
RR
D000596:Amino Acids / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D003470:Culture Media; D004789:Enzyme Activation; D005947:Glucose / Q000494:pharmacology*; D005978:Glutathione / Q000494:pharmacology; D007453:Iodide Peroxidase / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D009447:Neuroblastoma / Q000201:enzymology*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D014815:Vitamins / Q000494:pharmacology
D000596:Amino Acids; D003470:Culture Media; D007527:Isoenzymes; D011500:Protein Synthesis Inhibitors; D014815:Vitamins; D007453:Iodide Peroxidase; D005978:Glutathione; D005947:Glucose