Nutrition and somatomedin. XIX. Molecular regulation of insulin-like growth factor-1 in streptozotocin-diabetic rats.

Poor growth in diabetes involves low circulating levels of somatomedins/insulin-like growth factors (IGFs), largely reflecting decreased growth factor release by the liver. To define regulatory mechanisms, circulating IGF-1 was compared with levels of a high mol wt putative hepatic IGF-1 precursor and hepatic IGF-1 mRNA in a model of progressive severity of diabetes in rats. Streptozotocin administered at 36, 72, 144, and 288 mg/kg produced graded metabolic decompensation 2 days later, from minimal hyperglycemia with continued weight gain at 36 mg/kg, to marked hyperglycemia, ketonemia, and weight loss at 288 mg/kg (all P less than 0.001). Total serum IGF-1 measured by RIA was unchanged with the 36 and 72 mg/kg doses of streptozotocin (471 +/- 19 and 439 +/- 27 ng/ml, respectively, vs. 517 +/- 27 ng/ml in controls) despite serum glucose greater than 400 mg/dl. With streptozotocin 144 and 288 mg/kg, serum IGF-1 fell to 131 +/- 27 and 142 +/- 10 ng/ml, respectively (both P less than 0.005 vs. controls). Serum IGF-1 was correlated strongly with serum beta-hydroxybutyrate and body weight (r = -0.88 and 0.91, respectively, P less than 0.0001), and less strongly with serum glucose (r = -0.59, P less than 0.0002). Extractable hepatic content of a high mol wt form of immunoreactive IGF-1 (a putative precursor) was unchanged at the two lowest doses of streptozotocin (68 +/- 4 and 83 +/- 9 ngeq/g vs. 67 +/- 4 in controls), but decreased to 16 +/- 3 and 29 +/- 4 ng/g at the two highest doses (both P less than 0.001 vs. controls).(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of ovarian ornithine decarboxylase by human chorionic gonadotrophin.

Treatment of 29-day-old female Sprague-Dawley rats with human chorionic gonadotrophin (hCG) produced a large and rapid increase in the activity of ornithine decarboxylase. Measurements that use a specific radioimmunoassay showed that the increased activity could be accounted for by a parallel change in the amount of ornithine decarboxylase protein. The increased protein content was caused by an increased rate of synthesis, since the half-life of ornithine decarboxylase was not changed by the hormone treatment. The content of mRNA for ornithine decarboxylase was determined by hybridization with a cDNA probe, and it was found that the increased amount of protein was correlated with a change in the amount of mRNA. These results indicate that treatment with hCG induces ornithine decarboxylase in the rat ovary by increasing the production or the stability of the mRNA for this enzyme. The increased amount of ornithine decarboxylase led to an increase in putrescine in the ovary but did not increase the content of the polyamines spermidine and spermine. These findings show that, despite its rapid and large scale induction, ornithine decarboxylase is not the rate-limiting step that determines the content of these polyamines in this tissue. They also suggest that putrescine itself may play an important role in the ovary.

Polyamine administration reduces ornithine decarboxylase activity without affecting its mRNA content.

Ornithine decarboxylase, the first enzyme in the polyamine biosynthetic pathway, is induced by androgens in the mouse kidney. Enzyme activity, as well as enzyme protein levels are increased 100-400 fold. Utilizing a specific cDNA probe to ODC, mRNA levels in these cells were found to increase 7-25 fold as measured by densitometric scanning. Treatment of the mice for 5 hours with 2 mmol/kg doses of putrescine or 1,3-diaminopropane after androgen stimulation reduced enzyme activity to control levels, while mRNA levels remained elevated 18-30 fold above control. In a different system, serum starved SV-3T3 cells showed low ODC activity and amounts of ODC mRNA. Serum stimulation increased the 2.2 kilobase mRNA levels 6 fold and enzyme activity 13 fold in a coordinate fashion within 5 hours. While the rise in activity was blocked by the simultaneous addition of serum and 1 mM putrescine, ODC mRNA levels appeared unchanged. The addition of 50 microM spermidine or 100 microM spermine also had no effect on ODC mRNA levels, while at the same time reducing enzyme activity amounts. These results suggest that the mechanism by which polyamines regulate ODC activity in the mouse is primarily translational.

Altered polyamine metabolism in Chinese hamster cells growing in a defined medium.

Chinese hamster cells (line CHO) maintained in McCoy's 5A medium (modified) supplemented with insulin (10 micrograms/ml), transferrin (5 micrograms/ml), and ferrous sulfate (1.1 microgram/ml) proliferate at rates similar to cultures growing in the McCoy's medium supplemented with 10% fetal bovine serum. Colony-forming ability is similar in cultures supplemented with either serum or the combination of growth factors. By 6 hours after replacement of serum with growth factors, ornithine decarboxylase (ODCase) activity increases, reaching a maximum value by 24 hours after serum replacement. This maximum is cell density dependent and can exceed a 30-fold increase over enzyme activity in cultures supplemented with serum. The increased enzyme activity is due to a decrease in the turnover rate of the enzyme, based on protein synthesis inhibition studies, and an accumulation of active enzyme molecules rather than an activation of existing molecules, since the catalytic activity of ODCase, determined using the radiolabeled form of alpha-difluoromethylornithine (an enzyme-activated, irreversible inhibitor of ODCase) in concert with supplements. Intracellular putrescine and spermidine levels are substantially decreased when cultures are maintained in medium supplemented with insulin, transferrin, and ferrous sulfate, rather than serum, which is the sole source of exogenous ornithine. Titration of cultures growing in the defined medium with ornithine leads to a decrease in ODCase activity and an increase in intracellular putrescine and spermidine levels. Putrescine- and spermidine-dependent S-adenosyl-L-methionine decarboxylase activities are similar in cultures maintained in either medium. These data demonstrate that some, but not all, aspects of polyamine biosynthesis are affected by the availability of ornithine, the first substrate in the pathway.

Comparison of ornithine decarboxylase from rat liver, rat hepatoma and mouse kidney.

Comparisons were made of ornithine decarboxylase isolated from Morris hepatoma 7777, thioacetamide-treated rat liver and androgen-stimulated mouse kidney. The enzymes from each source were purified in parallel and their size, isoelectric point, interaction with a monoclonal antibody or a monospecific rabbit antiserum to ornithine decarboxylase, and rates of inactivation in vitro, were studied. Mouse kidney, which is a particularly rich source of ornithine decarboxylase after androgen induction, contained two distinct forms of the enzyme which differed slightly in isoelectric point, but not in Mr. Both forms had a rapid rate of turnover, and virtually all immunoreactive ornithine decarboxylase protein was lost within 4h after protein synthesis was inhibited. Only one form of ornithine decarboxylase was found in thioacetamide-treated rat liver and Morris hepatoma 7777. No differences between the rat liver and hepatoma ornithine decarboxylase protein were found, but the rat ornithine decarboxylase could be separated from the mouse kidney ornithine decarboxylase by two-dimensional gel electrophoresis. The rat protein was slightly smaller and had a slightly more acid isoelectric point. Studies of the inactivation of ornithine decarboxylase in vitro in a microsomal system [Zuretti & Gravela (1983) Biochim. Biophys. Acta 742, 269-277] showed that the enzymes from rat liver and hepatoma 7777 and mouse kidney were inactivated at the same rate. This inactivation was not due to degradation of the enzyme protein, but was probably related to the formation of inactive forms owing to the absence of thiol-reducing agents. Treatment with 1,3-diaminopropane, which is known to cause an increase in the rate of degradation of ornithine decarboxylase in vivo [Seely & Pegg (1983) Biochem. J. 216, 701-717] did not stimulate inactivation by microsomal extracts, indicating that this system does not correspond to the rate-limiting step of enzyme breakdown in vivo.