Steven Seelig

A rapid, inexpensive, quantitative technique for the analysis of two-dimensional electrophoretograms

Abstract We describe a rapid, reproducible, and relatively inexpensive method for quantitative analysis of two-dimensional gel radiofluorograms. The equipment required includes a mediumresolution black and white video camera for data acquisition, a video digitizing circuit for data translation, and a microcomputer for data analysis. The system described can also be used to quantitate protein- or DNA-stained one- or two-dimensional gels or chromatograms.

Hepatic fatty acid-binding protein mRNA is regulated by growth hormone

Hepatic fatty acid-binding protein (FABP) is one of several abundant proteins which may participate in fatty acid uptake and utilization. Using differential hybridization to screen for growth hormone-responsive gene products, a complementary deoxyribonucleic acid (cDNA) was isolated which proved to be a hepatic FABP cDNA fragment. Hypophysectomy caused a 60% reduction in hepatic FABP messenger ribonucleic acid (mRNA) levels in rat liver, and growth hormone administration to hypophysectomized rats resulted in restoration of the expression of hepatic FABP mRNA. Other pituitary hormones did not alter these changes in expression. The response to growth hormone occurred within 4 hours of administration. During development, expression of hepatic FABP mRNA in rat liver was low in late fetal life, with increases to 40% of adult values by day 2 of life. Significant increases to adult levels did not occur until after day 25, when weaning is essentially completed. Alteration of hepatic FABP mRNA expression by growth hormone in rat liver may be important in the complex regulation of fatty acid uptake and metabolism.

Hormonal regulation of serum alpha1-antitrypsin and hepatic alpha1-antitrypsin mRNA in rats

We evaluated the effects of pituitary dependent hormones on alpha 1-antitrypsin in male rats. Hepatic alpha 1-antitrypsin mRNA was measured by in vitro translation and by specific hybridization with a mouse cDNA alpha 1-antitrypsin probe. Hypophysectomy caused a 50-75% decrease in serum elastase inhibitory capacity (measuring functional alpha 1-antitrypsin) and hepatic alpha 1-antitrypsin mRNA content. In hypophysectomized animals, no increase in elastase inhibitory capacity or alpha 1-antitrypsin mRNA levels by translation was found when met-human growth hormone alone or corticosterone, dihydrotestosterone and thyroxine were given together. Growth hormone increased alpha 1-antitrypsin mRNA by hybridization to a small extent. Addition of growth hormone to the combination of corticosterone, dihydrotestosterone, and thyroxine increased serum elastase inhibitory capacity and alpha 1-antitrypsin mRNA. We conclude that growth hormone acts synergistically with the other pituitary dependent hormones to regulate serum and hepatic mRNA levels of alpha 1-antitrypsin.

Growth hormone acts at a pretranslational level in hepatocyte cultures

We have examined the effects of ovine growth hormone and recombinant DNA synthesized human growth hormone on hepatocytes maintained in serum free cultures. Both growth hormone preparations augmented or attenuated 3 specific mRNA sequences as revealed by two-dimensional gel electrophoresis of [35S] methionine labeled products synthesized in vitro in an mRNA dependent rabbit reticulocyte lysate system. The results clearly indicate that growth hormone, free of potential pituitary contaminants, acts directly on hepatocytes at a pretranslational level.

Effects of elevated serum insulinlike growth factor-II on growth hormone and insulinlike growth factor-I mRNA and secretion

The insulinlike growth factors (IGF) appear to exert feedback control over their own production. In an effort to determine the physiologic mechanisms for this feedback modulation, we utilized a previously developed in vivo model in which rIGF-II secreting tumor cells are transplanted into immunodeficient rats to form IGF-II secreting tumors. The tumor-bearing rat have serum IGF-II concentrations sevenfold greater than those in controls (119 +/- 16 ng/mL [mean +/- SE] v 17 +/- 2 ng/mL, P less than .0001). Serum IGF-I concentrations were reduced among the tumor-bearing rats (438 +/- 42 ng/mL v 606 +/- 32 ng/mL, P = .002) and were negatively correlated with IGF-II concentrations (r = -.47, P = .025), suggesting that IGF-II suppressed the secretion of IGF-I. Increased serum IGF-II concentrations, however, did not affect basal growth hormone concentrations (tumor-bearing, 44 +/- 12 ng/mL; control 33 +/- 6 ng/mL, P = 0.96). The GH response to GH releasing factor was likewise similar in both groups. Moreover, pituitary GH mRNA level were not different in the two groups, suggesting that IGF-II does not have a significant effect on GH secretion in this in vivo model. There was no association between serum glucose and serum IGF-I or IGF-II concentrations. To examine the effect of IGF-II on IGF-I production from the liver, we measured IGF-I mRNA levels in a subset of animals. Despite these differences in serum IGF-I concentrations, the tumor-bearing rats did not have significantly lower liver IGF-I mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Response of Serine Antiproteases to Growth Hormone Therapy in Growth Hormone Deficient Children

Growth hormone regulates the hepatic mRNA levels of alpha 1-antitrypsin and two contrapsin-like mRNAs in the rat. To determine whether growth hormone regulates similar serine protease inhibitors in humans, we measured serum alpha 1-antitrypsin, alpha 1-antichymotrypsin, and antithrombin III by radioimmunodiffusion in 16 growth hormone deficient children before and after growth therapy. Of the 19 determinations made, 17/19 showed an increase in alpha 1-antitrypsin after administration of growth hormone, 198.6 +/- 39.1 mg/dl before growth hormone and 239.4 +/- 44 mg/dl after growth hormone (p = 0.005). Specificity of the response for alpha 1-antitrypsin was indicated by the fact that neither alpha 1-antichymotrypsin or antithrombin III values changed after growth hormone (p = 0.6 and 0.5, respectively). These data are compatible with the hypothesis that growth hormone regulates serine protease inhibitors in humans and suggests that investigation of other members of the serpin gene family might prove fruitful in defining additional growth hormone target genes.

Methionyl- and Pituitary Derived Human Growth Hormone Have Identical Effects on the Expression of Growth Hormone Responsive Rat Hepatic mRNA

Pituitary extracts of human growth hormone have been used extensively for therapy of growth hormone deficiency, although they are known to contain a variety of contaminating polypeptides. Biosynthetic human growth hormone is now available for this use and appears to be functionally identical in promoting growth. To establish additional criteria of identity we compared the effect of these two hormone preparations on a family of hepatic messenger RNA sequences in hypophysectomized adult male rats. Total hepatic RNA from these animals was translated in a cell-free rabbit reticulocyte lysate. Five translational products previously demonstrated to be responsive to ovine and methionyl-human growth hormone were found to be equally induced by pituitary derived human growth hormone, despite demonstrable heterogeneity in pituitary derived preparations. In addition, no significant alterations in approximately 200 non-growth hormone responsive translational products were identified. Methionyl and pituitary derived growth hormone have identical effects on the expression of hepatic mRNA.

428 THE ONTOOENY OF THE RESPONSE TO GROWTH HORMONE

We defined the ontogeny of five hepatic mRNA sequences which are responsive to growth hormone (GH) in juvenile (35 d) hypophysectomized rats ay in vitro translation of mRNA sequences and two-dimensional gel electrophoresis of the 35[S]-methionine labeled products. Each of the five had a well defined ontogeny which did not parallel the ontogeny of hepatic GH receptors or serum GH. Major periods for transition of this family of mRNA sequences were weaning and puberty. The pattern of ontogeny for two of the products was similar to that observed for GH responsive serum proteins. Induction of relative GH deficiency in rat pups by administration of propylthiouracil resulted in alterations in the mRNA sequences suggestive of GH deficiency; however, in twelve day old animals GH was much less effective in reversing these alterations than in older animals. Augmentation of normal pups with pituitary dependent hormones (thyroxine corticosterone and GH) resulted in changes consistent with an overall increase in maturation of the liver. This effect was also observed in non-hormonally responsive products, suggesting the change is not specific to the GH responsive mRNA family. From these observations we conclude that: 1). factors other than GH or GH receptor content are of importance in the expression of GH responsive products in the 12 day old animal; 2) the ontogeny of GH responsive serum proteins may reflect pretranslational events; 3) GH and the other pituitary dependent hormones modify the cadence of expression of hepatic gene products;4) and that the ability of the liver to respond to GH is an age dependent phenomenon.