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Endocrinology Vol. 141, No. 3 910-921
Copyright © 2000 by The Endocrine Society

ARTICLES

Differential Cloning of Growth Hormone-Regulated Hepatic Transcripts in the Aged Rat1

Petra Tollet-Egnell, Amilcar Flores-Morales, Jacob Odeberg, Joakim Lundeberg and Gunnar Norstedt

Department of Molecular Medicine, Karolinska Institute, Karolinska Hospital (P.T.-E., A.F.-M., G.N.), 171 76 Stockholm; and the Department of Biochemistry and Biotechnology, Royal Institute of Technology (J.O., J.L.), 100 44 Stockholm, Sweden

Address all correspondence and requests for reprints to: Dr. Petra Tollet-Egnell, Department of Molecular Medicine, Karolinska Institute, CMM L8:01, Karolinska Hospital, 171 76 Stockholm, Sweden. E-mail: petra.tollet.egnell{at}molmed.ki.se


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
It has been suggested that aging or at least some of its symptoms are related to a physiological decline in GH levels with age. This study was performed to elucidate age-related changes in GH-dependent effects at the level of gene expression. Through the application of complementary DNA representational difference analysis (RDA) we have identified gene products that are reduced during aging in rat liver. The expression of these genes was restored upon GH treatment. Results from reverse Northern and ribonuclease protection analysis confirmed that the RDA products were truly differentially expressed. In addition to well characterized GH-regulated genes, including CYP2C12, CYP2C13, and {alpha}2u-globulin, we demonstrate the differential expression of at least 11 genes previously not known to be under GH control. Several hepatic transcripts encoding enzymes and receptors involved in the metabolism of protein, carbohydrates, and lipids were identified. Other RDA products consisted of transcripts encoding proteins involved in ATP synthesis, detoxification of reactive oxygen species, or immune responses. This list of GH-regulated genes in the old rat may shed further light on the action and mechanism behind the positive effects of GH on, for example, body composition and the immune system that have been observed in different animal and human studies.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GH, AS ITS name implies, regulates essential functions of body growth (1). In addition to growth promotion, GH is involved in regulating metabolic processes, such as lipid, protein, and carbohydrate metabolism (2, 3), and also in liver-specific metabolism of hydrophobic compounds, including drugs and hormones (4, 5). GH is also known to improve the function of all major immune cell types, including T and B lymphocytes, NK cells, and macrophages (6). Physiological conditions where the secretion of GH is reduced will lead to disturbances in the various biological effects mediated by GH.

GH deficiency in adults is associated with abnormalities in body composition, including reduced proportions of total lean body mass and increased proportions of body fat (7). Other changes in hypopituitary patients include decreased bone mass and increased prevalence of osteoporosis (8) as well as premature mortality due to cardiovascular disease (9). Replacement therapy with GH has shown distinct effects on body composition and physical performance (10, 11). Furthermore, some studies have revealed a beneficial/normalizing effect of GH therapy on parameters such as cardiac (12, 13) and renal function (14, 15), thyroid hormone metabolism (16), bone metabolism (17, 18), and total and regional fuel metabolism (19).

The levels of GH and insulin-like growth factor I (IGF-I; the main mediator of GH actions) progressively decline during aging (20, 21). There is also evidence that some of the age-related changes in body composition, serum lipids, and muscle performance closely resemble symptoms of adult-onset GH deficiency (21, 22). In addition to these physiological changes, aging is often associated with pathological changes that are generally considered to be related to altered immune functions (6). Thus, it can be suspected that aging or at least some of its symptoms are related to a physiological decline in GH levels with age. Strong support for this possibility was provided by results of treatment of elderly subjects with GH (23). GH treatment was shown to increase lean body mass, reduce body fat, improve general well-being, and reduce the rate of age-related decline in bone density (23). Although GH therapy has repeatedly shown to have beneficial effects on these conditions, there are still questions to be answered in connection with GH replacement therapy of the elderly. A greater understanding of the cellular and molecular mechanisms behind GH-induced changes in aged animals should provide clues for better diagnosis and design of GH analogs without side-effects.

IGF-I is an important regulator of animal growth and is believed to mediate many of the endocrine functions of GH (24). Several investigations have clearly indicated that plasma concentrations of IGF-I decrease with age, and it has been suggested that this might contribute to the decrease in tissue protein synthesis and tissue function characteristic of biological aging (25, 26). It has also been shown that GH receptor signal transduction leading to IGF-I gene expression is impaired in livers of aged C57BL6 mice (27), suggesting that the livers of old animals might be resistant to GH. The aims of the present study were to identify GH-dependent gene products that are reduced during aging in rat liver and to clarify the GH responsiveness in old rats in terms of hepatic gene expression. We have investigated the effects of continuous treatment with GH in 2-yr-old male rats through the application of complementary DNA (cDNA) representational difference analysis (RDA). RDA is a powerful technique for cloning the differences between genomes (28, 29) and has recently been adapted for cloning differentially expressed genes (30, 31). We report here the identification of 21 differentially expressed cDNAs, several of which represent novel GH-regulated genes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Old male Sprague Dawley rats (~2 yr of age; obtained from B&K Universal AB, Stockholm, Sweden) and younger (adult) male rats (~8 weeks of age, obtained from Möllegaards Breeding Center Ltd., Skensved, Denmark) were maintained under standardized conditions of light and temperature, with free access to animal chow and water. The adult rats were either intact or hypophysectomized (Hx) at 6 weeks of age. Old and Hx rats were treated with vehicle or recombinant human GH (hGH) by continuous infusion from osmotic minipumps (model 2004, Alza Corp., Palo Alto, CA). hGH, a gift from Pharmacia & Upjohn, Inc. (Stockholm, Sweden), was administered to animals at a daily dose of 0.34 µg/g BW. After 3 weeks of treatment, the rats were killed, and liver samples were removed and frozen in liquid nitrogen. There were five animals in each group of rats.

cDNA synthesis
Total hepatic RNA was isolated from vehicle or hGH-treated rats using Trizol reagent (Life Technologies, Inc., Grand Island, NY), according to the protocol supplied by the manufacturer. Messenger RNA (mRNA) was purified from 1 mg total RNA using oligo-(deoxythymidine) paramagnetic beads (DynAl AS, Oslo, Norway). cDNA was synthesized from 2 µg mRNA using a cDNA synthesis kit purchased from Promega Corp. (Madison, WI).

RDA
RDA was performed according to a protocol developed by J. Odeberg et al. (31). Hepatic cDNA obtained from vehicle- and GH-treated intact old male rats were used as driver (vehicle) and tester (GH), or vice versa, to generate gene products that were induced or repressed by GH. After two rounds of subtraction and amplification, using hybridization tester-driver ratios of 1:100 and 1:800, difference products were run in a 1.8% agarose gel and stained with ethidium bromide to visualize bands. After being excised and eluted from the gel, eight bands were cloned in the BamHI site of the pBluescript II SK+ vector (Stratagene, La Jolla, CA). Between 20–30 isolated colonies were picked for each excised gel slice, clones were checked for inserts by PCR, and vector-specific primers (T7 and T3 primers obtained from Stratagene), and plasmid minipreparations were made using the Wizard system (Promega Corp.). Sequence analysis of differentially expressed cDNA products was performed using cycle sequencing with dye-labeled nucleotides (Big-Dye, Perkin-Elmer Corp., Norwalk, CT), and loaded on a PE Applied Biosystems 377 DNA sequencer (Perkin-Elmer Corp.). The sequences were analyzed for homologies with published sequences in the nonredundant and EST divisions of the public databases (GenBank, EMBL, DOBJ, and PDP) using the BlastN software (32).

Reverse Northern analysis
PCR fragments corresponding to 84 distinct RDA clones were diluted in 0.4 M NaOH and 0.5 M NaCl and arrayed in duplicate onto duplicate nylon membranes (HyBond-N, Amersham Pharmacia Biotech, Arlington Height, IL). The arrays were produced by spotting DNA solutions onto the membranes with a Biomek 2000 Laboratory Robot (Beckman Coulter, Inc., Palo Alto, CA) and a custom-designed high density replica tool (96 pins) with an inverted cup at the top of the pins that carries approximately 0.5 µl solution. The robot was programmed to produce filters with a density of 864 spots in a 96-well microtiter plate area. Individual spots in the array were obtained by repetitive transfer of DNA to the same location in the arrays. The replicating tool transferred approximately 2 µl PCR product (50 ng/µl)/spot. When control experiments were performed to determine the accuracy of the technique, this spotting procedure was shown to generate hybridization signals with an average interassay variation of 10%. The filter membranes were denatured with 0.2 M NaOH and 0.5 M NaCl for 2 min, dried, UV-cross-linked for 7 sec (700 kJ), and neutralized with 1.5 M NaCl and 0.5 M Tris-HCl (pH 7.5) for 2 min. cDNA corresponding to the housekeeping gene ß-actin was used as an internal control.

To generate the probes, 32P-labeled cDNA was synthesized from 0.1 µg hepatic mRNA derived from vehicle- or GH-treated intact old male rats. The mRNA was incubated with 1 µg random hexamers (Promega Corp.) for 10 min at 70 C, chilled on ice, and added to a solution containing 50 U Moloney murine leukemia virus reverse transcriptase (New England Biolabs, Inc., Beverly, MA) in 50 mM Tris-HCl (pH 8.3); 8 mM MgCl2; 10 mM dithiothreitol; 40 U RNasin (Promega Corp.); 0.5 mM each of deoxy (d)-ATP, dTTP, and dGTP, 5 µM dCTP; and 50 µCi [32P]dCTP in a final volume of 25 µl. After 1 h of incubation at 37 C, 1 µl 5 mM dCTP was added to the reaction mixture, followed by incubation at 37 C for 30 min and, finally, for 15 min at 50 C. The reaction was stopped by adding 2 µl 100 mM EDTA. Parental mRNA was removed from the cDNA synthesis reaction by adding 2.5 µl 1 M NaOH and incubating for 10 min at 65 C. The reaction was neutralized by adding 25 µl 1 M NaH2PO4. Unincorporated nucleotides were removed by gel filtration on Sephadex G-25 preloaded spin columns (Amersham Pharmacia Biotech, Uppsala, Sweden). Using this procedure, a total recovery of approximately 700 x 106 cpm/[32P]dCTP-labeled probe was obtained routinely. The probe was denatured at 90 C for 5 min before adding it to the hybridization solution.

The filter membranes were washed in 5 x SSPE (1 x solution contains 0.18 M NaCl, 10 mM NaH2PO4 (pH 7.4), and 5 mM EDTA) and prehybridized for 5 h at 42 C in a buffer containing 5 x SSPE, 5 x Denhardt’s [0.1% (wt/vol) each of Ficoll 400, polyvinylpyrrolidone, and BSA], 50% formamide, 0.5% SDS, and 0.5 mg/ml denatured salmon sperm DNA. Hybridization was carried out for 18 h at 42 C using 32P-labeled cDNA probes added in fresh prehybridization buffer. After the hybridization, the filters were washed (0.2 x SSC and 0.1% SDS at 50 C for 1 h) and analyzed in a phosphorimager (BAS-2500, Fuji Photo Film Co., Ltd., Tokyo, Japan) using the Image Gauge program. The results are expressed in relation to ß-actin cDNA levels on the respective filter. The experiment was repeated twice with similar results.

Solution hybridization analysis
Total nucleic acids (tNA) were isolated by homogenization of tissue specimens, using a Polytron PT-2000 (Kinematica AG, Littau, Switzerland). Digestion of samples with proteinase K (Merck & Co., Darmstadt, Germany) and subsequent extraction with chloroform and phenol have been described previously (33). mRNA levels corresponding to the expression of individual RDA clones were measured in tNA samples, using a solution hybridization/RNase protection assay. Transcript-specific 35S-labeled complementary RNA (cRNA) probes were transcribed in vitro from the respective cDNA vector construct according to the method of Melton et al. (34). Reagents for in vitro transcription were obtained from Promega Corp.

Hybridization of aliquots of tNA samples was performed in 40 µl 0.6 M NaCl, 22 nM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% (wt/vol) SDS, 1 mM dithiothreitol, 20% formamide, 10 µg/ml transfer RNA (bakers yeast transfer RNA from Boehringer Ingelheim GmbH Bioproducts Partnership, Heidelberg, Germany) and 15,000–20,000 cpm probe/incubation. After overnight incubation at 70 C, the samples were exposed to ribonucleases (RNase A and RNase T1, Boehringer Ingelheim GmbH Bioproducts Partnership), and the hybrids were precipitated by the addition of 100 µl 6 M trichloroacetic acid, collected on a glass-fiber filter (GF/C, Whatman Ltd., Madison, UK), and counted in a liquid scintillation counter.

The concentration of nucleic acids in tNA samples was measured spectrometrically. Samples were analyzed in triplicate, and the results are expressed as counts per min of specific mRNA/µg tNA. To permit accurate comparison of specific mRNA levels (corresponding to the expression of a specific RDA product) between different hormonal statuses, the samples of interest were always analyzed in the same assay. The data presented were statistically analyzed using Student’s t test.

mRNA levels corresponding to CYP2C12 (35) and ß-actin (36) expression were measured using specific cRNA probes, as described previously. Quantitations of these specific mRNA species were achieved by comparison with a standard curve obtained from hybridizations to known amounts of in vitro synthesized mRNA. These results are expressed as picograms of specific mRNA per µg tNA and in relation to ß-actin mRNA levels.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
cDNA RDA
To study GH-dependent changes in hepatic gene expression of old rats 2-yr-old male rats were infused with hGH for 3 weeks using osmotic minipumps. The GH-induced gene CYP2C12 (35) was used as a positive control for treatment efficacy. As demonstrated in Fig. 1Go, the effect of GH treatment varied between animals, ranging from 10- to 100-fold induction of CYP2C12 mRNA. To identify GH-regulated genes in rat liver, liver tissues from rat-1 (untreated) and rat-8 (GH-treated) were selected for further analysis in the cDNA RDA protocol.



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  		Figure 1. Hepatic CYP2C12 mRNA expression demonstrating efficacy of GH treatment in individual rats. Male rats, about 2 yr of age, were treated for 3 weeks with vehicle (black bars) or recombinant hGH (gray bars) by continuous infusion from osmotic minipumps. hGH was administered to animals at a daily dose of 0.34 µg/g BW. tNA samples were prepared from livers and analyzed in solution hybridization assays specific for CYP2C12 mRNA. Results are expressed as p per µg TNA. Values are the mean ± SD of triplicate determinations.

 
Both GH-induced (GH-up) and GH-reduced (GH-down) expressions were analyzed, using liver tissue from GH-treated rat 8 as tester (GH-up) or driver (GH-down). Liver tissue from untreated rat 1 was used as driver (GH-up) and tester (GH-down), respectively. Two successive rounds of subtraction and amplification, using hybridization tester-driver ratios of 1:100 and 1:800, generated two second difference products, DP2-up and DP2-down. The representations (digested and amplified cDNA), originating from untreated or treated rat livers, are shown in Fig. 2Go together with the obtained difference products. DP2-up and DP2-down consisted of bands from 200–500 bp in size. Eight bands from each DP2 were excised from the gel (Fig. 2Go), cloned in the BamHI site of the pBluescript II SK+ vector, and sequenced for further identification. Between 20–30 single colonies were selected from each band, sequenced, and compared against GenBank and dbEST databases using the BlastN software (32). Each band generated between 6–23 hits to known or unknown gene products (Table 1Go). As indicated in Table 1Go, up to 13 distinct sequences could be obtained from a single band. In total, 50 discrete sequences were identified among the DP2-up clones, and 14 were identified from DP2-down. The identities of the known sequences are summarized in Table 2Go. It should be noted that additional products might still be identified from this experiment, as gene products such as CYP2C7, serine protease inhibitor-2, and the GH receptor, which are all induced by GH in rat liver, are missing from the list of DP2-up clones.



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  		Figure 2. Agarose gel electrophoresis of representations and difference products identified by cDNA RDA. Ethidium bromide-visible bands in a 1.8% agarose gel correspond to size markers (A), representations of vehicle- (B) or GH-treated (E) rat livers, difference products 1 (DP1) after one round of subtraction/amplification (C and F), and difference products 2 (DP2) after two rounds of subtraction/amplification (D and G). cDNA obtained from vehicle-treated rats was used as tester to generate DP1-down and DP2-down in lanes C and D. Similarly, cDNA obtained from GH-treated rats was used as tester to generate DP1-up and DP2-up in lanes F and G.

 

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  		Table 1. Distribution of different sequences in the excised and cloned DP2 bands

 

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  		Table 2. Identification of differential cDNA clones obtained in RDA

 
In addition to the cDNA clones encoding known proteins (Table 2Go), at least 27 DP2-up clones did not show identity with any known sequence in the databases. Similarly, 12 unknown sequences were derived from the DP2-down clones. These new potentially GH-regulated gene products, corresponding to 20 distinct sequences, are listed in Table 3Go. The majority of these sequences were homologous to EST sequences characterized from rat, mouse, or human tissues. Sequences derived from potentially new rat transcripts, listed in Tables 2Go and 3Go, have been deposited in GenBank and the new accession numbers are available from the authors upon request.


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  		Table 3. Unidentified differential cDNA clones obtained in RDA

 
The success of the RDA protocol was demonstrated by the amplification and identification of gene products already known to be regulated by GH (indicated by underlining in Table 2Go). As shown in Table 2Go, the DP2-up clones included the GH-inducible CYP2C12 transcript. Similarly, gene products known to be suppressed by the continuous presence of GH, such as CYP2C13, {alpha}2u-globulin, and carbonic anhydrase III, were identified among the DP2-down clones. In addition to known GH-regulated transcripts, various gene products were identified that have not previously been shown to be controlled by GH (Table 2Go). Several transcripts encoding enzymes and receptors involved in the metabolism of protein, carbohydrates, and lipids were identified as DP2-up transcripts, including glutaryl-coenyzme A (glutaryl-CoA) dehydrogenase, acetyl-CoA carboxylase (ACC), farnesyl diphosphate synthase, and the glucagon receptor. Another group of transcripts was identified among the DP2-up clones, consisting of gene products encoding proteins involved in the immune system, such as {alpha}1-macroglobulin ({alpha}1M), pre-{alpha}-inhibitor H3, inter-{alpha}-inhibitor H4, preprocomplement C3, and C4 complement protein. The DP2-down clones encoding known proteins were almost all male-specific gene products previously reported to be suppressed by the continuous presence of GH. However, two new potentially GH-repressed genes were identified: hyaluronic acid binding protein and s-Myc. Another DP2-down clone encoded alcohol dehydrogenase, which has been shown to be induced (37), rather than suppressed, by GH. This clone might, therefore, be a false positive RDA product.

Screening for false positive clones
Initially, reverse Northern analysis was performed to confirm that the obtained cDNA RDA difference products truly represented differentially expressed transcripts. The different DP2 products were PCR amplified with vector-based primers and arrayed onto nylon filters together with ß-actin as an internal control. Hepatic polyadenylated RNA was isolated from the individual rats used in the RDA protocol (rats 1 and 8), and 32P-labeled cDNA was synthesized and used to probe the filters. The level of expression was quantified from a phosphorimage of the filters and calculated in relation to ß-actin mRNA levels. The results are presented in Fig. 3Go and summarized in Table 4Go. In this preliminary screening, five false positive clones (1B, 1E, 2C, 6A, and 8F) were identified among the DP2-up clones, and one (d-2T) was found among the DP2-down clones. The expression of ADH (d-2T) was slightly induced by GH in reverse Northern experiments, which is in agreement with previously reported data showing that hepatic expression of the ADH gene is induced by GH (37). The ADH transcript should thus have been among the DP2-up clones rather than among the DP2-down clones. However, it cannot be excluded that GH suppresses a closely related ADH gene product, with high sequence homology to d-2T, in old male rats.



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  		Figure 3. Reverse Northern blot demonstrating differentially expressed RDA products. PCR fragments corresponding to 84 distinct RDA clones were arrayed in duplicate onto duplicate nylon membranes together with ß-actin as an internal control. The membranes were hybridized to 32P-labeled cDNA probes generated from hepatic mRNA obtained from vehicle-treated (A) or GH-treated (B) rats. The identity of the spots is described in C. The RDA clones corresponding to gene products encoding proteins of unknown function are indicated by italic letters. The results were analyzed in a phosphorimager, expressed in relation to ß-actin, and summarized in Table 4Go. The lower intensity of the ß-actin signal in B compared with that in A is due to slightly lower specific activity of the cDNA probe obtained from the GH-treated liver. The experiment was repeated twice.

 

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  		Table 4. GH-induced changes in hepatic expression of DP2 clones in old male rats

 
If a particular DP2 sequence on the filters used to hybridize to the labeled cDNA has high homology to another gene product, which is oppositely regulated by GH, the net result could be no GH regulation. This is exemplified by the hybridization results obtained for CYP2C12 and CYP2C13 (Table 4Go). These CYP genes are well known to be transcriptionally up- or down-regulated, respectively, by the continuous presence of GH (38). The CYP2C12 sequence amplified in the RDA (2N) shows 88% identity with CYP2C13. Although the CYP2C12 mRNA levels were shown to be induced 100-fold in the liver of the GH-treated rat selected for the RDA (rat 8), compared with those in the untreated rat (rat 1; Fig. 1Go), only a 5-fold difference in expression levels could be detected by the reverse Northern protocol. Similarly, the CYP2C13 sequence of clone d-1B is 84% homologous to CYP2C12, explaining the small effect of GH on CYP2C13 expression in the reverse Northern blot. In addition to the limitations due to cross-reactivity between different cDNAs, the reverse Northern protocol is not as sensitive, for example, RNase protection analysis, where the hybridization occurs in solution.

To further characterize our RDA results we next performed RNase protection/solution hybridization analysis with specific 35S-labeled cRNA probes for each gene product. tNA samples were prepared from the RDA rats (rats 1 and 8) and hybridized with the different probes. As demonstrated in Table 4Go, the results obtained with the solution hybridization protocol were slightly different from the reverse Northern data. In particular, the GH-induced changes in mRNA expression of {alpha}2u-globulin, CYP3A2, carbonic anhydrase III, and 3ß-hydroxysteroid dehydrogenase were larger according to this analysis (Table 4Go). Furthermore, 3 of the clones that were not differentially expressed according to the reverse Northern results (2C, 6A, and 8F) did show a small GH-dependent difference in mRNA levels when analyzed by solution hybridization. However, other gene products, such as fibrinogen, GDP dissociation inhibitor, and retinol-binding protein turned out to be less differentially expressed, and a total of 14 false positives were identified. Low abundant transcripts are easier to detect in solution hybridization protocols using total RNA rather than tNA samples, and it is therefore possible that slightly different results would have been obtained in this assay using total RNA instead of tNA. The lack of GH-regulated expression of the ADH gene suggests that this assay needs to be improved for the detection of certain gene products. Taken together, although the majority of the obtained DP2 clones were confirmed as differentially expressed transcripts, additional and more detailed analyses are required to definitely rule out that the false positives are not, in fact, GH regulated.

Effect of GH on putative GH-regulated genes in aged or hypophysectomized male rats
To study the biological significance of the above-discussed RDA results, the GH-regulated expression of the gene products listed in Table 2Go was initially determined in livers from a pool of GH-treated old male rats (rats 6–10) and compared with that in untreated rats (rats 1–5). As shown in Table 4Go, the GH-induced changes in mRNA levels were, in general, smaller between the 2 pools of rats compared with the differences observed between the 2 rats selected for the RDA protocol. These results are in line with the data presented in Fig. 1Go, showing a marked difference in GH-induced expression of CYP2C12 mRNA between the individual GH-treated rats. In addition, some gene products were equally well expressed in the 2 pools of rats, indicating that these transcripts are not truly GH regulated. These gene products were probably differentially expressed between the individual RDA rats due to other uncontrolled differences not related to GH treatment. In summary, 20 distinct DP2 clones (indicated by italics in Table 4Go) were identified as GH-regulated gene products in pooled rat liver samples, including 13 new potentially GH-regulated transcripts.

A more careful study of selected gene products was next performed in individual animals. The effects of aging (Fig. 4AGo) and hypophysectomy (Fig. 4BGo) as well as GH responsiveness in old or Hx rats were determined in solution hybridization assays. As shown in Fig. 4AGo, the expressions of glutathione-S-transferase (GST; 1R), D-dopachrome tautomerase (DDT; 1V), {alpha}1M (3I), ATP synthase subunit 9 (4Z), and s-Myc (d-6P) were reduced in aged (2-yr-old) compared with normal male rats. When the old rats were treated with GH for 3 weeks, the expressions of GST, DDT, {alpha}1M, and ATP synthase subunit 9 were induced, whereas s-Myc mRNA levels were reduced even further. GH treatment of old male rats thus normalized the mRNA levels of GST, DDT, {alpha}1M, and ATP synthase subunit 9, but suppressed s-Myc expression to 25% of the level in normal rats. Other gene products, including glutamate dehydrogenase (7S), ribosomal protein L23a (5N), farnesyl diphosphate synthase (1C), ACC (1I), H-rev107 (6R), and the glucagon receptor (8F), were less affected by age, but increased upon GH treatment, leading to elevated mRNA levels in GH-treated old rats compared with normal rats.



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  		Figure 4. Effects of aging, hypophysectomy, and GH treatment on hepatic mRNA expression of selected RDA clones in male rats. Old (2 yr of age) or Hx male rats (8 weeks of age) were treated for 3 weeks with vehicle or hGH by continuous infusion from osmotic minipumps. tNA samples were prepared from normal (8 weeks of age), old, or Hx male rats and analyzed in solution hybridization assays specific for the different RDA mRNA species. A, Effects of aging and GH treatment of old rats; B, effects of hypophysectomy and GH treatment of Hx rats. Results are expressed in relation to ß-actin, and are summarized in Table 5Go. Values are the mean ± SE of five animals. *, Significantly different from normal male; ¤, significantly different from the untreated old male (A) or the untreated Hx male (B), P < 0.05

 
Removal of the pituitary in combination with restoration of serum GH levels is the classical way to study GH-dependent gene expression in vivo. To further establish the role of GH in the expression of our differentially expressed RDA products, the effects of hypophysectomy and subsequent treatment with GH were evaluated in male rats (8 weeks of age). As presented in Fig. 4BGo, removal of the pituitary in male rats led to suppression of all gene products analyzed, except for ribosomal protein L23a and H-rev107. The observation that expression of these gene products was almost unaffected by removal of the pituitary suggests that neither GH nor other pituitary factors are important regulators of these genes in normal (not aged) rats. However, GH induced both mRNA species in Hx rats, mimicking the effect of GH in old rats. Two other RDA products, ATP synthase and the glucagon receptor, were reduced in Hx animals, but were not restored upon GH treatment, indicating that other pituitary factors are involved in the control of these genes in normal male rat liver.

GH treatment of Hx rats led to superinduction (above the levels in intact rats) of GST, DDT, and {alpha}1M. GH "only" restored the expression of these genes in the old rats, indicating that these transcriptional effects of GH are negatively modulated by one or several pituitary factors. In contrast, the mRNA expressions of glutamate dehydrogenase, farnesyl diphosphate synthase, and ACC were superinduced by GH in the old rats and only restored to normal levels in Hx rats. In this case, a pituitary factor that potentiates the GH effect could be postulated. The different effects of aging, hypophysectomy, and GH treatment are summarized in Table 5Go. Three main categories of gene products could be predicted. The first group, consisting of only one member, s-Myc, is characterized by an age-associated decline in mRNA expression and further reduction after GH treatment. The second group consists of gene products that were reduced in old rats and restored or superinduced in GH-treated animals. Members of the third group were not affected by age, but were induced by GH, leading to elevated mRNA levels compared with those in normal rats.


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  		Table 5. Classification of GH-regulated transcripts

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The data presented here clarify that the aged rat liver is responsive toward GH, although distinct target genes might differ in sensitivity. We also describe the identification of new GH-responsive genes in rat liver. GH treatment of 2-yr-old male rats led to the induction or suppression of several hepatic gene products. The identity could be described for 81% of these RDA clones. Among the identified gene products, 8 (4%) are previously characterized as GH regulated, whereas at least 12 (7%) constitute potentially new GH-regulated transcripts. Fifteen percent of the RDA clones turned out to be false positives. The unknown RDA products might also include new GH-regulated genes, but the identities of those have to await further analysis. Some of the identified RDA products were further characterized in terms of GH responsiveness, and different categories of gene products could be described. Seven RDA clones showed an age-dependent decline in mRNA levels, whereas 4 were unaffected, implicating that distinct GH-regulated gene products are differently modulated by age and the reduction in serum concentrations of GH that occurs during aging. Furthermore, the reduced expression of these genes might be involved in some of the age-related physiological and pathological changes in old rats.

One theory of aging proposes that reactive oxygen species (ROS) that are generated by metabolism cause cumulative damage over a lifetime (39). Roughly 2–3% of oxygen taken up is chemically reduced by the addition of single electrons and converted into ROS, which have been shown to cause molecular damage to proteins, lipids, and nucleic acids (40). However, tissues are protected against this damage by antioxidant enzymes, such as superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and GST (41, 42). One of our RDA products (1R) encoded a GST that was highly expressed in normal male rat livers, reduced in aged or Hx rats, and induced by GH in both old and Hx rats. The age-related decline in expression of this GST might thus serve as an example of how GH-regulated genes might be involved in age-related disorders.

The GSTs are products of a gene superfamily (43). These dimeric proteins catalyze the conjugation of glutathione with a variety of electrophilic compounds, rendering them nontoxic (42). They are composed of 3 classes of subunits according to protein sequence similarity and antibody cross-reactivity: {alpha}, µ, and {pi}. A fourth GST is a unique membrane-bound transferase that is structurally distinct from the cytosolic forms (44). Our RDA clone 1R is not identified as a rat GST, but shows 98% homology with an EST sequence (EST221686) from normalized rat placenta. The best alignment to sequences encoding known proteins was obtained with bovine GST class {pi} mRNA (45), which is 85% homologous to 1R. This indicates that we might have identified a new member of the GST superfamily.

The RDA clones 1V (DDT) and 3I ({alpha}1M) showed similar patterns of mRNA expression as GST (1R), with high mRNA levels in normal male rat livers and low levels in aged rats. These genes have not previously been shown to be regulated by GH, and very little is known about their precise physiological functions, although both DDT and {alpha}1M have been implicated to play a role in the immune system. DDT tautomerizes D-dopachrome, with concomitant decarboxylation to give 5,6-dihydroxyindole. The decolorization of D-dopachrome with the formation of 5,6-dihydroxyindole was used in monitoring the isolation of the protein from rat liver, and therefore, the enzyme was provisionally named D-dopachrome tautomerase (46). However, conversion of D-dopachrome does not appear to be the physiological function of the enzyme, as D-dopachrome is not present in the organism. The cDNA encoding DDT was subsequently cloned from rat (47), mouse (48) and human (49) liver. In all species DDT was shown to have an amino acid sequence homologous with that of macrophage migration inhibitory factor (MIF). In addition, the gene for DDT in human and mouse was identical in exon structure to MIF (48). MIF has been described to be an anterior pituitary hormone and to be released from immune cells stimulated by low concentrations of glucocorticoids (50). Once secreted, MIF acts to control, or counterregulate, the immunosuppressive effects of glucocorticoids on the immune system. MIF and DDT are not only related by sequence, but also by in vitro enzyme activity, as MIF has the potential to catalyze D-dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (51). Although the substrate does not occur naturally, the observation that MIF has tautomerase activity suggests that MIF may mediate its biological effects by an enzymatic reaction. Based on the homology with MIF, it has been suggested that DDT might play an important biological role in inflammation and immunity.

{alpha}1M has also been implicated as playing a role in the immune system. This macroglobulin occurs naturally at high concentrations in plasma (52), and its mRNA is expressed abundantly in various tissues (53). {alpha}1M belongs to the {alpha}M family, which is comprised of tetrameric, dimeric, and monomeric glycoproteins, including plasma proteinase inhibitors as well as the complement components C3, C4, and C5 (54). By binding to {alpha}Ms, proteinases and perhaps other biological targets become destined to rapid clearance and degradation (54). By controlling the activity of proteinases, they have been implicated in modulating reactions in the immune response and the defense mechanism against invading pathogens. Other target proteinases for {alpha}Ms may be found among those that function in the extracellular matrix, participating in growth and tissue remodeling, or those that are released during cellular turnover. In summary, although the precise pathophysiological functions of DDT and {alpha}1M remain to be elucidated, the suppression of DDT and {alpha}1M mRNA levels in aged rats and the restoration by GH treatment indicate that these gene products may participate in mediating the improved function of immune cells demonstrated in GH-treated animals. Thus, the roles of these proteins in aging and immune responses warrant further investigations.

The action of GH is anabolic and affects the intermediary metabolism of protein, carbohydrates, and lipids (3). It is therefore not surprising that GH treatment of old rats leads to the induction of transcripts encoding proteins involved in, for example, protein synthesis. Two RDA clones, encoding glutamate dehydrogenase (7S) and the ribosomal protein L23a (rp L23a; 5N), were induced about 2-fold in livers of old rats treated with GH. Glutamate dehydrogenase is a mitochondrial matrix enzyme that catalyses the interconversion between {alpha}-ketoglutarate, a citric acid cycle intermediate, and L-glutamate (55). The GH-induced expression of glutamate dehydrogenase mRNA might be one way by which the cell can increase the rate of protein synthesis during physiological conditions such as growth stimulation. The effect of GH on protein synthesis has also been shown to involve increased transport of amino acids, decreased usage of amino acids for energy expenditure, and an apparent increase in ribosomal efficiency (3, 56). The GH-induced activation of the translational machinery might be due at least in part to an increased expression of the ribosomal components, such as the rp L23a. Analysis of rp-gene promoters in higher eukaryotes have to date only elucidated features typical of housekeeping genes (57). Transcriptional regulation of rp-genes mediated by endocrine factors has to our knowledge not been reported previously. Whether the observed effects on glutamate dehydrogenase and rp L23a are direct effects of GH on the hepatocyte or indirect effects mediated by, for example, increased availability of amino acids has to await further investigations. It is also of importance to elucidate whether these effects are restricted to the liver or whether the GH-mediated increase in protein synthesis and lean body mass might include an elevated expression of glutamate dehydrogenase and rp L23a mRNA in extrahepatic tissues.

Another group of RDA clones (1I, 4Z, and 8F) encodes proteins that are important in fuel metabolism and ATP synthesis. As stated above, GH serves as an anabolic hormone that mediates the sparing of protein stores at the expense of fat. Both inhibited glucose utilization and increased lipolysis characterize the effect of GH. Thus, GH treatment of old rats would lead to a decrease in body fat and an increase in serum FFA levels. One RDA product (1I) encodes ACC, which is the rate-limiting enzyme in the biogenesis of long chain fatty acids (58). The expression of ACC mRNA in rat hepatocytes is inhibited by fatty acids and stimulated by insulin (59). The effect of GH on hepatic ACC mRNA levels observed in this study could thus be interpreted as GH being able to override the inhibitory effect mediated by fatty acids. It is also possible that the effect is secondary and due to an increased secretion of insulin, as elevated levels of FFA in serum stimulate the production of insulin (60). Although ACC activity is also controlled at the level of enzymatic activity, so that high levels of acyl-CoA suppresses the synthesis of new fatty acids (61), it seems strange that GH would stimulate the production of more ACC. However, ACC is also thought to play a role in the control of fatty acid oxidation (59). Mammalian ACC is present in two isoforms, both of which catalyze the formation of malonyl-CoA from acetyl-CoA and CO2 (62). ACC-{alpha} is the rate-limiting enzyme in the biogenesis of long fatty acids, but ACC-ß is also believed to control mitochondrial fatty acid oxidation (59). ACC-{alpha} is predominantly expressed in adipose tissue, whereas ACC-ß is found in tissues such as heart and skeletal muscle, which mainly use fatty acids for ATP production. Little is known about the regulation of ACC-ß, but it is not inconceivable that a lipolytic hormone such as GH could, directly or indirectly via increased FFA levels, induce the level of an enzyme involved in the oxidation of fatty acids. In the liver, the increased availability of FFA for energy metabolism would thus be accompanied by an increased ability to generate ATP in the mitochondria.

In this context, the observation that ATP synthase subunit 9 (clone 4Z) mRNA was induced by GH in livers of old rats, further strengthens the correlation between increased lipolysis in fat cells and an increased usage of fatty acids in the generation of ATP. Mitochondrial ATP synthase is a multisubunit enzyme that catalyses ATP synthesis during oxidative phosphorylation (63). This enzyme is responsible for the production of most of the ATP in mammalian organisms (64). ATP synthase consists of two major components, F1 and F0, linked by a slender stalk (65). The F0 part translocates the protons and transduces the energy of the proton electrochemical gradient to the catalytic F1 part. ATP synthase subunit 9 is a component of F0 part (63). In humans, three isoforms of subunit 9 have been identified, encoded by the nuclear genes P1, P2 (66), and P3 (67), whereas only P1 and P2 have been characterized in the rat (68). Based on the correlation between subunit 9 expression and adenosine triphosphatase content in tissues, it has been suggested that the expressions of the P1 and P2 genes play a pivotal role in the control of adenosine triphosphatase biosynthesis in mice (69). Our RDA clone 4Z showed highest homology with human P3 (ATP5G3) cDNA (67), indicating the presence of isoform 3 also in rats. To our knowledge nothing is known about the transcriptional regulation of the P3 gene.

Many molecular mechanisms of aging have been proposed, including oxidative damage, genome instability, teleomere shortening, and decreased levels of endocrine factors (70). In a recent publication by Lee et al. (71), a gene expression profile was described for the aging process in mouse skeletal muscle. The list of transcriptional changes induced by aging included reduced levels of transcripts involved in energy metabolism and increased expression of gene products involved in stress responses. It has previously been speculated that an age-associated increase in mitochondrial dysfunction might be the cause of elevated production of ROS in old animals. The results obtained by Lee et al. support this hypothesis. Furthermore, caloric restriction, which is known to slow the intrinsic rate of aging, was shown to contrast many of these effects. Interestingly, some of the gene products that were affected by CR in old mice were identical to GH-responsive transcripts described in the present study of old rats. This might be explained by the fact that the secretion of GH is increased in animals subjected to a reduction in caloric intake (72, 73). In summary, endocrine factors such as GH may turn out to play an important role in at least some effects of animal aging. Further investigations are needed, however, to clarify the physiological function of the gene products identified and the link between these products and age-related disorders. Future experiments, using reverse genetics in both animal and cell models, may provide such information.


   Acknowledgments
 
We thank Eva Johansson for skillful technical assistance, and Dr. Agneta Mode for the kind gift of the CYP2C12 probe.


   Footnotes
 
1 This work was supported by grants from the Swedish Medical Research Council, [13X-08556 and 03X-3972(LS)], the Swedish Society of Medicine, and the Knut and Alice Wallenberg Foundation. Back

Received August 3, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Isaksson OG, Edén S, Jansson J-O 1985 Mode of action of pituitary growth hormone on target cells. Annu Rev Physiol 47:483–499[CrossRef][Medline]
  2. Angelin B, Rudling M 1994 Growth hormone and hepatic lipoprotein metabolism. Curr Opin Lipidol 5:160–165[Medline]
  3. Berneis K, Keller U 1996 Metabolic actions of growth hormone: direct and indirect. Bailliere Clin Endocrinol Metab 10:337–352[CrossRef][Medline]
  4. Gustafsson J-Å, Mode A, Skett P 1983 Sex steroid induced changes in hepatic enzymes. Ann. Rev. Physiol. 45:51–60
  5. Zaphiropoulos PG, Mode A, Norstedt G, Gustafsson J-Å 1989 Regulation of sexual differentiation in drug and steroid metabolism. Trends Pharmacol Sci 10:149–153[CrossRef][Medline]
  6. Burgess W, Liu Q, Zhou J, Tang Q, Ozawa A, VanHoy R, Arkins S, Dantzer R, Kelley K 1999 The immune-endocrine loop during aging: role of growth hormone and insulin-like growth factor-I. Neuroimmunomodulation 6:56–68[CrossRef][Medline]
  7. Christiansen J, Jörgensen J, Pedersen S, Muller J, Jörgensen J, Muller J, Heickendorf L, Skakkebaek N 1991 GH-replacement therapy in adults. Horm Res 36:66–72
  8. Vandeweghe M, Taelman P, Kaufman J 1993 Short and long-term effects of growth hormone treatment on bone turnover and bone mineral content in adult growth hormone-deficient males. Clin Endocrinol (Oxf) 39:409–415[Medline]
  9. Rosen T, Bengtsson B 1990 Premature mortality due to cardiovascular disease in hypopituitarism. Lancet 336:285–288[CrossRef][Medline]
  10. Verhelst J, Abs R, Vandeweghe M, Mockel J, Legros J, Copinschi G, Mahler C, Velkeniers B, Vanhaelst L, Van Aelst A, De Rijdt D, Stevenaert A, Beckers A 1997 Two years of replacement therapy in adults with growth hormone deficiency. Clin Endocrinol (Oxf) 47:485–494[CrossRef][Medline]
  11. Vance M 1998 The Gordon Wilson Lecture. Growth hormone replacement in adults and other uses. Trans Am Clin Climatol Assoc 109:87–96[Medline]
  12. Beshyah S, Shahi M, Foale R, Johnston D 1995 Cardiovascular effects of prolonged growth hormone replacement in adults. J Intern Med 237:35–42[Medline]
  13. Pfeifer M, Verhovec R, Zizek B, Prezelj J, Poredos P, Clayton R 1999 Growth hormone (GH) treatment reverses early atherosclerotic changes in GH-deficient adults. J Clin Endocrinol Metab 84:453–457[Abstract/Free Full Text]
  14. Jörgensen J, Pedersen S, Thuesen L, Jörgensen J, Ingemann-Hansen T, Skakkebaek N, Christiansen J 1989 Beneficial effects of growth hormone treatment in GH-deficient adults. Lancet 1:1221–1225[Medline]
  15. Cuneo R, Salomon F, Wilmshurst P, Byrne C, Wiles C, Hesp R, Sonksen P 1991 Cardiovascular effects of growth hormone treatment in growth-hormone-deficient adults: stimulation of the renin-aldosterone system. Clin Sci (Colch) 81:587–592[Medline]
  16. Timsit J, Savino W, Safieh B, Chanson P, Gagnerault M, Bach J, Dardenne M 1992 Growth hormone and insulin-like growth factor-I stimulate hormonal function and proliferation of thymic epithelial cells. J Clin Endocrinol Metab 75:183–188[Abstract]
  17. Kann P, Piepkorn B, Schehler B, Andreas J, Lotz J, Prellwitz W, Beyer J 1998 Effect of long-term treatment with GH on bone metabolism, bone mineral density and bone elasticity in GH-deficient adults. Clin Endocrinol (Oxf) 48:561–568[CrossRef][Medline]
  18. Rodriguez-Arnao J, James I, Jabbar A, Trainer P, Perrett D, Besser G, Ross R 1998 Serum collagen crosslinks as markers of bone turn-over during GH replacement therapy in growth hormone deficient adults. Clin Endocrinol (Oxf) 48:455–462[CrossRef][Medline]
  19. Jörgensen J, Muller J, Alberti K, Schmitz O, Christiansen J, Orskov H, Moller N 1993 Marked effects of sustained low growth hormone (GH) levels on day-to-day fuel metabolism: studies in GH-deficient patients and healthy untreated subjects. J Clin Endocrinol Metab 77:1589–1596[Abstract]
  20. Kelijman M 1991 Age-related alterations of the growth hormone/insulin-like-growth-factor I axis. J Am Geriatr Soc 39:295–307[Medline]
  21. Corpas E, Harman S, Blackman M 1993 Human growth hormone and human aging. Endocr Rev 14:20–39[CrossRef][Medline]
  22. Strobl J, Thomas M 1994 Human growth hormone. Pharmacol Rev 46:1–34[Abstract]
  23. Rudman D, Feller A, Nagraj H, Gergans G, Lalitha P, Goldberg A, Schlenker R, Cohn L, Rudman I, Mattson D 1990 Effects of human growth hormone in men over 60 years old. N Engl J Med 323:1–6[Abstract/Free Full Text]
  24. Daughaday W, Rotwein P 1989 Insulin-like growth factors I and II. Peptide, messenger ribonucleic acid and gene structures, serum, and tissue concentrations. Endocr Rev 10:68–91[CrossRef][Medline]
  25. Florini J, Harned J, Richman R, Weiss J 1981 Effect of rat age on serum levels of growth hormone and somatomedins. Mech Ageing Dev 15:165–176[CrossRef][Medline]
  26. Velasco B, Cacicedo L, Escalada J, Lopez-Fernandez J, Sanchez-Franco F 1998 Growth hormone gene expression and secretion in aging rats is age dependent and not age-associated weight increase related. Endocrinology 139:1314–1320[Abstract/Free Full Text]
  27. Xu X, Bennett S, Ingram R, Sonntag W 1995 Decreases in growth hormone receptor signal transduction contribute to the decline in insulin-like growth factor I gene expression with age. Endocrinology 136:4551–4557[Abstract]
  28. Lisitsyn N, Lisitsyn N, Wigler M 1993 Cloning the differences between two complex genomes. Science 259:946–951[Abstract]
  29. Hubank M, Schatz D 1994 Identifying differences in mRNA expression by representational difference analysis of cDNA. Nucleic Acids Res 22:5640–5648[Abstract/Free Full Text]
  30. Melia M, Bofill N, Hubank M, Meseguer A 1998 Identification of androgen-regulated genes in mouse kidney by representational difference analysis and random arbitrarily primed polymerase chain reaction. Endocrinology 139:688–695[Abstract/Free Full Text]
  31. Odeberg J, Wood T, Blucher Å, Rafter J, Norstedt G, Lundeberg J A cDNA RDA protocol based on solid-phase technology suited for analysis of gene expression in small tissue samples. BioTechniques, in press
  32. Altschul S, Gish W, Miller W, Myers E, Lipman D 1990 Basic local alignment search tool. J Mol Biol 215:403–410[CrossRef][Medline]
  33. Durnam DM, Palmiter RP 1983 A practical approach for quantitating specific mRNA by solution hybridization. Anal Biochem 131:383–393
  34. Melton DA, Krieg PA, Rebagliati MR, Maniatis T, Zinn K, Green MR 1984 Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promotor. Nucleic Acids Res 12:7035–7056[Abstract/Free Full Text]
  35. Mode A, Wiersma-Larsson E, Gustafsson J-Å 1989 Transcriptional and posttranscriptional regulation of sexually differentiated rat liver cytochrome P-450 by growth hormone. Mol Endocrinol 3:1142–1147[CrossRef][Medline]
  36. Nudel U, Zakut R, Shani M, Neuman S, Levy Z, Yaffe D 1983 The nucleotide sequence of the rat cytoplasmic ß-actin gene. Nucleic Acids Res 11:17759–1771
  37. Potter J, Yang V, Mezey E 1993 Regulation of the rat class I alcohol dehydrogenase gene by growth hormone. Biochem Biophys Res Commun 191:1040–1045[CrossRef][Medline]
  38. Legraverend C, Mode A, Westin S, Ström A, Eguchi H, Zaphiropoulos P, Gustafsson J-Å 1992 Transcriptional regulation of rat P-450 2C gene subfamily members by the sexually dimorphic pattern of growth hormone secretion. Mol Endocrinol 6:259–266[Abstract]
  39. Harman D 1981 The aging process. Proc Natl Acad Sci USA 78:7124–7128[Abstract/Free Full Text]
  40. Wei Y, Lu C, Lee H, Pang C, Ma Y 1998 Oxidative damage and mutation to mitochondrial DNA and age-dependent decline of mitochondrial respiratory function. Ann NY Acad Sci 854:155–170[CrossRef][Medline]
  41. Mates J, Sanchez-Jimenez F 1999 Antioxidant enzymes and their implications in pathophysiologic processes. Front Biosci 4:D339–345
  42. Ketterer B 1998 Glutathione S-transferases and prevention of cellular free radical damage. Free Radical Res 28:647–658[Medline]
  43. Hayes J, Strange R 1995 Potential contribution of the glutathione S-transferase supergene family to resistance to oxidative stress. Free Radical Res 22:193–207[Medline]
  44. Morgenstern R, Guthenberg C, DePierre J 1982 Microsomal glutathione S-transferase. Purification, initial characterization and demonstration that it is not identical to the cytosolic glutathione S-transferases A, B and C. Eur J Biochem 128:243–248[Medline]
  45. Hernando N, Martin-Alonso J, Ghosh S, Coca-Prados M 1992 Isolation of a cDNA encoding a glutathione S-transferase (GST) class-pi from the bovine ocular ciliary epithelium. Exp Eye Res 55:711–718[CrossRef][Medline]
  46. Odh G, Hindemith A, Rosengren A, Rosengren E, Rorsman H 1993 Isolation of a new tautomerase monitored by the conversion of D-dopachrome to 5,6-dihydroxyindole. Biochem Biophys Res Commun 197:619–624[CrossRef][Medline]
  47. Zhang M, Aman P, Grubb A, Panagopoulos I, Hindemith A, Rosengren E, Rorsman H 1995 Cloning and sequencing of a cDNA encoding rat D-dopa-chrome tautomerase. FEBS Lett 373:203–206[CrossRef][Medline]
  48. Esumi N, Budarf M, Ciccarelli L, Sellinger B, Kozak C, Wistow G 1998 Conserved gene structure and genomic linkage for D-dopachrome tautomerase (DDT) and MIF. Mamm Genome 9:753–757[CrossRef][Medline]
  49. Nishihira J, Fujinaga M, Kuriyama T, Suzuki M, Sugimoto H, Nakagawa A, Tanaka I, Sakai M 1998 Molecular cloning of human D-dopachrome tautomerase cDNA: N-terminal proline is essential for enzyme activation. Biochem Biophys Res Commun 243:538–544[CrossRef][Medline]
  50. Donnelly S, Bucala R 1997 Macrophage migration inhibitory factor: a regulator of glucocorticoid activity with a critical role in inflammatory disease. Mol Med Today 3:502–507[CrossRef][Medline]
  51. Rosengren E, Bucala R, Aman P, Jacobsson L, Odh G, Metz C, Rorsman H 1996 The immunoregulatory mediator macrophage migration inhibitory factor (MIF) catalyzes a tautomerization reaction. Mol Med 2:143–149[Medline]
  52. Gordon A 1976 The {alpha} macroglobulins of rat serum. Biochem J 159:643–650[Medline]
  53. Eggertsen G, Hudson G, Shiels B, Reed D, Fey G 1991 Sequence of rat {alpha}1-macroglobulin, a broad-range proteinase inhibitor from the {alpha}-macroglobulin-complement family. Mol Biol Cell 8:287–302[Abstract]
  54. Sottrup-Jensen L 1989 {alpha}-Macroglobulins: structure, shape, and mechanism of proteinase complex formation. J Biol Chem 264:11539–11542[Free Full Text]
  55. Nelson R, Povey M, Hopkinson D, Harris H 1977 Electrophoresis of human l-glutamate dehydrogenase: tissue distribution and preliminary population survey. Biochem Genet 15:87–91[CrossRef][Medline]
  56. Kostyo J 1968 Rapid effects of growth hormone on amino acid transport and protein synthesis. Ann NY Acad Sci. 148:389–407
  57. Mager W 1988 Control of ribosomal protein gene expression. Biochim Biophys Acta 949:1–15[Medline]
  58. Ha J, Kim K 1994 Inhibition of fatty acid synthesis by expression of an acetyl-CoA carboxylase-specific ribozyme gene. Proc Natl Acad Sci USA 91:9951–9955[Abstract/Free Full Text]
  59. Kim K 1997 Regulation of mammalian acetyl-coenzyme A carboxylase. Annu Rev Nutr 17:77–99[CrossRef][Medline]
  60. Madison L, Seyffert WJ, Unger R, Barker B 1968 Effect on plasma free fatty acids on plasma glucagon and serum insulin concentrations. Metabolism 17:301–304[CrossRef][Medline]
  61. Kim K, Lopez-Casillas F, Bai D, Luo X, Pape M 1989 Role of reversible phosphorylation of acetyl-CoA carboxylase in long-chain fatty acid synthesis. FASEB J 3:2250–2256[Abstract]
  62. Thampy K 1989 Formation of malonyl coenzyme A in rat heart. Identification and purification of an isozyme of A carboxylase from rat heart. J Biol Chem 264:17631–17634[Abstract/Free Full Text]
  63. Walker J, Lutter R, Dupuis A, Runswick M 1991 Identification of the subunits of F1F0-ATPase from bovine heart mitochondria. Biochemistry 30:5369–5378[CrossRef][Medline]
  64. Attardi G, Schatz G 1988 Biogenesis of mitochondria. Annu Rev Cell Biol 4:289–333[CrossRef]
  65. Walker J, Collinson I 1994 The role of the stalk in the coupling mechanism of F1F0-ATPases. FEBS Lett 346:39–43[CrossRef][Medline]
  66. Dyer M, Walker J 1993 Sequences of members of the human gene family for the c subunit of mitochondrial ATP synthase. Biochem J 293:51–64
  67. Yan W, Lerner T, Haines J, Gusella J 1994 Sequence analysis and mapping of a novel human mitochondrial ATP synthase subunit 9 cDNA (ATP5G3). Genomics 24:375–377[CrossRef][Medline]
  68. Sangawa H, Himeda T, Shibata H, Higuti T 1997 Gene expression of subunit c(P1), subunit c(P2), and oligomycin sensitivity-conferring protein may play a key role in biogenesis of H+-ATP synthase in various rat tissues. J Biol Chem 272:6034–6037[Abstract/Free Full Text]
  69. Houstek J, Andersson U, Tvrdik P, Nedergaard J 1995 The expression of subunit c correlates with and thus may limit the biosynthesis of the mitochondrial F0F1-ATPase in brown adipose tissue. J Biol Chem 270:7689–7694[Abstract/Free Full Text]
  70. Johnson F, Sinclair D, Guarente L 1999 Molecular biology of aging. Cell 96:291–302[CrossRef][Medline]
  71. Lee C, Klopp R, Weindruch R, Prolla T 1999 Gene expression profile of aging and its retardation by caloric restriction. Science 285:1390–1393[Abstract/Free Full Text]
  72. Arce V, Cella S, Locatelli V, Muller E 1991 Studies of growth hormone secretion in calorically restricted dogs: effect of cholinergic agonists and antagonists, glucose and thyrotropin-releasing hormone. Neuroendocrinology 53:467–472[Medline]
  73. Sonntag W, Xu X, ngram R, D’Costa A 1995 Moderate caloric restriction alters the subcellular distribution of somatostatin mRNA and increases growth hormone pulse amplitude in aged animals. Neuroendocrinology 61:601–608[Medline]



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