Gene Expression Profile of the Aging Process in Rat Liver: Normalizing Effects of Growth Hormone Replacement

Petra Tollet-Egnell, Amilcar Flores-Morales, Nina Ståhlberg, Renae L. Malek, Norman Lee and Gunnar Norstedt

Department of Molecular Medicine (P.T.-E., A.F.-M., N.S., G.N.) Karolinska Institutet Karolinska Hospital 171 76 Stockholm, Sweden
The Institute for Genomic Research (N.L., R.L.M.) Rockville, Maryland 20850


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The mechanisms that control life span and age-related phenotypes are not well understood. It has been suggested that aging or at least some of its symptoms are related to a physiological decline in GH levels with age. To test this hypothesis, and to improve our understanding of the cellular and molecular mechanisms behind the aging process, we have analyzed age-induced changes in gene expression patterns through the application of DNA chip technology. In the present study, the aging process was analyzed in rat liver in the presence or absence of GH replacement. Out of 3,000 genes printed on the microarrays, approximately 1,000 were detected in the rat liver. Among these, 47 unique transcripts were affected by the aging process in male rat livers. The largest groups of age-regulated transcripts encoded proteins involved in intermediary metabolism, mitochondrial respiration, and drug metabolism. Approximately 40% of the differentially expressed gene products were normalized after GH treatment. The majority of those transcripts have previously not been shown to be under GH control. The list of gene products that showed normalized expression levels in GH-treated old rats may shed further insight on the action and mechanism behind the positive effects of GH on, for example, fuel metabolism and body composition observed in different animal and human studies.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
It is well established that biological aging is associated with functional deficits at the cellular, tissue, organ, and system levels, but the mechanisms that control life span and age-related phenotypes are still not well understood. Several molecular models of aging have evolved from current research, including damage by reactive oxygen species (ROS) generated by metabolism, genome instability, genetically programmed extension mechanisms, cell death, and systemic aging. It has been proposed that the life span of an organism is the sum of deleterious changes and counteracting repair and maintenance mechanisms that respond to the damage (1). The decline in tissue function and increased incidence of disease during aging are thus likely to be the result of several interacting factors. Furthermore, cell-autonomous and systemic aging may coexist or trigger each other.

Age-dependent cellular and molecular alterations in specific subpopulations of neurons within the hypothalamus and pituitary have been linked to the neuroendocrine alterations that occur during aging (2). Since hormones have an important trophic and integrative role in maintaining tissue function, it has been suggested that aging or at least some of its symptoms are related to a physiological decline in hormonal levels with age (3). A number of effects of normal aging closely resemble features of hormonal deficiency, which in mid-adult patients are successfully reversed by replacement therapy with the appropriate hormone (4). This could be exemplified by the age-related changes in body composition, serum lipids, and muscle performance that closely resemble symptoms of adult-onset GH deficiency (5, 6) and the fact that GH treatment has been shown to normalize these changes in both GH-deficient adult patients and healthy elderly subjects (7).

However, the role of GH in aging processes is not without controversy since both accelerated and decelerated aging have been reported to be associated with GH (8). Ames dwarf mice (df/df), which are deficient in GH, PRL, and TSH, live longer than their normal siblings (9) whereas transgenic mice overexpressing GH exhibit reduced life spans and various indices of premature aging (10, 11). Reduced life span has also been reported in patients with supraphysiological levels of GH (12, 13). A greater understanding of the cellular and molecular mechanisms behind GH-induced changes in aged individuals should provide clues to explain these opposing effects of GH.

The major aim of this study was to identify gene products that are affected both by aging and GH therapy in old rats in such a way that their levels of expression become normalized by GH treatment. It is not inconceivable that such gene products might be involved in mediating the reported beneficial/normalizing effect of GH therapy on parameters such as fuel metabolism (14), body composition (15), plasma LDL cholesterol concentrations (16, 17), and physical performance (18). The liver is a main target for GH action where this hormone is involved in regulating general processes, such as intermediary metabolism and tissue growth (19, 20), but also in the liver-specific metabolism of hydrophobic compounds (21, 22). Furthermore, many GH-regulated hepatic transcripts have been described in the literature. The liver was therefore the tissue of choice for this study.

To identify GH-responsive genes that might mediate positive effects of GH treatment, we have recently analyzed GH-mediated effects on hepatic gene expression in old male rats through the application of cDNA representational difference analysis (RDA) (23). Several transcripts encoding enzymes and receptors involved in the metabolism of protein, carbohydrates, and lipids were differentially expressed in livers from GH-treated 2-yr-old rats. Those findings were confirmed and extended in the present study, using a high-through-put analysis of gene expression. The survey of 3,000 genes, represented on cDNA microarrays, revealed that aging resulted in an altered gene expression for intermediary metabolism, as well as ATP synthesis, and that GH therapy reversed many of these effects. However, some gene products were superinduced or superreduced in GH-treated old rats as compared with untreated young rats, indicating a final pattern of altered gene expression.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
cDNA microarrays were used to examine the molecular events behind aging and GH replacement therapy in old rats. Initially, transcriptional responses were determined by comparing livers from young (10 weeks of age) and old (2 yr of age) male rats. Of 3,000 genes printed on the microarrays, approximately 1,000 were detected in the rat liver. Among these, 17 unique transcripts displayed a greater than 1.7-fold induction (170% of control) in expression levels during aging, whereas 30 were reduced (60% of control). Of the 47 gene products affected by the aging process, seven were found to encode proteins with as yet unknown functions, but the rest were divided into various functional classes (Table 1GoGo). The largest group consisted of gene products involved in fuel metabolism. Transcripts encoding enzymes participating in the oxidation of glucose and fatty acids were increased, whereas proteins involved in mitochondrial respiration were reduced. Various transcripts encoding proteins involved in lipogenesis were also increased in the aged liver.


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Table 1. Effects of Old Age and GH Therapy on Hepatic Gene Expression

 

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Table 1A. (Continued)

 
To elucidate the molecular mechanisms behind GH-dependent changes in aged animals, we next performed similar experiments to compare patterns of hepatic mRNA expression in old untreated rats to rats treated for 3 weeks by continuous infusion with GH. The resulting list of GH-responsive genes is included in Table 1Go, and the normalizing effect of GH therapy on different functional classes of genes is illustrated in Fig. 1aGo. In addition to already known GH-regulated transcripts, such as CYP2C12 (24) and {alpha}-2 u-globulin (25), several gene products were identified as GH responsive that have not previously been shown to be controlled by GH. As much as 36% of the unique gene products affected during aging showed normalized expression levels in response to GH replacement therapy. In particular, several genes encoding proteins involved in ATP synthesis were reduced during aging and induced to normal levels by GH. The level of peroxisome proliferator activated receptor-{alpha} (PPAR{alpha}) mRNA expression was increased in the old rats, together with PPAR{alpha}-activated genes such as 3-ketoacyl-coenzyme A (CoA) peroxisomal thiolase and CYP4A3. These transcripts were reduced to normal levels in the GH-treated rats. However, other transcripts such as fatty acid synthase and stearyl CoA desaturase, which were already induced in the old rats, were further induced (superinduced) upon GH treatment. The number of unique gene products that were affected by age and/or GH treatment is schematically illustrated in Fig. 1bGo. These data indicate that although 17 of 47 age-affected transcripts were normalized by GH treatment, many more were affected by GH without being influenced by old age.



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Figure 1. Overview of Results Obtained from cDNA Microarray Analysis of the Aging Process in Male Rat Liver, in the Absence or Presence of GH Therapy

A, Functional classification of differentially regulated transcripts during aging. The white areas within each pie sector represent the percent of normalized transcripts obtained through GH treatment of old animals. B, Comparison between the total number of gene products affected by aging, GH treatment, or both. The larger circle (blue, dark green, and light green) represents the GH-dependent liver transcripts, and the smaller circle (yellow, dark green, and light green) represents the age-dependent transcripts. The blue area illustrates the number of gene products affected by GH but not by age. The yellow area illustrates the number of gene products only affected by age but not by GH. The shared areas represent age-dependent transcripts normalized by GH (dark green) and age-dependent transcripts influenced but not normalized by GH (light green).

 
Continuous treatment of male rats with GH through osmotic minipumps, which mimics the female-specific pattern of GH secretion, is well known to feminize the liver at the level of gene expression. This feminizing effect of GH was illustrated in the present study by the induction of CYP2C12 and CYP2C7, which represent GH-regulated female-specific (24) or female-predominant (26) gene products. Similarly, the male-specific carbonic anhydrase III (27) and {alpha}-2 u-globulin (25) were reduced in the old rats and further reduced by GH. The expression of these genes is dependent on the male-specific pulsatile pattern of GH secretion in the rat and repressed by continuous infusion of GH. Taken together, these results showed that the old GH-treated male rats in our study obtained normalized expression levels for several age-affected transcripts, in addition to others that were superinduced or superreduced.

To verify the data obtained in the chip experiments, we next performed RNase-protection/solution hybridization analysis with specific [35S]-labeled cRNA probes for selected gene products. As demonstrated in Fig. 2Go, the age- and GH-induced changes in expression levels of these hepatic transcripts were very similar in the two different protocols. It should be noted that 20% of the gene products listed in Table 1Go have recently been described by us as being affected by GH treatment in old male rats (23), as determined by RDA. The results presented here were thus confirmed by solution hybridization analysis and are overlapping with previous data obtained through RDA.



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Figure 2. Effects of Aging and GH Treatment on Hepatic mRNA Expression of Selected Gene Products in Male Rats

Old male rats, about 2 yr of age, were treated for 3 weeks with recombinant hGH by continuous infusion from osmotic minipumps. hGH was administered to animals at a daily dose of 0.34 µg/g body weight. Total nucleic acid (tNA) samples were prepared from normal (10 weeks of age), old, or GH-treated old male rats and analyzed in solution hybridization assays specific for the different mRNA species. Results were calculated as counts per min per µg tNA and expressed in relation to mRNA levels in the normal rat liver. Values are the mean ± SD of triplicate determinations.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Aging of the liver is associated with a variety of functional alterations (28) such as reduced liver mass, declined hepatic flow, accumulation of lipofuscin (the aging pigment), and a decline in the hepatic clearance of metabolized hydrophobic compounds. Many studies have shown a decreased hepatic protein turnover, including impaired protein synthesis and protein degradation (29). Aging has been shown to alter the expression and activity for hepatic gluconeogenic, glycolytic, and nitrogen-metabolizing enzymes (30). The ability of insulin to inhibit hepatic glucose production is impaired with aging (31), and it has been suggested that changes in hepatic insulin sensitivity may account for the age-dependent increase in plasma insulin concentrations in animal models of aging. Furthermore, the number of hepatic mitochondria has been shown to decrease in aging rodents (32, 33, 34) and man (35), and several biochemical studies (36, 37, 38, 39), but not all of them (40), reveal a decline of respiratory functions with age in normal livers. As demonstrated in this study, many of these age-related changes can be correlated to an altered expression pattern of specific transcripts. In addition, we identified gene products that were affected both by aging and GH therapy so that their levels of expression became normalized by GH treatment.

Several kinds of evidence support a role for glucose and altered carbohydrate metabolism in many of the pathologies of aging. Based on the results presented here and elsewhere (41, 42), it is apparent that the aged rat liver has a reduced ability to supply glucose. Previous reports of reduced hepatic dephosphorylation of glucose-6-phosphate (G6P) in old rats (43) are confirmed by results obtained in this study, where an age-induced decline in glucose-6-phosphatase mRNA levels was observed. The decline of glucose-6-phosphatase in old rats may be due to the age-related decrease in serum GH levels, since GH treatment reversed the effect of age on hepatic glucose-6-phosphatase mRNA levels. The enzymatic activity and mRNA expression of glucose-6-phosphatase, which is the final enzyme in the gluconeogenic pathway, has previously been shown to be normalized (induced) by GH in hypophysectomized rats (44) and by calorie restriction in old rats (43). Since the enzyme hexokinase is inhibited by G6P it may be speculated that an increased accumulation of G6P represents a cause for the diminished sensitivity to insulin in aged animals.

A reduced ability to dephosphorylate G6P may not only reduce the hepatic synthesis and secretion of glucose, but also increase glucose oxidation and lipogenesis in the aged liver. Accumulation of G6P in the liver has been shown to induce the expression of genes belonging to these pathways (45). In this study, this can be exemplified by the age-associated increase in transcripts such as pyruvate kinase, spot 14, fatty acid synthase, and stearyl CoA desaturase, which might be a consequence of decreased glucose-6-phosphatase activity. The GH-mediated increase (normalization) in glucose-6-phosphatase levels did not, however, reverse the expression of these transcripts. In contrast, a further increase of spot 14, fatty acid synthase, and stearyl CoA desaturase was observed in the GH-treated rats.

However, G6P is not the only regulator of lipogenesis. Genes related to cholesterol and fatty acid metabolism have been shown to be induced when cellular sterol levels drop, through a mechanism involving activation of sterol regulatory element-binding proteins (SREBPs) (46). Since GH has been reported to decrease the concentration of hepatic cholesterol (47), it might be speculated that the observed effect of GH on spot 14, fatty acid synthase, stearyl CoA desaturase, and farnesyl diphosphate synthase may be mediated through GH-induced changes in intracellular sterol levels. GH has been shown to be an important regulator of 7{alpha}-hydroxylase activity and bile acid synthesis in rat (47) and human (48) liver. A consequence of GH deficiency in aged rats might therefore be a reduced fecal excretion of bile acids, as was described in the hypophysectomized rat (47). In GH-treated rats, an increased synthesis of bile acids would instead lead to reduced hepatic cholesterol and induced (normalized) expression of genes involved in the synthesis of cholesterol and fatty acids.

Induced lipogenesis is often observed in states of reduced expenditure of energy, and the opposite regulation of fatty acid synthesis and oxidation is a well known concept (49). It has been shown that an intermediate of fatty acid synthesis, malonyl-CoA, inhibits the first step in mitochondrial and peroxisomal ß- oxidation, the transfer of fatty acyl groups to carnitine (50). Carnitine octanoyltransferase (COT), a member of the family of carnitine acyltransferases, facilitates the transport of medium chain fatty acids through various organelles for further metabolism (51). The COT mRNA levels were reduced in old rats and normalized upon GH replacement, consistent with the picture of GH being a lipolytic hormone that increases levels of FFA in serum, as well as hepatic uptake and degradation thereof. The level of peroxisomal ß-oxidation has been shown to be reduced during aging (52), but, to our knowledge, a major effect of GH has not been observed. However, GH has been shown to increase the activity (53) and mRNA (our own unpublished results) levels of 2,4-dienoyl-CoA reductase in livers of hypophysectomized rats, indicating that the mitochondrial oxidation of polyunsaturated fatty acids is dependent on normal levels of GH. The stimulatory effect of GH on this enzyme transcript was confirmed in the present study. Whether the small effect observed in mRNA levels for COT will lead to a major change in the degradation of fatty acids must await further analysis. It may, however, be hypothesized that the stimulatory effects of GH on hepatic lipid catabolism are at least partly mediated by GH-induced levels of carnitine acyltransferases.

Suprisingly, both the mitochondrial and the peroxisomal 3-ketoacyl-CoA thiolase mRNA levels were induced in the old rats and normalized by GH treatment, indicating that the last step in the oxidation of fatty acids into acetyl-CoA would be repressed by GH. This might be explained by the levels of PPAR{alpha} mRNA, which showed the same pattern of expression. PPAR{alpha} is a member of the steroid/nuclear receptor superfamily and mediates the biological and toxicological effects of peroxisome proliferators (54). The discovery that several fatty acids, including arachidonic acid and linoleic acid, activate PPAR{alpha} suggests that fatty acids could represent biological activators (55, 56, 57). According to this hypothesis, PPAR{alpha} could function as a fatty acid sensor, which would allow the fatty acids to regulate their own metabolism. Studies on mice lacking the PPAR{alpha} have shown that this receptor/transcription factor modulates constitutive expression of genes encoding several mitochondrial fatty acid-catabolizing enzymes, in addition to mediating inducible mitochondrial and peroxisomal fatty acid ß-oxidation (58). Both the mitochondrial (59) and the peroxisomal (58) 3-ketoacyl-CoA thiolase genes have been shown to be induced by PPAR{alpha}.

In situations where the secretion of GH is reduced, as in the hypophysectomized or aged rat, a reduced degree of adipose lipolysis and unhindered lipogenesis would lead to reduced levels of FFA in the circulation. Since fatty acids are thought to be endogenous activators of PPAR{alpha}, the activity of PPAR{alpha}-regulated genes would be lower than in animals with normal GH secretion. This would be reversed because of stimulated lipolysis after administration of GH. At the same time, GH treatment leads to a reduced hepatic expression of PPAR{alpha}, which might be a way to negatively feedback-regulate the degree of inducible ß-oxidation in the liver. Absence of GH would lead to elevation of PPAR{alpha} and an increased ability to sense changes in circulating FFA. In line with this, the secretion of GH from the pituitary is reduced by high levels of circulating FFA in rats as well as in humans (60).

PPAR{alpha} also participates in the control of fatty acid uptake, transport, and esterification by stimulating genes such as the liver cytosolic fatty acid-binding protein, fatty acid transport protein, and acyl-CoA synthase. Other PPAR{alpha}-regulated cellular events include microsomal fatty acid {omega}-hydroxylation through the induction of genes encoding cytochrome P450 {omega}-hydroxylases such as CYP4A1 and CYP4A3. Dicarboxylic fatty acid metabolites of accumulated long chain fatty acids, formed via the P450-catalyzed {omega}- hydroxylation pathway, have been suggested to be among the primary inducers of fatty acid-binding protein and peroxisomal ß-oxidation (61). In the present study, mRNA levels of CYP4A3 were elevated in the livers from old rats. Taken together, these results indicate that in addition to increased sensitivity toward fatty acids, the aging liver may also have a higher capacity to {omega}-hydroxylate them into potent activators of PPAR{alpha}.

PPAR{alpha} expression (62) as well as peroxisome proliferator-induced ß-oxidation (63, 64) have recently been shown to be induced after hypophysectomy of male rats and repressed by GH administration. The GH-mediated decline in PPAR{alpha}, 3-ketoacyl-CoA thiolase, and CYP4A3 expression observed in this study, confirms an inhibitory effect by GH on PPAR{alpha}-mediated biological effects. Furthermore, GH-stimulated Janus kinase-signal transducer and activator of transcription 5b (JAK2/STAT5b) signaling has been reported to inhibit PPAR{alpha}-dependent reporter gene transcription (65). The cross-talk between STAT5b and PPAR{alpha} signaling pathways further suggests a connection between GH- and fatty acid-mediated events.

GH serves as an anabolic hormone, sparing protein stores at the expense of fat in situations of caloric restriction. Thus, GH promotes lipolysis and prevents lipogenesis in adipose tissue, which increases the availability of FFA for energy expenditure. The anabolic effects of GH include a decreased degradation of whole body protein and a reduced catabolism of amino acids (66). GH administration to hypophysectomized rats decreases the hepatic uptake of amino acids (67), as well as the gene expression (68) and activities of urea cycle enzymes in the liver (69). In the present study, GH treatment led to a decreased expression of alanine aminotransferase. This finding is compatible with a GH-mediated down-regulation of urea synthesis, since a GH-mediated decline in alanine aminotransferase expression may lead to reduced deamination of alanine to pyruvate and the concomitant amination of {alpha}-ketoglutarate to glutamate.

Both the number of mitochondria (34) and the respiratory function (36, 37) of rat livers have been shown to decrease during aging. Mitochondria are the major ATP producer of the mammalian cell. Moreover, mitochondria are also the main intracellular source and target of ROS that are continually generated as byproducts of aerobic metabolism in animal cells. It has been suggested that an age-associated defect in the respiratory chain may increase the production of ROS and free radicals in mitochondria. Results obtained in the present study indicate a reduced expression of the mitochondrial genome in rat liver during aging. Several regions of the mitochondrial genome were shown to be less expressed in livers from old rats, including gene products encoding F1-ATPase, cytochrome b, and cytochrome c oxidase (I, II, and III). The same transcripts were found to be normalized in the GH-treated rats. Whether the observed effects on mRNA levels reflect a reduced number of mitochondria in the aged liver or a reduced level of mitochondrial gene expression must await further investigations. However, some nuclear-encoded components of the respiratory chain were also reduced by age. Taken together, these results support previous reports of a defective respiratory chain in the aged liver and suggest that the age-associated decline in serum GH levels might contribute to this effect.

Our observations that GH treatment of old rats increased the levels of several transcripts encoding various components of the electron transport chain are in agreement with previously published studies on rat liver mitochondria. As early as 1972, Matsubara et al. (70) reported that the respiratory cytochrome content of isolated mitochondria decreased in GH-deficient (hypophysectomized) animals in parallel with a decrease in respiratory activities, and that subsequent administration of GH increased the cytochrome contents. Others have shown that GH induces the concentration and/or activity of proteins such as cytochrome a, a3, b, c, and c1 (71), NADH dehydrogenase (72), cytochrome c oxidase (71), and ATP synthase (73) in hypophysectomized rats. GH-induced levels of transcripts encoding these proteins may thus lead to the increase in mitochondrial respiratory rate that has been described in hypophysectomized rats substituted with GH (72, 73, 74). However, thyroid hormones are considered to be more important regulators of mitochondrial respiration.

In the liver, effects of GH are also exerted on functions that are not primarily involved in intermediary metabolism and growth. It has been shown that GH regulates the synthesis of many proteins that are specifically or preferentially expressed in the liver, such as plasma proteins, secretory proteins, serine protease inhibitors, P450 enzymes, and other drug-metabolizing enzymes. The effects of GH on hepatic drug metabolism, for example, are complicated by the fact that the mode of GH administration exerts different effects on different genes (75). This is explained by the sexually differentiated pattern of GH secretion (76), which in rodents leads to a sex-specific expression of many hepatic proteins (22). These proteins include {alpha}-2 u-globulin, 5{alpha}-reductase, P450 enzymes (phase I enzymes), and glutathione S-transferases (phase II enzymes). In this study, the continuous infusion of GH through osmotic minipumps, which mimics the female-specific pattern of GH secretion, reduced the expression of male-specific proteins such as {alpha}-2 u-globulin, CYP3A2, and carbonic anhydrase III. At the same time, female-specific proteins such as CYP2C7, CYP2C12, and glutathione S-transferases were induced. In fact, most transcripts encoding drug-metabolizing enzymes and secretory proteins were up-regulated by GH in the old rat, leading to a final picture of increased hepatic activity. Such effects may alter the hepatic metabolism and activation or detoxification of drugs, carcinogens, and endogenous substrates.

Although aging is not simply the result of different hormonal deficiencies, the development of hormone replacement therapies might successfully prevent or delay some aspects of the aging process. However, these treatments have not been uniformly proven to be safe and beneficial. Although GH therapy has repeatedly been shown to have beneficial effects upon fuel metabolism and body composition, there are still questions to be answered in connection with GH replacement therapy of the elderly. Data obtained in this study suggest that GH treatment of old rats has multiple effects on the hepatic phase I and II drug-metabolizing enzymes, intermediary metabolism, and mitochondrial respiration. These GH-induced changes in expression levels of specific mRNAs may lead to both positive and negative effects on hepatic function and age-related phenotypes. The fact that the final picture of hepatic gene expression in old male rats substituted with GH was not the same as in younger rats can partly be explained by the mode of GH administration applied in this study. It will therefore be interesting to perform a similar study with old male rats treated intermittently with GH, mimicking the male-specific pattern of GH secretion. Among the positive (normalizing) effects of GH on specific gene products, the normalized expression of glucose-6-phosphatase, PPAR{alpha}, and the mitochondrial genome deserve further investigation. It will be of interest to explore whether these effects are due to a direct action of GH on the hepatocyte or are secondary to GH-induced changes in other tissues. In vitro studies, utilizing primary rat and human hepatocytes, will thus be useful tools in future analysis of GH action in the aged liver.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Generation of cDNA Microarrays
Approximately 3,000 cDNA clones were selected from the TIGR Rat Gene Index (www.tigr.org) and our own collection of rat cDNA libraries (23). Bacterial colonies were grown overnight in 1.5 ml LB medium in 96-deep-well-plates (Beckman Coulter, Inc., Fullerton, CA), and plasmid minipreparations were followed by a PCR amplification of the inserts using vector-specific primers T3 (5'-AATTAACCCTCACTAAAGGG-3') and T7 (5'-GTAATACGACTCACTATAGGGC-3'). The amplified inserts, each produced by pooling two 100 µl PCR-reactions, were purified by ethanol precipitation, resuspended in 40 µl 3x SSC, and purity checked on an agarose gel. From the gel it could be concluded that approximately 10% of the array elements, representing more than one clone per well contained DNA. The sequences of all genes mentioned here by name were reverified by single-pass 3'-sequencing. CMT GAPS Amino silane-coated slides (Corning, Inc., Corning, NY) were used as adhesive surface for printing, using a GMS 417 arrayer (Affimetrix, CA). The slides were postprocessed as described previously (77) and stored in a dust-free dark box until hybridization.

RNA Preparation and mRNA Isolation
Old male Sprague Dawley rats (~2 yr of age,) and younger male rats [~10 weeks of age, obtained from B&K Universal AB (Stockholm, Sweden)], were maintained under standardized conditions of light and temperature, with free access to animal chow and water. Old 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 kind gift from Pharmacia & Upjohn, Inc. AB (Stockholm, Sweden), was administered to animals at a daily dose of 0.34 µg/g body weight. After 3 weeks of treatment, the rats were killed, and liver samples were removed and frozen in liquid nitrogen. The animal experiments were approved by the local ethical committee.

Total RNA was isolated using TRIzol Reagent (Life Technologies, Inc. , Gaithersburg, MD), according to the protocol supplied by the manufacturer. The quality of the RNA samples was examined on a denaturing agarose gel. Equal amounts of total RNA from five animals in the same experiment group were pooled before mRNA purification. mRNA was purified from 1 mg of total RNA using 35 mg oligo(dT)-cellulose (Amersham Pharmacia Biotech, Uppsala, Sweden) equilibrated in a RNAse-free buffer consisting of 20 mM Tris-HCl, 0.5 M NaCl, and 1 mM EDTA. Poly(A)+ RNA was bound to the cellulose, washed four times using the same buffer, washed once in a low-salt buffer (0.1 M NaCl), and eluted in prewarmed elution buffer (20 mM Tris-HCl, 1 mM EDTA). Finally, the eluted Poly(A)+ RNA was ethanol precipitated and resuspended in water. The yield from this mRNA purification was typically about 5%.

cDNA Labeling, Purification, and Hybridization
The protocol employed for probe labeling and purification was essentially as described previously (77). Four micrograms of mRNA were used from each group of animals for each experiment. Labeled cDNA was produced by a random primed reverse transcription reaction using Superscript II (Life Technologies, Inc.). Random hexamer primers and Cy-labeled nucleotides were obtained from Amersham Pharmacia Biotech. In the first set of experiments, each hybridization compared Cy3-labeled cDNA reverse transcribed from hepatic mRNA isolated from young male rats (10 weeks of age), with Cy5-labeled cDNA isolated from old animals (2 yr of age). In another set of experiments, Cy3-labeled cDNA derived from livers of nontreated old animals were compared with Cy5-labeled cDNA from old animals that had been continuously treated with hGH for 3 weeks.

The labeled and purified cDNA was added to the array at a final volume of 15 µl hybridization buffer [5x SSC, 0.2% SDS, 10 µg poly (A) RNA, 10 µg yeast tRNA]. The array was covered by a plastic 22 x 22 mm cover slip (Grace Biolabs, OR) and put in a sealed hybridization chamber (Corning, Inc.). After the hybridization, which took place at 65 C for 15–18 h, the array was washed and dried. The array was immediately scanned using a GMS 418 scanner (Affimetrix). Image analysis was performed using the GenePix Pro software (Axon Instruments, Foster City, CA). An arbitrary value of 1.7 was chosen to denote differences in the level of hybridization between control and experimental cDNA. Each experiment was repeated twice and the results are expressed as the mean between two ratios ± range.

Solution Hybridization Analysis
Total nucleic acids (tNA) were isolated by homogenization of tissue specimens, using a polytrone PT-2000 (Kinematica AG, Switzerland). Digestion of samples with proteinase K (Merck, Darmstadt, Germany) and subsequent extraction with chloroform and phenol have been described previously (78). mRNA levels corresponding to the expression of individual clones were measured in tNA samples, using a solution hybridization/RNase protection assay. Transcript-specific 35S-labeled cRNA probes were transcribed in vitro from the respective cDNA vector construct, according to the method of Melton et al. (79). Reagents for in vitro transcription were obtained from Promega Corp. (Madison, WI).

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 tRNA (bakers yeast tRNA 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 digested with RNases (RNase-A and RNase-T1 from 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 (Whatman GF/C from Whatman Ltd. Madison, Kent, UK), and counted in a liquid scintillation counter. The concentration of nucleic acids in tNA samples was measured spectrophotometrically. Samples were analyzed in triplicate and the results determined as counts per min specific mRNA per µg tNA.


    ACKNOWLEDGMENTS
 
We would like to thank Eva Johansson for skillful technical assistance.


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

This work was supported by grants from the Swedish Medical Research Council (13X-08556), the Swedish Society of Medicine, the Fredrik and Ingrid Thuring Foundation, and the Knut and Alice Wallenberg Foundation. This work has been funded in part by a National Heart, Lung and Blood Institute Grant, HL-59781.

Received for publication August 7, 2000. Revision received October 2, 2000. Accepted for publication October 18, 2000.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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