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
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ABSTRACT |
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INTRODUCTION |
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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.
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RESULTS |
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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. 2, 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 1
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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DISCUSSION |
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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-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 mRNA, which showed the same pattern of
expression. PPAR
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
suggests
that fatty acids could represent biological activators (55, 56, 57).
According to this hypothesis, PPAR
could function as a fatty acid
sensor, which would allow the fatty acids to regulate their own
metabolism. Studies on mice lacking the PPAR
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
.
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, the activity of PPAR
-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
,
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
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 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
-regulated cellular events include
microsomal fatty acid
-hydroxylation through the induction of genes
encoding cytochrome P450
-hydroxylases such as CYP4A1 and
CYP4A3. Dicarboxylic fatty acid metabolites of accumulated
long chain fatty acids, formed via the P450-catalyzed
-
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
-hydroxylate them into potent
activators of PPAR
.
PPAR 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
, 3-ketoacyl-CoA thiolase, and CYP4A3
expression observed in this study, confirms an inhibitory effect by GH
on PPAR
-mediated biological effects. Furthermore, GH-stimulated
Janus kinase-signal transducer and activator of transcription 5b
(JAK2/STAT5b) signaling has been reported to inhibit PPAR
-dependent
reporter gene transcription (65). The cross-talk between STAT5b and
PPAR
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
-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 -2 u-globulin,
5
-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
-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, 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.
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MATERIALS AND METHODS |
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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 1518 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 15,00020,000 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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