(Received for publication, February 21, 1995; and in revised form, December 24, 1995)
From the
We have studied the role of the mitochondrial 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) synthase gene in regulating ketogenesis. The gene exhibits expression in various tissues and it is regulated in a tissue-specific manner. To investigate the underlying mechanisms of this expression, we linked a 1148-base-pair portion of the mitochondrial HMG-CoA synthase promoter to the human growth hormone (hGH) gene and analyzed the expression of the hGH reporter gene in transgenic mice. mRNA levels for hGH were observed in liver, testis, ovary, stomach, colon, cecum, brown adipose tissue, spleen, adrenal glands, and mammary glands from adult mice, and also in liver and stomach, duodenum, jejunum, brown adipose tissue, and heart of suckling mice. There was no expression either in kidney or in any other nonketogenic tissue. The comparison between these data and those of the endogenous mitochondrial HMG-CoA synthase gene suggests that the 1148 base pairs of the promoter contain the elements necessary for expression in liver and testis, but an enhancer is necessary for full expression in intestine of suckling animals and that a silencer prevents expression in stomach, brown adipose tissue, spleen, adrenal glands, and mammary glands in wild type adult mice. In starvation, transgenic mice showed higher expression in liver than did wild type. Both refeeding and insulin injection reduced the expression. Fat diets, composed in each case of different fatty acids, produced similar expression levels, respectively, to those found in wild type animals, suggesting that long-, medium-, and short-chain fatty acids may exert a positive influence on the transcription rate in this 1148-base-pair portion of the promoter. The ketogenic capacity of liver and the blood ketone body levels were equal in transgenic mice and in nontransgenic mice.
Ketogenesis in mitochondria is mainly controlled by two enzymes:
carnitine palmitoyl transferase I and mitochondrial HMG-CoA ()synthase. The activity of the latter increases with
fasting, fat feeding, diabetes, glucagon administration, and with the
transition from the fetal to the suckling
state(1, 2) , and these effects are accompanied by an
increase in the mRNA levels for this gene(3) . The close
correspondence between the increase in mRNA levels, enzyme activity,
and ketogenesis has also been observed in the intestine and liver of
suckling pups(4, 5) , which reinforces the hypothesis
that it is the main regulatory point in ketogenesis, both in adult and
neonatal rats. Mitochondrial HMG-CoA synthase is also expressed in
specialized cells of testis and ovary, although it appears that the
function in these gonadal tissues is not concerned with ketogenesis but
with the gonadal function in the synthesis of sexual
hormones(6) .
The long term goal of our research is to understand the molecular mechanisms underlying mitochondrial HMG-CoA synthase gene expression, its tissue-restricted distribution, and its accurate hormonal control. The potential regulatory properties of the 1148 bp of the 5` sequence have been studied. This promoter is sufficient to direct the tissue-specific expression of a reporter gene in vitro, as shown by transient chloramphenicol acetyltransferase expression assays in hepatoma cells(7) . In addition a promoter fragment located at -104 bp has very recently been shown to contain a cis-responsive element that activates transcription by fatty acids and clofibrate through the peroxisome proliferator activated receptor in transfected Hep G2 cells(8) . However, the various cells cultured in vitro are imperfect models of the situation in vivo, and these models do not reveal the influence of the chromatin structure on gene expression, or the hormonal response. Therefore, the investigation of the promoter elements that regulate the expression of the mitochondrial HMG-CoA synthase gene in transgenic mice is of interest.
Transgenic animal technology is a powerful tool with which to study the contribution of regulatory genes to the control of metabolic pathways. We have studied the expression in transgenic mice of human growth hormone in different tissues under the control of a 1148-bp portion of the proximal promoter of mitochondrial HMG-CoA synthase. Our results show that adult transgenic animals express the reporter gene in liver, testis, ovary, colon, and cecum as in wild type mice, although quantitative differences in mRNA levels were observed. It is also expressed in stomach, brown adipose tissue, spleen, adrenal glands, and mammary glands at variance with the natural promoter, from which it is concluded that a silencer may be located outside the -1148 bp. Suckling transgenic mice also express the reporter gene in different parts of the thin intestine, confirming previous data on expression in wild type mice. In addition, we have also studied, in the transgenic animal, the influence of fasting-refeeding processes, of feeding with a fat diet, and of insulin on the expression. Results confirm previous studies in rat liver.
Figure 1:
Mitochondrial HMG-CoA synthase/human
growth hormone (mtHMG-CoAS/hGH) chimeric gene used to create transgenic
mice and genomic characterization of transgenic founders. A,
structure of the mtHMG-CoAS/hGH fusion construct in which DNA derived
from mtHMG-CoA synthase gene is indicated by hatched areas,
and hGH sequences are indicated by solid boxes (exons) and open boxes (introns). The transcription initiating site and
direction of transcription from the mtHMG-CoA synthase promoter are
indicated by the arrow. B, Southern blot of genomic DNA
isolated from each of the transgenic F mice and from a
nontransgenic mouse. Genomic DNA (10 µg) was digested with PvuII restriction enzyme. The 1.4-kb fragment was produced by
the restriction of PvuII at the site of hGH gene and the site
of mtHMG-CoAS promoter inserted in the genome in a head-to-tail manner.
Control and transgenic mice showed a 4.8-kb fragment, coming from the PvuII restriction of the endogenous mtHMG-CoAS gene, which
hybridized with the 1.4-kb PvuII probe. Lanes 1, 2, 3, and 4 are F
mice. Lane
5 is a control. Lane 6 is a control DNA plus 10
copies.
The transgene
was identified with primers that yield a fragment of 1095 bp (upstream
primer from HMG-CoA synthase gene 5`-CTCACTCAGCGTGTGCTCATCTGCCTGC;
downstream primer from hGH gene, 5`-GGGCTACATAGGAAGAACGGGGATTGCAGG).
PCRs were typically carried out as follows. A standard 100-µl
mixture contained 1 µg of DNA from tail samples, 10 µl of 10
PCR buffer (Mg
-free), 1.5 mM MgCl
, 0.2 mM deoxynucleoside triphosphates,
1.25 units of Taq polymerase (Life Technologies, Inc.), and 50
pmol of each primer. PCR was performed for 30 cycles. Each cycle
consisted of denaturation at 94 °C for 1 min, primer annealing at
65 °C for 1 min, and primer extension at 72 °C for 90 s. Ten
microliters of the PCR sample were electrophoresed in a 1% agarose gel.
The transgene copy number was subsequently determined by Southern
blot analysis of the tail DNA prepared from F mice. 10
µg of DNA were digested with PvuII, electrophoresed in 1%
agarose, and transferred to Hybond-C membrane (Amersham Corp.) in 10
SSC (1
SSC = 0.15 M NaCl, 0.015 M sodium citrate). The DNA was fixed to the membrane at 80 °C
for 2 h. The membrane was prehybridized in buffer containing 0.7 M NaCl, 40 mM NaH
PO
, pH 7.6, 0.4
mM EDTA, 0.2% poly(vinylpyrrolidone), 0.2% Ficoll, 0.1% SDS,
and 0.2 mg/ml salmon sperm DNA and hybridized in the same solution plus
9% (w/v) dextran sulfate and the
P-labeled 1.4-kb PvuII fragment from pMShGH (2
10
cpm/ml).
Washes were performed in 0.2
SSC, 0.1% SDS at 68 °C. The
intensity of the signal generated from genomic DNA was measured by
densitometric scanning of the autoradiograms in a Bioprofil
(Vilber-Lourmat) photodensitometer and compared with the intensity of
signal produced from a known amount of a 1.4-kb PvuII fragment
from pMShGH DNA.
All tissues were
rapidly frozen in liquid nitrogen, then powdered, and total cellular
RNA was extracted with guanidine isothiocyanate and then purified by
centrifugation through a CsCl cushion(10) . Aliquots of 15
µg were fractionated on 1% agarose gel containing formaldehyde, and
subjected to Northern transfer on NY13 nytran filter (Schleicher &
Schuell). Filters were fixed at 80 °C at 254 nm for 2 h. After 6 h
of prehybridation using high stringency conditions (at 42 °C in 1 M NaCl, 50% formamide, 7.5 Denhardt's solution,
0.1% SDS, 50 mM NaH
PO
, pH 6.3, 10%
dextran sulfate, and heat denatured salmon sperm DNA, 0.2 mg/ml), the
filters were hybridized overnight using the random-primed
P-labeled 150-bp partial cDNA probe (see below). The
radioactivity was 2
10
cpm/ml. Filters were washed
briefly at 42 °C in 300 mM NaCl, 30 mM sodium
citrate, pH 7.0, and 1% SDS, followed by three 20-min washes at 65
°C in 30 mM NaCl, 3 mM sodium citrate, pH 7, and
0.1% SDS. Filters were autoradiographed at -70 °C in contact
with Kodak x-ray film with an intensifying screen.
The levels of mRNA were determined by densitometric scanning of the autoradiograms in a Bioprofil (Vilber-Lourmat) photodensitometer. Densitometry values were corrected by using a fragment from the cDNA clone (pRSA13) for rat serum albumin as a constitutive probe (11) . Filters were dehybridized for 30 min at 100 °C with the same washing system and then rehybridized. Statistical analysis was carried out by Student's t test with significance levels chosen as p < 0.005 and p < 0.01.
The copy number of the transgene in the positive mouse was determined by Southern blot analysis (see ``Materials and Methods''). The founder and the first generation offspring of the founder indicated that the mtHMG-CoA synthase/hGH gene was integrated in one or two copies/haploid genome (Fig. 1B). Matings between two heterozygous animals which were offspring of the founder female resulted in the death of the total progeny within a few hours of birth. It was observed that many pups were eaten by their mothers, in a much greater proportion than usual. Neonatal mortality in the transgenics differed from that in the nontransgenic animals, and there were no adults deaths.
Histological evaluation of the mammary fat pads from 6-8-month-old transgenic animals killed 1 day after delivery revealed moderate epithelial hyperplasias when compared to nontransgenic lactating animals. These hyperplasias resembled in some extend to previously described by Webster et al.(16) in transgenic mice expressing a murine mammary tumor virus promoter/activated c-src fusion gene. The alveolar lumen and mammary ducts of the transgenic lactating rats presented an accumulation of particulate material and reduced lipid content. According to Bernirschke et al.(17) this type of lesion could be considered as preneoplastic in mice, and furthermore most mammary carcinomas are preceded by these lesions. However, our transgenic animals did not develop grossly detectable mammary tumors. It is worthwhile, however, to remark that in one of four transgenic lactating rats examined, we observed a large inflammation area within the mammary fat pad. These lesions were probably produced by the high expression of hGH (see Fig. 2); suckling mice were rejected by their mothers, probably as a results of the pain induced. Indeed, transgenic mothers were seen to eat their pups in a far greater proportion than usual. This interpretation is confirmed by the finding that pups from transgenic males but suckling from a nontransgenic female gained weight as control mice of the same age. Recently Cecim et al.(18) reported that mammary tumors are produced in transgenic mice with high serum levels of hGH in which the chimeric genes were either PEPCK/hGH or metallothionein/hGH.
Figure 2: Expression of the mthGH-CoAS/hGH chimeric gene. The expression of the chimeric gene was analyzed by Northern blot from RNA isolated from different tissues indicated under ``Materials and Methods.'' A, samples obtained from four adult transgenic mice were pooled, and a representative result is presented. This also applied to the testis and ovary obtained from four males and females, respectively. B, samples obtained from four 12-day-old transgenic mice were pooled and analyzed.
The results obtained in this study, which is the first to assay the mitochondrial HMG-CoA synthase promoter in transgenic mice, provide evidence that from the comparison between the expression of hGH and of the endogenous mitochondrial HMG-CoA synthase either in transgenic or in nontransgenic animals (data not shown), five different patterns of expression can be seen in adult mice: 1) positive, similar expression, such as in ovary; 2) negative expression, such as in duodenum, jejunum, ileum, white adipose tissue, heart, lung, brain and skeletal muscle; 3) far superior expression in hGH than in the endogenous gene, such as in testis; 4) lower expression but still appreciable levels in hGH, such as in liver, cecum, and colon, in comparison to mitochondrial HMG-CoA synthase; and 5) appreciably high mRNA levels for hGH and no expression at all for the HMG-CoA synthase, such as in stomach, brown adipose tissue, spleen, adrenal glands, and mammary glands.
We conclude from these results that the 1148-bp
fragment contains the cis-acting sequences which are required
for specific expression in liver. This suggests that an enhancer
outside the -1148-bp sequence stimulates the transcription rate
in this tissue, since in its absence the mRNA levels are decreased.
Studies of several other mammalian genes in transgenic mice have
revealed that regulatory elements in the proximal promoter are
sufficient to confer liver-specific expression, although maximal
expression requires a distal DNA sequence that enhances the expression.
For example, this arrangement is found for the gene that encodes
transthyretin, where 300 bp of the 5`-flanking region sequence alone
results in relatively low levels of expression in the liver, and high
levels of expression require the presence of an enhancer found 2 kb
upstream(19) . The same occurs in albumin and -fetoprotein
genes, where both promoters contain proximal elements that direct low
levels of expression in the liver, but high expression levels in the
liver are controlled by distinct liver-specific enhancer elements that
are located farther upstream from each gene(20, 21) .
The distal liver-specific enhancer for the albumin gene is located 10
kb upstream of its promoter. For the
-fetoprotein gene, three
enhancer elements located between 1 and 7 kb upstream of the
transcription start site are required for proper levels of tissue
expression in transgenic mice.
The expression pattern seen in testis suggests that outside of the -1148 bp of mitochondrial HMG-CoA synthase promoter there must be negative sequences that partially overcome the expression in this tissue. A similar higher expression in testis in comparison to control mice was also observed by Short et al.(22) with the transgene PEPCK/hGH. We did not find any different expression between hGH and HMG-CoA synthase in ovary.
The results in cecum and colon suggest that an enhancer, outside the -1148-bp sequence, stimulates the transcription rate in these tissues, since in its absence the mRNA levels are decreased. Studies of fatty acid binding protein expression in colon and cecum of transgenic mice whose reporter gene was also human growth hormone show that the tissue specific cis-elements that promote the expression in these tissues are localized to small DNA sequences, between -1600 and +21 bp. The absence of these sequences strongly modifies the mRNA pattern(23) . Analogous results were also observed with intestinal fatty acid binding protein in the sequences comprised between -1178 and +28(24) .
An unexpected result was found: there was an intense hGH expression in brown adipose tissue, stomach, and mammary gland of adult mice, but not so intense in adrenal glands and spleen, at variance with what happens in the adult endogenous gene. The cis-acting sequences responsible for the regulation of mitochondrial HMG-CoA synthase expression in stomach, brown adipose tissue, and mammary gland appear to be present in the proximal 1148 bp of the promoter of mitochondrial HMG-CoA synthase. A similar expression in stomach in transgenic but not in control mice was observed by Short et al.(22) with the transgene PEPCK/hGH. It appears that some elements, which may repress the transcription, are not present in the 1148 bp of the promoter. On the other hand, if the absence of expression in other tissues, such as muscle heart, brain, lung, kidney, white adipose tissue, and the different parts of the gut is produced under the same mechanism, i.e. the occurrence of regulatory sequences, we could affirm that the cis-element of other nonexpressed tissues is probably different from those responsible for transcription in brown adipose tissue, stomach, adrenal glands, spleen, and mammary gland.
Analogous experiments of expression were carried out in neonatal mice. As seen in Fig. 2, Panel B, in addition to the results seen in adult mice, we can observe bands corresponding to duodenum, jejunum, ileum, and kidney tissues, as expected(4) . It was noteworthy, however, that the intensity of the mRNA bands for hGH in small intestine were much lower than that of the endogenous mitochondrial HMG-CoA synthase, suggesting that a possible enhancer or cis-acting element for intestinal expression is not present in this promoter fragment. We have also observed that the mRNA levels for hGH in liver are the same than in the mitochondrial HMG-CoA synthase endogenous gene, at variance with what happens in adult transgenic mice. This could be interpreted as a partial repression of the gene, produced in transgenic animals after weaning. Brown adipose tissue was also only expressed in transgenic mice as happens in adult.
Figure 3: Regulation of the expression of mtHMG-CoAS/hGH in fasting/feeding conditions. Mice were starved for 24 h and then refed for up to 150 min. At the indicated time mice were decapitated, and their livers quickly removed and frozen in liquid nitrogen. Total RNA was isolated and subjected to electrophoresis and to Northern transfer. Data are expressed as a percentage of the mRNA signal of control animals fed a standard diet (means and S.E. of four mice in each group). A representative Northern blot is shown in the inset.
Figure 4: Regulation of the expression of mtHMG-CoAS/hGH by insulin. Starved mice (24 h) were injected intraperitoneally with insulin (40 units/kg), and the specific mRNA levels corresponding to different times of action of hormone were determined. A representative Northern blot is shown in the inset.
Figure 5: Effect of fatty acids in the expression of mtHMG-CoAS/hGH. Three mice were fed with different fatty acid diet for 5 days. A representative Northern blot is shown. C, control animals; O, oleate (5% w/w); Oc, octanoate (8% w/w); B, butyrate (5% w/w).
Figure 6: Serum hGH concentrations under different treatments. Serum hGH concentrations were determined as indicated under ``Material and Methods.'' C, control; S, starved; S-R, starved and refed; S-I, starved and insulin-treated; O, oleic acid-treated. Results are means plus S.E. of four animals in each group.
In conclusion, the use of transgenic mice has allowed us to extend our knowledge of an important regulatory site in ketogenesis. These results opened the possibility to elucidate the functional characteristics of the mitochondrial HMG-CoA synthase promoter outside 1148 bp and how this gene may modulate ketogenesis in liver and other tissues.