1 Division of Endocrinology and 2 Department of Psychiatry, Stanford University, Stanford 94305; 3 Mental Illness Research, Education, and Clinical Center, and 4 Geriatric Research, Education and Clinical Center, Veterans Affairs Palo Alto Health Care System, Palo Alto, California 94304; and 5 Department of Biochemistry, University of Nijmegen, 6500 HB Nijmegen, The Netherlands
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ABSTRACT |
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Hormone-sensitive lipase
(HSL) hydrolyzes triglyceride (TG) in adipose tissue. HSL is also
expressed in heart. To explore the actions of cardiac HSL,
heart-specific, tetracycline (Tc)-controlled HSL-overexpressing mice
were generated. Tc-responsive element-HSL transgenic (Tg) mice were
generated and crossed with myosin heavy chain (MHC)-tTA Tg mice,
which express the Tc-responsive transactivator (tTA) in the heart. The
double-Tg mice (MHC-HSL) were maintained with doxycycline (Dox) to
suppress Tg HSL. Upon removal of Dox, cardiac HSL activity and protein
increased 12- and 8-fold, respectively, and the expression was heart
specific. Although cardiac TG content increased twofold in control mice
after an overnight fast, it did not increase in HSL-induced mice.
Electron microscopy showed numerous lipid droplets in the myocardium of
fasted control mice, whereas fasted HSL-induced mice showed virtually
no droplets. Microarray analysis showed altered expression of cardiac
genes for fatty acid oxidation, transcription factors, signaling
molecules, cytoskeletal proteins, and histocompatibility antigens in
HSL-induced mice. Thus cardiac HSL plays a role in controlling
accumulation of triglyceride droplets and can affect the expression of
a number of cardiac genes.
hormone-sensitive lipase; gene expression; microarray analysis
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INTRODUCTION |
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FREE FATTY ACIDS
(FFA) are a major energy source in the heart. FFA provide up to 75% of
the heart's energy requirement in the resting state (13).
Fatty acids utilized by cardiomyocytes are derived from circulating FFA
and from the hydrolysis of intracellular stores of triglycerides
(19). Circulating FFA are derived from either the
hydrolysis of triglyceride stored in peripheral adipocytes or
triglyceride in very low density lipoproteins or chylomicrons. Hormone-sensitive lipase (HSL) is responsible for the hydrolysis of
triglyceride in adipocytes, whereas lipoprotein lipase (LPL) plays a
role in hydrolyzing triglyceride in lipoproteins. The released FFA are
taken up by cardiomyocytes and immediately utilized for -oxidation
in mitochondria or reesterified and stored as cytosolic triglyceride
droplets. On the basis of the fact that HSL is expressed in the heart
(11) and that the characteristics of the enzyme involved
in cardiac lipolysis are similar to those of adipose HSL
(27), intracellular triglyceride lipolysis is thought to
be catalyzed by myocardial HSL. Thus adipose HSL, cardiac LPL, and
cardiac HSL seem to control cardiac lipid supply in a coordinate
manner, although the precise mechanism remains to be elucidated.
In addition to triglyceride hydrolase activity, HSL has neutral cholesterol esterase activity and also catalyzes the hydrolysis of diacylglycerol into monoacylglycerol (3). Diacylglycerol can function as a second messenger in signal transduction, mediating intracellular Ca2+ mobilization and protein kinase C activation in a variety of tissues and cells (14). Because diacylglycerol can be a substrate of HSL in the heart, HSL might potentially play a role in cardiac signal transduction by controlling diacylglycerol concentration.
Cardiomyocytes accumulate lipid droplets with fasting, wherease no lipid droplets can be observed in the heart from fed animals (8). Ischemia stimulates intracellular lipolysis, resulting in decreased triglycerides and increased FFA in the myocardium (24). However, the intracellular mechanism of accumulation and hydrolysis of lipid droplets within cardiomyocytes has not yet been clarified.
Although FFA are an essential energy source for the heart, excess FFA have detrimental effects on cardiac function. Patients with elevated serum FFA are more likely to develop arrhythmias at the onset of myocardial infarction (16), and high plasma FFA concentrations have been shown to be related to the size of a myocardial infarction (18). In addition, in vitro and ex vivo studies have shown that FFA diminish contractility (17) and that FFA overload causes breakdown of membrane phospholipids and mitochondrial dysfunction (9). To investigate the effect of aberrant expression of HSL on cardiac lipid metabolism, we generated heart-specific HSL-overexpressing mice by utilization of the tetracycline-controlled gene expression (Tet) system. In this system, transgene expression is controlled by a tetracycline-responsive element (TRE), which is activated by the binding of a hybrid protein "tetracycline-responsive transcriptional activator" (tTA). The tTA binds to TRE in the absence of tetracycline, resulting in the initiation of HSL transcription. Our data show the tight regulation of foreign HSL in transgenic mice and the effects of HSL overexpression in the heart.
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MATERIALS AND METHODS |
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Chemicals and Reagents
All chemicals were from Sigma Chemical (St. Louis, MO) unless otherwise indicated. Restriction enzymes were from GIBCO-BRL (Grand Island, NY). DNA purification reagents were from Qiagen (Santa Clarita, CA). pTRE plasmid was from Clontech (Palo Alto, CA). Cholesteryl [1-14C]oleate and [Construction of Transgenes
A full-length cDNA of rat HSL was subcloned into pTRE plasmid at the EcoRI/XbaI site. A 3.5-kb fragment containing TRE, HSL, and the SV40 poly A signal was excised by HindIII/XhoI digestion, separated in agarose gel, and purified using QIAEX II.Generation of Double-Transgenic Mice
The TRE-HSL gene was injected into fertilized oocytes derived from C57B/6J × CBA F1 background in the Transgenic Core Facility at Stanford University. Four identified TRE-HSL founders were crossed with C57BL/6J mice to expand the strain. Heterozygous TRE-HSL mice were then crossed with heterozygous MHC
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Control of Transgene Expression
Mating MHCGenotyping
Genotypes were identified by Southern blot analyses and PCR with the use of genomic DNA from tail biopsies. DNA (10 µg) from each animal was digested with BamH1 and fractionated on 1% agarose. DNA was transferred to a nylon membrane (Hybond N, Amersham) and probed with 32P-labeled rat HSL cDNA and tTA cDNA. Probed membranes were washed and visualized by a Phosphorimager 445 SI (Molecular Dynamics, Sunnyvale, CA). Primers used for PCR are (forward/reverse) TRE-HSL: GGC GTG TAC GGT GGG AGG/GCA AAG ACG TTG GAC AGC C; MHCImmunoblot Analysis
Immunoblot analyses were performed as previously described (11, 20). Two to twenty micrograms of protein from tissue homogenates were subjected to SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was incubated with rabbit anti-HSL antibody, anti-p27 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), or heart-type fatty acid binding protein (FABP) antibody and then with horseradish peroxidase-linked anti-rabbit IgG (Amersham). The membranes were visualized with chemiluminescence reagent ECL (Amersham), exposed to Kodak XAR film, and then analyzed by a Fluor-S multi-image analyzer (Bio-Rad, Hercules, CA).HSL Activity
HSL activity was measured as neutral cholesteryl esterase activity according to a method previously described (11). The results are expressed in nanomoles of cholesteryl oleate hydrolyzed per milligram of protein per hour.Tissue Lipid Content
Mice were anesthetized with 0.11 mg/g ketamine and 0.013 mg/g xylazine, and hearts were perfused with 3 ml of PBS via the left ventricle to wash out red blood cells. Lipids were extracted from 30-40 mg of the heart with 42 volumes of chloroform-methanol-PBS (10:5:6) and analyzed by TLC as previously described by Ruiz and Ochoa (23). Briefly, an internal standard of cholesteryl formate (0.2 µg) was added to each sample, and aliquots of extracted lipids that had been dried and redissolved in toluene were spotted onto EDTA-treated TLC plates. Standards for each of the lipid classes were spotted at various concentrations to construct a calibration curve. The plates were developed in a stepwise fashion with chloroform-methanol-water 60:40:5 (vol/vol/vol) to 2 cm, ethyl acetate-2-propanol-ethanol-chloroform-methanol-0.25% KCl 35:5:20:22:15:9 (vol/vol/vol/vol/vol/vol) to 5 cm, toluene-diethyl ether-ethanol 60:40:3 (vol/vol/vol) to 7.5 cm, heptane-diethyl ether 94:8 (vol/vol) to 10.5 cm, and pure heptane to 12.5 cm. The plates were charred by dipping in 10% cupric sulfate in 8% phosphoric acid and were heated to 200°C for 2 min. The charred plates were then analyzed on a Fluor-S multi-image analyzer.Serum Lipid Analysis
Blood was drawn by a puncture of the heart. Animals of both genders were used. Serum was separated immediately, and total cholesterol, triglyceride, and free fatty acid concentrations were determined with commercially available kits (Sigma, Wako Chemicals).Morphological Studies
Tissues were fixed with 1.5% glutaraldehyde, 4% polyvinylpyrrolidone, 0.05% calcium chloride, and 0.1 M sodium cacodylate, pH 7.4, for 16 h. Light microscopy was performed as previously described (21). For electron microscopy, the fixed tissue was washed with 0.15 M imidazole, treated with 4% osmium tetroxide (30 min) and 2% uranyl acetate (1 h at 4°C), and then dehydrated and embedded. Thin sections were stained with lead citrate and uranyl acetate and examined.Microarray Expression Analysis
Experiments were basically performed following the Affymetrix GeneChip expression analysis protocol by use of Affymetrix Hybridization Oven 640 and Fluidics Station. Cardiac ventricular RNA was prepared from fed or fasted MHCSemiquantitative PCR Analysis
Expression of alcohol dehydrogenase-1 (ADH-1), CAAT box enhancer binding protein (C/EBP)-Statistical Analysis
Values were compared among three groups: MHC ![]() |
RESULTS |
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Generation of Transgenic Strains
From the first set of injections, four animals of 37 live births were identified as TRE-HSL positive by Southern blot analysis. These four founders (TRE-HSL1, -2, -3, and -4) contained three, one, three, and two copies of the integrated TRE-HSL gene, respectively. All founders were crossed with C57BL/6J mice to expand the strain. TRE-HSL2, -3, and -4 strains were successfully bred, although only two of the integrated three copies of TRE-HSL gene were transmitted in the TRE-HSL3 strain. None of the three copies of TRE-HSL was transmitted in the TRE-HSL1 strain. Heterozygous F1 offspring of the TRE-HSL2, -3, and -4 mice (HSL+/HSL expression was induced by removal of Dox from the drinking water
for 3 wk. Animals were then killed for tissues to be harvested and for
investigation of HSL expression. As shown in Fig.
2A, cardiac HSL protein
expression was induced eightfold in Dox() MHC-HSL4
(MHC+/
/HSL+/
) mice compared with control
MHC
-tTA (MHC+/
/HSL
/
) mice. In
contrast, transgenic HSL was completely suppressed in Dox(+) MHC-HSL4
mice. Cardiac HSL activity in Dox(
) mice was 12-fold higher than in
Dox(+) mice or control mice. A similar level of cardiac HSL induction
was seen in MHC-HSL2 mice, whereas none of the MHC-HSL3 mice displayed
inducible cardiac HSL expression. There was no significant difference
in HSL induction between genders. The MHC-HSL4 strain was mainly used
in subsequent experiments unless otherwise stated.
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To test the tissue specificity of the induction of HSL expression, HSL
activity was investigated in various tissues. As shown in Fig.
2B, removal of Dox from the drinking water induced HSL activity only in the heart. In Dox() MHC-HSL mice, HSL activity in
the heart was 4-18 times higher than in other tissues except fat.
Fat showed the highest HSL expression (60-70 nmol · mg
protein
1 · h
1) among any of the
tissues in all three groups. Dox(+) MHC-HSL mice and control MHC
-tTA
mice showed no HSL induction in any tissues examined. These data
indicate that the inducible expression of HSL is cardiac specific in
MHC-HSL transgenic mice.
Effect of Cardiac HSL Overexpression
Microscopy.
A histological examination of tissues from left ventricle muscle from
fed control and 4-wk HSL-induced mice revealed no special changes. The
wet weights of the hearts were also not affected by HSL overexpression
for 4 wk (male: 162 ± 6 vs. 153 ± 5 mg for control and
HSL-induced, respectively). To bring out potential differences, the
mice were fasted for 16 h. Heart muscle was removed and processed
for both light and electron microscopy. Light microscopy staining with
toluidine blue showed numerous small blue droplets (presumed to be
lipid) dispersed in the muscle of the fasted control MHC-tTA mice,
but there were no such droplets in the muscle of the HSL-induced
MHC-HSL mice (data not shown). When sections of the fasted animals were
viewed with the electron microscope, a similar story was found; i.e.,
many lipid droplets were located within clusters of mitochondria in the
muscle fibers of control MHC
-tTA mice (Fig.
3A), but not within the
mitochondrial clusters, or elsewhere, in cardiac muscle fibers of the
HSL-induced MHC-HSL mice (Fig. 3B). No other morphological
differences were observed in the samples taken from the mice.
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Lipid analysis.
To biochemically analyze the changes observed by microscopy, cardiac
lipid content was then assessed. As shown in Table
1, cardiac triglyceride content was
increased approximately twofold in control MHC-tTA mice after an
overnight fast. In contrast, cardiac triglyceride content was not
changed in HSL-induced heart. Cardiac free cholesterol and FFA content
were increased with fasting in both MHC
-tTA and HSL-induced MHC-HSL
mice but were not affected by overexpression of HSL. Total
phospholipids were not changed by fasting in hearts from either control
or HSL-induced mice (data not shown). No cholesteryl esters were
detected in the hearts of control or HSL-induced mice. Serum lipid
concentrations were analyzed after 3 wk of HSL induction. In both fed
and fasted conditions, serum triglyceride, total cholesterol, and FFA
were not changed by the overexpression of HSL in the heart (Table
2).
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Gene expression.
To study the effects of HSL-overexpression on cardiac gene expression,
microarray analysis was performed. From an analysis comparing control
and HSL-induced mice, cardiac expression of ~100 genes was changed at
least twofold in either the fed or fasted condition. The known genes
whose expression was altered greater than twofold are listed in Tables
3 and 4.
Among these genes, the expression of ADH-1, C/EBP-,
HMG-CoA synthase, M-STP1, MT-I and -II, p27, and tubulin
-4 and -5 were altered by HSL overexpression under both fed and fasted
conditions. These nine genes were investigated by semiquantitative PCR
analysis to confirm the consistency of the results of microarray
analysis. As shown in Fig. 4, all of the
changes seen in microarray analysis were consistent with the results
from semiquantitative PCR analysis.
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DISCUSSION |
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In this study, we have established heart-specific,
tetracycline-controlled, HSL-overexpressing mice. Transgenic HSL was
expressed specifically in the heart, and its expression was tightly
regulated by the administration of Dox. Cardiac HSL activity was
induced 12-fold and protein mass 8-fold in heterozygous MHC-HSL mice. Dox-dependent suppression of transgenic HSL was complete in our transgenic mice, as documented by the fact that cardiac HSL activity and protein levels in Dox-treated MHC-HSL mice were as low as those of
control MHC-tTA mice. Although some leakage of the target gene has
been reported in transgenic mice with the Tet system (30),
our data showed no significant leakage of transgene expression.
Upon fasting, serum FFA increase, and cardiomyocytes accumulate triglyceride droplets. It is thought that an imbalance between influx and utilization of FFA causes cardiac lipid droplet formation. In this study, we have demonstrated that cardiac HSL is capable of hydrolyzing accumulated lipid droplets in vivo, preventing lipid accumulation. Cardiac lipid accumulation can be harmful; excess FFA can lead to cytotoxicity, and it has been reported that diabetic Zucker rats accumulate lipid droplets in cardiomyocytes, leading to lipoapoptosis (31). Lipid droplet accumulation is also observed in certain diseases such as dilated and hypertrophic cardiomyopathies (12, 29). Controlling cardiac lipid accumulation might possibly be beneficial in these conditions.
Overexpression of heart HSL affected the expression of several cardiac
genes associated with metabolism, cell growth, immune response, and the
cytoskeleton. We propose that the overexpression of HSL increased
intracellular fatty acid flux by hydrolyzing any triglycerides produced
by esterification and that the change in fatty acid metabolism is a
common mechanism responsible for the alteration in these genes. Thus,
under both fed and fasted conditions, ADH, C/EBP-, and HMG-CoA
synthase were upregulated in HSL-induced mice. The mouse ADH-1 product
ADH-A2 is a class 1 ADH that catalyzes the oxidation of retinol into
retinalaldehyde. Although the physiological function of cardiac
ADH is not fully understood, it also catalyzes
-oxidation of
-hydroxy fatty acids and cytotoxic aldehydes as physiological
substrates (1). Because fatty acids can be substrates for
ADH, it is possible that the induction of cardiac ADH occurred to
compensate for the presumed increased release of intracellular fatty
acids brought about by the action of HSL. C/EBPs are a highly conserved
family of DNA-binding proteins implicated in the transcriptional
control of genes involved in cell growth, differentiation, and lipid
metabolism. C/EBP-
and -
can bind to regulatory sites within the
ADH-promoter region, activating transcription (28). This
is consistent with the idea that HSL overexpression might lead to
transcriptional activation of ADH by increased expression and binding
of C/EBP-
to the ADH promoter. Because fatty acids do not affect
C/EBP expression either in vitro or in vivo (10), HSL
might play a role in modifying the signaling pathways that regulate
C/EBP transcription.
HMG-CoA synthase, which is located in both the cytosol and mitochondria, catalyzes the condensation of acetoacetyl-CoA and acetyl-CoA to form HMG-CoA. The HMG-CoA produced by cytosolic HMG-CoA synthase is converted to mevalonate by HMG-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis, whereas the HMG-CoA produced within mitochondria is converted to acetoacetate by HMG-CoA lyase and then to hydroxybutyrate and acetone. In this manner, mitochondrial HMG-CoA synthase is an important enzyme for ketogenesis. Fatty acids increase the transcription of mitochondrial HMG-CoA synthase both in vitro and in vivo (2, 5), and this is mediated by peroxisome proliferator-activated receptors (22). The upregulation of HMG-CoA synthase by HSL overexpression suggests that chronic FFA release stimulated intracellular ketogenesis. Accordingly, other ketogenic and fatty acid-oxidative genes, i.e., carnitine palmitoyltransferase I, cytochrome P450, steroid dehydrogenase, and uncoupling protein-2 were also upregulated in fasted HSL-induced mice. On the other hand, the cholesterol biosynthetic pathway does not seem to be activated by HSL overexpression, because mRNA levels of other enzymes involved in cholesterol synthesis were not affected.
A number of genes involved in cell growth or intracellular signaling were also affected by HSL overexpression. Among this category, p27 was downregulated in both fed and fasted conditions, and this was confirmed by Western blots. Although the upregulation of genes involved in cell growth or intracellular signaling could suggest the possibility that HSL overexpression might result in cardiac hypertrophy, we did not observe any evidence of cardiac hypertrophy. However, HSL overexpression was maintained for only 3-4 wk in the current studies, and longer time periods might be required for these changes to be seen. FFA can affect cell growth, differentiation, and intracellular signaling (15). In addition, linoleic acid induces depletion of p27 and increases CDK2 activity, which is required for G1/S transition (7), and a fatty acid inhibitor, cerulenin, increases expression of the CDK inhibitors p21 and p27 (4). These reports are consistent with our data and suggest that chronically overreleased intracellular FFA possibly led to these changes in our transgenic mice.
Previous studies have also shown that unsaturated fatty acids are able
to cause alterations in the normal distribution pattern of cytoskeletal
proteins, including tubulins, actins, and myosins (6). In
both fed and fasted conditions, expression of tubulin -4 and -5 was
decreased in HSL-induced heart. These data also support the idea that
overexpression of HSL, causing chronic excess exposure of FFA, affects
cardiac gene expression of these antigens and cytoskeletal molecules.
Thus the changes in gene expressions seen in HSL-induced mice
convincingly suggest the existence of chronic exposure to high
intracellular fatty acid flux. However, cardiac fatty acid content was
not changed in HSL-induced mice compared with pair-fed or pair-fasted
control mice. An increase in intracellular FFA release by the action of
HSL might have been compensated for by an accelerated efflux or
oxidation of FFA in HSL-induced mice.
HSL and LPL are thought to be coordinately regulated. Shimada et al. (26) reported that HSL activity and mRNA expression are elevated in adipose tissue in LPL-transgenic mice, which overexpress LPL in adipose tissue, skeletal muscle, and heart. In contrast, our data show stable LPL mRNA expression despite high expression of HSL in the hearts of transgenic mice. These data might indicate independent roles of LPL and HSL in cardiac lipid metabolism. It is possible that overexpressed HSL only accelerates the rate of intracellular hydrolysis and reesterification of triglyceride and does not affect the demand for extracellular FFA provided by the action of LPL.
In summary, in the current study, we created heart-specific HSL-overexpressing mice with a Tet system. The results show a role for cardiac HSL in controlling the accumulation of triglyceride droplets in the heart. HSL overexpression also affected the expression of a number of genes involved in lipid/energy metabolism, cell growth/cell cycle, and cellular antigen presentation. This animal model will be an excellent tool to study further the function of cardiac HSL.
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ACKNOWLEDGEMENTS |
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We thank Vanita Natu and Ann Numoto for technical assistance and Dr. Andrew Greenberg for helpful discussions.
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FOOTNOTES |
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This work was supported in part by research grants from the Research Service of the Department of Veterans Affairs (F. B. Kraemer, G. M. Murphy Jr.), and by Grants DK-46942 and DK-49705 from National Institute of Diabetes and Digestive and Kidney Diseases (F. B. Kraemer) and a Dean's Fellowship from the Stanford University School of Medicine (S. P. Selwood).
Address for reprint requests and other correspondence: Fredric B. Kraemer, Division of Endocrinology, S-005, Stanford Medical Center, Stanford, CA 94305-5103 (E-mail: fbk{at}stanford.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 10 January 2001; accepted in final form 7 June 2001.
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