Cardiac gene expression profile and lipid accumulation in response to starvation

Jinya Suzuki1, Wen-Jun Shen2, Brett D. Nelson2, Simon P. Selwood3, Greer M. Murphy Jr.3,4, Hideo Kanefara1, Sadao Takahashi1, Koji Oida1, Isamu Miyamori1, and Fredric B. Kraemer2,5

1 Third Department of Internal Medicine, Fukui Medical University, Fukui 910-1193, Japan; 2 Division of Endocrinology and 3 Department of Psychiatry, Stanford University, Stanford 94305-5103; and 4 Mental Illness Research and 5 Geriatric Research, Education and Clinical Center, Veterans Affairs Palo Alto Health Care System, Palo Alto, California 94304


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Starvation induces many biochemical and histological changes in the heart; however, the molecular events underlying these changes have not been fully elucidated. To explore the molecular response of the heart to starvation, microarray analysis was performed together with biochemical and histological investigations. Serum free fatty acids increased twofold in both 16- and 48-h-fasted mice, and cardiac triglyceride content increased threefold and sixfold in 16- and 48-h-fasted mice, respectively. Electron microscopy showed numerous lipid droplets in hearts of 48-h-fasted mice, whereas fewer numbers of droplets were seen in hearts from 16-h-fasted mice. Expression of 11,000 cardiac genes was screened by microarrays. More than 50 and 150 known genes were detected by differential expression analysis after 16- and 48-h-fasts, respectively. Genes for fatty acid oxidation and gluconeogenesis were increased, and genes for glycolysis were decreased. Many other genes for metabolism, signaling/cell cycle, cytoskeleton, and tissue antigens were affected by fasting. These data provide a broad perspective of the molecular events occurring physiologically in the heart in response to starvation.

microarray analysis; differential expression; lipid droplet


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

STARVATION INDUCES PROFOUND metabolic changes in the body. Fasting leads to a shift in energy supply from glucose to free fatty acids (FFA) in many tissues. The heart preferably uses fatty acids as an energy source, although its energy balance shifts widely depending on physiological conditions, such as fasting, diabetes, or ischemia (22). Fasting has been reported to lead to a number of biochemical and histological changes in the heart. Biochemically, fasting increases myocardial glycerides and glycogen content through inhibition of the glycolytic pathway with enhanced oxidation of fatty acids (10, 25). Yaffe et al. (30) reported that starvation induces changes in cardiac FFA composition, i.e., 20- to 22-carbon polyunsaturated fatty acids accumulate in the heart of starved rats. Interestingly, it has been reported that starvation diminishes myocardial damage in rats and rabbits (9, 26); glycogen accumulation might contribute to this protection, although this is controversial (8).

Histologically, fasting induces intracellular lipid droplet accumulation in cardiomyocytes. In experimental rodent models, lipid droplets accumulate around clustered mitochondria during fasting, whereas almost no lipid droplets are visible in the normal, fed condition (1, 18). Although cardiac lipid droplet accumulation is a physiological phenomenon, it is also a pathological event in certain conditions, such as cardiomyopathies (20, 28) or in diabetic Zucker rats, in which cardiac lipid accumulation leads to lipo-apoptosis (31). It has been thought that cardiac lipid accumulation is due to an imbalance between fatty acid influx and beta -oxidation (23). It is unclear whether the lipid accumulation is a reaction to protect the tissue from toxic FFA or preparation for upcoming lethal starvation.

As described above, many biochemical and histological changes are known to occur in the heart in response to starvation. However, a global, systematic evaluation of the molecular events occurring in the starved heart has not been investigated. We thought that identification of the gene expression profiles induced by nutritional intervention would provide insights into the molecular events underlying their physiological response to starvation. To explore the molecular response to starvation, we have analyzed cardiac gene expression profiles utilizing oligonucleotide microarrays, together with biochemical and histological changes.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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). pGEM-T Easy plasmid was from Promega (Madison, WI). [alpha -32P]dCTP was from Amersham Life Sciences Products (Arlington Heights, IL). Organic solvents were from J. T. Baker (Phillipsburg, NJ).

Animal study. Experiments were basically performed using groups of control C57B/6J mice (27). Mice were maintained on a chow diet (PMI5012) with a 12:12-h dark-light cycle. On the day of experiments, animals were fasted for 16 h (5 PM to 9 AM) or 48 h (9 AM to 9 AM) and killed by cervical dislocation. The heart ventricles were immediately harvested, rinsed with PBS, and homogenized in TRIzol reagent (GIBCO-BRL) for RNA extraction.

Serum lipid analysis. Blood was drawn by a puncture of the inferior vena cava. Animals of both genders were used. Serum was separated immediately, and total cholesterol, triglyceride (TG), and FFA concentrations were determined with commercially available kits (Sigma, Wako).

Tissue lipid content. Mice were anesthetized and hearts were exposed. Immediately, the 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 vol of chloroform-methanol-PBS (10:5:6). Cholesteryl formate (0.2 mg/ml) was added in chloroform-methanol (2:1) to serve as an internal standard for recovery. After centrifugation, 100 µl of the organic solvent phase were transferred, air-dried, and dissolved in 50 µl of toluene. An aliquot (2 µl) was then applied in duplicate to 20 × 20-cm TLC plates (Whatman, Clifton, NJ) and developed sequentially with 1) chloroform-methanol-water 60:40:10 (vol/vol/vol) to 1 cm and 2) hexane-ether-acetic acid 85:15:2 (vol/vol/vol) to 13 cm. Separated lipids were visualized and analyzed as previously described (24).

Electron microscopy. Experiments were basically performed as previously described (27). Briefly, mouse hearts were excised and fixed in 2.5% glutaraldehyde for 2 h, followed by 2% osmium tetroxide for 2 h. Samples were then dehydrated and embedded. Thin sections (80 nm) were stained with lead citrate and uranyl acetate. Microscopic examination was performed with a transmission electron microscope (Hitachi H-7500) at Pathology & Cytology Laboratories (Saitama, Japan). Three animals per group were used, and more than 10 positions per sample were studied.

Microarray expression analysis. Experiments were basically performed by following the Affymetrix GeneChip expression analysis protocol with the Affymetrix Hybridization Oven 640 and Fluidics Station (27). Cardiac ventricular RNA from four animals per group was pooled and used for subsequent preparation. Four hundred micrograms of pooled total RNA (100 µg/animal) were used for mRNA preparation, double-strand (ds) cDNA synthesis, and biotin-labeled cRNA synthesis. Labeled cRNA (10 µg) was applied to an Affymetrix oligonucleotide array GeneChip Murine 11K set. Comparison analyses were performed for fed vs. 16-h-fasted mice and fed vs. 48-h-fasted mice.

Northern blot analysis. RNA was extracted from hearts of C57B/6 mice by TRIzol reagent (GIBCO-BRL). Total RNA (20 µg) was size-fractionated on 1% agarose, transferred to a nylon membrane, and probed with 32P-labeled mouse alcohol dehydrogenase (ADH)-1, aldehyde dehydrogenase-2 (AD-2), fructose 1,6-bisphosphatase (F16B), hexokinase-II (HK-II), hormone-sensitive lipase (HSL), 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase, perilipin, pyruvate carboxylase (PvCx), uncoupling protein-2 (UCP2), beta -actin, and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA. The exposed plates were scanned and analyzed by Mac BAS software on BAS1500 (Fuji Film).

cDNA probes. Total RNA was isolated from mouse heart, and first-strand cDNA was synthesized with oligo(dT) primer by Super Script II (GIBCO-BRL). The cDNAs of targeted genes were amplified by PCR, cloned into pGEM-T easy vector (Promega), and sequenced. The sequences of the primers for PCR are (forward/reverse) ADH-1: TGG CTC TGC CGT CAA AGT/CCA GAA CGA AGC AGG TCA; AD-2: CCT TCG GTG GAT TCA AGA TG/GAC AAG CAG GCA TGA CAT TC; F16B: CTC AAC GAG GGC TAT GCC AA/CCG TGA CCT GTG TCA CTT GA; HK-II: GCT GAG CAG AGC CTA GTT AC/AAC GCT CAC TAG ACC GAG TG; HMG-CoA synthase: CTC ACC ACT CTG CCC AAG AA/AGC TCT GGT CCA CAG CAG CA; PvCx: GCA GGG CTA CAT TGG CAT TC/CCA CCT TGA TGT CTA TGA CC; UCP2: GCC AAC CTC ATG ACA GAT GA/TAG AAA GGG CTG AGG GCT CA.

Statistical analysis. Values were compared among the three groups. Three to six animals per group were normally used for experiments. ANOVA was used to determine a significant difference by use of STATVIEW (Abacus Concepts, Berkeley, CA) on a Macintosh computer. A value of P < 0.05 was considered significant. Results are presented as means ± SE.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Weight and serum lipid concentrations. Mice were fasted for 16 or 48 h to investigate changes in body weight and serum lipid levels. As shown in Fig. 1, body weight of male mice decreased 13% after 16 h of fasting and 22% after 48 h of fasting. Female mice decreased their body weights 11% after 16 h of fasting and 18% after 48 h of fasting. Serum TG concentration was decreased 32% after 16 h of fasting and 35% after 48 h of fasting, but the difference was not statistically significant (Table 1). Serum FFA level was increased more than twofold in both 16- and 48-h-fasted mice compared with fed mice. These data indicate that the protocol has a significant impact on body weight and serum lipid profiles.


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Fig. 1.   Body weight of mice. Animals were weighed and fasted for up to 48 h. Data represent means ± SE; n = 5 (male) or 4 (female). * P < 0.05 vs. fed group.


                              
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Table 1.   Serum lipid profile

Cardiac lipid content. To investigate the effect of fasting on cardiac lipid composition, tissue lipids were extracted and analyzed by TLC. As shown in Fig. 2, cardiac TG content was increased threefold in 16-h-fasted mice and sixfold in 48-h-fasted mice. Cardiac free cholesterol content and FFA content were not significantly changed in either 16- or 48-h-fasted mice compared with fed mice. Cardiac cholesteryl ester content was below the detectable level in each group of animals.


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Fig. 2.   Cardiac lipid composition. Hearts were perfused with 3 ml of PBS via the left ventricle, and lipids were extracted. Lipids were separated by TLC and analyzed as described in MATERIALS AND METHODS. Animals of both genders were used. Results represent means ± SE; n = 5. TG, triglyceride; FFA, free fatty acid; FC, free cholesterol. * P < 0.05 vs. fed group.

Electron microscopy. To investigate histological changes in accordance with cardiac TG accumulation, electron microscopy was performed. As shown in Fig. 3, A and D, hearts from fed animals contain few, if any, lipid droplets. The maximal diameter of detected lipid droplets was about one-half the size of mitochondria. In contrast, hearts from 16-h-fasted mice showed numerous lipid droplets in close proximity to clusters of mitochondria (Fig. 3, B and E). The maximal diameter of the droplets was about the size of mitochondria. In the hearts from 48-h-fasted mice, more lipid droplets were observed than in 16-h-fasted mice (Fig. 3, C and F). The size of the lipid droplets was the same as those seen in 16-h-fasted mice. No morphological changes were observed in either 16- or 48-h-fasted mice.


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Fig. 3.   Low-magnification electron micrograph of left ventricle muscle from fed and fasted mice. Whereas muscle fibers from fed mice show few lipid droplets (A and D), muscle fibers from 48-h-fasted mice show numerous lipid droplets (arrows) associated with clusters of mitochondria (C and F). Mice fasted for 16 h show fewer lipid droplets than 48-h-fasted mice (B and E).

Cardiac gene expression profile. To explore the effect of fasting on cardiac gene expression profiles, microarray analyses were performed. Differential expression was analyzed between fed and 16-h-fasted mice and between fed and 48-h-fasted mice. The known genes whose expressions were altered more than twofold are listed in Tables 2 and 3. After a 16- or 48-h fast, >70 and 180 cardiac genes, respectively, were changed more than twofold.

                              
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Table 2.   Differential expression of cardiac genes in 16-h-fasted mice


                              
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Table 3.   Differential expression of cardiac genes in 48-h-fasted mice

Among genes involved in lipid/glucose/energy metabolism, only PvCx was decreased in 16-h-fasted mice, whereas many genes were changed in the 48-h-fasted heart. Genes for fatty acid oxidation, such as adipose differentiation-related protein (ADRP), ADH-1, AD-2, HMG-CoA synthase, UCP2, HSL, and CAAT box enhancer-binding proteins (C/EBPs), were increased. Genes for glycolysis, such as HK-II and PvCx, were decreased, accompanied by an increase in expression of F16B, an enzyme for gluconeogenesis. Likewise, more genes involved in other aspects of metabolism were affected in 48-h-fasted mice than in 16-h-fasted mice. Interestingly, aryl sulfotransferase (M-STP1) and metallothioneins (MTs), which were also increased in HSL-transgenic (Tg) mice (27), were increased in 48-h-fasted hearts.

In the category of cell cycle/growth/signaling, a number of genes were affected by both periods of fasting, although the expression profiles are somewhat different. With 16 h of fasting, many early-response genes, such as c-fos, Cyr61, and zif/268, were increased, whereas few such genes were changed in 48-h-fasted mice. Instead, more genes for signaling, cell cycle, and apoptosis, such as Fas, cyclin G, and mitogen-activated protein kinases, were affected by prolonged fasting. After 48 h of fasting, many genes for cellular basic functions, such as dipeptidase and SKD1, were affected. Most of these genes are involved in intracellular protein processing or catabolism. In contrast, only two such genes were changed after 16 h of fasting.

Genes related to the cytoskeleton, such as collagens, fibrillin, myosins, and tubulins, were almost all decreased in the 48-h-fasted heart. Previous studies have shown that cis unsaturated FFA are able to cause alterations in the normal distribution pattern of cytoskeletal proteins, including tubulins, actins, and myosins (17), and that starvation leads to a reduction of actin and myosin (6). Consistent with those reports, our data suggest that extreme fasting may cause structural damage to cardiac cells.

The expression profiles of immune-related genes showed remarkable differences between short- and long-term fasting. After a 16-h fast, a number of genes for major histocompatibility complex (MHC) antigens were increased, whereas only a few genes of MHC were affected after a 48-h fasting. However, in general, more cardiac genes were affected by 48 h of fasting than by 16 h of fasting, as shown in Tables 2 and 3.

Northern blot analysis. To confirm the changes identified by microarray analysis, Northern blot analysis was performed on genes for lipid/glucose/energy metabolism by use of C57B/6J mice. The transcripts were normalized by GAPDH, because its transcript was not affected by fasting in preliminary experiments. As shown in Fig. 4, transcripts of ADH-1, AD-2, HSL, F16B, HMG-CoA synthase, and UCP2 were increased, and the HK-II transcript was decreased with fasting. The induction or suppression of all genes was more profound in 48-h-fasted mice except ADH-1, whose expression was increased more than fourfold in both 16- and 48-h-fasted mice. Expression of HMG-CoA synthase showed the most drastic changes, with a 9- to 10-fold induction by the 16-h fast and a 22- to 27-fold induction by the 48-h fast. The PvCx transcript did not have a sufficient signal for quantitative analysis, and the beta -actin transcript was decreased with fasting, as previously reported (6) (data not shown). It was of interest to see whether perilipin expression was induced by fasting in the heart, because perilipins are lipid droplet-associated proteins whose expression is induced concomitant with lipid accumulation in 3T3-L1 adipocytes (14, 15). However, our Northern analysis did not detect perilipin transcripts in mouse hearts under any conditions (data not shown).


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Fig. 4.   Northern blot analysis of genes for lipid/glucose/energy metabolism. Total RNA from 3 mice/group was pooled, and 20 µg of each were size-fractionated, transferred to a membrane, and probed with 32P-labeled cDNA probes. ADH, alcohol dehydrogenase-1; AD2, aldehyde dehydrogenase-2; F16B, fructose 1,6-bisphosphatase; HK-II, hexokinase II; HMG, HMG-CoA synthase; HSL, hormone-sensitive lipase; UCP2, uncoupling protein-2. Signals were normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and degrees of change from fed mice are shown beneath blots.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the current study, we investigated the effect of starvation on cardiac gene expression by use of oligonucleotide arrays. We screened 11,000 genes, including EST clones, on the array. According to the genome projects, the number of total mammalian genes was estimated to be ~30-40,000 (7). Because we analyzed ~25-30% of the total genes, and the array was designed to select genes randomly, our data represent, or at least highlight, the expression profile of cardiac genes. Indeed, changes in the expression of some genes were consistent with previous reports.

Among 11,000 genes, 75 genes (16-h fast) and 182 genes (48-h fast) were affected more than twofold. Because the threshold of the differential expression on microarray analysis is set much higher than conventional Northern blot analyses for consistency and specificity (Affymetrix comment), even more genes may have been affected by starvation than detected by microarray analysis. Indeed, our Northern analyses showed significant changes in 16-h-fasted mice that were not detected by differential expression on microarray analysis. Our Northern analysis also demonstrated the accuracy of our microarray data, but additional samples will be required to confirm the broad generalizability of these results.

To our surprise, the expression pattern in 48-h-fasted mice was quite similar to that in cardiac-specific, HSL-overexpressing mice (27). Affected genes were consequently categorized into the same functional groups as was done for the HSL-Tg mice. Among those genes, ADH-1, C/EBP-beta , HMG-CoA synthase, M-STP1, metallothionein (MT)-I and -II, and tubulin alpha -4 and alpha -5 were all changed similarly in fed HSL-Tg mice, fasted HSL-Tg mice, and 48-h-fasted control mice. Thus transcriptional regulation of these genes may be fairly sensitive to fatty acid (FA), presumably sharing similar FA-sensitive transcriptional factors. Further investigation into the 5'-regulatory regions of these genes may clarify the mechanism.

In accordance with the accumulation of TG droplets, many genes for lipid, glucose, and energy metabolism were changed with prolonged fasting. ADRP, which mediates FA uptake and/or lipid droplet formation (12); ADH-1, which catalyzes the beta -oxidation of omega -hydroxy fatty acids (2); HSL, a rate-limiting enzyme for lipolysis; UCP2, which dissipates the proton gradient of the inner mitochondrial membrane and reduces ATP synthesis; and HMG-CoA synthase, a mitochondrial ketogenic enzyme, were all increased. In contrast, the glycolytic enzymes HK-II and PvCx were decreased, and F16B, an enzyme for gluconeogenesis, was increased. All of these changes are consistent with the shift of the energy source in the heart during fasting from glucose to FA. Among these genes, HMG-CoA synthase was upregulated the most drastically by fasting. Continuous exposure to FFA likely led to this induction, because it has been reported that FA increase the transcription of mitochondrial HMG-CoA synthase both in vitro and in vivo (5, 13). The HMG-CoA produced by mitochondrial HMG-CoA synthase is transformed into acetoacetate by the action of HMG-CoA lyase and then into hydroxybutyrate and acetone. In this manner, mitochondrial HMG-CoA synthase is the rate-limiting enzyme of the ketogenic pathway (16). Although it has been thought that ketogenesis occurs exclusively in the liver, the drastic induction of cardiac HMG-CoA synthase suggests that intracellular ketogenesis occurs also in cardiac myocytes.

In the category of genes involved in other aspects of metabolism, M-STP1 and MT-I and -II were increased with 48 h of fasting. These genes were also increased in the hearts of cardiac-specific, HSL-overexpressing transgenic mice, presumably due to chronic intracellular exposure to FFA (27). M-STP1 is a member of the sulfotransferase multigene family, whose function is involved in a number of metabolic pathways, including sulfonation of catecholamines, thyroid hormones, and phenolic drugs (11, 29). Studies of this enzyme have focused mainly on liver, kidney, and lung, and its expression in the heart has not been studied in detail. Because prolonged fasting stimulates the release of catecholamines and steroid hormones, the induction of M-STP1 in the heart might occur to metabolize the increased levels of these hormones as a protective mechanism for the heart. Alternatively, the induction of M-STP1 in the heart might be the direct effect of elevated FFA.

MTs are metal-binding proteins that play a role in the homeostasis of essential metals. Their expression has been shown to be stimulated by short-chain FA (19). In addition to their function in metal metabolism, it has been suggested that MTs play a role in energy metabolism, because their expression in brown adipose tissue is stimulated by cold temperature (3), and MT-null mice are obese with increased adipose mass (4). Our data also lend support to a role for MTs in energy metabolism.

The expression profile of genes involved in cell cycle/growth/signaling demonstrates a transitional change from early-response genes to a variety of signaling molecules. With 48 h of fasting, in addition to many genes for cell cycle regulation, such as cyclin G, Fas, and FHL1, general signaling molecules, such as MAP kinases and serine threonine tyrosine kinase, were changed. These changes likely reflect the profound reaction of cardiac cells to survive critical starvation.

It is of interest that genes for MHC antigens and antigen processing were upregulated by the 16 h of fasting. It has been reported that cardiomyocytes and endothelial cells express MHC class I and II antigens, and their expression is affected by cardiac inflammatory reactions (21). The fact that 16 h of fasting stimulated cardiac genes involved with antigen presentation is in contrast to the repressed expression of MHCs in fasted HSL-Tg mice (27); this suggests that the mechanism responsible for induction might not be an effect of FA. Further study will be required to elucidate the detailed mechanisms.

Electron microscopy demonstrated almost no lipid droplets in hearts from fed mice, whereas cardiac TG content increased threefold after 16 h of fasting and sixfold after 48 h of fasting. The increase in cardiac TG content was due to accumulation of lipid droplets around clusters of mitochondria without any changes in membrane structures. Electron microscopy also demonstrated that, even with marked TG accumulation, cardiomyocytes do not accumulate droplets as large as those seen in adipocytes or hepatocytes. The maximum size of lipid droplets was similar to the size of mitochondria, and the number of lipid droplets increased with longer fasting. In contrast to dramatic changes in gene expression profiles, 48 h of fasting did not cause any visible pathological findings detectable by microscopy. Longer starvation might be required to cause pathological changes, although 48 h is the ethical limit for scientific investigation. Other interventions, such as a high-fat diet or induction of a diabetic state, might be needed for the induction of pathological changes, i.e., cardiac lipoapoptosis (31).

In summary, we report the expression profile of cardiac genes induced by physiological starvation. Concomitant with TG droplet accumulation, many genes for lipid/glucose/energy metabolism were changed by fasting. In addition, many genes for signaling molecules, cell structure, and cellular antigens were also affected. Studying transcriptional responses to nutritional interventions will be beneficial for understanding pathophysiological events in the heart.


    ACKNOWLEDGEMENTS

We thank Vanita Natu, Shailja Patel, and Naoyo Yamaguchi for technical assistance.


    FOOTNOTES

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.), National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-46942 and DK-49705 (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: J. Suzuki, Third Dept. of Internal Medicine, Fukui Medical Univ., Fukui 910-1193, Japan (E-mail: jinya{at}fmsrsa.fukui-med.ac.jp).

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.

First published March 5, 2002;10.1152/ajpendo.00017.2002

Received 15 January 2002; accepted in final form 27 February 2002.


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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Endocrinol Metab 283(1):E94-E102
0193-1849/02 $5.00 Copyright © 2002 the American Physiological Society




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