We found that Chongkukjang, traditional unsalted fermented soybean, has an antiobesity effect in mice with diet-induced obesity and examined the changes in hepatic transcriptional profiles using cDNA microarray. High-fat diet-induced obese C57BL/6J mice were divided into three groups: normal-diet control group (NDcon, 10% of total energy from fat), high-fat diet control group (HDcon, 45% of total energy from fat), and HDcon plus 40% Chongkukjang (HDC) and were fed for 9 weeks. The HDC group mice were pair-fed (isocalorie) with mice in the HDcon group. Final body weight, epididymal fat accumulation, serum total cholesterol, and LDL-cholesterol were improved in HDC group. The cDNA microarray analyses revealed marked alterations in the expression of about 800 genes. Several genes involved in fatty acid catabolism (Acaa2, Mgll, Phyh, Slc27a2, and Slc27a5) were normalized by Chongkukjang consumption. This study showed beneficial effects of Chongkukjang consumption in preventing diet-induced obesity and related metabolic abnormalities.
The incidences of various chronic diseases and diseases of aging in industrialized societies are increased by excess energy intake and lack of exercise. One example is metabolic syndrome, characterized by obesity, insulin resistance, glucose intolerance, dyslipidermia, and hypertension. Numerous studies suggest that excessive energy intake is the most fundamental cause of obesity, and high-energy intake is not only related to the absolute amount of lipid but also to the energy density of food [
It has been reported that beans modulate or prevent chronic and acute diseases of adults, including hardening of the arteries, heart disease, diabetes, senile dementia, cancer, and osteoporosis [
Chonkukjang is prepared by cooking soybeans and inoculating with microorganism to initiate fermentation. Chonkukjang has distinct characteristics when compared to other fermented soybean products. It is fermented predominantly with
Therefore, in this study, we investigated the effects of Chonkukjang consumption on weight gain, epididymal fat accumulation, lipid metabolism, and hepatic mRNA expression in mice with diet-induced obesity. To obtain a more comprehensive picture of the diet-induced hepatic transcriptional adaptation in the C57BL/6J mouse, we used cDNA microarray, containing ~10,000 mouse transcripts.
Male 4-week-old C57BL/6J mice were fed a chow diet for 1 week and then divided into 3 groups (
Composition of experimental diet.
NDcon | HDcon | High-fat diet | HDC | |
*A (10% fat) | *B (45% fat) | *C (60% fat) | C plus CKJ | |
1000 g | 1000 g | 1000 g | 600 g | |
Casein, 80 mesh | 189.56 | 233.06 | 258.45 | 155.07 |
L-Cystine | 2.84 | 3.50 | 3.88 | 2.33 |
Corn starch | 298.56 | 84.83 | 0.00 | 0.00 |
Maltodextrin 10 | 33.17 | 116.53 | 161.53 | 96.92 |
Sucrose | 331.74 | 201.36 | 88.91 | 53.34 |
Cellulose, BW200 | 47.39 | 58.26 | 64.61 | 38.77 |
Soybena oil | 23.70 | 29.13 | 32.31 | 19.38 |
Lard | 18.96 | 206.84 | 316.60 | 189.96 |
Mineral mix S10026 | 9.48 | 11.65 | 12.92 | 7.75 |
DiCalcium phosphate | 12.32 | 15.15 | 16.80 | 10.08 |
Calcium carbonate | 5.21 | 6.41 | 7.11 | 4.26 |
Potassium citrate, 1 H2O | 15.64 | 19.23 | 21.32 | 12.79 |
Vitamin mix V10001 | 9.48 | 11.65 | 12.92 | 7.75 |
Choline bitartrate | 1.90 | 2.33 | 2.58 | 1.55 |
FD&C yellow dye #5 | 0.05 | — | — | — |
FD&C red dye #5 | — | 0.06 | — | — |
FD&C blue dye #5 | — | — | 0.06 | 0.04 |
Chongkukjang (CKJ) | 0.00 | — | — | 400 |
kcal/1000 g | 3845.32 | 4727.61 | 5242.62 | 4929.57 |
*Modified AIN-93 diet (Research Diets, Inc., USA).
Normal diet | High-fat diet | ||
NDcon(1) | HDcon(2) | HDC(3) | |
Carbohydrates (energy %) | 70 | 35 | 31 |
Protein (energy %) | 20 | 20 | 22 |
Fat (energy %) | 10 | 45 | 48 |
Kcal/g | 3.85 | 4.73 | 4.93 |
(1)AIN-93 modified diet with 4% fat (10% fat calorie) content. (2)AIN-93 modified diet with 24% fat (45% fat calorie) content. (3)AIN-93 modified high-fat diet (60% fat calorie) plus 40% fermented soybean paste.
To reduce the effects of differences in dietary intake, blood samples were collected after 12 hours overnight fasting by orbital venipuncture. Blood samples were left on ice for one hour and centrifuged at 1100 ×g for 15 minutes, and serum was separated and collected. Serum samples were stored at −80°C before analysis. After blood collection, liver and epididymal fat were surgically removed, washed in saline to remove foreign substances, dried on filter paper, and quick frozen in liquid nitrogen after weighing.
Serum triglyceride, total cholesterol, and HDL-cholesterol levels were estimated by an enzymatic colorimetric method using a commercial assay kit (Asan Pharm. Co, Seoul, Korea). LDL-cholesterols were analyzed by the Friedwald method [LDL-cholesterol = total cholesterol − HDL-cholesterol − (triglyceride)/5] [
Mouse 10K cDNA microarray used in this study consisted of 10336 spots. It included 6531 transcripts from National Institute of Aging (NIA), 1243 transcripts from Brain Molecular Anatomy Project (BMAP), 2060 transcripts from InCyte Pharmaceuticals (Fremont, CA, USA), and yeast DNA and housekeeping genes as negative controls. Total RNA was prepared from livers using Trizol (Invitrogen, Carlsbad, CA, USA). Fluorescence-labeled cDNA probes were prepared from 20
The two fluorescent images (Cy3 and Cy5) were scanned separately by a GMS 418 Array Scanner (Affymetrix, Santa Clara, CA, USA), and the image data were analyzed using ImaGene 4.2 (Biodiscovery, Santa Monica, CA, USA) and MAAS (Gaiagene, Seoul, Korea) softwares [
4
Primers for RT-PCR.
Gene | Sense | Anti-sense |
---|---|---|
Acaa2 | 5′-TGTGTCAGAAATGTGCGCTTC-3′ | 5′-CAAGGCGTATCTGTCACAGTC-3′ |
Dhcr24 | 5′-GCACAGGCATCGAGTCATC-3′ | 5′-GGCACGGCATAGAACAGGTC-3′ |
Dpagt1 | 5′-CTCGCTGTTGGGATTCGTG-3′ | 5′-GCTGAGCTTGTTGAGGTCCTG-3′ |
Hsd11b1 | 5′-CTCCAGAAGGTAGTGTCTCGC-3′ | 5′-CCTTGACAATAAATTGCTCCGCA-3′ |
Mgll | 5′-CGGACTTCCAAGTTTTTGTCAGA-3′ | 5′-GCAGCCACTAGGATGGAGATG-3′ |
Phyh | 5′-ACTGCCTTCTCCCCGAGATT-3′ | 5′-CCGGGATGTCTTCTTGCCA-3′ |
Pon1 | 5′-TACTGGTGGTAAACCATCCAGA-3′ | 5′-GCAGCTATATCGTTGATGCTAGG-3′ |
Prkag1 | 5′-GAAGCAGTGTTTTGTGGGCAT-3′ | 5′-ACGTCTCTATCTTGTGCTCCT-3′ |
Slc27a2 | 5′-GAGTCGTGGAGGTCTGAAGTC-3′ | 5′-ACCTTAGGCGATGATGATTGATG-3′ |
Slc27a5 | 5′-CTACGCTGGCTGCATATAGATG-3′ | 5′-CCACAAAGGTCTCTGGAGGAT-3′ |
Cpt1l | 5′-AGAATCTCATTGGCCACCAG-3′ | 5′-CAGGGTCTCACTCTCCTTGC-3′ |
5′-GGGTCAGAAGGACTCCTATG-3′ | 5′-GATACAATGCCATGTTCAAT-3′ |
Mice data from individual experiments were expressed as the mean ± standard deviation. SAS version 8 (SAS Institute, Cary, NC USA) was used for all statistical analyses. Significance of differences were analyzed by Duncan’s multiple rage test, and the accepted level of significance was
Dietary and energy intakes and weight changes during the experimental period are shown in Table
Body weight and food intake in mice fed experimental diets for 9 weeks.
Normal diet | High fat diet | ||
NDcon | HDcon | HDC | |
Dietary intake (g/day) | 2.40 ± 0.27 | 2.27 ± 0.30 | 2.11 ± 0.39 |
Energy intake (kcal/day) | 9.15 ± 1.03b | 10.69 ± 1.43a | 10.72 ± 1.79a |
Initial body weight (g) | 22.67 ± 1.14 | 23.23 ± 1.33 | 23.99 ± 1.19 |
Final body weight (g) | 27.10 ± 1.44b | 30.02 ± 2.47a | 27.96 ± 1.73b |
Epididymal fat | 2.21 ± 0.58b | 3.54 ± 1.32a | 1.84 ± 0.18c |
Values are means ± SD. Values with different superscript letters (a, b, c) indicate significant differences among groups at
Serum lipid concentrations. Values are means ± SD. Values with different superscript letters (a, b, c) indicate significant differences among groups at
The gene expression profile of liver tissues from the NDcon, HDcon, and HDC mice were compared. Only the genes for which changes in mRNA levels were 2.0-fold or more and found to be statistically significant by the SAM method were designated as differentially expressed genes (Figures
The numbers of genes differentially expressed in HDcon Mice but normalized or reversed by Chongkukjang consumption (HDC)(1).
Molecular function | Increase | Decrease | Total |
---|---|---|---|
Metabolism | 19 | 34 | 53 |
Defense/stress/inflammation responses | 0 | 13 | 13 |
Signal transduction/apoptosis/cell cycle | 66 | 31 | 97 |
Transcription regulation | 28 | 22 | 50 |
Protein synthesis and modification | 24 | 19 | 43 |
Transport | 23 | 20 | 43 |
Cellular adhesion/cytoskeleton/trafficking | 11 | 5 | 16 |
Chromosome remodeling | 14 | 6 | 20 |
DNA replication | 5 | 4 | 9 |
RNA processing | 10 | 2 | 12 |
Microtubule-based movement | 7 | 4 | 11 |
Unclassified | 13 | 15 | 28 |
Total | 220 | 175 | 395 |
(1)Data represent a summary of results of six independent hybridizations. In three of six replicated experiments, the labeling of HDcon and NDcon samples was reversed to compensate for any nonlinearity in the emission signal intensity response curve for each fluorophore. Genes designated as differentially expressed in the HDcon mice were those for which the normalized signal increased or decreased at least 2.0-fold and detected as significant change by SAM method.
Representative
SAM scatter plot of observed relative difference versus the expected relative difference. The genes showing significant difference in expression between the NDcon and the HDcon or the HDcon and the HDC mice were identified. Broken lines represent
The largest number of genes expressed differentially in the HDcon group but normalized by Chongkukjang consumption were those involved in signal transduction, protein synthesis and modification, and metabolism. The genes involved in metabolism, such as carbonic anhydrase 1, squalene epoxidase, lipolysis stimulated lipoprotein receptor, acyl-CoA synthetase long-chain family member 3, 24-dehydrocholesterol reductase, isocitrate dehydrogenase 3 (NAD+), and gamma, were augmented in the HDcon mice, compared to the NDcon mice. These upregulated genes in the HDcon mice were normalized by Chongkukjang consumption (Data not shown).
The genes involved in protein synthesis and modification such as disintegrin and metallopeptidase domain 10, ubiquitin specific peptidase 25, cathepsin 7, ubiquitin-conjugating enzyme E2N, serine (or cysteine) peptidase inhibitor, clade E, member 2, ring finger protein 103, ubiquitin-conjugating enzyme E2E 3, UBC4/5 homolog (yeast), ubiquitin-conjugating enzyme E2I, and ring finger protein 17 were also increased by at least 2-fold in the HDcon mice and normalized by Chongkukjang consumption. The genes implicated in transcription regulation showed a similar pattern: nuclear factor, erythroid derived 2,-like 1, FBJ osteosarcoma oncogene, c-myc-binding protein, retinoic acid receptor, and gamma. The genes which play roles in signal transduction/apoptosis/cell cycle and transcription regulation also showed similar expression patterns as those described above: Src homology 2 domain-containing transforming protein C1, mitogen-activated protein kinase 13, phospholipase C, delta 1, Src homology 2 domain-containing transforming protein D, cyclin L1, sphingosine kinase 1, mitogen-activated protein kinase 14, cell division cycle 6 homolog (S. cerevisiae), cyclin B1.
Several genes encoding metabolic enzymes such as acetyl coenzyme A acyltransferase 2 (Acaa2), hydroxysteroid 11-beta dehydrogenase 1, monoglyceride lipase, phytanoyl-CoA hydroxylase, and isocitrate dehydrogenase 3 (NAD+) alpha were decreased by at least 2-fold in the HDcon mice and normalized by Chongkukjang consumption. Genes for lipid transporter such as solute carrier family 27 (fatty acid transporter), and member 2 (Slc27a2) and solute carrier family 27 (fatty acid transporter), member 5 (Slc27a5) were also decreased in the HDcon mice and normalized by Chongkukjang consumption. Other genes showing a similar expression pattern included three genes for ATP synthase (ATP synthase, H+ transporting mitochondrial F1 complex, beta subunit, ATP synthase, H+ transporting, mitochondrial F0 complex, subunit g, ATP synthase, H+ transporting, mitochondrial F1 complex, and gamma polypeptide 1), cytochrome c oxidase subunit VIIa polypeptide 2-like and electron transferring flavoprotein, and dehydrogenase.
Changes in gene expression observed by DNA microarray analysis were further confirmed with a small set of known genes by real-time RT-PCR (SYBR Green I), using the same mice liver samples used in the above microarray hybridization. When gene expression profiles obtained by both microarray analysis and RT-PCR were compared, their patterns were very similar with regard to the direction (up- or downregulation) and degree of differences in expression (Table
Comparison between cDNA microarray analysis and real-time RT-PCR.
Genbank No. | Gene | cDNA microarray | Real time RT-PCR | ||
HDcon | HDC | HDcon | HDC | ||
BG085346 | Acetyl-Coenzyme A acyltransferase 2 (Acaa2) | −4.18 | 0.28(2) | 0.94 | |
AA277495 | Carnitine palmitoyltransferase 1a, liver (Cpt1a) | ND(3) | ND | 1.73 | 3.41 |
BF457090 | 24-dehydrocholesterol reductase (Dhcr24) | 2.1 | 1.45 | 0.78 | |
BG063933 | Dolichyl-phosphate (UDP-N-acetylglucosamine) acetylglucosaminephosphotransferase 1 (Dpagt1) | 2.3 | 3.02 | 1.91 | |
AA023077 | Hydroxysteroid 11-beta dehydrogenase 1 (Hsd11b1) | −8.95 | 0.23 | 1.14 | |
AI835105 | Monoglyceride lipase (Mgll) | −8.99 | 0.29 | 0.62 | |
W82212 | Phytanoyl-CoA hydroxylase (Phyh) | −5.78 | 0.37 | 1.21 | |
AI893897 | Paraoxonase 1 (Pon1) | 2.0 | 1.68 | 0.69 | |
AA259329 | Protein kinase, AMP-activated, gamma 1 noncatalytic subunit (Prkag1) | 2.2 | 2.20 | 1.41 | |
AA108401 | Solute carrier family 27 (fatty acid transporter), member 2 (Slc27a2) | −4.06 | 0.25 | 0.86 | |
AA254935 | Solute carrier family 27 (fatty acid transporter), member 5 (Slc27a5) | −11.73 | 0.25 | 0.83 |
(1)The symbol double-headed arrow (
To assess the effects of Chongkukjang consumption on high-fat diet-induced obesity in C57BL6J mice, we investigated diet and energy intakes, body fat distribution, and lipid profiles. Body weight was increased by high-fat diet in the HDcon group, but it remained normal in the HDC group. Although diet intake was not significantly different, unsaturated fatty acids (linoleic acid and oleic acid) intake was three times higher in HDC group than HDcon group, and the increased caloric density of the high-fat diet led to significantly higher weight gain compared with the normal diet, which resulted in higher feed efficiency. Thus, mice fed a high-fat diet showed a more rapid growth and greater epididymal fat accumulation than the normal-diet-fed mice. However, the higher weight gain and greater epididymal fat accumulation exhibited by the high-fat diet group were reduced by Chongkukjang consumption. Changes in epididymal fat accumulation reflected body weight changes. These results suggest that Chongkukjang consumption can suppress the increase of weight gain induced by a high-fat diet.
In this study, a high-fat diet significantly increased triglyceride and LDL-cholesterol concentration in serum, which has been shown in other studies [
Because a global analysis of gene expression in response to changes in physiological status appears to be essential for understanding the molecular mechanisms underlying the alterations, we investigated the hepatic transcriptional profiles in a diet-induced obesity mouse model and their alteration by Chongkukjang consumption using a cDNA microarray.
Mammalian tissues have four known types of thiolase which differ in intracellular localization. Cytosolic acetoacetyl-CoA thiolase catalyzes the formation of acetoacetyl-CoA required for cholesterol biosynthesis [
In a study by Takahashi et al., hepatic Acaa gene expression was upregulated by CLA with a decrease in body weight gain and epididymal white adipose tissue weight [
In this study, Slc27a2 and Slc27a5 genes encoding fatty acid transport proteins (FATPS/solute carrier family 27) were also upregulated by Chongkukjang consumption. Fatty acid transport proteins are integral transmembrane proteins that enhance the uptake of long-chain and very long-chain fatty acids into cells [
Hepatic Phyh gene was downregulated by high-fat diet but was normalized by Chongkukjang consumption. The Phyh gene product, Phytanoyl-CoA hydroxylase (Phyh), catalyzes the conversion of phytanoyl-CoA to 2-hydroxyphytanoyl-CoA, which is the first step in the phytanic acid
Cholesterol accounts for 99% of all sterols in mammals, playing multiple biological roles as a major constituent of membranes, a precursor to numerous signaling molecules, and an inducer of the Hedgehog family of morphogens [
In summary, we have demonstrated that Chongkukjang consumption has beneficial effects on body weight, epididymal fat accumulation, serum lipid profile and hepatic gene expression in mice fed a high-fat diet. The Chongkukjang consumption decreased weight gain, epididymal fat accumulation, serum triglyceride, total cholesterol, and LDL-cholesterol. In addition, the majority of genes expressed differentially in the high-fat diet group were normalized by Chongkukjang consumption. We propose that Chongkukjang consumption improves serum lipid profiles and body fat accumulation, probably by modulating transcriptional levels of enzymes for utilization of fatty acid.
Therefore, Chongkukjang may prevent obesity induced by long-tem high-fat diet. We expect that our microarray result will provide useful basic data in research that investigates the effect of Chongkukjang consumption on the metabolic disease by diet-induced obesity.
This study was supported by research grants from Korea Science and Engineering Foundation (KOSEF) for Biofoods Research Program, Ministry of Science and Technology (M10510120001-05N1012-00110).