Anti-hypertensive effect of Korean fermented soybean paste (Doenjang) through regulation of the renin-angiotensin system (RAS) in male rats fed with high-fat and/or high-salt diet

Background: Korean fermented food, doenjang, is questioned due to its high salt content, although it has been reported an abundance of benecial effects. Therefore, we investigated its impact on renin-angiotensin system (RAS) hypertension using 3T3-L1 adipocytes and male Sprague-Dawley rats. Results: Among the rats fed with normal diet (ND), high-fat diet (HD), high-fat diet with 8% table salt (HDS), or high-fat diet with doenjang containing 8% table salt (HDJ) for 13 weeks, the HDJ group showed signicantly lower blood pressure, lesser body weight, and reduced levels of serum angiotensin II and aldosterone, compared to the HD and HDS groups. In addition, mRNA expressions levels of angiotensinogen, angiotensin converting enzyme, angiotensin II receptor type 1, and angiotensin II receptor type 2 were downregulated in epididymal fat of the HDJ group. Conclusions: In spite of its high salt content, Doenjang appears to inhibit obesity-induced hypertension through modulation of RAS by blocking the angiotensin converting enzyme, even with high fat intake.

in vitro and in vivo studies have shown the health bene ts of doenjang and its active compounds. Our previous clinical studies have reported its antiobesity, antioxidant effects, and its bene cial effects on overall health index. However, consumption of doenjang is still questioned, because of its high salt content [12]. Salt is added during the manufacturing of doenjang to control the harmful microbial growth and improve its preservation. It promotes the growth of bene cial microbe, inhibits undesirable fermentation, and improves its taste. [11]. However, excessive sodium intake is generally known to increase the risk of hypertension through hypervolemia, promote heart and kidney disease, and contribute to stroke and gastric cancer [14,15].
In our previous study, we had investigated how doenjang regulates hypertension caused by a high-salt diet. Doenjang, when fed along with normal diet, caused a signi cant reduction in the blood pressure and lowered the expression of sodium transferase-related genes, as compared to the same amount of salt alone. [16,17].
In the present study, the antiobesity and antihypertensive effects of traditional fermented foods in the obese condition were studied in various ways. Here, we considered the salt content, which is different from the previous research, which focused on the effects of doenjang effects in normal conditions. We included high-fat as well as high-salt diets in our experiments, which have investigated few other studies, and the results were inconclusive yet [18]. High-salt and high-fat diets synergistically promote obesity and high blood pressure [3]. Purpose of this study was to examine the effect of a combination of high-salt and highfat diet on RAS in adipocytes and how doenjang containing high amount of salt, regulates obesity and blood pressure, even when consumed together with a high-fat diet.

Materials And Methods
Preparation of Doenjang Doenjang was produced by the Sunchang Sauce Corporation of South Korea (Sunchang-gun, Jeollabuk-do, Korea). This fermented food is prepared using soybean after maturing it for 6 months, by traditional Korean fermentation process (Fig. 1). After steamed soybeans, it was made into blocks, and fermented with Aspergillus oryzae and Bacillus subtilis for one month. After this, it was mixed with brine (saltwater, 26%, w/v) in a 1:3 ratio and further fermented for 2 more months. Once matured, doenjang was dried using a freeze-dryer (FD12008, Ilshin Biobase, Gyeonggi-do, Korea), and its salinity was adjusted with NaCl (Samchun, Pyeongtaek-si, Gyeonggi-do, Korea) to 8%, using Mohr's method.

Animals and treatments
Three-week-old male Sprague-Dawley rats were purchased from Central Lab. Animal, Inc. (Seoul, Korea), and acclimated at 12 h light and 12 h dark cycles at 25 ± 2°C and humidity 50% ± 5% conditions. After 7 days adaptation, they were randomly (no diffrence in body weight and initial SBP) divided into 4 groups (n=6); normal diet control (ND; AIN76A, Research Diets, Inc. New Brunswick, NJ, USA), high-fat diet control (HD; 60% fat by weight, D12492, Research Diets, New Brunswick, NJ, USA), high-fat diet with 8% table salt (HDS), and a high-fat diet with doenjang containing 8% table salt (HDJ). All animals were fed with their corresponding experimental diets (Table 1) for 13 weeks, with water supplied. Body weights were noted once a week and food intake was measured each day. Systolic blood pressure (SBP) was recorded weekly by the indirect tail-cuff method (BP-2000, Visitech Systems, Inc., Apex, NC, USA) 30 min after placing them at 37°C. The mean SBP was recorded after 7 measurements. During the last 4 weeks, the animals were individually housed in metabolic cages 24 h for 3 consecutive days in a week, with water provided through drinking bottles and food in the cage. Food and water intake, and fecal and urine output were measured and collected for analyses. Mean value of each criterion was recorded for each rat. All animal procedures were approved by the Animal and Use Committee of Chonbuk National University (CBNU 2018-052).

Tissue collection
At the end of 13 weeks, rats were fasted for 12 h before anesthetizing with 2 mg/kg BW alfaxan (Jurox, Australia) and 0.5 mL/kg BW rompun (Bayer, Seoul, Korea) through intramuscular injection to collect blood and tissues. Liver, epididymal adipose tissue, and one of the kidneys were rinsed with saline, weighed, and immediately frozen in liquid nitrogen and stored at -80°C for further analyses. The other kidney was xed in 10 % formaldehyde and embedded in para n. Blood was drawn by orbital vein puncture and centrifuged at 3,000 rpm for 15 min at 4°C to collect serum.

Biochemical analysis
The serum levels of renin, angiotensin II and aldosterone were measured using assay kits (Rat Renin ELISA Kit, MyBioSource, San Diego, CA, USA; Angiotensin II ELISA Kit, and Aldosterone ELISA Kit, Enzo Life Sciences, Inc., Farmingdale, NY, USA), following the manufacturer's protocols.

Histology of fat cryosections
Frozen epididymal adipose tissues were cryopreserved in OCT (Scigen Scienti c Gardena, CA, USA), and frozen in liquid nitrogen. Sections of 10 μm thickness were cut with cryomicrotome (Shandon Cryotome FE, Thermo Scienti c, MA, USA), transferred onto glass slide (Marienfeld, Germany) at -30°C. Sections were stained with hematoxylin and eosin (H&E) and mounted in glycerol gelatin. Cells were observed using an Axiophot Zeiss Z1 microscope (Carl Zeiss, Gottingen, Germany) at X200 magni cation, and adipocytes were counted. Difference in cell size in each group were noted.

RNA extraction and real-time PCR
Total RNA was isolated from after homogenizing the tissues in TRIzol reagent (Invitrogen, Grand Island, NY, USA), and the concentration of total RNA was equalized using quantifying on Biodrop Duo (Biochrom, Holliston, MA, USA). cDNA was synthesized using PrimeScript™ RT Master Mix (Takara Bio Inc., Shiga, Japan) according to the manufacturer's instructions. RNA expression was measured by quantitative realtime polymerase chain reaction (qPCR) using the SYBR Green real-time PCR master mix (TOYOBO, Osaka, Japan), on a 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA), and quantitative analysis of PCR data were calculated through 2 −ΔΔCt method, using beta-actin as an internal control. Primers used for the qPCR are listed in Table 2.

Urine and feces analyses
Urine and feces samples, collected from metabolic cages, were analyzed for their Na + and K + contents, by inductively coupled plasma-mass spectroscopy (ICP-MS; 7500A, Agilent Technologies, Germantown, MD, USA), the center for University-Wide Research Facilities (CURF) at Jeonbuk National University.

Analyses of serum ion levels
The Na + and K + concentrations in the serum were determined using Fuji Dri-Chem Slide Na-K-Cl (FUJIFILM, Tokyo, Japan) with FDC 3500i chemistry analyzer (Fuji Dri-Chem Analyzer, Tokyo, Japan).
Preparation of doenjang samples for treating the cell line Doenjang was purchased from the Sunchang Sauce Corporation of South Korea (Sunchang-gun, Jeollabuk-do, Korea). The 1 gram of each freeze-dried doenjang was mixed with 10 mL of solvent (80% ethanol) at RT for 24 h on a shaker. The supernatants were collected and ltered through ADVANTEC No. 2 lter paper, and 1 mL of each ltrate was freeze-dried in a speed vacuum concentrator (FD12008, Ilshin Biobase, Gyeonggi-do, Korea).

Study design and cell culture
The 3T3-L1 preadipocyte cell line ((CL-173)-ATCC, VA, USA) was maintained in DMEM (Hyclone, USA) containing 10% bovine serum (Gibco, NY, USA) and 100 U/mL 1% penicillin-streptomycin (Hyclone, USA) at 37°C under 5% CO 2 in a humidi ed incubator. RNA was extracted from 3T3-L1 cells at different times after differentiation. In order to observe the effect of RAS blockers, the 3T3-L1 cells were seeded in 6-well plates and upon reaching 100% con uence (day 0), they were continued in culture for 48 h. Then the growth medium was replaced with differentiation medium, containing DMEM, 10% fetal bovine serum (FBS), 0.5 μM isobutylmethylxanthine (IBMX), 1 μM dexamethasone (DEXA), and 10 μg/mL insulin (Sigma-Aldrich The initial body weight of the animals did not vary signi cantly among different groups. After 13 weeks on treatment diets, mean body weight of the HD group rats had signi cantly increased, as compared to those from HDS and HDJ groups, while the ND group had the lowest body weight (Fig. 2).The group treated with doenjang with high-salt, displayed a protective effect against high-fat induced obesity. Diet intake among different groups fed normal diet (ND), and high-fat diet (HD, HDS, HDJ) was not signi cantly different, respectively (Table 3). Compared to the ND group, the blood glucose levels were elevated in the HD and HDS groups, whereas doenjang supplementation markedly attenuated this high-fat diet induced increase in blood glucose (Table 3).

Adipocyte morphology
Animals from the HD and HDS groups showed a signi cantly higher weight of epididymal fat, compared to HDJ and ND groups (Fig. 3a). However, doenjang treated rats from the high-fat diet group showed signi cantly reduced epididymal fat mass, as well as reduced size of adipocytes in it (Fig. 3b). The ratio of the number of adipocytes to area, was signi cantly increased in the HDJ group as shown in the histological analysis (Fig. 3c).

Expression of obesity related genes
In order to study the effects of doenjang on obesity, the expression of genes related to lipid-regulating enzymes and transcription factors were quanti ed in the epididymal adipose tissue. Doenjang treated rats showed signi cantly lower expression of leptin mRNA in the epididymal adipose tissue. In contrast, adiponectin expression was upregulated due to doenjang supplementation (Fig. 4).

Systolic blood pressure
The base-line SBP in all groups was 110±10 mm Hg, which was signi cantly increased with high-salt intake over the experimental period, in spite of weight loss. However, SBP of the HDJ group was comparable to that of ND, even though the HDJ diet had the same amount of salt as HDS, indicating that the high-salt content in doenjang did not alter the SBP, unlike HDS. Animals from HDJ and ND groups had steady and normal SBP compared to those from HD group also, HDS group showed signi cantly higher SBP in the last week of the treatment, and it appeared to have reached a plateau at a high of 169.31 mm Hg (Fig. 5).
Renin, angiotensin II, and aldosterone levels in serum Serum renin level was the highest in the ND group, while the high-fat diet groups showed slightly lower level compared to ND group, still the difference was not signi cant, whereas only HDS group had signi cantly less serum rennin than ND group (Fig. 6). Although serum angiotensin II did not signi cantly decrease in the HDS group compared to the HDJ group, it showed at reducing trend in the latter (Fig. 6). Serum aldosterone also decreased signi cantly in HDJ (Fig. 6).
Expression of RAS-related genes in adipose tissue Expression levels of RAS related genes in adipose tissue, angiotensinogen (Agt), renin, and angiotensinconverting enzyme (Ace) mRNA were notably downregulated in HDJ group, whereas they were comparable in HD and HDS groups ( Fig. 23-25). Furthermore, animals from the doenjang group showed reduced expression of lipogenic genes, including angiotensin II receptor type 1 (Agtr1) and angiotensin II receptor type 2 (Agtr2), as compared to those from the high salt group (Fig. 7).
Expression of RAS-related genes in kidney and liver Doenjang treatment reduced Agt expression in the liver (Fig. 8), and Agtr1 in the kidney (Fig. 8), which indicated vasodilation of blood vessels, whereas Ace and Agtr2 in the kidney did not change signi cantly (Fig. 8). mRNA expression of kidney renin, a feedback controller of angiotensinogen, was upregulated in the doenjang-fed group compared to the high-fat, and high-salt groups (Fig. 8). Aldosterone-related genes in kidney, including Star, Hsd3b1, Cyp11a1, Cyp21, and MR, were downregulated by doenjang. In particular, transcription of Cyp21 and MR was drastically reduced in the doenjang group (Fig. 9a). Level of renalase de ciency of which increases the blood pressure, was suppressed in both HD and HDS groups, while in HDJ group, it was enhanced (Fig. 9b).

Serum Na + / K + levels and their excretion
After 24 h in metabolic cages, animals from the HDS and HDJ groups showed a greater amount of water intake and urine excretion than those from the ND and HD groups (Fig. 10). Concentrations of sodium and potassium in both urine and feces were signi cantly higher in the HDJ group than in the HDS group.
However, the ratio of Na + /K + was the highest in the latter.
Serum analysis showed that HD group had the highest concentration of sodium in their serum. In the HDJ group serum sodium levels showed decreasing tendency among the high-fat diet groups, while the serum potassium level decreased signi cantly in the HDJ group unlike in the HDS group ( Table 4).
Effects of Doenjang extract on 3T3-L1 cell line As shown in Fig. 11, treatment with only doenjang could lower the key adipogenic transcription factor, Pparg, signi cantly in the differentiated adipocyte after 4 days of exposure, and this was similar to the effects of losartan and captopril. Expression of Agt also reduced due to doenjang and captopril over the levels seen in the control. Similarly Ace gene expression decreased signi cantly due to treatment with doenjang as in the positive control group, treated with Captopril, an Ace inhibitor (Fig. 11).

Discussion
Both high-salt and high-fat diets are closely linked and synergistically promote obesity and hypertension. Consumption of high-fat is associated with abnormal accumulation of fat, resulting in an increase in the adipocyte size and/or number [19]. High intake of salt induces hypertension, and extracellular uid accumulation when the osmolality increases in the extracellular space, due to high Na + concentration [20]. Previous studies have shown that doenjang inhibits lipid accumulation in the adipocytes. High sodium content also has been reported to suppress the weight gain with the intake of high-fat diet [21]. Present study showed that doenjang and high-salt, lowered the body weight to a level comparable to that of the normal diet fed animals (Fig. 2). Salt affects the activity of renin and angiotensin, and controlled digestive e ciency, and reduces the ability of the gastrointestinal tract to extract calories from the food [21]. This appears to be the underlying mechanism of the body weight reduction in the HDS group. However, bodyweight and obesity-related biomarkers in the HDS group showed an inverse relationship. Adipocyte hypertrophy and hyperplasia were observed in the HD and HDS groups. Interestingly, in the HDJ group had signi cantly decreased adipocyte size, epididymal fat weight; simultaneously it signi cantly increased the number of adipocytes to area ratio compared to the HD and HDS group (Fig. 3).
Doenjang, which has soybean as its main ingredient, contains different soy proteins, soy saponin, phospholipids, and iso avones. These components in soybeans are responsible for its hypolipidemic and antiobesity effects, through increasing β-oxidation and inhibiting fatty acid synthesis [10,22]. Our previous study showed that doenjang has been lowered bodyweight and improved obesity-related parameters such as insulin, leptin, Pparg, and Acc [15]. The results of the present study also con rmed the antiobesity effect of doenjang evident by the decreased gene expression of leptin, while increased adiponectin levels (Fig. 4). Chronic intake of high-salt diet may increase the risk of renal injury, kidney dysfunction, and hypertension in normal rats [23]. Interestingly, in this study, blood pressure was signi cantly different in the HDS and HDJ groups, in spite of similar body weights and the same amount of salt intake (Fig. 5). The biopeptides present in doenjang may be responsible for its action on RAS, reducing the blood pressure in HDJ group [24].
Obesity is known to induce greater sensitivity towards the changes in blood pressure. Our previous study had indicated that the Korean traditional fermented soybean foods, doenjang and ganjang, reduce the blood pressure in normal SD rats [16,17]. RAS is a critical homeostatic regulator that relies on a feedback regulation to achieve and sustain the delicate balance in the blood pressure, required for healthy physiological function [25]. It is activated in the adipose tissue in diet-induced obesity. Expression of systemic RAS is also upregulated in obesity [26]. Therefore, we fed the rats with a high-fat diet to induce obesity and treated them with doenjang to study the effects of doenjang on RAS expression in adipocytes. Our results showed that expression of adipocyte RAS related genes was signi cantly downregulated by doenjang compared to the salt group. Expression of Agt, the rst component of the adipocyte RAS, is positively correlated with adipose mass. Renin, Ace, and Agtr1 genes are also upregulated in adipose tissue in obesity [27,28]. Ang II promotes lipogenesis by activating Agtr2, and simultaneously, by decreasing lipolysis by activating Agtr1 [29]. The results of the current study also con rmed that doenjang reduced lipid accumulation through Agtr1 and Agtr2, leading to suppression of obesity. Adipose-derived angiotensin is also released into the systemic circulation, which regulates the activation of bioactive Ang II, leading to increased levels of plasma Ang II, ultimately resulting in elevated blood pressure. Adipocyte RAS contributes to control of fat mass and can affect systemic functions such as metabolism [27,30]. Increased excretion of Agt from fat tissue into the plasma may elevate plasma Agt, resulting in increased blood pressure in the obese [6,30]. Our results indicated that adipocyte Agt had a pattern similar to that of plasma angiotensin, which was derived from the liver, and we found that the hepatic Agt was also elevated (Fig. 7).
In this study, we evaluated serum levels of RAS and its mRNA expression in kidney. Serum level of renin in the ND group was higher than that in the HDS and HDJ groups, despite the low blood pressure. This is because, in response to the low sodium level in blood, renin is activated [32]. Lower renin levels may be due to abnormal reabsorption of excess renal sodium, decreased renin secretion, and genetic abnormalities in RAS or related genes. High renin hypertension, as well as low renin hypertension appear to be linked with end-stage organ damage, and worsen the prognosis of hypertension than the hypertension with normal renin levels [33]. High concentration of renin is not always associated with high enzyme activity, but it is directly proportional to renin mRNA expression, renin secretion, and plasma renin activity [16,34]. A previous study has also shown that renin mRNA expression tended to be suppressed by the high-salt diet [35]. It appears that renal renin may affect serum renin. Non-proteolytically enhanced catalyzing activity of renin or pro-renin, when bound to pro-renin receptor (PRR), elevates angiotensin I production from angiotensinogen, thereby enhancing RAS [36]. Reduced renin can be explained through a negative feedback loop involving Ang II, which regulates expression and secretion of renin [37]. The present study showed that serum Ang II and renal Ace were higher in the high-salt group as a result of upregulated Ang II, leading to the downregulation of renin through the negative feedback loop. The patterns of Ace gene expression are similar to systemic Ace activity [8]. Systemic Ace is the main enzyme responsible for the production of Ang II from Ang I in the intravascular space [38]. Ang II enhances hypertension by increasing oxidative stress and activating angiotensin II receptors [39]. In this study, the levels of Agtr1 in the kidney showed a decreasing trend in the HDJ group, while Agtr2 increased signi cantly in the doenjang group, compared to the high-salt group (Fig. 8). Agtr1 is distributed throughout the kidney and contributes to most of the Ang II actions [40]. Ang II activates Agtr1 to bring about a variety of biological responses, such as vasoconstriction, renal sodium reabsorption, cell proliferation, cell dedifferentiation, and growth, and increased aldosterone secretion, which contribute to increased blood pressure and the development of hypertension [41]. Actions mediated through Agtr2 generally counterbalance the actions of Agtr1 [40]. Aldosterone levels in serum, catalyzed by Ang II, were signi cantly decreased in the ND and HDJ groups, compared to the levels in HD and HDS groups in the present study (Fig. 6). Similar trends were also observed in aldosterone-related mRNA expression in the kidney. Expression of Star, Hsd3b1, Cyp11a1, Cyp21, and MR genes decreased in the HDJ group (Fig. 9a). In particular, doenjang reduced the expression of Cyp21 and MR genes signi cantly. Aldosterone is synthesized by the action of Cyp21 in the adrenal cortex [42]. Mineralocorticoid receptor (MR), a ligand-dependent transcription factor, mediates the actions of aldosterone in different tissues [43]. Ang II causes vasoconstriction and activates aldosterone, a mineralocorticoid that reabsorbs sodium in the distal tubules and collecting ducts of the kidney at the expense of potassium excretion. This increased vasoconstriction and sodium reabsorption contribute to higher blood pressure and uid retention [44]. In accordance with this mechanism, our study showed that serum Na + levels were only marginally higher in the HDS group than in the HDJ group, but no signi cant difference was observed, whereas serum K + levels were signi cantly higher in the HD and HDS groups ( Table 4). Release of aldosterone from the adrenal cortex is enhanced by high serum concentration of potassium, regardless of the action of Ang II [40]. Consumption of high-salt diets increases the frequency of urination, because of the stimulated thirst, and excessive water intake [45]. Our study also demonstrated that water intake and urine output increased signi cantly in the high-salt and doenjang groups due to 8% of salt was contained in the HDS and HDJ diets (Fig. 10).
Potassium excretion in urine and feces was signi cantly lower in the HDS group than in the HDJ group in the present study, while sodium excretion was not noticeably different. Moreover, the Na + /K + ratio was the highest in the HDS group (Table 4). This ratio in urine may contribute to the deterioration of renal function. It was found that the higher the sodium excretion through urine, the lower the potassium excretion, which means that the elevated urine Na + /K + ratio is associated with increased blood pressure, leading to faster deterioration of renal function [46]. Kidney RAS is essential for Na + excretion, and for controlling blood pressure [41]. It can also act independent of plasma RAS level upon high-salt intake. Aberrant activation of the kidney RAS generates several factors responsible for hypertension, regulated by a variety of pathophysiological mechanisms related to hypertension and kidney damage [17,39]. The kidney RAS was found and identi ed to be possibly related to modulation of renalase enzyme. Increased dietary sodium intake decreases the renalase levels in kidney and plasma, and signi cantly increases the expression of Agtr1 in kidney, a critical element of kidney RAS [47]. Similarly, in the present study, high-salt intake induced kidney RAS, and it reduced the expression of renalase gene in the renal tissue (Fig. 9b).
Exact mechanism of action and the target of doenjang were determined using 3T3-L1 cell line, and compared them with those of Losartan and Captopril, which are antihypertensive drugs that inhibit Agtr1 and Ace, respectively, in the adipocytes. Pparg, Agt, and Ace reduced signi cantly in the 3T3-L1 adipocytes treated with doenjang (Fig. 11), indicating that fat production was inhibited by doenjang. Therefore, all these data revealed that doenjang regulates blood pressure through RAS, by inhibiting Ace in adipocytes.

Conclusion
Results of this study revealed that doenjang exerts its bene cial effects by reducing body weight, white adipose tissue, and lipid contents of serum and liver, and improves the obesity-induced high blood pressure. Doenjang supplementation downregulated the expression of genes related to adipocyte RAS, leading to the regulation of systemic RAS. This, in turn, affects the kidney RAS, which ameliorates kidney dysfunction and metabolic syndrome. In the doenjang treated rats, serum ions and excretion of Na + and K + ions differ drastically from those treated with high salt. To sum up, it is clear that doenjang ameliorates hypertension by suppressing the elevated blood pressure, even though it contains high-salt. Our data suggests that doenjang may inhibit Ace in adipocytes, through a mechanism similar to that of Captopril.

Consent for publication: Not applicable
Availability of data and materials: All of the data are available with reasonable request from the corresponding author.
Competing interests: No potential con ict of interest was reported by the authors.    Manufacturing process diagram for Doenjang.

Figure 2
Growth curve of body weight. Body weight growth curve of the four groups of rats. The body weights of each group of rats recorded weekly are shown. Values are the mean ± SEM, with different letters signi cantly different (p < 0.05) by Duncan's multiple range test (a > b). Six rats were assigned to each group. ND; normal diet group, HD; high-fat diet group, HDS; high-fat diet with salt group, HDJ; high-fat diet with doenjang group.     with Duncan's multiple range test (a > b). *p < 0.05 for HDS versus HDJ. Six rats were assigned to each group. Six rats were assigned to each group. ND; normal diet group, HD; high-fat diet group, HDS; high-fat diet with salt group, HDJ; high-fat diet with doenjang group. Agt; Angiotensinogen, Ace; Angiotensinconverting enzyme, Agtr1; Angiotensin II receptor type 1, Agtr2; Angiotensin II receptor type 2.

Figure 8
Effect of doenjang treatment on mRNA levels of RAS-related genes in liver and kidney of rats. Values are given as mean ± SEM. Values with different superscripts are signi cantly different (p < 0.05) by ANOVA with Duncan's multiple range test (a > b). *p < 0.05 for HD versus HDS, #p < 0.05 for HDS versus HDJ. Six rats were assigned to each group. Six rats were assigned to each group. ND; normal diet group, HD; high-fat diet group, HDS; high-fat diet with salt group, HDJ; high-fat diet with doenjang group. Agt; Angiotensinogen, Ace; Angiotensin-converting enzyme, Agtr1; Angiotensin II receptor type 1, Agtr2; Angiotensin II receptor type 2.  Urine output and water intake in 24 h metabolic cages. Values are given as mean ± SEM. Values with different superscripts are signi cantly different (p < 0.05) by ANOVA with Duncan's multiple range test (a > b). Six rats were assigned to each group. ND; normal diet group, HD; high-fat diet group, HDS; high-fat diet with salt group, HDJ; high-fat diet with doenjang group.

Figure 11
Effects of Doenjang extract on 3T3-L1 cell line. Values with different superscripts are signi cantly different (p < 0.05) by ANOVA with Duncan's multiple range test (a > b). Pre; preadipocytes, Con; adipocytes without