Effect of Continuous Feeding of Ayu-Narezushi on Lipid Metabolism in a Mouse Model of Metabolic Syndrome

Ayu-narezushi, a traditional Japanese fermented food, comprises abundant levels of lactic acid bacteria (LAB) and free amino acids. This study aimed to examine the potential beneficial effects of ayu-narezushi and investigated whether ayu-narezushi led to improvements in the Tsumura Suzuki obese diabetes (TSOD) mice model of spontaneous metabolic syndrome because useful LAB are known as probiotics that regulate intestinal function. In the present study, the increased body weight of the TSOD mice was attenuated in those fed the ayu-narezushi-comprised chow (ayu-narezushi group) compared with those fed the normal rodent chow (control group). Serum triglyceride and cholesterol levels were significantly lower in the Ayu-narezushi group than in the control group at 24 weeks of age. Furthermore, hepatic mRNA levels of carnitine-palmitoyl transferase 1 and acyl-CoA oxidase, which related to fatty acid oxidation, were significantly increased in the ayu-narezushi group than in the control group at 24 weeks of age. In conclusion, these results suggested that continuous feeding with ayu-narezushi improved obesity and dyslipidemia in the TSOD mice and that the activation of fatty acid oxidation in the liver might contribute to these improvements.


Introduction
Metabolic syndrome (MS) is a serious global health problem that is expected to worsen in the future because of the increasing prevalence of obesity, sedentary lifestyles, and unbalanced diets [1][2][3]. MS initiates with visceral fat accumulation and progresses to insulin resistance, abnormal lipid metabolism, hypertension, and various other diseases throughout the body [4]. Furthermore, MS may lead to/ related to type 2 diabetes mellitus, vascular inflammation, atherosclerosis, and renal, liver, and heart diseases. Although the pathogenesis of MS is very complicated and controversial, the imbalance of the gut microbiota, i.e., dysbiosis, is suggested to be associated with MS [5][6][7]. Numerous studies have reported the beneficial effects of probiotics in improving dysbiosis, and probiotics have been increasingly introduced as health-promoting supplements in our daily diet. In fact, beneficial commensal microbes, such as Bifidobacterium spp. and Lactobacillus spp. have long been consumed as supplements [8,9].
Many traditional fermented foods and beverages worldwide, such as yoghurt and kimuchi, have been reported as probiotic sources [10,11]. Narezushi, one of the traditional fermented foods that is also a prototype of sushi in modern Japanese cuisine, is mainly made from salted and fermented rice and various fish types, and Funazushi made from crucian carp in Shiga prefecture is the most famous narezushi in Japan. In Toyama prefecture, ayu-narezushi is known since olden times, and it is made from boiled rice, malted rice, and salted ayu (Plecoglossus altivelis). Nomura et al. [12] analyzed the ingredients of ayu-narezushi made by the method handed down and reported that it comprised high levels of lactic acid bacteria (LAB) and free amino acids (FAAs), as shown in Table S1 (Supplementary Materials, SM1). Certain LAB and Bifidobacteria are considered as probiotics based on their health-promoting effects involving metabolism and immunity [13]. LAB reportedly lowers serum cholesterol levels in animals and humans [14,15]. Furthermore, certain FAAs have been recognized for their biological activities; e.g., taurine has effective actions on MS, which include reducing triglycerides to prevent obesity and improving insulin resistance to regulate glucose metabolism [16]. e abovementioned studies have, therefore, focused on the relationship between pathophysiology and specific components, such as LAB and FAA, regarding improving disorders. Accordingly, we considered that ayu-narezushi containing abundant LAB and FAAs might become not only a mere traditional food but also a novel candidate for functional food as probiotics.
Tsumura Suzuki obese diabetes (TSOD) mice models (TSOD mice) are polygenic models of spontaneous type 2 diabetes mellitus in which the mice develop moderate degrees of obesity, which is particularly apparent in animals older than 11 weeks of age [17,18]. Male TSOD mice exhibit glycosuria, hyperglycemia, and hyperinsulinemia within the absence of any special treatment [19] and are valuable models for examining disease mechanisms and drug efficacy in MS treatment. erefore, we hypothesized that feeding TSOD mice with ayu-narezushi is abundant in LAB and FAAs improves the pathophysiology of MS.
is study aimed to investigate the effect of ayu-narezushi on MS through the feeding experience of TSOD mice. We also examined a part of the action mechanism, improving lipid metabolism in mice by using gene expression analysis.

Preparation of Ayu-Narezushi.
e main components of ayu-narezushi are ayu (Plecoglossus altivelis), cooked rice, and salt. Briefly, the internal organs and gills of the fish caught from the Shou River in Toyama prefecture were removed and soaked in natural salt at 5°C in a refrigerator for 3 weeks. ereafter, the meat was rinsed under running water for 5 minutes to remove excess salt and slime. en, cooked rice and malted rice were placed in the bottom of a keg and layered on top with the meat and cooked rice, in order; this procedure was repeated 7-8 times to create layers of rice and meat. e inner lid of the keg was then placed, and weights were placed on top of the lid. e keg was preserved in a cold and dark place for fermentation (temperature range: 5°C−10°C), and ayu-narezushi can be consumed after approximately 1 month. In the present study, ayu-narezushi that was fermented for 60 days in the keg was used.

Experimental Animals.
Overall, 20 male TSOD mice at 4 weeks of age were purchased from the Institute for Animal Reproduction (Ibaraki, Japan). During the acclimation period, the animals were provided a normal rodent chow with Labo MR Stock (Nosan Corporation, Yokohama, Japan) and chlorinated water ad libitum. e mice were housed in plastic cages in a nonbarrier-sustained animal room maintained at 22°C ± 2°C with 60% ± 10% relative humidity and a 12/12 h light/dark cycle. After acclimation, the TSOD mice were weighted and assigned to two groups (n � 10): the control group that was fed the normal chow and ayu-narezushi group that was fed the chow comprising 10% (w/w) ayu-narezushi, which was prepared by mixing the normal chow with ayu-narezushi including the meat, cooked rice, and malted rice. e experimental diet was fed to the TSOD mice starting from 5 weeks of age. During the experiment, there was no notable difference in food intake between the two groups. e study was performed in accordance with the animal experiment guidelines specified by the University of Toyama. All experimental protocols were approved by the Committee on Animal Experimentation of the University of Toyama (A2015MED-54).
During the experiment, the body weights of all mice were measured once a week from the start of the experiment (5 weeks of age) to the end of the experiment (24 weeks of age). For histological and biochemical analyses, 5 mice in each group were sacrificed at 8 and 24 weeks of age. Additionally, after anesthesia with sodium pentobarbital, blood samples were collected from the posterior vena cava and centrifuged at 1,200 × g for 15 minutes to collect serum. e samples were maintained at −80°C until further use for biochemical analysis. Additionally, the main internal organs, including the liver, pancreas, kidney, lungs, and spleen, as well as visceral fat, were rapidly excised and rinsed in ice-cold saline, followed by fixation in 10% neutral-buffered formalin, and embedded in paraffin for histopathological analysis. Remaining liver and visceral fat tissues were also stored at −80°C until further use for gene expression analysis.

Histological and Biochemical Analyses.
e formalinfixed paraffin-embedded tissues were processed using standard procedures, and 4 μm-thick serial sections were stained with hematoxylin and eosin for histological analysis using an optical microscope (Olympus BX41; Olympus Corporation, Tokyo, Japan). e number of visceral adipocytes was determined using the WinROOF image analysis software (Mitani Corporation, Fukui, Japan). e digital images of a minimum of three fields of hematoxylin-/eosinstained slides of visceral fat were captured at 200x magnification using a digital camera (Olympus DP21, Olympus Corporation, Tokyo, Japan), and the software counted the number of visceral adipocytes.

RNA Preparation and Real-Time PCR.
e mRNA expression levels of three genes related to fatty acid oxidation (peroxisomal proliferator-activated receptor α; PPARα, acyl-CoA oxidase; ACO, and carnitine-palmitoyl transferase; CPT1) were assessed by real-time PCR. Total RNA was isolated from the liver and visceral fat samples by using Isogen2 reagent (Nippon Gene, Japan). Complementary DNA was synthesized from 500 ng of total RNA by using a ReverTra Ace ® qPCR RT Master Mix with a gDNA Remover (Toyobo, Japan). PCR reactions and analyses were carried out using a LightCycler ® Nano System (Roche, Japan) with denaturation for 10 min at 95°C, followed by 45 PCR cycles of denaturation at 95°C for 10 s, annealing at 60°C for 10 s, and extension at 72°C for 15 s. All primers used for analysis were designed by Eurofins Genomics (Japan). e primer sequence details are presented in Table 1 [20]. e amount of mRNA was calculated using glyceraldehyde-3-phosphate dehydrogenase as an endogenous control.

Statistical
Analysis. Values were expressed as means ± standard deviation. Means were compared using Student's t-test. A P value of <0.05 was considered statistically significant.

Changes in Body Weight in the Ayu-Narezushi and the Control Groups from 4 to 24 Weeks of Age.
e mean body weight in both the ayu-narezushi and control groups increased gradually from 4 to 24 weeks of age, when the experiment was concluded. However, there was a tendency of attenuation in the increase of mean body weight starting approximately at 11 weeks of age in the ayu-narezushi group compared with the control group (Figure 1). In statistical analysis, there was not the statistical significant difference between ayu-narezushi and control group.

Comparison of Histopathological Findings in Visceral Fat in the Two Groups.
e size of visceral adipocytes was remarkably smaller in the ayu-narezushi group than in the control group at 24 weeks of age (Figures 2(a) and 2(b)); however, no remarkable difference was observed in their size between the two groups at 8 weeks of age (Figures 2(c) and  2(d)). e quantification of the images captured at identical magnification revealed that the mean number of visceral adipocytes at 24 weeks of age was significantly higher in the ayu-narezushi group (100.3 ± 18.2 cells) than in the control group (85.2 ± 14.5 cells) ( Table 2, P < 0.05).
On the other hand, no remarkable differences were observed in other organs, including the liver and spleen, between the ayu-narezushi and control groups.

Serum Levels of Triglycerides and Cholesterol in the Ayu-Narezushi and the Control Groups.
e serum triglyceride and cholesterol levels, which were not significantly different at 8 weeks of age between the two groups, were significantly lower in the ayu-narezushi group than in the control group at 24 weeks of age (Figures 3(a) and 3(b), P < 0.05).

Validation of Gene Expression with Real-Time PCR.
Hepatic mRNA expression levels of PPARα tended to increase in the ayu-narezushi group compared with the control group at 24 weeks of age (Figure 4(a)), although significant difference were not observed. In addition, hepatic mRNA expression levels of ACO and CPT1 were significantly increased in the ayu-narezushi group compared with the control group at 24 weeks of age (Figures 4(b) and 4(c), P < 0.05). ese data suggest that an overexpression of ACO and CPT1 induced improvement of lipid metabolism through activation of fatty acid oxidation. e visceral fat mRNA expression level of PPARα was significantly increased in the ayu-narezushi group compared with the control group at 24 weeks of age (Figure 4(d), P < 0.01).

Discussion
In the current study, we investigated the effect of ayunarezushi on conditions associated with MS including weight gain and hyperlipidemia using TSOD mice feeding ayu-narezushi because ayu-narezushi comprised abundant levels of LAB and FAA that are extensively known for their probiotic activity. During the experiment period, we found that the increase in mean body weight slowed down gradually from 11 to 24 weeks of age in mice that were fed the ayu-narezushi-comprising chow compared with that in mice fed the control chow. e size of the visceral adipocytes was smaller in the ayu-narezushi group than in the control group at 24 weeks of age. Furthermore, the serum triglyceride and cholesterol levels were significantly lower in the ayu-narezushi group. Overall, these data suggested that the longterm consumption of ayu-narezushi is beneficial for TSOD mice.
Next, we investigated the action mechanism that lipid metabolism was improved in the ayu-narezushi group using gene expression analysis. In this study, we showed that the hepatic mRNA expression level of PPARα tended to increase in the ayu-narezushi group, although significant difference was not observed. Furthermore, hepatic mRNA levels of PPARα target genes, such as CPT1 and ACO, were significantly increased in the same group. PPARα is expressed principally in the liver and is a key regulator of lipid metabolism [21,22]. PPARα regulates the expression of many genes, such as ACO and CPT1, that were involved in fatty acid oxidation [23,24]. Accordingly, these data e Scientific World Journal 3 suggested that ayu-narezushi induced fatty acid oxidation in the liver of TSOD mice.
In addition, we also found that visceral fat mRNA expression levels of PPARα were significantly increased in the ayu-narezushi group at 24 weeks of age. Although PPARα is expressed at high levels in the liver, Takahashi et al. [25] reported that overexpression of PPARα in obese mice adipose tissue improves insulin sensitivity and suggested that PPARα activation in adipose tissue contributes to the improvement of glucose metabolism disorders via the enhancement of free fatty acid metabolism. Although further studies are needed to investigate, PPARα activation in visceral fat of TSOD mice feeding ayu-narezushi might play an important role in the improvement of disease related to lipid metabolism dysfunction.
In this study, the specific component candidate substances of ayu-narezushi that were related to these beneficial effects remain unclear. Nomura et al. [12] have reported that ayu-narezushi fermented for 60 days comprised LAB (3.7 × 10 7 cfu/g), organic acids (mainly lactic acid; 3,114 mg/ 100 g), and FAA (1,169 mg/100 g). In the further study, they had also found that the LAB sufficiently fermented ayunarezushi over 40 days primarily comprised L. sakei (90%). erefore, the ayu-narezushi in the current study, which was fermented longer (60 days), should comprise comparable levels of L. sakei. Although few studies have investigated this bacterium, Lim et al. [26] have reported that L. sakei OK67 ameliorated high-fat-diet-induced hyperglycemia and obesity by reducing inflammation and increasing the expression of colon tight junction proteins in mice. is LAB species might be functionally effective as an antiobesity agent in TSOD mice.
Recently, studies have revealed the novel functional roles of D-amino acids. Amino acids have an alpha-carbon that is connected to four functional groups, including amine and carboxyl groups, a hydrogen, and a side chain. Amino acids, except for glycine, have two stereoisomers: levorotatory (L) and dextrorotatory (D). Although L-amino acids are essential for life as the building blocks of proteins, D-amino acids reportedly impact important physiological events in mammals, such as neurotransmission, testosterone synthesis, and fertilization [27][28][29]. We have performed mass spectrometry analysis to quantify the L-and D-amino acids [30] and found six D-amino acids such as D-Ala and D-Asp in ayu-narezushi ( Figure S1: Supplementary Materials, SM2). Kato and Oikawa [31] have also reported that L. sakei LK-145 produces high levels of D-amino acids. erefore, it  e mean body weight of the ayu-narezushi group (n � 5, white circles) was lower than that of the control group (n � 5, black circles) over 10 weeks of age. is trend continued until 24 weeks of age, with a gradual increase in the difference in mean body weight between the two groups over the weeks.    , and CPT1 (c) mRNA levels in liver and PPARα (d) mRNA levels in visceral fat. Each tissue was isolated at 24 weeks of age. e mRNA levels were determined by real-time PCR, normalized to glyceraldehyde-3-phosphate dehydrogenase. e data represent the mean ± standard deviation of 5 animals. * Significantly different from the control group with * P < 0.05; * * P < 0.01. PPAR, peroxisome proliferator-activated receptor; ACO, acyl-CoA oxidase; and CPT1, carnitine-palmitoyl transferase. e Scientific World Journal is possible that the D-amino acids in ayu-narezushi contribute to the improvements in the body weight and serum levels of triglycerides and cholesterol in TSOD mice. However, further investigation is warranted to identify the specific D-amino acids in ayu-narezushi and their functional roles. Two studies have examined the components of the gut microbiome in TSOD mice at different ages. One study comparing the intestinal flora of TSOD mice with that of control mice at 5 and 12 weeks of age has observed that some species were detected only in the TSOD mice such as the genera Turicibacter and SMB53, a member of the Clostridiaceae family, and suggested that these bacteria contribute to type 2 diabetes in TSOD mice [32]. e second study has reported that there was a correlation among gut dysbiosis, short-chain fatty acids, and MS in 24-week-old TSOD mice [33]. Enriched levels of Gram-positive bacteria and corresponding decreases in Gram-negative bacteria were also detected in TSOD mice. Moreover, the abundance of Bacteroidetes spp. was decreased, whereas that of the Firmicutes spp. was increased, and the total plasma shortchain fatty acid level was significantly lower in the TSOD mice than that of the controls. ese results suggested the dysbiosis of the gut microbiome in TSOD mice as early as 12 weeks of age, which was observed until 24 weeks of age. LAB intake has been reported to modulate the gut microbiota in some studies [34,35]. Although L. sakei in ayu-narezushi might have improved the gut microbiome of TSOD mice, further studies clarifying the association between ayu-narezushi feeding and dysbiosis in this animal model are warranted.

Conclusions
Feeding TSOD mice with ayu-narezushi, which comprises abundant levels of LAB and FAA, improved the change in body weight and serum levels of triglycerides and cholesterol. In these mice, it was considered that the activation of fatty acid oxidation in the liver had contributed to the improvement of diseases related to MS. Traditional fermented food items comprising LAB, such as yoghurt and sauerkraut, are available worldwide. e continuous use of drugs for the prevention of MS is not ideal. erefore, we propose that the continuous feeding of traditional fermented foods like ayu-narezushi may be expected to have great potential for the improvement of MS.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare no conflicts of interest.