Obesity has emerged as an important health problem through a rapid increase in incidence worldwide. Excessive body weight gain and body fat accumulation pose a serious threat, leading to the onset of obesity and metabolic disorders such as diabetes and cardiovascular disease (Bastien et al., 2014). A high-fat diet (HFD) affects elevated serum cholesterol and triglycerides (TG) and increases the risk of chronic low-grade inflammation from obesity, along with adipose tissue enlargement (Dewulf et al., 2011). The influence of metabolic disorders in combination with the HFD can also damage the liver. Recently, the association and risk of obesity with nonalcoholic fatty liver disease (NAFLD) has received a lot of attention. NAFLD includes a variety of symptoms ranging from simple steatosis to nonalcoholic fatty hepatitis, cirrhosis, or hepatocellular carcinoma (Wong et al., 2015), and more than 80% of obese individuals in western countries are affected by NAFLD (Younossi, 2019). Recently, several studies have focused on gut microbiota as a new environmental factor contributing to the relationship between obesity and NAFLD (Le Roy et al., 2013; Tilg et al., 2016; Da Silva et al., 2018). The gut microbial community consists of thousands of bacterial species, coexisting with the host, and has a significant influence on the physiology and metabolism of the host (Turnbaugh et al., 2006; Ridaura et al., 2013). Recently, a link between the effects of choline deficiency on fatty liver development and changes in the human gut microbiota was confirmed (Spencer et al., 2012), and it was revealed that changes in the gut microbiota regulate the progression of NAFLD (Buzzetti et al., 2016). Thus, the regulation of gut microbial communities may suggest new therapeutic strategies in the management of metabolic diseases such as obesity and NAFLD. Probiotics regulate the gut microbiome and have proven beneficial effects on metabolic symptoms. They are also known to be effective in improving lipid profiles and hyperlipidemia, while affecting lipid metabolism (Roller et al., 2004; Sun and Buys, 2015).

In our previous study, a lactic acid bacterium, designated ADM14, was found to have anti-adipogenic effect by reducing significantly intracellular triglyceride content on 3T3-L1 adipocytes and by decreasing the expression of five adipogenic marker genes (Won et al., 2020a). The strain ADM14 was identified as a member of Lactobacillus sakei in the study of Won et al. (2020a), but L. sakei has been recently reclassified as L. sakei (Zheng et al., 2020). The aim of this study was to investigate the effect of L. sakei ADM14 on host lipid metabolism, fatty liver, and metabolic problems in HFD mouse model. In addition, changes in gut microbiota structure on the host were analyzed through fecal microbiome analysis the relationship between the gut-liver axis was evaluated.

The incidence and severity of obesity and metabolic complications are constantly rising, and drug therapy has the potential to cause adverse effects (Sonnenburg and Bäckhed, 2016; Dahiya et al., 2017). The necessity for alternative therapy is increasing, and attention has been drawn by the effectiveness and utilization of probiotic lactobacilli (Aryana and Olson, 2017). Animal studies and clinical studies have reported the anti-obesity effects of probiotic lactic acid bacteria and improvements in metabolic disorders (Dahiya et al., 2017). In this study, the administration of L. sakei AMD14 suppressed weight gain and reduced body fat mass in a high-fat mouse model as shown in our previous study (Won et al., 2020b). Although, there is no significant difference in total caloric consumption, the difference in physical indicators is confirmed to be an inhibitory effect by L. sakei ADM14 (Figure 1). The reduction in the weight of subcutaneous fat tissue, as well as reduction in the weight of the epididymal fat tissue also observed previous study (Won et al., 2020b), appears to induce the overall decrease in lipid accumulation in mice administered with L. sakei AMD14. In this study, it was found that the weight of the liver was decreased, and as a result of microscopic observation through Oil Red O staining of the liver tissue, a decrease in the size of the adipocytes was observed (Figures 1D,E). This is seen as evidence that the expansion of adipocytes and accumulation of lipids in the liver were inhibited. Cardiovascular disease and lipidemia are major metabolic complications that occur and intensify because of obesity (Cani and Delzenne, 2009). These metabolic disorders are mainly caused by an increase in blood cholesterol and TGs (Yoo and Kim, 2016). The significant increases in total cholesterol, TG, and glucose in serum of the HD group were consistent with the predicted characteristics of obesity. Administration of L. sakei ADM14 suppressed and improved these indicators and confirmed the possibility of preventing symptoms related to lipidemia. The improvement in blood lipids after administration of L. sakei ADM14 could be evaluated on the basis of another case that the probiotic lactic acid bacteria effectively improved the serum markers of high-fat diet mice (Wang et al., 2012).

To confirm the potential mechanism by which L. sakei ADM14 inhibits obesity in a high-fat diet mouse model, genetic expression analysis of epididymal adipose tissue was performed (Figure 2). Adipose tissue is one of the important organs that regulate lipid metabolism (Schneeberger et al., 2015). Adipocyte transcription factors Pparγ and C/EBPα play an important role in the development of adipocytes (Rosen et al., 1999; Jeong et al., 2008), and our results showed a tendency to decrease in the expression of both genes. It has been reported that these transcription factors have a positive correlation with lipid accumulation rates (Park et al., 2009). Additionally, the decreased expression of Srebp1C, which is involved in fatty acid synthesis, and Fas, which is involved in regulating lipid production, suggests that the accumulation of lipids in adipocytes was inhibited. Likewise, aP2, which is related to adipose production, and CD36 and Lpl, which are related to lipid storage showed decreased expression and through this, it could be confirmed that a change occurred in adipose tissue metabolism. The expression of Pparγ, C/EBPα, Fas, aP2, and CD36 in epididymal fat was confirmed to decrease also in this study as shown previously (Won et al., 2020b). From these results, it could be confirmed that administration of L. sakei ADM14 may cause changes in adipose tissue metabolism by affecting transcription factors, lipid production, or lipid storage. Low-grade chronic inflammation of adipose tissue is one of the major factors in obesity-induced insulin resistance and metabolic disorders (Gregor and Hotamisligil, 2011). As the expression of inflammation-related genes in adipose tissue was decreased, it was also confirmed that low-grade inflammation was alleviated (Figure 2; Won et al., 2020b).

Nonalcoholic fatty liver disease is representative of the symptoms induced by obesity (Canfora et al., 2019). The balance of lipid metabolism in the liver is disrupted, causing excessive lipogenesis and hepatic lipid accumulation (Rotman and Sanyal, 2017; Friedman et al., 2018). NAFLD can lead to liver damage and develop into fatty hepatitis, cirrhosis, or hepatocellular carcinoma (Le Roy et al., 2013). Fundamentally, the increase in the expression of major genes related to lipogenesis in liver tissue seems to be one of the biggest causes. Glucose absorbed in the intestine regulates the transcription of lipogenic genes through ChREBP (Kawaguchi et al., 2001). In our results, it appears that the decreased expression of ChREBPα/β resulted in the decreased expression of adipogenic genes, thus affecting the reduction of hepatic lipid accumulation in the liver. The expression of fatty acid synthesis-related genes such as Pparγ, Fas, and Acc1 decreased with the inhibition of glucose catabolism. The expression of Srebp1C targeting Fas and Acc1 also decreased. The main characteristic of NAFLD is the accumulation of TG in the liver (Eslamparast et al., 2013). In a state of excess energy such as obesity, the activity of Dgat, which catalyzes TG synthesis, increases (Choi and Diehl, 2008). The increase in free fatty acid delivered to the liver also upregulates the synthesis of TG and ultimately increases the likelihood of developing NAFLD (Choi and Diehl, 2008). The results of our study showed that the expression of Dgat1 and Dgat2 was reduced in the final stage of TG synthesis. Likewise, a reduction in the uptake of free fatty acid through the reduction of Lpl and CD36 expression seems to affect the process of TG synthesis. In our results, it was confirmed that the expression of Dgat1 and Dgat2, which catalyzes TG synthesis, was decreased. Likewise, it appears that a reduction in the uptake of free fatty acid to the liver through a decrease in Lpl and CD36 expression influenced the TG synthesis. β-oxidation is one of the key pathways for fatty acid metabolism in the liver (Lu et al., 2014). Pparα and Cpt1α were upregulated in the HDA group compared to the HD group. This has been shown to upregulate lipolysis and fatty acid oxidation. In this study, although NAFLD is shown not to be caused by high-fat diet, genetic analysis in the mechanisms of lipid metabolism in the liver suggests that L. sakei ADM14 downregulates lipid production and upregulates fatty acid oxidation-related genes, while regulating hepatocytes lipid accumulation and a reduction in TG synthesis (Figure 3).

A high-fat diet can contribute to changes in the gut microbiome and can lead to metabolic imbalances caused by gut microbiota dysbiosis (Wang et al., 2011; Wu et al., 2017). In addition, gut microbiota dysbiosis has recently been reported to be associated with liver diseases as well as metabolic diseases such as obesity (Da Silva et al., 2018; Kong et al., 2018). Therefore, for the prevention and treatment of metabolic diseases, an analysis targeting the gut microbiota must be conducted (Li et al., 2020). As a result of analyzing the fecal microbiota, we observed a significant decrease (p<0.05) in the Firmicutes to Bacteroidetes ratio in the HDA group compared to the HD group at the phylum level as also shown in cecal microbiota from our previous study (Won et al., 2020b; Figure 2). The main cause was the difference in the relative abundance of the Bacteroidetes between HDA and HD groups over 10weeks. In particular, relative abundance of the Bacteroidetes after 10weeks from the HDA group was also similar with that of cecum shown in the study of Won et al. (2020b). In the analyses of the fecal microbiota at three different times (1, 6, and 10weeks), the increase in Firmicutes ratios from 1 to 6weeks was much greater than that from 6 to 10weeks in both the HD and HDA groups, whereas the decrease in Bacteroidetes ratios was greater from 6 to 10weeks in the HD group but was greater from 1 to 6weeks in the HDA group (Figure 4A). The Proteobacteria ratios from 6 to 10weeks were decreased in the HDA group unlike in the HD group (Figure 4A). The administration of L. sakei ADM14 from 1 to 10weeks does not affect Firmicutes ratios between the HD and HDA groups but affect the ratios of Bacteroidetes and Proteobacteria in the HDA group. In the study of Won et al. (2020b), administration of L. sakei ADM14 was shown to increase Firmicutes ratios in the cecal microbiota. These results suggest that the failure to increase the Firmicutes ratio in fecal microbiota despite the administration of L. sakei might be due to colonization of L. sakei ADM14 in the intestine. The Firmicutes can produce more harvestable energy than Bacteroidetes, and when the relative abundance of Firmicutes increases and Bacteroidetes decreases, the absorption of calories in the digestive tract increases (Turnbaugh et al., 2006; Komaroff, 2017). This difference in terms of energy harvesting promotes metabolic diseases such as obesity. In our results, the administration of L. sakei ADM14 induces a change in the proportion of the dominant taxa at the phylum level and suggests that differences in energy resources may occur. At the genus level, Bacteroides, Oscillibacter, and Alistipes, which have higher proportions in the HDA group compared to the HD group, were reported to have a negative correlation with obesity (Dahiya et al., 2017; Thingholm et al., 2019). Bacteroides and Alistipes were also observed to have higher proportions in cecal sample of the HDA group in previous study by Won et al. (2020b). Bacteroides and Alistipes are known to be the major producers of short chain fatty acids such as acetic acid and butyric acid (Borton et al., 2017; Yin et al., 2018). In fecal microbiota, members of genus Alistipes, family Muribaculaceae and species Bacteroides acidifaciens were notable bacteria whose relative abundance was significantly changed by the administration of L. sakei ADM14 (Figure 4E). The genus Alistipes is found at low levels in the intestines of patients with hepatocellular carcinoma, colitis, and NAFLD and has been reported to have protective effects against some diseases including liver fibrosis, colitis, and cardiovascular disease (Parker et al., 2020). Muribaculaceae, previously known as family S24-7, was reported as representative bacteria with low abundance in mice fed a high-fat diet (Lagkouvardos et al., 2016). Bacteroides acidifaciens has been reported to be effective in preventing metabolic disorders such as obesity by controlling the host lipid metabolism (Yang et al., 2017). Overall, L. sakei ADM14 changed the composition of gut microbiota, suggesting the possibility of preventing metabolic disorders caused by dysbiosis. In particular, an increase in the abundance of Alistipes through gut microbiota modification helps to prevent diseases such as fatty liver through gut-liver axis modulation.

In this study, Latilactobacillus sakei ADM14 was found to inhibit adipogenesis by regulating the expression level of lipid metabolism in adipose tissue and liver tissue in a high-fat diet mouse model. In addition, fecal microbial community analysis suggested that the composition and ratio of gut microbiota were modified to induce changes in energy harvest, and the possibility of preventing metabolic disorders through modulation of the gut-liver axis. To our knowledge, there are few studies to evaluate the relationships among obesity, lipid metabolism in both epididymal fat and liver, and fecal microbiomes at different times by probiotic strain, particularly lactic acid bacteria isolated from kimchi. Further research and clinical studies are needed to evaluate their applicability and effectiveness in human.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: https://www.ncbi.nlm.nih.gov/ (PRJNA751416, PRJNA751419, PRJNA751420, PRJNA751421, PRJNA751431, and PRJNA751433).

The animal study was reviewed and approved by Animal Care and Use Committee of the College of Biotechnology at Sungkyunkwan University (approval number: SKKUIACUC-18-04-14-3).

S-MW and MS performed the experiments of this study and reviewed the manuscript. MK supported some experiments of this study. KP discussed the results of this study. J-HY designed this study and wrote manuscript. All authors contributed to the article and approved the submitted version.

This work was supported by the “Cooperative Research Program for Agriculture Science and Technology Development (project no. PJ015247)” of the Rural Development Administration, Republic of Korea and a Research Initiative Program of Sungkyunkwan University, Republic of Korea.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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