Egg oil from Portunus trituberculatus alleviated obesity and regulated gut microbiota in mice

Egg oil from Portunus trituberculatus (Pt-egg oil) can overcome insulin resistance resulting from abundant bioactive lipids. However, its effects on obesity and gut microbiota were unclear. Here, we evaluated whether Pt-egg oil could improve obesity and gut microbiota or not in high-fat diet feeding mice. Results exhibited that Pt-egg oil markedly reduced body weight and adipose weight gain, improved lipid accumulation and circulatory cytokines, inhibited epididymal adipose cell size. Moreover, Pt-egg oil modified gut microbiota, involving decreases in the ratio of Firmicutes to Bacteroidetes, Proteobacteria, Actinobacteria, and increase in Verrucomicrobia phylum. Pt-egg oil reduced serum and fecal lipopolysaccharide (LPS) levels and down-regulated Toll-like receptor 4 pathway in both epididymal adipose and liver tissues. Meanwhile, Pt-egg oil increased short chain fatty acids and up-regulated of G-protein-coupled receptors in both epididymal adipose and liver tissues. These suggest that Pt-egg oil could be alternative food supplement for the prophylactic effects on anti-obesity and improvement in human gut health.

. Effects of Pt-egg oil on the histology of adipose and liver tissues using H&E staining (n = 10). The adipose cell size and hepatic lipids area were measured by CaseViewer 2.0, and the size or area of the control was defined as 1. ## P < 0.01 vs control; ** P < 0.01 vs HFD.
Pt-egg oil restored gut microbiota dysbiosis. Gut microbiota dysbiosis contributes positively to obesity. Figure 2A showed the data of Venn diagram analysis, and three groups showed the own distinct OTUs. PCA score plot showed an oberious different microbiota distribution between all experimental groups (Fig. 2B,C). Moreover, gut microbiota at the Phylum was significantly different (Fig. 2D). F/B ratio was statistically increased in HFD group compared with control group, which was significantly reduced in Pt-egg oil-treated animals compared with obese mice. Moreover, Pt-egg oil decreased Proteobacteria, while increased Verrucomicrobia. In addition, the abundance of Actinobacteria in Pt-egg oil-treated mice was remarkably decreased compared with obese mice. Figure 3 showed that 48 genus exhibited remarkably different abundances in HFD mice compared with control, and 41 genus different in Pt-egg oil mice compared with HFD, implying that Pt-egg oil may alleviate obesity through regulating bacterial subset. Pt-egg oil reduced the numbers of Ruminiclostridium_5, Ruminiclostridium, Ruminococcaceae_UCG-013, Anaerotruncus, Oscillibacter, Faecalibaculum, norank_f_Erysipelotrichaceae, (all belonging to Firmicutes), Helicobacter, (belonging to Proteobacteria) unclasslfied_Coriobacteriaceae and Coriobacteriaceae_UCG-002, (belonging to Actinobacteria), Bifidobactenium, and Desulfovibrio compared with obese mice. While the relative abundances of Rikenellaceae_RC9_gut_group, Parabacteroides, and Paraprevotella (belonging to Bacteroidetes), Lactobacillus, Marvinbryantia, Adlercreutzia, Candidatus_Saccharimonas, Family_ XIII_AD3011_group, Asllobaculum, and Romboutsia were increased in Pt-egg oil mice. In addition, the abundances of the SCFAs-producing microbiota Lachnospiraceae_NK4A136_group, norank_f_Lachnospiraceae, Ruminiclostridium_9, Prevotellaceae_UCG-001, Butyricimonas, Alloprevotella, Clostridium_sensu_stricto_1, Allobaculum, and Bacteroides were increased in HFD-fed mice supplemented with Pt-egg oil. Notably, unclassi-fied_f_Ruminococcaceae and Akkermansia (belonging to Verrucomicrobia), were enriched by Pt-egg oil treatment in obese mice.
LEfSe analysis (LDA score log 10 > 4) was conducted to identify specific phylotypes which were changed by Pt-egg oil treatment. Firmicutes was increased in HFD mice, mainly including Erysipelotrichia and unclassified_p_ Firmicutes at class level (Fig. 4A). HFD also caused an increase in Epsilonproteobacteria at class level, belonging to Proteobacteria (Fig. 4A). HFD feeding decreased the levels of Bacteroidetes, in which Bacteroidia was the dominant strain at class level (Fig. 4A). Supplementation with Pt-egg oil significantly reduced Epsilonproteobacteria abundance, and increased Bacteroidia at class level (Fig. 4B), but no significant difference in Firmicutes Phylum.
Pt-egg oil regulated secondary metabolites of gut microbiota. Obesity is regulated by gut microbiota through the secondary metabolites, including LPS and SCFAs. As shown in Fig. 5A,B, HFD feeding caused obvious increases in LPS concentrations in serum and in feces (P < 0.01). Pt-egg oil significantly decreased serum and fecal LPS concentrations by 50.16% and 31.19%, respectively. Fecal and serum acetate, propionate, and butyrate contents were all remarkably reduced in obese mice compared with control group (Fig. 5C-H, P < 0.01). Interestingly, the three fecal SCFAs were significantly increased in Pt-egg oil-receiving mice by 89.56%, 1.13 fold, and 74.60%, respectively. Moreover, Serum acetate and butyrate concentrations were remarkably increased by www.nature.com/scientificreports www.nature.com/scientificreports/ 39.72% and 69.40% in Pt-egg oil group compared with HFD group. However, there is not significant difference in serum propionate level between Pt-egg oil group compared with HFD group.
Pt-egg oil down-regulated LpS-dependent pathway and up-regulated ScfAs-dependent pathway. LPS and SCFAs affect obesity through spurring specific cascades, including LPS-dependent TLR4 pathway and SCFAs-dependent GPRs pathway in both adipose and the liver tissues. Table 2 showed that HFD feeding elevated TLR4 and CD14 mRNA relative expression, while Pt-egg oil down-regulated the levels of TLR4 and CD14 mRNA in adipose tissues of obese mice (P < 0.05, P < 0.01). Moreover, HFD induced strong decreases in GPR41 and GPR43 mRNA expression in adipose tissues, and Pt-egg oil significantly reversed the reductions (P < 0.01). In addition, Pt-egg oil markedly lowered TLR4 and CD14 mRNA expression and increased GPR41 and GPR43 mRNA expression in the liver of obese mice (P < 0.05, P < 0.01).

Discussion
Gut microbiota has been considered as an key environmental factor in the development of obesity 15 . In this study, the effects of Pt-egg oil on antiobesity and regulation of gut microbiota were investigated. The data showed that Pt-egg oil reduced body and adipose weight, serum and hepatic lipids, and epididymal adipose cell size, which suggesting the significant antiobese effects of Pt-egg oil. Gut microbiota analysis showed that Pt-egg oil prevented the loss of Bacteroidetes and Verrucomicrobia and restrained the increase of Firmicutes, Proteobacteria, and Actinobacteria in obese mice. This study also demonstrated that Pt-egg oil mediated LPS and SCFAs production.
Numerous studies have shown that gut microbiota of obese individual is characterized by an abnormal gut microbiota composition 16,17 . We measured community structures of each group by PCA, and it was clear separation between the three group. These suggested that Pt-egg oil could help to shape gut microbiota community through natural selection and competing 18 . Some contradictory conclusions on the change of F/B ratio were shown in obese individual. For example, Cui et al. reported that an increase F/B ratio developed obesity in HFD mice, and fish and frill oil mixture alleviated the ratio 19 . However, other papers showed a low F/B ratio in obese mice 5,20 . In the present study, HFD feeding elevated F/B ratio in mice, which was restored by Pt-egg oil Univariate differential abundance of OTUs at the Phylum level was tested by incorporating Fisher's exact test and the false discovery rate (FDR) among control, HFD, and Pt-egg oil groups and between mouse genotypes. P values were corrected with the Benjamini-Hochberg method to correct for the false discovery rate across multiple comparisons, which were generated using Metastats and considered significance at P < 0.05. ## P < 0.01 vs control; * P < 0.05, ** P < 0.01 vs HFD.  [21][22][23] . Pt-egg oil treatment reduced Proteobacteria and Actinobacteria, and increased Verrucomicrobia at the Phylum level. Further, Helicobacter, belonging to Proteobacteria, unclasslfied_Coriobacteriaceae and Coriobacteriaceae_UCG-002, belonging to Actinobacteria, were lowered by Pt-egg oil. The numbers of unclassified_f_Ruminococcaceae and Akkermansia, belonging to the Phylum Verrucomicrobia, were also elevated in obese mice when treated with Pt-egg oil. Noticeably, the Gram-negative Desulfovibrio genus was significantly reduced in Pt-egg oil-treated mice, which is responsible for inflammation and obesity resulting from lipid A structures of LPS 24 . Lactobacillus and Bacteroides, the beneficial intestinal bacteria, were also promoted by Pt-egg oil, which is proved to positively relate to intestinal integrity, glucose tolerance or attenuated obesity 25,26 . In addition, Pt-egg oil reversed HFD-decreased Bacteroidia at class level, which is negatively correlated with obesity. Meanwhile, Erysipelotrichia and unclassified_p_Firmicutes, belonging to Firmicutes, was reduced by Pt-egg oil, which could positively stimulate obesity and hyperlipemia 27 . Similar results can also be found in studies on other marine bioactive lipids 28,29 . These indicate that Pt-egg oil can alleviate obesity by directly modulating gut microbiota.
Special microbiota can produce SCFAs, including Bacteroides, Lactobacillus, Bifidobacterium, Prevotella, Lachnospiraceae, Butyricimonas, Alloprevotella, Clostridium, Allobaculum etc. [30][31][32] . Our results showed that Pt-egg oil promoted the abundance of the SCFAs-producing microbiota Lachnospiraceae_NK4A136_group, norank_f_ Lachnospiraceae, Prevotellaceae_UCG-001, Ruminiclostridium_9, Butyricimonas, Alloprevotella, Clostridium_ sensu_stricto_1, Allobaculum, and Bacteroides, but lowered Bifidobacterium. After transporting into blood, SCFAs can be taken up by body tissues and subsequently act as substrates and signal melecules 33 . Acetate could promote cholesterol synthesis, and propionate and butyrate could modulate lipid/cholesterol metabolisms 34 . Pt-egg oil significantly enhanced fecal acetate, propionate, and butyrate contents in obese mice, and also increased serum acetate and butyrate concentrations. These changes may be associated with the improvement in many factors in Pt-egg oil-treated mice, such as regulation of gut microbiota composition, decrease in body weight gain, and others 35 . As SCFAs receptors, GPR41 and GPR43 take part in such metabolic pathways, including lipolysis and lipogenesis 36 . Many studies proved that the increases in GPR41 and GPR43 expression could mitigate serum lipids and obesity 37,38 . In this study, Pt-egg oil increased SCFAs and GPR41 and GPR43 mRNA expression. Pt-egg oil-treated mice also showed significant improvement on serum and hepatic lipids levels, body weight gain, adipocyte size, and adipocytokines. These demonstrate that Pt-egg oil-inducted SCFAs generation by regulating special gut flora positively contributes to antiobese effects in mice.
LPS can provoke obesity, inflammation, and even diabetes with the most potent capability 39 . Significantly, the abundances of Proteobacteria Phylum, Desulfovibrio and Enterorhabdus, LPS producing bacteria, were reduced by www.nature.com/scientificreports www.nature.com/scientificreports/ Pt-egg oil treatment. And this regulation was accompanied with decreases in serum and fecal LPS concentrations. Thus, we suggest that the inhibition of pathogenic LPS-producing bacteria by Pt-egg oil might result in a decrease of the decrease of the LPS load into the systemic circulation, and may revealed that the antiobese effects of Pt-egg oil is, in part, responsible for the dramatically reduction in LPS. Previous study has shown that LPS could induce intestinal barrier integrity impared 40 . Pt-egg oil stimulated the elevations in intestinal barrier protectors abundance, such as Lachnospiraceae_NK4A136_group and norank_f_Lachnospiraceae 41 . In addition, LPS reduction has been repeatedly shown to improve obesity and obesity-related cytokines 42 , and in this study, Pt-egg oil decreased LPS concentrations which were associated with enhanced adiponectin, and lowered leptin, resistin, and TNF-α. TLR4/CD14 pathway triggered by LPS is the primary mechanism linking gut bacteria to alleviate obesity 43 . In HFD-fed TLR4-deficient mice, the epididymal adipose weight and blood LPS level were only 69% and 18% of HFD mice, respectively 44 . In the present study, Pt-egg oil inhibited the elevation of TLR4 and CD14 gene mRNA expression in adipose and liver tissues, which is conjuncted with the regulation of gut microbiota, declines in LPS levels, and decreases in body and fat weight. All of these demonstrate that Pt-egg oil alleviates obesity by improving gut microbiota and LPS.
In summary, this paper demonstrated that Pt-egg oil alleviated HFD-induced obesity and improved lipids metabolism in mice. These were directly related with the modulation of gut microbiota community. Pt-egg oil-regulated specific bacteria could improve body weight and lipids metabolism by down-regulation of LPS/ TLR4 pathway and up-regulation of SCFAs/GPRs signaling. In short, it suggested that Pt-egg oil may be an alternative food supplement in alleviating obesity and improving other intestinal diseases.

Materials and methods
Statement. All methods in these experiments were conducted according to the relevant guidelines and regulations of Qingdao University. Moreover, the animals' experimental protocols were approved by the ethical committee for experimental animal care at Qingdao University.

preparation of Pt-egg oil. Tongqu Aquatic Food Company (Zhoushan, Zhejiang, China) provided
Portunus trituberculatus eggs. Pt-egg oil was prepared according to our previous study 14  Animal experiments. C57BL/6J mice (male, 16-18 g, 4-5 weeks) were from Vital River Laboratory Animal Center (Beijing, China; licensed ID SCXK2014-0004). They were fed in a 12:12 h light-dark condition at 22-24 °C in normal cages. The mice were randomized into three groups (10 per group): control group (fed with normal chow diet: 74% corn starch, 16% casein, and 10% corn oil based on weight, 4.09 kcal/g), high fat diet (HFD)-feeding group (fed with HFD: 29% corn starch, 16% casein, 10% corn oil, and 45% lard based on weight, 6.57 kcal/g), and Pt-egg oil group (administrated with HFD and 600 mg/kg Pt-egg oil intragastrically). After 16 weeks treatment, faeces were collected form each mouse feeding in metabolism cages. The animals were sacrificed after 12 h fasting and serum was collected to measure serum lipids and adipokines contents. The liver and epididymal adipose tissues were separated rapidly for hematoxylin and eosin (H&E) stain or measurement of hepatic lipids.
Hepatic TG and TC levels measured by the same kits as used in serum analysis after the lipids in liver extracted by the method of Folch et al.   Serum and fecal ScfAs determination. Serum and fecal SCFAs levels were evaluated according to our previous study 45 . fecal DnA extraction. DNA (n = 4 per group) was extracted from feces by QIAamp DNA Stool Mini Kit (Qiagen, Dusseldorf, Germany).
Intestinal microflora analysis. PCR amplify, sequences analysis, taxonomic identification, alpha and beta diversities were all performed according to our previous stidies 45,46 .
Statistical analysis. OTUs univariate differential value at Phylum level was tested according to Fisher's test. P < 0.05 is considered significance after Benjamini-Hochberg method correcting. Data are shown as mean ± S.D and statistically analyzed by SPSS 17.0 software (SPSS Inc., Chicago, IL, USA). Difference between three groups is conducted by Student's test and P < 0.05 is considered significance.