Cooked pork-derived exosome nanovesicles mediate metabolic disorder—microRNA could be the culprit

doi:10.21203/rs.3.rs-2326155/v1

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Cooked pork-derived exosome nanovesicles mediate metabolic disorder—microRNA could be the culprit

https://doi.org/10.21203/rs.3.rs-2326155/v1

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Journal Publication

published 09 Mar, 2023

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In this study, we extracted exosomes from cooked meat by ultra-high-speed centrifugation. Exosome had an average of size of 70.29 nm. Flow cytometry demonstrated that the positive rate of exosomal surface marker CD63 and CD81 were 84.5% and 95.9%. microRNA sequencing revealed the exosomal microRNA were differences among porcine muscle, fat and liver. The mice plasma levels of miR-1, miR-133a-3p, miR-206 and miR-99a were increase with varying degrees after drinking water with exosomes. GTT and ITT suggest that abnormal glucose metabolism and insulin resistance in mice. Moreover, the lipid droplets were significant increased in the liver were also observed. Transcriptome analysis identified 446 differentially expressed genes in liver. Functional enrichment analysis found that metabolic pathway were most significantly enriched. microRNA may function as a critical regulator involved in the metabolic disorder of mice. This study suggests that the exosomal microRNAs from meat products has the potential to adversely affect health.

meat

pork

exosome

microRNA

transcriptome

Extracellular vesicles (EVs), could be produced by all types of cells in the organism, are lipid bilayer membrane vesicles [1]. Despite its continuous classification change, EVs are currently mainly divided into two categories: ectosomes and exosomes [2]. In which, Exosome is a discrete population of 40–160 nm-sized (~ 100 nm on average) vesicles [3]. For more than a decade, the field of exosome research has been developing rapidly. Exosomes play important roles in intercellular communication by exchange substances between cells [4]. Moreover, exosome contents have been revealed to be a potential biomarkers for a number of diseases, for instance, in various cancers [5], Parkinson's disease [6], and Alzheimer's disease [7].

The exosome mainly contain lipids, proteins and nucleic acids, predominantly microRNA (miRNA) [8]. At present, exosomal miRNA has recently received tremendous attention. microRNA is a non-coding small RNA with highly conserved mechanism of action, which makes it possible to play a role cross-species. Increasing evidences in recent years have suggested exosomal miRNAs in food can be absorbed by animal circulatory system, where exert various biological functions. Xiao et al. reported the existence of exosome in 11 edible fruits and vegetables and successfully identified miRNA profiles [9]. Exosomal miRNA could be a key factor in functional food. Experiments have demonstrated that ginger-derived exosome can promote the mucosal tissue healing of mice induced by colitis [10]. In addition, exosome vesicles containing miRNA are found in milk. Gu et al. reported that the exosomes in pig colostrum contain rich immune related miRNAs [11].

Pork has been an important human food source and is the most consumed meat globally. Raw pork commonly are fabricated into cooked dishes after high-temperature cooking. Moreover, previous study indicate that exosomes and microRNAs are stable at high temperatures [12]. Therefore, we try to identified the exosomes in the cooked porcine muscle, fat and liver and further investigate the microRNA profiles in cooked meat. Besides, we further evaluated the effect of feeding exosomes from cooked pork on the growth of mice.

2.1. Isolation of exosomes

Exosomes were isolated from cooked porcine muscle, fat and liver by the modifying previous exosome extraction method [13]. Briefly, a certain amount of meat was minced in a meat grinder and cooked for 0.5-1 hour in one times the quantity of simmering water. Afterward, the broth was filtered through filter paper and cooled. The filtrate was centrifuged at 2,000× g for 30min. Then, supernatant was collected, centrifuged for 45 min at 12,000× g and filtered through a 0.2 µm filter. The filtrate was centrifuged at 160,000× g for 2 h. Precipitate was collected and washed with PBS, and again centrifuged at 160,000× g for 1 h. Final, precipitation was re-suspended in PBS to make exosome suspensions.

2.2. Characterization of cooked meat-derived exosomes

Exosomal morphology were visualized using transmission electron microscope (TEM, HT7800, Hitachi, Japan). Samples were prepared as described previously [14]. Atomic force microscopy image capture were performed as previously described [9]. The particle size of exosomes were determined by dynamic light scattering analysis (DLS) using Zetasizer Nano ZS (Malvern Instruments, Worchestershire, UK). Flow cytometry was used to examine the exosome characteristic protein markers CD63 (#557288, BD Pharmingen™, USA) and CD81 (#555676, BD Pharmingen™, USA) using a flow cytometer (BD Accuri C6 Flow Cytomenter).

2.3. Mice and feeding

All mice and diets were purchased from Dashuo Company (Chengdu, China). The ICR female mice of ∼14 g were randomized into two groups. A group of mice was supplemented with porcine muscle-derived exosomes in drinking water for 80 days (EXD, n = 6). Each mice was fed exosomes equivalent to the quantity contained in 5 g of pork every day. A separate group was added with phosphate-buffered saline (PBS) as a control (CON, n = 6).

2.4.Tissue section staining and GTT and ITT

The methods of HE staining and Oil Red O staining were performed according to our previous research work [15]. Glucose tolerance test (GTT) and insulin tolerance test (ITT) also were performed according to our previous research work (Shen et al., 2021).

2.5. RNA extraction, library construction, sequencing and RT-qPCR

RNA was isolated using Trizol LS (Ambion, Carlsbad, CA) according to the product instructions. Library construction and sequencing of exosomal miRNA and liver mRNA was performed by Lianchuan BioTech Co., Ltd (Lianchuan Bio, Hangzhou, China). The sequencing data were analyzed referring to our previous publication [16, 17]. Differentially expressed genes (DEGs) were identified using the edgeR software (|logFC| > 1, pvalue < 0.05).

Reverse transcription-quantitative PCR (RT-qPCR) were performed using Mir-X™ miRNA First Strand synthesis kit (Takara, Dalian, China) and TB Green Premix Ex Taq II kit (Takara, Dalian, China) on a CFX96 system (Bio-Rad, CA, USA). Following PCR, absolute quantification was performed by a standard curve method.

2.6. Everted intestinal sac assay

The everted intestinal sac as an in vitro model was prepared by reference to Liu et al [18]. Female SD rats were fasted overnight and anesthetized to excise jejunum. Each intestinal segment was cut to 4 cm and rinsed with cold saline and immediately placed in 37°C Tyrode's buffer oxygenated with 95% O2 and 5% CO2. The intestinal sacs were ligated at one end and gently everted with a glass rod. Exosomes extracted from 250 g meat were added to 20 mL Tyrode's buffer. The control group was conducted without exosomes. The intestinal sac model was incubated in a thermostatic shaker at 37°C with 50 rpm for 2h.

2.7. miRNA target gene prediction and functional enrichment analysis

The target gene of exosomal miRNAs were analyzed and predicted by online platform OmicStudio (https://www.omicstudio.cn/analysis) by using Sus scrofa and Mus musculus as species background, respectively. Gene Ontology (GO) and Kyoto Gene and Encyclopedia of Genomes (KEGG) enrichment analysis were performed using DAVID knowledgebase (v2022q3, https://david.ncifcrf.gov). Protein–protein interaction (PPI) networks were constructed using the STRING database for Cytoscape software 3.9.1.

2.8. Statistical Analysis

Data were visualized using GraphPad Prism 8.0 software. The results were represented as mean ± SEM. Comparisons between two groups were performed using a two-tailed Student's t-test. p < 0.05 was considered statistical significance (*p < 0.05, **p < 0.01).

3.1. Identification of exosomes isolated from cooked meat

As shown in Fig. 1A, exosomes were isolated from cooked meat (CM-Exo), including porcine muscle (PME), fat (PFE) and liver (PLE), using the ultracentrifugation method. The purified samples were further characterized by transmission electron microscopy (TEM) and atomic force microscopy (AFM). Vesicles with exosome-like features were confirmed by (Fig. 2BC). We further analyzed the distribution of vesicles size by Zetasizer Nano (Fig. 2D). The average particles size of those exosome-like vesicles showed 70.29 nm. Approximately 80% of vesicles were within 20–200 nm. Flow cytometry results showed that the positive rate of exosome-specific markers CD63 and CD81 were 84.5% (Fig. 1EF) and 95.9% (Fig. 1GH), respectively.

3.2. microRNA profiles of CM-Exo

The PME, PLE and PFE were examined the miRNA expression profile by high throughput sequencing. miR-1, miR-133a-3p and miR-206 contribute to the total miRNA more than 80% in the PME (Fig. 2A). The relative abundance of miR-122 (37.8%) was found to be highest in the PLE followed by miR-451 (8.9%), miR-99a (5.3%) and other miRNAs (Fig. 2B). For the PFE, the first five were miR-125b, miR-26a, miR-143-3p, miR-27a and miR-99a, respectively (Fig. 2C). KEGG pathway enrichment analysis revealed that miRNAs from PME were mostly involved in MAPK signaling pathway, insulin resistance and autophagy (Fig. 2D). The top three significantly enriched pathways for PLE miRNAs were insulin resistance, TNF signaling pathway and MAPK signaling pathway (Fig. 2E). T cell receptor signaling pathway, autophagy and TNF signaling pathway were the most significantly represented pathway for PFE miRNAs (Fig. 2F).

3.3. Effect of supplementary feeding cooked pork-derived exosomes on growth of mice

We then selected cooked porcine muscle-derived exosomes to feed mice. Mice were given exosomes (equivalent to the quantity contained in 5 g of pork) in their drinking water daily for up to 80 days (Fig. 3A). After the exosomes intake, we assessed several miRNAs levels in mice plasma with a higher relative abundance in exosomes (Fig. 3C-F). The results indicate that the plasma miR-1, miR-133a-3p, miR-206 and miR-99a levels were increased within 0–8 hour in mice after exosomes ingestion. In addition, the everted intestinal sac assay was performed (Fig. 3B). A significant increase in the four miRNAs level in everted intestinal sac was observed after adding exosomes to the buffer (Fig. 3G).

Figure 3H showed that the body weights of exosomes supplemented mice were significantly increased compared to control group mice from day 10. Following intraperitoneal glucose injection, EXD group had significantly higher blood glucose levels at 15–120 minutes compared to CON group (Fig. 3J). ITT showed impaired blood glucose consumption and reduced insulin sensitivity in EXD mice (Fig. 3K). In addition, histomorphological analysis showed that small vacuoles were observed in the EXD mice liver (Fig. 3I). Oil Red O staining revealed more abundant lipid droplets in EXD mice liver (Fig. 3L).

3.4. Cooked pork-derived exosome cause transcriptome changes in mice liver

We further investigated mouse liver transcriptomic changes. Overall, we detected 466 differentially expressed genes (DEG) with 292 genes upregulated and 174 genes downregulated in EXD mouse (Fig. 4A). Heatmap showing DGEs in EXD and CON mouse livers was shown in Fig. 4B. GO enrichment analysis revealed that DEGs were mainly enriched in steroid metabolic process, cholesterol, sterol and steroid biosynthetic process, cholesterol and lipid metabolic process (Fig. 4C). KEGG enrichment indicated that metabolism pathways enriched the most genes followed by MAPK signaling pathway, lipid and atherosclerosis (Fig. 4D).

3.5. Potential regulatory networks involved in lipid metabolic process

Through PPI network analysis and hub genes screening, a PPI network including top 100 of the hub genes identified was constructed (Fig. 5). Itgax, Tlr4, Itgb2, Csf1r and Tyrobp were top hub gene in this network. Given the altered lipid metabolism in the results of Fig. 3, we constructed a potential miRNA-mRNA-lipid metabolism pathways regulatroy network (Fig. 6). The network demonstrated a significant interconnection between exosomal miRNA and liver lipid metabolism pathways.

Current studies have shown that exosome is widely found in many foodstuffs such as vegetable, fruit [9], milk [19], etc,. However, few studies on the exosomes in meat products have been reported. Exosomes are present in muscle and fat of animals in vivo is an indisputable fact [20]. The process of exosomegenesis involves the invaginations of the plasma membrane and the formation of intracellular multivesicular bodies [3]. This indicates that exosome-like vesicles is widespread in cells before the exosomes secretion. Thus, exosomes exist in meat products of our daily diet is conceivable. According to this, we attempt the separation and identification of the cooked meat-derived exosomes (CM-exo). CM-Exo was observed with both TEM and AFM imaging (Fig. 2BC). In this study, the average diameter of the CM-exo was 70.29 nm. Yun et al. reported that the average size of exosomes extracted from milk is 75.7 nm, which is similar to our results [21]. However, plant-derived exosome nanoparticles have a diameter of approximately 50 to 500 nm [22]. This indicated that there could be differences between exosomes in animals and plants. Moreover, we detected the relative high positivity rates for markers of exosomes CD63 and CD81. These results confirm the existence of exosomes in cooked meat.

The exosome mainly contain lipids, proteins and nucleic acids, predominantly microRNA (miRNA) [8]. Recently, growing evidence indicates that exosomal miRNA in food can be absorbed in mammalian circulatory system and regulate consumers various biological processes. Therefore, we evaluated the miRNA expression profile in CM-Exo through miRNA sequencing. We observed great differences in exosomal miRNA composition across the three different sites: muscle, fat and liver. In the PME, miR-1 and miR-133a-3p is predominated, while for PLE, miR-122 and miR-451 is predominated. The miRNA was relatively uniformly distributed in the PFE. Subsequently, we perform miRNA functional enrichment analysis for the top ten miRNAs. Interestingly, PME and PLE miRNAs were found to be significantly enriched with insulin resistance. Meanwhile, metabolic pathways were the pathway with the highest number of target genes. Previously, exosomes are reported to participate in multiple biological processes including chronic inflammation, insulin resistance metabolic disorders [23]. Therefore, we further investigated the effect of cooked meat-derived exosomes of food on metabolism of mice.

Following oral administration in the drinking water, some highly-expressed exosomal miRNAs were altered in plasma. The plasma levels of miR-1, miR-133a-3p, miR-206 and miR-99a were increase with varying degrees. Previous studies have proved milk exosomal miRNA can be absorbed into the organism [24]. In our study, everted intestinal sac assay also confirmed this. Mice chronically drinked with PME grew significantly faster compared to control mice. GTT and ITT suggest that abnormal glucose metabolism and insulin resistance in EXD mice. Moreover, the lipid droplets were significant increased in the liver. These results all indicate EXD mice have abnormal metabolism. The liver is a major metabolic organ and plays important roles in lipids and glucose metabolism [25]. The transcriptomic analysis that we performed further established this. DEGs in liver were predominantly enriched in the metabolic pathways. PPI network analysis revealed top 100 DEGs were divided into three cluster. We observed numerous genes linked to lipid metabolism were centrally located, such as Insig1 [26], Tlr4 [27], Scd1 [28], etc. The relative abundance of miR-1 in PME was the highest. Previous studies reported miR-1 was able to induce lipogenesis in hepatocytes [29]. miR-143-3p had the maximum number of target genes in network (Fig. 6). miR-143-3p was involved in protection of against insulin resistance and lipid deposition in liver metabolic syndrome [30]. The miRNA-mRNA-pathway regulatory network indicated PME miRNA may function as a critical regulator involved in the metabolic disorder of mice.

In the present study, we demonstrated the presence of exosome derived from cooked meat products, including porcine muscle, fat and liver. The exosomal miRNA were differences among the three different tissues. The mice exhibited insulin resistance and lipid metabolic disorder in liver after long-term cooked-pork derived exosomes supplementation. This work suggested the importance of exosomes derived from meat products could not be ignored, where miRNA may not be beneficial for our health.

Ethics approval and consent to participate

All experiments in the present study were conducted in accordance with the Regulations on the Administration of Laboratory Animals (Ministry of Science and Technology of China). All procedures in the present study were approved by the Animal Ethical and Welfare Committee of the Sichuan Agricultural University, Sichuan, China (approval number DKY-B20131403).

Consent for publication

All authors consent to publish.

Availability of data and materials

The datasets used and analysed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that there is no competing interest.

Funding

This work was supported by Sichuan Science and Technology Program (2021ZDZX0008, 2021YFYZ0007, 2021YFYZ0030, 2020YFN0147, scsztd-2022-08-09); China Agriculture Research System of MOF and MARA; National Center of Technology Innovation for Pigs.

Authors' contributions

Linyuan Shen: Conceptualization, Writing – original draft, Investigation, Formal analysis. Jianfeng Ma: Conceptualization, Writing – original draft, Investigation, Formal analysis. Yiting Yang and Tianci Liao: Formal analysis, Data curation, Software. Lei Chen, Shunhua Zhang and Lili Niu: Investigation, Validation, Methodology. Jinyong Wang, Ye Zhao, Xiaoxia Hao and Anan Jiang: Data curation, Investigation, Visualization, Resources. Xuewei Li: Supervision, Project administration. Mailin Gan and Li Zhu: Writing – review & editing, Supervision, Methodology, Funding acquisition.

Acknowledgements

Not applicable.

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No competing interests reported.

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Journal Publication

published 09 Mar, 2023

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Editorial decision: Major revision
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You are reading this latest preprint version

Cooked pork-derived exosome nanovesicles mediate metabolic disorder—microRNA could be the culprit

Status:

Journal Publication

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Isolation of exosomes

2.2. Characterization of cooked meat-derived exosomes

2.3. Mice and feeding

2.5. RNA extraction, library construction, sequencing and RT-qPCR

2.6. Everted intestinal sac assay

2.7. miRNA target gene prediction and functional enrichment analysis

2.8. Statistical Analysis

3. Result

3.1. Identification of exosomes isolated from cooked meat

3.2. microRNA profiles of CM-Exo

3.3. Effect of supplementary feeding cooked pork-derived exosomes on growth of mice

3.4. Cooked pork-derived exosome cause transcriptome changes in mice liver

3.5. Potential regulatory networks involved in lipid metabolic process

4. Discussion

Conclusions

Declarations

References

Additional Declarations

Status:

Journal Publication

Version 1