Porcine muscle-derived extracellular vesicles participate in hepatic glucose and lipid metabolism via promoting AMPK signaling pathway
Porcine 肌肉来源的细胞外囊泡 通过促进 AMPK 信号通路参与肝脏糖脂代谢

Lipeng Xing^1,#, Yufei Cui^1,#, Xinyuan Jiang^1,#, Junyi Luo¹,Ting Chen¹, Jiajie Sun¹, Yongliang Zhang¹, Qianyun Xi^1,*
兴立鹏 ^1，#， 崔宇飞 ^1，# 江新元 ^1，#， 罗俊怡 ¹，陈婷 ¹， 孙佳杰 ¹， 张永亮 ¹， 倩云习 ^1，*

¹State Key Laboratory of Livestock and Poultry Breeding, Guangdong Provincial Key Laboratory of Animal Nutrition Control, National Engineering Research Center For Breeding Swine Industry, Guangdong Province Research Center of Woody Forage Engineering and Technology, College of Animal Science, South China Agricultural University, No. 483 Wushan Road, Guangzhou 510642, China.
¹ 广州 510642 五山路 483 号，广东省动物营养控制重点实验室，养猪业国家工程研究中心 ，广东省木本牧草工程技术研究中心，华南农业大学动物科学学院， 畜禽养殖国家重点实验室。

Email addresses: xqy0228@163.com (Qianyun Xi)
邮箱：xqy0228@163.com（倩云习）.

^#These authors contributed equally to this work.
^# 这些作者对这项工作做出了同等的贡献。

Porcine muscle-derived extracellular vesicles participate in hepatic glucose and lipid metabolism via promoting AMPK signaling pathway
Porcine 肌肉来源的细胞外囊泡通过促进 AMPK 信号通路参与肝脏糖脂代谢

Abstract
抽象

The liver, a crucial metabolic regulatory organ, is pivotal in maintaining glucose and lipid homeostasis, energy distribution, and nutrient utilization. However, intensive breeding practices and short-term profit motives have led to various issues impacting pig health. These challenges notably disrupt liver glycolipid metabolism, impair production efficiency and pork quality, and result in substantial economic losses for the swine industry. Consequently, there is an urgent need to identify safe and efficacious strategies to enhance glycolipid metabolism in pig livers. In this investigation, exosomes and primary porcine liver cells were successfully isolated from pig muscle tissue. Upon intravenous administration of pig muscle-derived exosomes to mice, a marked enhancement in glucose utilization and liver gluconeogenesis was observed. Additionally, mice exhibited elevated serum glucose levels, reduced serum triglyceride concentrations, enhanced Treatment of primary pig liver cells with pig muscle exosomes led to a notable elevation in glucose levels in the culture medium, a significant decrease in intracellular triglyceride content, enhanced AMPK phosphorylation, and upregulation of protein expression of HK1, G6PC, ATGL, and CPT1. Subsequent treatment of AML12 cells with pig muscle exosomes yielded results consistent with those observed in primary pig liver cells. In summary, our findings demonstrate the capacity of pig muscle exosomes to ameliorate glycolipid metabolism in liver cells. AMPK phosphorylation, and upregulated protein expression of HK1, G6PC, ATGL, and CPT1.
肝脏是重要的代谢调节器官，对于维持葡萄糖和脂质稳态、能量分配和营养利用至关重要。然而，集约化的养殖实践和短期的利润动机导致了影响猪健康的各种问题。这些挑战显著扰乱了肝脏糖脂代谢，损害了生产效率和猪肉质量，并给养猪业带来了巨大的经济损失。因此，迫切需要确定安全有效的策略来增强猪肝脏中的糖脂代谢。在这项研究中，成功地从猪肌肉组织中分离出外泌体和原代猪肝细胞。在向小鼠静脉注射猪肌肉来源的外泌体后，观察到葡萄糖利用和肝脏糖异生显着增强。此外，小鼠表现出血清葡萄糖水平升高、血清甘油三酯浓度降低、增强猪肌肉外泌体对原代猪肝细胞的处理导致培养基中葡萄糖水平显着升高，细胞内甘油三酯含量显着降低，AMPK 磷酸化增强，以及 HK1、G6PC、ATGL 和 CPT1 的蛋白质表达上调。随后用猪肌肉外泌体处理 AML12 细胞产生的结果与原代猪肝细胞中观察到的结果一致。总之，我们的研究结果证明了猪肌肉外泌体改善肝细胞中糖脂代谢的能力。AMPK 磷酸化，并上调 HK1、G6PC、ATGL 和 CPT1 的蛋白表达。

Keywords: Pig; muscle-derived extracellular vesicles; glucose and lipid metabolism; liver; AMPK
关键词： 猪; 肌肉来源的细胞外囊泡; 糖脂代谢;肝脏;AMPK 的

Introduction
我介绍

The swine industry is rapidly evolving towards larger scale and higher intensity, resulting in ongoing enhancements in pig production efficiency. However, this trend is accompanied by challenges such as increased stocking density, utilization of high-energy feed, and emphasis on rapid weight gain in pig genetic selection, which are exacerbating hepatic glucose-lipid metabolic disorders in swine populations. Prolonged consumption of high-energy diets, inadequate feeding practices, and other factors can precipitate metabolic ailments like porcine fatty liver syndrome and insulin resistance in swine herds. These conditions not only affect production efficiency and pork quality but also lead to substantial economic repercussions for the livestock sector.
养猪业正在迅速向更大规模、更高强度发展，导致生猪生产效率不断提高。然而，这一趋势伴随着放养密度增加、高能饲料的利用以及猪基因选择中对体重快速增加的重视等挑战，这些挑战正在加剧猪种群的肝脏糖脂代谢紊乱。长期食用高能量饮食、喂养习惯不足和其他因素会引发猪群猪脂肪肝综合征和胰岛素抵抗等代谢疾病。这些条件不仅影响生产效率和猪肉质量，还对畜牧业造成重大经济影响。

Research has shown that metabolic disorders of glucose and lipids in the liver can lead to not only fat deposition and inflammatory damage in the liver but also to a decline in immune function within the organism[8; 22] Regardless of birth weight, HFD-fed pigs exhibited higher body weight, plasma triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) concentrations[24]. Chinese Guizhou miniature pigs were fed either a normal control diet or a high-fat/high-sucrose diet (HFSD) for 6 months. At the end of 6 months, adipocyte hypertrophy and hepatic steatosis were observed in the HFSD-fed animals; miniature pigs fed the HFSD diet without the addition of dietary cholesterol developed insulin resistance, mild diabetes, and atherosclerotic lesions[21].This significantly increases the risk of disease in pig populations, which can result in mortality or premature culling in severe cases. Additionally, the disruption of glucose and lipid metabolism in fattening pigs leads to a notable decline in meat quality post-slaughter (such as the intramuscular fat content and the fatty acid composition ratio), affecting consumer acceptability and industry competitiveness. Mice were chronically administered exosomes derived from boiled pork through drinking water. The mice exhibited abnormal glucose metabolism and insulin resistance. Furthermore, there was a significant increase in lipid droplets in the livers of the mice[17].Exosomes secreted by porcine soleus (SOL) muscle promote adipogenic activity in intramuscular adipocytes[26]. Porcine muscle exosomes show metabolic regulation of target tissues. Therefore, we are discussing whether exosomes derived from pig muscle can improve glucose and lipid metabolism in the liver at both in vivo and cellular levels.
研究表明，肝脏中葡萄糖和脂质的代谢紊乱不仅会导致肝脏脂肪沉积和炎症损伤，还会导致机体内免疫功能下降 [8;22] 无论出生体重如何，HFD 喂养的猪表现出较高的体重、血浆甘油三酯（TG）、高密度脂蛋白胆固醇（HDL-C）、 和低密度脂蛋白胆固醇（LDL-C）浓度 [24]。 中国贵州小型猪饲喂正常对照日粮或高脂肪/高蔗糖日粮 （HFSD） 6 个月。在 6 个月结束时，在 HFSD 喂养的动物中观察到脂肪细胞肥大和肝脂肪变性;饲喂 HFSD 日粮而不添加膳食胆固醇的小型猪出现胰岛素抵抗、轻度糖尿病和动脉粥样硬化病变 [21]。 这显着增加了猪群患病的风险，严重时可能导致死亡或过早扑杀。此外，育肥猪糖脂代谢紊乱导致屠宰后肉质（如肌内脂肪含量和脂肪酸组成比）明显下降，影响消费者的接受度和行业竞争力。 通过饮用水长期给予小鼠从煮猪肉中提取的外泌体。 小鼠表现出异常的葡萄糖代谢和胰岛素抵抗。 此外，小鼠肝脏中的脂滴显着增加 [17]。 猪比目鱼肌（SOL）肌肉分泌的外泌体促进肌内脂肪细胞的成脂活性 [26]。 猪肌肉外泌体显示靶组织的代谢调节。 因此，我们正在讨论源自猪肌肉的外泌体是否可以在体内和细胞水平上改善肝脏中的葡萄糖和脂质代谢。

Results
结果

Porcine muscle-derived extracellular vesicles did not alter mouse body weight but enhanced glucose metabolism.
猪肌肉来源的细胞外囊泡不会改变小鼠体重，但会增强葡萄糖代谢。

CANX protein was detected in pig muscle tissue but not in pig muscle exosomes, where exosome marker proteins TSG101, CD63, and CD9 were enriched (Fig. 1A). Pig muscle exosomes exhibited a concave red blood cell shape under transmission electron microscopy, with a particle size notably enriched at 129.8 nm (Fig. 1B&C). This successful isolation of pig muscle exosomes establishes their potential for subsequent functional investigations. Upon injecting PKH67-stained pig muscle exosomes into mice via the tail vein, their enrichment in the liver was observed (Fig. 1D). Initially, pig muscle exosomes led to a significant reduction in the weight and feed intake of mice, although these effects were not sustained in the later stages of the experiment (Fig. 1E-G). Notably, insulin sensitivity in mice injected with pig muscle exosomes did not significantly differ from the control group (Fig. 1H), despite increased glucose utilization (Fig. 1I) and liver gluconeogenesis capacity (Fig. 1J). Additionally, these mice exhibited elevated serum glucose levels (Fig. 1K) and reduced serum triglyceride levels (Fig. 1L) compared to the control group.
在猪肌肉组织中检测到 CANX 蛋白，但在猪肌肉外泌体中未检测到，其中外泌体标记蛋白 TSG101、CD63 和 CD9 富集（图 1A）。猪肌肉外泌体在透射电镜下呈现凹形红细胞形状，粒径在 129.8 nm 处明显富集（图 1B&C）。猪肌肉外泌体的成功分离为其后续功能研究奠定了潜力。通过尾静脉将 PKH67 染色的猪肌肉外泌体注射到小鼠体内后，观察到它们在肝脏中的富集（图 1D）。最初，猪肌肉外泌体导致小鼠的体重和采食量显着减少，尽管这些影响在实验的后期阶段没有持续（图 1E-G）。值得注意的是，尽管葡萄糖利用率（图 1I）和肝脏糖异生能力（图 1J）增加，但注射猪肌肉外泌体的小鼠的胰岛素敏感性与对照组（图 1H）没有显着差异。此外，与对照组相比，这些小鼠表现出血清葡萄糖水平升高（图 1K）和血清甘油三酯水平降低（图 1L）。

Porcine muscle-derived extracellular vesicles enhanced glucose and lipid metabolism in liver.
猪肌肉来源的细胞外囊泡增强了肝脏中的葡萄糖和脂质代谢。

Subsequent experimental findings revealed a significant decrease in the liver index of mice injected with pig muscle exosomes, accompanied by a notable increase in the gastrocnemius index, while showing no significant impact on subcutaneous fat and epididymis fat indices (Fig. 2A). Notably, there was a marked reduction in muscle glycogen content in the gastrocnemius muscle (Figure 2B) and a significant increase in hepatic glycogen content (Figure 2C), as confirmed by PAS staining (Figure 2D). Histological examination of liver sections stained with hematoxylin and eosin (HE) did not indicate any liver damage in either group of mice (Fig. 2E). Moreover, the liver triglyceride (TG) level was significantly decreased in mice treated with pig muscle exosomes (Fig. 2F). While the expression levels of HK1, PK, PCK1, G6PC, ATGL, HSL, MAGL, and CPT1 genes in the liver remained unchanged (Fig. 2G-H), the protein levels of HK1, G6PC, ATGL, and CPT1 exhibited a significant increase. Additionally, the analysis of AMP-activated protein kinase (AMPK) signaling revealed a significant elevation in phosphorylated AMPK protein levels (p-AMPK) (Fig. 2I).
随后的实验结果显示，注射猪肌肉外泌体的小鼠肝脏指数显着下降，腓肠肌指数显着增加，同时对皮下脂肪和附睾脂肪指数没有显着影响（图 2A）。值得注意的是，腓肠肌中的肌糖原含量显着降低（图 2B），肝糖原含量显着增加（图 2C），PAS 染色证实（图 2D）。用苏木精和伊红（HE）染色的肝切片的组织学检查均未表明两组小鼠有任何肝损伤（图 2E）。此外，用猪肌肉外泌体处理的小鼠的肝脏甘油三酯（TG）水平显着降低（图 2F）。虽然肝脏中 HK1、PK、PCK1、G6PC、ATGL、HSL、MAGL 和 CPT1 基因的表达水平保持不变（图 2G-H），但 HK1、G6PC、ATGL 和 CPT1 的蛋白水平表现出显着增加。此外，对 AMP 活化蛋白激酶（AMPK）信号传导的分析显示磷酸化 AMPK 蛋白水平（p-AMPK）显着升高（图 2I）。

Porcine muscle-derived extracellular vesicles promote glucose and lipid metabolism in primary porcine hepatocytes.
猪肌肉来源的细胞外囊泡促进猪原代肝细胞的葡萄糖和脂质代谢。

Primary pig liver cells were isolated, revealing a distinctive dense cobblestone-like structure when observed under a microscope, with outward clustering (Fig. 3A). Immunostaining showed evident expression of CK18 keratin (Fig. 3B) and glycogen accumulation (Fig. 3C). Treatment of these cells with pig muscle exosomes resulted in a significant increase in glucose content in the cell culture medium (Figure 3D) and a notable decrease in cellular triglyceride levels (Figure 3E), consistent with oil red O staining results (Figure 3F). Furthermore, pig muscle exosome treatment upregulated the gene expression of HK1, ATGL, and MAGL in primary pig liver cells, while showing no significant impact on the expression of PK, PCK1, G6PC, and HSL genes (Figure 3G-H). At the protein level, there was a significant increase in the expression of HK1, G6PC, ATGL, and CPT1, along with a notable elevation in p-AMPK protein levels (Figure 3I).
分离出原代猪肝细胞，在显微镜下观察时显示出独特的致密鹅卵石状结构，并具有向外聚集（图 3A）。免疫染色显示 CK18 角蛋白（图 3B）和糖原积累（图 3C）的明显表达。用猪肌肉外泌体处理这些细胞导致细胞培养基中葡萄糖含量显着增加（图 3D）和细胞甘油三酯水平显着降低（图 3E），与油红 O 染色结果一致（图 3F）。此外，猪肌肉外泌体处理上调了原代猪肝细胞中 HK1、ATGL 和 MAGL 的基因表达，而对 PK、PCK1、G6PC 和 HSL 基因的表达没有显着影响（图 3G-H）。在蛋白质水平上，HK1、G6PC、ATGL 和 CPT1 的表达显着增加，p-AMPK 蛋白水平显着升高（图 3I）。

Porcine muscle-derived extracellular vesicles promote glucose and lipid metabolism in AML12 cells.
猪肌肉来源的细胞外囊泡促进 AML12 细胞中的葡萄糖和脂质代谢。

The impact of pig muscle exosomes on AML12 cells was confirmed through validation experiments. Our findings demonstrated that treatment of AML12 cells with pig muscle exosomes mirrored the outcomes observed in pig liver primary cells treated with the same exosomes. Specifically, there was a significant increase in glucose content in the cell culture medium of pig muscle exosome-treated AML12 cells (Figure 4A), accompanied by a notable decrease in cellular triglyceride levels (Figure 4B) and a reduction in the presence of lipid droplets as evidenced by oil red O staining (Figure 4C). Furthermore, treatment with pig muscle exosomes led to a significant upregulation in the gene expression of key metabolic regulators including PK, PCK1, G6PC, ATGL, and MAGL in AML12 cells, while showing no significant impact on the expression of HK1 and HSL genes (Figure 4D-E). At the protein level, the expression of HK1, G6PC, ATGL, and CPT1 was significantly elevated, along with a notable increase in p-AMPK protein levels (Figure 4F).
通过验证实验证实了猪肌肉外泌体对 AML12 细胞的影响。我们的研究结果表明，用猪肌肉外泌体处理 AML12 细胞反映了在用相同外泌体处理的猪肝原代细胞中观察到的结果。具体来说，猪肌肉外泌体处理的 AML12 细胞的细胞培养基中的葡萄糖含量显着增加（图 4A），伴随着细胞甘油三酯水平的显着降低（图 4B）和脂滴存在的减少，如油红 O 染色（图 4C）所证明的那样。此外，用猪肌肉外泌体处理导致 AML12 细胞中 PK、PCK1、G6PC、ATGL 和 MAGL 等关键代谢调节因子的基因表达显着上调，而对 HK1 和 HSL 基因的表达没有显着影响（图 4D-E）。在蛋白质水平上，HK1、G6PC、ATGL 和 CPT1 的表达显着升高，p-AMPK 蛋白水平显着增加（图 4F）。

Porcine muscle exosomes inhibit liver cell proliferation
猪肌肉外泌体抑制肝细胞增殖

Our findings demonstrated a significant decrease in the liver index of mice following the administration of pig muscle exosomes via the tail vein compared to the control group. Subsequently, we examined the impact of pig muscle exosomes on liver proliferation. The administration of pig muscle exosomes led to a notable reduction in the expression of Cyclin D1 and Cyclin E1 genes in mice, with no significant alteration observed in PCNA gene expression (Fig. 5A). Moreover, there was a significant decrease in the levels of phosphorylated mTOR, Cyclin D1, and Cyclin E1 proteins (Fig. 5B). At the cellular level, pig muscle exosomes inhibited the proliferation of primary pig liver cells (Fig. 5C) and suppressed the expression of PCNA, Cyclin D1, and Cyclin E1 genes (Fig. 5D), along with downregulating the expression of PCNA, Cyclin D1, and Cyclin E1 proteins through the inhibition of mTOR signaling (Fig. 5E). Consistent with the outcomes observed in primary pig liver cells, treatment with pig muscle exosomes in AML12 cells hindered cell proliferation (Fig. 5F), reduced the expression of PCNA and Cyclin E1 genes (Fig. 5G), and inhibited the expression of PCNA, Cyclin D1, and Cyclin E1 proteins by suppressing mTOR signaling (Fig. 5E).
我们的研究结果表明，与对照组相比，通过尾静脉施用猪肌肉外泌体后，小鼠的肝脏指数显着降低。随后，我们研究了猪肌肉外泌体对肝脏增殖的影响。猪肌肉外泌体的施用导致小鼠细胞周期蛋白 D1 和细胞周期蛋白 E1 基因的表达显着降低，PCNA 基因表达没有观察到显着改变（图 5A）。此外，磷酸化 mTOR、细胞周期蛋白 D1 和细胞周期蛋白 E1 蛋白的水平显着降低（图 5B）。在细胞水平上，猪肌肉外泌体抑制原代猪肝细胞的增殖（图 5C），抑制 PCNA、细胞周期蛋白 D1 和细胞周期蛋白 E1 基因的表达（图 5D），同时通过抑制 mTOR 信号传导下调 PCNA、细胞周期蛋白 D1 和细胞周期蛋白 E1 蛋白的表达（图 5E）。与在原代猪肝细胞中观察到的结果一致，在 AML12 细胞中用猪肌肉外泌体处理可阻碍细胞增殖（图 5F），降低 PCNA 和细胞周期蛋白 E1 基因的表达（图 5G），并通过抑制 mTOR 信号传导抑制 PCNA、细胞周期蛋白 D1 和细胞周期蛋白 E1 蛋白的表达（图 5E）。

Discussion
D 地震

Disorders in hepatic glucose and lipid metabolism are complex and can initiate a cascade of diseases, impacting breeding efficiency. Porcine non-alcoholic fatty liver is caused by high-energy diets and lack of exercise [5]. Neonatal piglet hypoglycemia arises due to minimal glycogen reserves and immature gluconeogenesis enzymes, relying on lactose from breast milk for glucose. Insufficient breast milk or impaired gluconeogenesis rapidly decreases blood glucose, increasing piglet mortality [2; 15]. The dietary supplementation of the diet with Chitosan oligosaccharide during late gestation and lactation reduced piglet hypoglycemia by stimulating hepatic gluconeogenesis and improved the growth rate of suckling piglets [23]. Sow ketosis results from heightened energy demands during late pregnancy and lactation. Insufficient dietary energy or disrupted hepatic fat metabolism leads to ketone body accumulation, causing ketosis [19]. Thus, maintaining stable hepatic glucose and lipid metabolism is vital for efficient pig breeding. This study demonstrates that porcine muscle exosomes enhance hepatic gluconeogenesis and lipid clearance, suggesting a novel strategy for addressing glucose and lipid metabolism disorders in production. Although the in vivo validation in this study was only conducted on mice, the research results hold promise for further functional validation in pigs.
肝脏葡萄糖和脂质代谢紊乱很复杂，可引发一系列疾病，影响育种效率。猪非酒精性脂肪肝是由于高能量饮食和缺乏运动引起的 [5]。 新生仔猪低血糖是由于糖原储备极少和糖异生酶不成熟，依赖母乳中的乳糖获取葡萄糖而引起的。母乳不足或糖异生受损会迅速降低血糖，增加仔猪死亡率 [2; 15]。 在妊娠晚期和泌乳期补充壳聚糖低聚糖通过刺激肝脏糖异生降低仔猪低血糖，提高乳仔猪的生长速度 [23]。 母猪酮症是由于怀孕后期和哺乳期能量需求增加造成的。饮食能量不足或肝脏脂肪代谢紊乱导致酮体堆积，引起酮症 [19]。 因此，维持稳定的肝脏糖脂代谢对于生猪的高效养殖至关重要。 这项研究表明，猪肌肉外泌体可增强肝脏糖异生和脂质清除，为解决生产中的葡萄糖和脂质代谢紊乱提出了一种新策略。 尽管本研究中的体内验证仅在小鼠身上进行，但研究结果有望在猪身上进行进一步的功能验证。

Freshly isolated primary hepatocytes retain most of their liver-specific functions. Therefore, in addition to using conventional liver cell lines, researchers prefer to obtain primary liver cells to verify drug functions. For a long time, researchers have made many attempts at the isolation and culture of hepatocytes, mainly including non-perfusion methods (such as tissue block method or homogenization method) and perfusion methods. Currently, the most widely used and preferred method is Seglen's in situ two-step collagenase method. Meng et.al established a method of porcine hepatocyte isolation with a modified four-step retrograde perfusion technique [13] . Li et.al adopted a three-step perfusion method to isolate porcine hepatocytes [6] . For weaned piglet (12 kg), the entire liver was perfused retrogradely with cold saline and then excised, followed by a five-step ex vivo open-loop recirculation perfusion method to isolate functional porcine hepatocytes [14]. The perfusion method was used to isolate porcine hepatocytes from Bama miniature pigs. The prepared collagenase Serva NB 8 and N-acetylcysteine (NAC) can improve the isolation of porcine hepatocytes, thereby increasing the yield of viable cells [7]. The perfusion method is overly complex in operation and requires a relatively high level of operational skills from experimenters.
新鲜分离的原代肝细胞保留了大部分肝脏特异性功能。因此，除了使用常规肝细胞系外，研究人员更倾向于获取原代肝细胞来验证药物功能。长期以来，研究人员在肝细胞的分离和培养方面进行了多次尝试，主要包括非灌注法（如组织阻滞法或均质法）和灌注法。目前，使用最广泛和首选的方法是 Seglen 原位两步胶原酶法。孟 et.al 建立了一种采用改良的四步逆行灌注技术分离猪肝细胞的方法 [13]。Li et.al 采用三步灌注法分离猪肝细胞 [6]。 对于断奶仔猪（12 kg），用冷盐水逆行灌注整个肝脏，然后切除，然后采用五步离体开环再循环灌注法分离功能性猪肝细胞 [14]。 采用灌注法从巴马小型猪中分离猪肝细胞。制备的胶原酶 Serva NB 8 和 N-乙酰半胱氨酸（NAC）可以改善猪肝细胞的分离，从而提高活细胞的产量 [7]。 灌注方法作过于复杂，对实验者作技能要求相对较高。

Researchers used a single-cell preparation instrument to extract neonatal rat cardiac myocytes from rat hearts for research [12; 25]. Healthy adipose tissue was harvested and disassociated using fat dissociation solution, the Single-cell Suspension Preparation System (SSPS) was used to obtain a mixture of single adipocytes, ADSCs and stromal vascular fraction (SVF) to verify the long-term survival rate of single adipocyte graft in vivo [11]. Therefore, we attempted to isolate primary liver cells from 4-week-old Landrace piglets using a single-cell preparation instrument. The entire process only took half an hour and was easy to operate, and we successfully isolated primary porcine liver cells. Using a single-cell preparation instrument is expected to become an efficient method for obtaining primary cells from various tissues in subsequent scientific research.
研究人员使用单细胞制备仪器从大鼠心脏中提取新生大鼠心肌细胞进行研究 [12;25]。 使用脂肪解离溶液收获并分离健康的脂肪组织，使用单细胞悬浮液制备系统（SSPS）获得单个脂肪细胞、ADSC 和基质血管部分（SVF）的混合物 ， 以验证单个脂肪细胞移植物在体内的长期存活率 [11]。 因此，我们尝试使用单细胞制备仪器从 4 周龄长白仔猪中分离原代肝细胞。整个过程仅用了半小时，作简单，我们成功分离出原代猪肝细胞。在后续的科学研究中，使用单细胞制备仪器有望成为从各种组织中获取原代细胞的有效方法。

AMPK is a key regulator of energy metabolism and a critical target for metabolic disease treatment. It detects cellular metabolic status, activating under low nutrient and energy conditions to inhibit anabolism and promote catabolism. This activation enhances ATP synthesis and suppresses cell growth, ensuring energy homeostasis. Coenzyme Q10 is a well-known anti-adipogenic factor that can act as an AMPK activator, regulate hepatic lipid metabolism, and inhibit abnormal lipid accumulation in the liver [1]. Fufang Zhenzhu Tiaozhi formula (FTZ), treatment attenuated hepatic steatosis and fibrosis via the adenosine monophosphate-activated protein kinase (AMPK) pathway. In vitro studies showed that FTZ also attenuated intracellular lipid accumulation in HepG2 cells exposed to palmitic acid (PA) and oleic acid (OA) [20]. Long-term AMPK activation in the liver can enhance lipid oxidation, thereby reducing hepatic lipid content and body fat [3]. Licochalcone A activates the sirt-1/AMPK pathway, improves hepatic steatosis, reduces liver tissue weight and lipid droplet accumulation in liver tissue, and alleviates obesity in mice [10]. BabaoDan can increase the expression of p-AMPK and the weights of the body and tissues (retroperitoneal fat pads, kidneys, and liver) of mice are significantly lower than those of the control group mice [18]. Taurine exerts a lipid-lowering effect by activating the SIRT1/AMPK/FOXO1 signaling pathway and regulating lipid metabolism in obese C57BL6 mice [4]. In hepatic glucose and lipid metabolism, injection of AICAR (an AMPK activator) in normal or insulin - resistant Zuker rats can inhibit hepatic gluconeogenesis, thereby reducing blood glucose levels. Treatment of primary cultured hepatocytes with metformin can activate AMPK to inhibit glucose production [16]. A 4-week exercise program reversed steatosis, reduced insulin levels, and improved glucose tolerance. Substituting exercise with AICAR was sufficient to replicate the above benefits [9]. In this study, we found that the gene and protein expression of G6PC also increased significantly when AMPK was activated, which seems inconsistent with previous literature reports. However, G6PC is an important rate-limiting enzyme in the final step of endogenous glucose output and plays a crucial role in directly determining gluconeogenesis and maintaining glucose homeostasis in the body. Therefore, we speculate that there are components in porcine muscle exosomes that can directly target G6PC for regulation without being affected by the AMPK signal, which requires further investigation for validation.
AMPK 是能量代谢的关键调节因子，也是代谢性疾病治疗的关键靶点。它检测细胞代谢状态，在低营养和能量条件下激活以抑制合成代谢并促进分解代谢。这种激活增强了 ATP 合成并抑制细胞生长，确保能量稳态。 辅酶 Q10 是一种众所周知的抗脂肪生成因子，可以作为 AMPK 激活剂，调节肝脏脂质代谢，抑制肝脏异常脂质积累 [1]。 扶方真珠条芝配方（FTZ）， 通过单磷酸腺苷活化蛋白激酶（AMPK）途径治疗减轻肝脂肪变性和纤维化。体外研究表明，FTZ 还减弱了暴露于棕榈酸（PA）和油酸（OA）的 HepG2 细胞中的细胞内脂质积累 [20]。 肝脏中 AMPK 的长期激活可以增强脂质氧化，从而降低肝脏脂质含量和体脂 [3]。 柠檬苦虫 A 可激活 sirt-1/AMPK 通路，改善肝脂肪变性，减少肝组织重量和肝组织中脂滴积累，减轻小鼠肥胖[10]。BabaoDan 可以增加 p-AMPK 的表达，小鼠的身体和组织（腹膜后脂肪垫、肾脏和肝脏）的重量明显低于对照组小鼠 [18]。 牛磺酸通过激活 SIRT1/AMPK/FOXO1 信号通路和调节肥胖 C57BL6 小鼠的脂质代谢来发挥降脂作用 [4]。 在肝糖和脂质代谢中，在正常或胰岛素抵抗的祖克大鼠中注射 AICAR（一种 AMPK 激活剂）可以抑制肝脏糖异生，从而降低血糖水平。用二甲双胍处理原代培养的肝细胞可以激活 AMPK 以抑制葡萄糖的产生 [16]。 为期 4 周的锻炼计划逆转了脂肪变性，降低了胰岛素水平，并改善了葡萄糖耐量。用 AICAR 代替运动足以复制上述益处 [9]。 在这项研究中，我们发现当 AMPK 被激活时，G6PC 的基因和蛋白质表达也显着增加，这似乎与以往的文献报道不一致。然而，G6PC 是内源性葡萄糖输出最后一步中重要的限速酶，在直接决定体内糖异生和维持葡萄糖稳态方面起着至关重要的作用。因此，我们推测猪肌肉外泌体中存在一些成分可以直接靶向 G6PC 进行调控，而不受 AMPK 信号的影响，这需要进一步研究以进行验证。

Conclusion
共含

Our study concludes that porcine muscle exosomes do not influence body weight or feed intake in mice but enhance hepatic gluconeogenesis and lipid clearance via the AMPK signaling pathway. These findings were corroborated in primary porcine liver cells and AML12 cells. Additionally, porcine muscle exosomes inhibit liver cell proliferation by suppressing the mTOR signaling pathway. This highlights the functional diversity of porcine muscle exosomes in vivo, offering a theoretical and experimental basis for their potential application in managing liver metabolic disorders in pig production.
我们的研究得出结论，猪肌肉外泌体不会影响小鼠的体重或采食量，但通过 AMPK 信号通路增强肝脏糖异生和脂质清除。这些发现在原代猪肝细胞和 AML12 细胞中得到了证实。此外，猪肌肉外泌体通过抑制 mTOR 信号通路来抑制肝细胞增殖。这凸显了猪肌肉外泌体在体内的功能多样性，为其在管理生猪生产中肝脏代谢紊乱的潜在应用提供了理论和实验基础。

Materials and Methods
M 材料和方法

Animals
动物

A four-week-old Landrace piglet was purchased from a local farm in Guangzhou City, Guangdong Province, China. Six-week-old mice were purchased from Guangdong Provincial Experimental Animal Center. The mice were housed under a 12 h light/12 h dark cycle at a constant temperature (25 ± 1 °C) with free access to food and water. After one week of acclimatization, twenty mice with similar weights were randomly divided into two groups. The experimental group received tail vein injections of 100 μg protein in extracellular vesicles per mouse per three days, while the control group received injections of the same volume of PBS buffer.
一头四周龄长白仔猪购自中国广东省广州市的一个当地农场 。 从广东省实验动物中心购买 6 周龄小鼠 。将小鼠饲养在恒温（25 ± 1 °C）下 12 小时光照/12 小时黑暗循环下，并自由获取食物和水。 适应一周后，将 20 只体重相似的小鼠随机分为两组。 实验组每三天在每只小鼠的细胞外囊泡中接受 100 μg 蛋白质的尾静脉注射 ，而对照组则接受相同体积的 PBS 缓冲液注射。

Glucose, insulin and pyruvate tolerance tests
葡萄糖、胰岛素和丙酮酸耐量试验

Before the GTT and PTT experiments, mice were fasted overnight and intraperitoneally injected with glucose or pyruvate (1g/kg). Before the ITT experiment, mice were fasted for 6 hours, and insulin (0.7U/kg) was intraperitoneally injected. Blood glucose was measured at 0, 15, 30, 60, and 120 minutes after injection, respectively.
在 GTT 和 PTT 实验之前，小鼠禁食过夜，腹腔注射葡萄糖或丙酮酸（1g/kg）。ITT 实验前，小鼠禁食 6 小时，腹腔注射胰岛素（0.7U/kg）。分别在注射后 0、15、30、60、120 分钟测得血糖。

Glucose assay
葡萄糖测定

The glucose content in mouse serum or cell culture medium was detected using a glucose assay kit (#F006-1-1, Njjcbio), and the specific instructions for use were referred to the manufacturer's guidelines.
使用葡萄糖测定试剂盒 （#F006-1-1，Njjcbio） 检测小鼠血清或细胞培养基中的葡萄糖含量 ，具体使用说明参考制造商指南。

Triglycerides assay
甘油三酯测定

The triglycerides content in mouse serum or cell was detected using a triglycerides assay kit (#F001-1-1, Njjcbio), and the specific instructions for use were referred to the manufacturer's guidelines.
使用甘油三酯检测试剂盒 （#F001-1-1，Njjcbio） 检测小鼠血清或细胞中的甘油三酯含量 ，具体使用说明参考制造商指南。

Liver / muscle glycogen assay
肝脏/肌糖原测定

The liver / muscle glycogen content in mouse was detected using a liver / muscle glycogen assay kit (#A043-1-1, Njjcbio), and the specific instructions for use were referred to the manufacturer's guidelines.
使用 live/ muscle 糖原检测试剂盒 （#A043-1-1，Njjcbio） 检测小鼠体内 liver/muscle 糖原含量 ，具体使用说明参考制造商指南。

Extraction of Extracellular Vesicles from Porcine Muscle Tissue
从猪肌肉组织中提取细胞外囊泡

Gastrocnemius muscle tissue was collected for muscle exosome extraction after humane exsanguination euthanasia of piglet. Mince the muscle tissue with surgical scissors and transfer to a digestion solution containing 2 mg/mL type IV collagenase. The ratio of tissue to digestive fluid volume is 1:2. Incubate in a 37 °C water bath for 1 hour, repeat 3 times, and combine the resulting solution. Centrifuge at 12000 g, 4 °C for 30 minutes to remove precipitate, filter through 0.22 μm filter, ultracentrifuge at 120000g, 4 °C for 90 minutes, resuspend the pellet with an appropriate amount of PBS buffer and store at -80°C.
采集腓肠肌组织，用于仔猪人道放血安乐死后肌肉外泌体提取。 用手术剪刀将肌肉组织切碎，然后转移到含有 2mg / mL IV 型胶原酶的消化溶液中。 组织与消化液体积的比例为 1：2。在 37°C 水浴中孵育 1 小时，重复 3 次，并混合所得的溶蚀液 。12000g，4°C 离心 30m，除去沉淀，经 0.22 μm 过滤器过滤，120000g 超速离心，4°C90m，用适量 PBS 缓冲液重悬沉淀 ，-80°C 贮存。

Transmission electron microscopy analysis of EVs
电动汽车的透射电镜分析

The intestinal EVs were placed on ice and diluted to a ratio of 1:10 for subsequent analysis. After thorough mixing, 10 μL of the intestinal EVs were placed on a copper grid coated with poly (methyl methacrylate), and the excess EVs solution at the edges of the copper grid was removed. The grid was incubated at room temperature for 5 min, after which uranyl acetate was added dropwise to the grid for negative staining for 1 min. The prepared copper grid was then placed under a transmission electron microscope (Talos L120C, FEI Company) to observe the morphology of the isolated intestinal EVs
将肠道 EV 置于冰上并稀释至 1：10 的比例以进行后续分析 。彻底混合后，将 10μL 肠道 EVs 放置在涂有聚甲基丙烯酸甲酯的 铜网格上 ，并去除铜网格边缘多余的 EVs 溶液。将网格在室温下孵育 5 分钟，然后滴加醋酸铀酰到网格中进行阴性染色 1 分钟。然后将准备好的铜栅架置于透射电子显微镜（Talos L120C，FEI 公司）下，观察分离的肠道 EV 的形态.

Nanoparticle tracking analysis of EVs
电动汽车的纳米颗粒跟踪分析

The intestinal EVs were diluted to a ratio of 1:50 for nanoparticle tracking analysis. A NanoSight LM10 particle size analyzer was used to determine the concentration and analyze the particle size of the intestinal EVs. A constant rate injection pump was used to inject 1 mL of intestinal EVs into the instrument. Tracking was performed at room temperature for 1 min, with manual adjustments made to the parameters and values read to maintain consistency within the sample. Each sample was measured three times, and the NanoSight analysis tool was used to analyze the captured particle number and concentration.
将肠道 EV 稀释至 1：50 的比例进行 n 无颗粒跟踪分析使用 NanoSight LM10 粒度分析仪确定肠道 EV 的 浓度并分析粒径 。 使用恒速注射泵将 1 mL 肠道 EV 注入仪器中。 在室温下进行 T 架 1 分钟，手动调整参数和读取的值以保持样品内的一致性。对每个样品进行多次测量 ，并使用 NanoSight 分析工具 分析捕获的颗粒数和浓度。

Exosome tracing
外泌体追踪

Porcine muscle exosomes were fluorescently labeled with PKH67 (#UR52303, Umibio), and the specific instructions for use were referred to the manufacturer's guidelines. Mice were injected with 100 μg of PKH67-labeled porcine muscle exosomes via the tail vein. Six hours later, muscle and liver tissue samples were collected and observed and photographed using an IVIS Lumina LT Series III^®
用 PKH67（#UR52303，Umibio）荧光标记猪肌肉外泌体，具体使用说明参考制造商指南。 通过尾静脉注射小鼠 100μgPKH67 标记的猪肌肉外泌体。六小时后，使用 IVIS Lumina LT 系列 III^® 收集肌肉和肝组织样本并观察和拍照.

Primary Hepatocyte Isolation and Culture
原代肝细胞分离和培养

Primary porcine hepatocytes were isolated using a single-cell suspension preparation device (Docsence, #SC-L1C) and dissociation reagent (Docsence, #TM003). Follow the manufacturer's instructions for specific operations. The isolated primary porcine hepatocytes were seeded into 25 cm² flasks for culture. The culture medium formulation is 100 mL DMEM/F12 basal medium supplemented with 10 mL fetal bovine serum, 1 mL ITS liquid media supplement, 4 μg dexamethasone, and 1 mL penicillin/streptomycin. Overgrown primary hepatocytes were trypsinized, resuspended, and counted. 150,000 cells were seeded per well in 24-well plates. After the cells reached 70-80% confluency, they were treated with 10 µg/mL of porcine muscle-derived extracellular vesicles.
使用单细胞悬浮液制备装置（Docsence，#SC-L1C）和解离试剂（Docsence，#TM003）分离原代猪肝细胞。 具体作请遵循制造商的说明。 将分离的原代猪肝细胞接种到 25 cm² 的烧瓶中进行培养。 培养基配方为 100mL DMEM / F12 基础培养基，补充有 10mL 胎牛血清，1mLITS 液体培养基补充剂，4μg 地塞米松和 1mL 青霉素/链霉素。将过度生长的原代肝细胞胰蛋白酶消化、重悬并计数。每孔接种 150,000 个细胞，在 24 孔板中。细胞达到 70-80%汇合度后，用 10 μg/mL 猪肌肉来源的细胞外囊泡处理。

AML12 Culture
AML12 文化

The culture of AML12 cells is the same as that of primary liver cells.
AML12 细胞的培养与原代肝细胞的培养相同。

CCK8 assay
CCK8 测定

Primary porcine liver cells or AML12 cells were seeded into 96-well plates at a density of 5000 cells per well. When the cells reached 60 - 70% confluence, they were treated with 10 µg/mL muscle exosomes for 24 h. After 24 h, the medium was replaced with complete medium containing 10% CCK8. The cells were then incubated in the incubator for another 2 h, and the absorbance at 450 nm was measured for data analysis.
将原代猪肝细胞或 AML12 细胞以每孔 5000 个细胞的密度接种到 96 孔板中。当细胞达到 60-70%汇合时，用 10μg/mL 肌肉外泌体处理 24 小时。24 h 后，用含有 10%CCK8 的完全培养基替换培养基。然后将细胞在培养箱中再孵育 2 小时，并测量 450nm 处的吸光度以进行数据分析。

PAS staining
PAS 染色

The cells were stained using a PAS staining kit (#C0142S, Beyotime), and the specific instructions were referred to the manufacturer's guidelines. Frozen sections of mouse liver were prepared following the conventional procedures including tissue collection, rapid freezing, sectioning, and slide baking. Oxidation with periodic acid solution, staining with Schiff reagent and hematoxylin, dehydration, clearing, and mounting were performed for observation.
使用 PAS 染色试剂盒（#C0142S，Beyotime）对细胞进行染色 ，具体说明参考制造商的指南。按照常规程序制备小鼠肝脏的冷冻切片，包括组织采集、快速冷冻、切片和载玻片烘烤。用过碘酸溶液氧化，用 Schiff 试剂和苏木精染色，脱水，澄清，封片观察。

CK18 staining
CK18 染色

Porcine primary liver cells were fixed in 4% paraformaldehyde for 30 min, permeabilized with 0.2% (v/v) Triton X - 100 for 15 min, and then blocked with 5% goat serum for 1 h. Subsequently, the cells were incubated overnight with the primary antibody CK18 (#10830-1-AP; Proteintech) at 4°C. After washing three times in PBS, the cells were stained with YF488 goat anti - rabbit immunoglobulin G (IgG) (#Y6105L; UElandy) for 1.5 h at room temperature. Nuclear staining was performed using DAPI (#C0065; Solarbio) for 10 min. Immunofluorescence (IF) staining was observed and imaged using a fluorescence microscope (Nikon Ti2-U).
将猪原代肝细胞固定在 4%多聚甲醛中 30 min，用 0.2%（v/v）Triton X-100 透化 15 min，然后用 5%山羊血清封闭 1 h。随后，将细胞与一抗 CK18（#10830-1-AP;Proteintech）在 4°C 下。 在 PBS 中洗涤 3 次后，用 YF488 山羊抗兔免疫球蛋白 G（IgG）染色细胞（#Y6105L;UElandy）在室温下 1.5 小时。使用 DAPI（#C0065 进行核染色;Solarbio）10 分钟。使用荧光显微镜（Nikon Ti2-U）观察免疫荧光（IF）染色并成像。

Hematoxylin-eosin (HE) staining
苏木精-伊红 （HE） 染色

Mouse liver specimens were fixed with 4% paraformaldehyde, embedded in paraffin, and then cut into 5-μm-thick sections for hematoxylin and eosin (HE) staining. After dewaxing and rehydration, the sections were stained with hematoxylin solution and eosin solution in sequence. Subsequently, the embedded slides were photographed using a microscope (ML31, Mshot).
用 4%多聚甲醛固定小鼠肝脏标本，包埋在石蜡中，然后切成 5μm 厚的切片进行苏木精和伊红（HE）染色。脱蜡复水后，依次用苏木精溶液和伊红溶液染色切片。随后，使用显微镜（ML31，Mshot）拍摄嵌入的载玻片。

Oil Red O staining
油红 O 染色

Primary porcine liver cells or AML12 cells treated with 10 µg/mL porcine muscle exosomes were washed three times with PBS buffer, fixed in 4% formaldehyde for 30 min at room temperature, washed three more times with PBS for 5 min each time, and then stained with Oil Red O for 1 h. The original Oil Red O solution was first diluted with water (3:2) and filtered through filter paper to prepare the working solution. After staining, the cells were washed three times with PBS for 5 min each time. Finally, the cell plates were photographed under a microscope.
用 10 μg/mL 猪肌肉外泌体处理的原代猪肝细胞或 AML12 细胞用 PBS 缓冲液洗涤 3 次，在室温下用 4%甲醛固定 30 min，再用 PBS 洗涤 3 次，每次 5 min，然后用油红 O 染色 1 h。先将原油红 O 溶液用水（3：2）稀释，经滤纸过滤，制备工作溶液。染色后，用 PBS 洗涤细胞 3 次，每次 5 分钟。最后，在显微镜下拍摄细胞板。

Quantitative real-time PCR
定量实时荧光定量 PCR

Total RNA was extracted with TRIzol (Thermo Fisher). One microgram of RNA was reverse-transcribed into cDNA using the Color Reverse Transcription Kit (EZBioscience, Cat No. A001CGQ). Quantitative real-time PCR (qPCR) was conducted on a QuantStudio Real-Time PCR System (Bio-Rad C100 Touch) employing 2× Real Star Fast SYBR qPCR Mix (GenStar, A301). GAPDH served as the internal reference for calibration.
用 TRIzol（Thermo Fisher）提取总 RNA。使用彩色逆转录试剂盒（EZBioscience，货号 A001CGQ）将 1 微克 RNA 逆转录为 cDNA。定量实时 PCR （qPCR） 是在采用 2× Real Star Fast SYBR qPCR 混合物（GenStar，A301）的 QuantStudio 实时荧光定量 PCR 系统 （Bio-Rad C100 Touch） 上进行的。GAPDH 作为校准的内部参考。

Protein extraction and Western blot analysis
蛋白质提取和蛋白质印迹分析

Radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors (BestBio Cat No. BB-3101) was used to extract proteins. The protein concentration was assessed using the Rapid Gold BCA Protein Assay Kit (Thermo Fisher). Western blotting analysis was performed by loading 15 µg of lysate onto sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, transferred the gels to polyvinylidenedifluoride (PVDF) membranes (Millipore), and incubated with rabbit anti-CANX (1:1000, #340144; ZEN-BIO), rabbit anti-TSG101 (1:1000, #381538; ZEN-BIO), rabbit anti-CD63 (1:1000, #340219; ZEN-BIO), rabbit anti-CD9 (1:1000, #R380441; ZEN-BIO), rabbit anti-p-AMPK (1:1000, #340763; ZEN-BIO), rabbit anti-AMPK (1:1000, #R380431; ZEN-BIO), rabbit anti-HK1 (1:1000, #121854; ZEN-BIO), rabbit anti-G6PC (1:1000, #YP-Ab-17245; UpingBio), rabbit anti-CPT1 (1:1000, #R381255; ZEN-BIO) , rabbit anti-ATGL (1:1000, #R23392; ZEN-BIO), rabbit anti-PCNA (1:1000, #R25294; ZEN-BIO), rabbit anti-Cyclin D1 (1:1000, #R380999; ZEN-BIO), rabbit anti-Cyclin E1 (1:1000, #340298; ZEN-BIO) and rabbit anti-Tubulin (1:5000, #bs-20694R; Bioss), goat anti-rabbit secondary antibody (1:50000, #BS13278, Bioworld) conjugated with HRP was used. Tubulin levels served as the loading control.
含有蛋白酶和磷酸酶抑制剂的放射免疫沉淀测定 （RIPA） 缓冲液（BestBio Cat No. BB-3101）用于提取蛋白质。使用 Rapid Gold BCA 蛋白检测试剂盒 （Thermo Fisher） 评估蛋白质浓度。 蛋白质印迹分析方法是将 15μg 裂解物上样到十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）凝胶上，将凝胶转移到聚偏二氟乙烯（PVDF）膜（Millipore）上，并与兔抗 CANx（1：1000，#340144;ZEN-BIO）， 兔抗 TSG101（1：1000，#381538;ZEN-BIO）， 兔抗 CD63（1：1000，#340219;ZEN-BIO）、 兔抗 CD9（1：1000，#R380441;ZEN-BIO）、 兔抗 p-AMPK（1：1000，#340763;ZEN-BIO）、兔抗 AMPK（1：1000，#R380431;ZEN-BIO）、兔抗 HK1（1：1000，#121854;ZEN-BIO）、兔抗 G6PC（1：1000，#YP-Ab-17245; UpingBio）、兔抗 CPT1（1：1000，#R381255;ZEN-BIO）， 兔抗 ATGL（1：1000，#R23392;ZEN-BIO）， 兔抗 PCNA（1：1000，#R25294;ZEN-BIO）， 兔抗细胞周期蛋白 D1（1：1000，#R380999;ZEN-BIO）、 兔抗细胞周期蛋白 E1（1：1000，#340298;ZEN-BIO） 和兔抗微管蛋白（1：5000，#bs-20694R;Bioss），使用与 HRP 偶联的山羊抗兔二抗（1：50000，#BS13278，Bioworld）。 微管蛋白水平作为负载控制。

Statistical analysis
统计分析

SPSS 25 and Graphpad prism 9.0 were used for stand-alone sample t-test analysis and plotting. The results were presented as mean ± standard error of the mean (SEM). The significance of difference was judged by a level of *p <0.05 or **p <0.01.
SPSS 25 和 Graphpad prism 9.0 用于独立样本 t 检验分析和绘图。结果以均值±平均值标准误差 （SEM） 表示。差异的显着性通过*p <0.05 或**p <0.01 的水平来判断。

Author Contributions
作者贡献

LX, YC and XJ contributed equally to this work. QX conceived the project. LX and YC conducted the cell and animal trial and collected samples, and analysis of data throughout the experiment; LX and XJ wrote the manuscript. LX, JL, TC, JS, YZ and QX revised and edited the manuscript, all authors put forward relevant insights and contributed to the discussion.
LX、YC 和 XJ 对这项工作做出了同等贡献。QX 构思了这个项目。LX 和 YC 进行了细胞和动物试验，并收集了样本，并在整个实验过程中分析了数据;LX 和 XJ 撰写了手稿。LX、JL、TC、JS、YZ 和 QX 对稿件进行了修改和编辑，所有作者都提出了相关见解，并为讨论做出了贡献。

Ethics approval and consent to participate
道德批准和参与同意

This study was approved by Institutional Animal Care and Use Committee of South China Agricultural University. All procedures involving animals were performed in accordance with the animal research committee of South China Agricultural University and the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.
本研究获得华南农业大学机构动物护理与使用委员会的批准。所有涉及动物的程序均按照华南农业大学动物研究委员会和美国国立卫生研究院 （NIH） 的《实验动物护理和使用指南》进行。

Funding
资金

This work was supported by the Project of Guangdong Provincial Nature Science Foundation (2023A151502511 and 2025A1515010952), Biological Breeding-National Science and Technology Major Project (2023ZD04068), and the National Natural Science Foundation of China (32072814).
本工作得到了广东省自然科学基金项目（2023A151502511 和 2025A1515010952）、生物育种-国家科技重大专项（2023ZD04068）和国家自然科学基金（32072814）的支持。

Declaration of competing interest
竞争利益声明

The authors declare that they have no conflicts of interest with the contents of this article.
作者声明，他们与本文的内容没有利益冲突。

Availability of data and material
数据和材料的可用性

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
当前研究期间使用和/或分析的数据集可根据合理要求从通讯作者处获得。

Fig.1. Porcine muscle exosomes did not alter mouse body weight but enhanced glucose utilization and glyconeogenesis in mice. (A) Western blot analysis of exosomal protein labeling, (B) Electron microscopy imaging of exosomes (scale bar = 200 nm), (C) Analysis of exosome size distribution, (D) Fluorescence imaging of PKH67-labeled porcine muscle exosomes in gastrocnemius muscle and liver 12 hours post tail vein injection, (E) Comprehensive visualization of whole-body distribution of porcine muscle exosomes in mice one month post-injection, (F) Monitoring of body weight changes in mice over the course of one month following porcine muscle exosome administration, (G) Assessment of food intake alterations in mice during the one-month period of porcine muscle exosome treatment, (H) Results of insulin tolerance tests, (I) Findings from glucose tolerance tests, (J) Outcomes of pyruvate tolerance tests, (K) Measurement of glucose levels in serum, and (L) Evaluation of triglyceride levels in serum.
图 1.猪肌肉外泌体没有改变小鼠体重，但增强了小鼠的葡萄糖利用和糖源生成。（A）外泌体蛋白标记的蛋白质印迹分析，（B）外泌体的电子显微镜成像（比例尺= 200 nm），（C）外泌体尺寸分布分析，（D）腓肠肌和肝脏中 PKH67 标记的猪肌肉外泌体的荧光成像尾静脉注射后 12 小时，（E）注射后一个月小鼠猪肌肉外泌体全身分布的全面可视化， （F）监测猪肌肉外泌体给药后一个月内小鼠的体重变化，（G）评估猪肌肉外泌体治疗一个月期间小鼠的食物摄入量变化，（H）胰岛素耐量试验的结果，（I）葡萄糖耐量试验的结果，（J）丙酮酸耐量试验的结果，（K）血清中葡萄糖水平的测量， （L） 评估血清中的甘油三酯水平。

Fig.2. Porcine muscle-derived extracellular vesicles enhanced glucose and lipid metabolism in liver. (A) Proportion of GAS, Liver, iWAT and eWAT. (B) Serum glucose level. (C) Serum TG level. (D) Liver HE-stained section (scale bar = 100 µm). (D) Relative mRNA expression of HK1, PK, G6PC and PCK1. (E) Relative mRNA expression of ATGL, HSL, and MAGL. (F) Western blot detection of p-AMPK, AMPK, HK1, G6PC, ATGL and CPT1.
无花果。2 猪肌肉来源的细胞外囊泡增强了肝脏中的糖脂代谢。（a） GAS、肝脏、iWAT 和 eWAT 的比例。（B）血清血糖水平。（C）血清 TG 水平。（D）肝脏 HE 染色切片（比例尺= 100μm）。（D）HK1、PK、G6PC 和 PCK1 的 mRNA 相对表达。（E）ATGL、HSL 和 MAGL 的相对 mRNA 表达。（F）蛋白质印迹检测 p-AMPK、AMPK、HK1、G6PC、ATGL 和 CPT1。

Fig.3. Porcine muscle-derived extracellular vesicles promote glucose and lipid metabolism in primary porcine hepatocytes. (A) Morphology of primary pig hepatocytes (scale bar = 200 µm). (B) Immunofluorescence image (scale bar = 20 µm). (C) Glycogen staining image (scale bar = 100 µm). (D) Glucose content in primary porcine hepatocytes culture medium. (E) TG content in primary porcine hepatocytes. (F) Oil Red O Staining Images (scale bar = 100 µm). (G) Relative mRNA expression of HK1, PK, G6PC and PCK1. (H) Relative mRNA expression of ATGL, HSL, and MAGL. (I) Western blot detection of p-AMPK, AMPK, HK1, G6PC, ATGL and CPT1.
图 3 猪肌肉来源的细胞外囊泡促进猪原代肝细胞的糖脂代谢。（一） 原代猪肝细胞的形态（比例尺= 200μm）。（B）免疫荧光图像（比例尺= 20μm）。（C）糖原染色图像（比例尺= 100μm）。（D）原代猪肝细胞培养基中的葡萄糖含量。（E）原代猪肝细胞中的 TG 含量。（F）油红 O 染色图像（比例尺= 100μm）。（G）HK1、PK、G6PC 和 PCK1 的 mRNA 相对表达。（H）ATGL、HSL 和 MAGL 的相对 mRNA 表达。（I）蛋白质印迹检测 p-AMPK、AMPK、HK1、G6PC、ATGL 和 CPT1。

Fig.4. Porcine muscle-derived extracellular vesicles promote glucose and lipid metabolism in AML12 cells. (A) Glucose content in AML12 cells culture medium. (B) TG content in AML12 cells. (C) Oil Red O Staining Images (scale bar = 100 µm). (D) Relative mRNA expression of HK1, PK, G6PC and PCK1. (E) Relative mRNA expression of ATGL, HSL, and MAGL. (F) Western blot detection of p-AMPK, AMPK, HK1, G6PC, ATGL and CPT1.
无花果。4 猪肌肉来源的细胞外囊泡促进 AML12 细胞中的糖脂代谢。（一）AML12 细胞培养基中的葡萄糖含量。（B）AML12 细胞中的 TG 含量。（C）油红 O 染色图像（比例尺= 100μm）。（D）HK1、PK、G6PC 和 PCK1 的 mRNA 相对表达。（E）ATGL、HSL 和 MAGL 的相对 mRNA 表达。（F）蛋白质印迹检测 p-AMPK、AMPK、HK1、G6PC、ATGL 和 CPT1。

Fig.5. Inhibition of cell proliferation by porcine muscle exosomes. (A) The impact of porcine muscle exosomes on the expression of PCNA, Cyclin D1, and Cyclin E1 genes and (B) the protein expression levels of p-mTOR, mTOR, Cyclin D1, and Cyclin E1in the liver of mice post-injection porcine muscle exosomes. (C) the effects of porcine muscle exosomes on porcine liver cells using CCK8 assays. (D) The gene expression profiles of PCNA, Cyclin D1, and Cyclin E1, and (E) the protein expression levels of p-mTOR, mTOR, PCNA, Cyclin D1, and Cyclin E1 in the liver post-injection of porcine muscle exosomes. (F) The impact of porcine muscle exosomes on AML12 cells was evaluated through CCK8 assays, (G) PCNA, Cyclin D1, and Cyclin E1 gene expression, and (F) p-mTOR, mTOR, PCNA, Cyclin D1, and Cyclin E1 protein expression levels.
无花果。5 猪肌肉外泌体抑制细胞增殖。（A）猪肌肉外泌体对小鼠肝脏中 PCNA、细胞周期蛋白 D1 和细胞周期蛋白 E1 基因表达的影响，以及（B）注射猪肌肉外泌体后小鼠肝脏中 p-mTOR、mTOR、细胞周期蛋白 D1 和细胞周期蛋白 E1 的蛋白表达水平。（C）使用 CCK8 测定法对猪肌肉外泌体对猪肝细胞的影响。（D）猪肌肉外泌体注射后肝脏中 PCNA、细胞周期蛋白 D1 和细胞周期蛋白 E1 的基因表达谱，以及（E）p-mTOR、mTOR、PCNA、细胞周期蛋白 D1 和细胞周期蛋白 E1 的蛋白表达水平。（F）通过 CCK8 测定、（G）PCNA、细胞周期蛋白 D1 和细胞周期蛋白 E1 基因表达，以及（F）p-mTOR、mTOR、PCNA、细胞周期蛋白 D1 和细胞周期蛋白 E1 蛋白表达水平评估猪肌肉外泌体对 AML12 细胞的影响。

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Table 1. Primer sequence
表 1. P 赖默序列

Table 2 Primer sequence
表 2P 利入体序列