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Flavonoids from mulberry leaves inhibit fat production and improve fatty acid distribution in adipose tissue in finishing pigs
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Yingying Liua, b, c, Yinglin Pengb, c, Chen Chenb, c, Huibo Renb, c, Ji Zhub, c, Yuan Dengb, c, Qingming Cuib, c, Xionggui Hub, c, Jianhua Hea, Huali Lib, c, Xinghui Zhud, Yulong Yina, e, *, Jun Hea, Yi Xiaod, *
Animal Nutrition | 2024, 16(1) : 147 - 157
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Animal Nutrition | 2024, 16(1): 147-157
• Original Research Article •
Flavonoids from mulberry leaves inhibit fat production and improve fatty acid distribution in adipose tissue in finishing pigs
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Yingying Liua, b, c, Yinglin Pengb, c, Chen Chenb, c, Huibo Renb, c, Ji Zhub, c, Yuan Dengb, c, Qingming Cuib, c, Xionggui Hub, c, Jianhua Hea, Huali Lib, c, Xinghui Zhud, Yulong Yina, e, *, Jun Hea, Yi Xiaod, *
Affiliations
  • aCollege of Animal Science and Technology, Hunan Agricultural University, Changsha, 410128, China
  • bHunan Institute of Animal and Veterinary Science, Changsha, 410131, China
  • cKey Laboratory of Conservation and Genetic Analysis of Local Pig Breed Germplasm Resources, Changsha, 410131, China
  • dCollege of Information and Intelligence, Hunan Agricultural University, Changsha, 410128, China
  • eInstitute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, 410125, China
Published: 2024-03-10 doi: 10.1016/j.aninu.2023.11.003
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This study evaluated the effects of flavonoids from mulberry leaves (FML) on plasma biochemical indices, serum activities of lipid metabolism-related enzymes, fat morphology, fatty acid composition, and lipid metabolism in different adipose tissues of finishing pigs. We used 120 Chinese hybrid barrows of Berkshire and Bama mini-pigs with an average initial body weight of 45.11 ± 4.23 kg. The pigs were randomly assigned to five treatment groups and fed a control diet based on corn, soybean meal, and wheat bran or a control diet supplemented with 0.02%, 0.04%, 0.08%, or 0.16% FML. Each experimental group had six replicates (pens), with four pigs per pen. After a 7-d adaptation period, the feeding trial was conducted for 58 d. Blood and adipose tissue samples were collected from 30 pigs (one pig per pen) at the end of the test. The results showed that FML supplementation significantly decreased the feed intake to body gain ratio, the plasma concentrations of total cholesterol and free fatty acids, and the serum activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (linear or quadratic effects, P < 0.05), and decreased the plasma triglyceride concentration (quadratic, P = 0.07). Increasing FML supplementation increased the average daily gain and serum activities of lipoprotein lipase (linear and quadratic effects, P < 0.05) and adipose triglyceride lipase (linear, P < 0.05). Dietary FML supplementation decreased the adipocyte area in the dorsal subcutaneous adipose (DSA) tissue of finishing pigs (linear, P = 0.05) and increased the adipocyte area in the visceral adipose tissue (quadratic, P < 0.01). Increasing FML supplementation decreased the C20:1 content in DSA, abdominal subcutaneous adipose, and visceral adipose tissues of finishing pigs (P < 0.05) and increased the C18:3n3 and n-3 PUFA contents (P < 0.05). The lipid metabolism genes were regulated by the PPARγ-LXRα-ABCA1 signaling pathway, and their expressions differed in different adipose tissues. These findings suggest that FML improved growth performance, regulated lipid metabolism, inhibited fat production, and improved fatty acid distribution in the adipose tissue of finishing pigs, thereby improving pig fat's nutritional quality and health value.

Finishing pig  /  Mulberry leaf  /  Flavonoid  /  Lipid metabolism  /  Fatty acid  /  Lard
Yingying Liu, Yinglin Peng, Chen Chen, Huibo Ren, Ji Zhu, Yuan Deng, Qingming Cui, Xionggui Hu, Jianhua He, Huali Li, Xinghui Zhu, Yulong Yin, Jun He, Yi Xiao. Flavonoids from mulberry leaves inhibit fat production and improve fatty acid distribution in adipose tissue in finishing pigs[J]. Animal Nutrition, 2024 , 16 (1) : 147 -157 . DOI: 10.1016/j.aninu.2023.11.003
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1.　Introduction
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Pork is one of the most consumed meats worldwide; however, its production and quality face severe challenges, including feeding sustainability and a perceived negative image of fat (McGlone, 2013). Adipose tissue, the largest energy storage and supply site in the animal body, can be divided into subcutaneous, abdominal, visceral, intermuscular, and intramuscular fat. The order of fat deposition in different parts of the animal body varies (Kolstad, 2001). Subcutaneous fat deposition is the earliest, followed by visceral fat (abdominal and perirenal fat) and fat deposition in the skeletal muscle tissue. Chinese people have eaten lard for thousands of years, and it still remains an important ingredient on the Chinese dining table. Approximately 40% of the fatty acids in lard are saturated fatty acids (SFA), and approximately 60% are unsaturated fatty acids (UFA) (Al-Rashood et al., 1996). Overeating SFA leads to lipid accumulation in the blood and increases the risk of cardiovascular diseases; however, UFA are healthy fats. Proper intake of UFA benefits cardiovascular, cerebrovascular, skin, and hormone secretion. From a health perspective, fat can be good or bad. The so-called “devil fat” is trans-fatty acids, which is more harmful than cholesterol and is the culprit in cardiovascular and cerebrovascular diseases (Denke, 2006). Encouragingly, very few trans-fatty acids are found in lard. Additionally, a well-balanced fatty acid intake can reduce the risk of cardiovascular and related diseases (Shahidi and Ambigaipalan, 2018).
Obesity and its related metabolic diseases caused by excessive accumulation of adipose tissue seriously threaten human health worldwide. Improving glycolipid metabolism using diet therapy/herbal remedies is an effective approach for ameliorating obesity (Kaila and Raman, 2008; Cao et al., 2022). Mulberry leaves are one of the earliest plant resources identified by the Ministry of Health of China as “food and medicine homology.” They are rich in amino acids, carbohydrates, vitamins, calcium, iron, manganese, zinc, and other minerals. Moreover, they are rich in metabolites, including flavonoids, polyphenols, alkaloids, polysaccharides, steroids, and other natural active ingredients beneficial for animal health (Zhu et al., 2019; Li et al., 2020). Mulberry leaves have many physiological functions, such as blood sugar and lipid-reducing properties (Han et al., 2018), and antioxidant (Zeng et al., 2019), antiviral, and anticancer properties (Fallah et al., 2016; Thaipitakwong et al., 2018; Zhang et al., 2018; Liu et al., 2019). Furthermore, Morus spp. extracts have no toxic effects and have nutraceutical potential (Rodrigues et al., 2019). Over the years, mulberry leaves have gained considerable attention as traditional medicines and have been widely used as adjuvants for weight loss (Chan et al., 2016). The reported benefits of mulberry leaves include inhibiting adipogenesis, improving the lipid profile, and reducing fat deposit and body weight (Sheng et al., 2017; Liu et al., 2021). These effects are likely due to abundant functional components such as flavonoids (Sheng et al., 2019). Mulberry leaves contain a range of flavonoids, including flavone glycosides and flavonoid derivatives, collectively referred to as flavonoids from mulberry leaves (FML). These compounds constitute approximately 10–30 g/kg of the dry weight of mulberry leaves (Jia et al., 1999), and rutin and isoquercetin are primary components (Ju et al., 2018). In recent studies, FML administration decreased lipid accumulation, alleviated liver steatosis and hepatic injury in a non-alcoholic fatty liver disease rat model (Hu et al., 2020), reversed the whitening of brown adipose tissue (BAT), and induced a dramatic shift in the gut microbiota of high-fat diet (HFD)-fed mice (Zhong et al., 2020). Similarly, rutin activated BAT in genetically obese and HFD-induced obese mice (Yuan et al., 2017). Additionally, studies have shown that FML can enhance growth performance and exhibit hypolipidemic effects in rodents and other animals (Kong et al., 2019; Hassan et al., 2020), improve antioxidant capacity, and regulate lipid metabolism in the skeletal muscles of pigs (Liu et al., 2022). Based on these findings, we hypothesized that FML could improve lipid metabolism in adipose tissue by regulating the distribution of fatty acids. Although mulberry leaves have been suggested as a potential intervention to improve lipid traits in finishing pigs, direct experimental or clinical evidence to validate this hypothesis is limited. Therefore, we aimed to investigate whether the FML administration could influence the fatty acid profile and lipid metabolism in finishing pigs.
2.　Materials and methods
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2.1.　 Animal ethics statement
The animal experiment conducted in this study was approved by the Institutional Animal Care and Use Committee of Hunan Agricultural University and was conducted in accordance with the Chinese Guideline for Ethical Review of Animal Welfare (ACC2019006, Changsha, China).
2.2.　 Preparation of flavonoids from mulberry leaves
Mulberry leaves were obtained from the Sericultural Research Institute of Hunan Province (Changsha, China), dried, and crushed into powder. Next, flavonoids were extracted from the leaves using 60% ethanol at 80 °C for 3 h and purified using AB-8 macroporous resin (Sun et al., 2014); a yield of 1.90% total flavonoid was obtained.
2.3.　 Study animals and experimental design
We used 120 crossbred F1 barrows of Berkshire and Bama mini-pigs with an average initial body weight of 45.11 ± 4.23 kg. All pigs were randomly allocated to five treatment groups, with six replicates (pens) per group and four pigs per pen. Next, the pigs were fed a control diet based on corn, soybean meal, and wheat bran or a control diet supplemented with 0.02%, 0.04%, 0.08%, or 0.16% FML, meeting the recommendations of the Chinese National Feeding Standard of Swine (NY/T 65-2004). Nutrient levels in the experimental diet were analyzed using the methods of the Association of Official Analytical Chemists (2005), or calculated according to the Chinese National Feeding Standard of Swine (NY/T 65-2004) (Table 1). Briefly, crude protein, crude fat, and ash values were determined, using the method 954.01 of Kjeldahl, method 920.39 of Soxhlet extraction, and method 942.05 of ashing, respectively, following the procedures outlined by the Association of Official Analytical Chemists (2005). Moreover, digestible energy and other nutrients were calculated according to the Chinese National Feeding Standard of Swine (NY/T 65-2004). Pellet feed was provided thrice daily, and the pigs had ad libitum access to drinking water and were fed throughout the 58-d experiment after a 7-d adaptation period. Each pen was equipped with a stainless-steel feeder and a nipple drinker. The feed consumption of each pen was recorded daily, and the pigs were weighed at the end of the experiment to determine the average daily gain (ADG), average daily feed intake (ADFI), and feed intake to body gain ratio (F:G).
2.4.　 Sample collection
At the end of this test, one pig per pen was randomly selected for sample collection and slaughtered after fasting for 12 h. To obtain blood samples, jugular venipuncture was performed, and two samples were collected in 10-mL heparin and heparin-free tubes. After centrifugation at 3,000 × g and 4 °C for 15 min, plasma and serum samples were obtained. Subsequently, the pigs were electrically stunned, exsanguinated, dehaired, eviscerated, and split down at the midline following standard commercial slaughtering procedures. Immediately after, samples of 1.0 cm3 section from the dorsal subcutaneous adipose (DSA), abdominal subcutaneous adipose (ASA), and visceral adipose tissues on the right side of the carcass were cut and flashed frozen using liquid N2 and stored at −80 °C for quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) analysis. One sample of a 1.0-cm3 section from the DSA tissue was collected for Western blot (WB) analysis. Moreover, approximately 100 g of the DSA, ASA, and visceral adipose tissues were collected and stored at −20 °C to determine the fatty acid profile. Finally, samples of 1.0 cm3 section from the DSA, ASA, and visceral adipose tissues were fixed in 4% paraformaldehyde for histological analysis.
2.5.　 Analysis of plasma biochemical indices
Plasma concentrations of glucose, triglyceride, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), free fatty acid (FFA), and insulin were detected using a CX-4 Automatic Biochemical Analyzer (Beckman Coulter Inc., Brea, CA, USA) and commercial kits from Leadman Biochemistry Technology Company (Beijing, China), following the manufacturer's instructions.
2.6.　 Analysis of serum enzyme activities
The activities and concentrations of several enzymes in the serum were analyzed using colorimetric methods with a spectrophotometer (Biomate 5; Thermo Electron Corporation, Rochester, NY, USA). Specifically, the activities of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, and lipoprotein lipase (LPL) and concentrations of lecithin-cholesterol acyltransferase and adipose triglyceride lipase (ATGL) were measured using commercial kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) according to the manufacturer's instructions.
2.7.　 Morphological study of adipose tissues
Adipocyte areas in the DSA, ASA, and visceral adipose tissues were measured using the classic method of Oil Red O (ORO) staining (Yin et al., 2018). Briefly, the adipose tissues were fixed in a solution containing formalin (10%) and calcium (2%), and incubated with ORO working solution at room temperature (18–28 °C) for 5 min. Finally, the sections were cleared in 60% isopropyl alcohol for 5 min and counterstained with Mayer's hematoxylin for 15 s.
2.8.　 Analysis of fatty acid composition
To determine the fatty acid composition of the DSA, ASA, and visceral adipose tissues, lipids were extracted from approximately 0.5 g of adipose tissue using chloroform. Lipid methyl esters were obtained by saponification with 2 mL hexane, 40 μL methyl acetate, and 100 μL sodium methoxide solution (Postaue et al., 2020). Next, the fatty acids were analyzed using an Agilent 7890A Gas Chromatograph (Agilent Technologies Inc., Santa Clara, CA, USA) with a chromatographic column, according to the method previously described by Liu et al. (2015). Afterward, individual fatty acid peaks were identified by comparing their retention times with those of standards. The results were expressed as a percentage of total fatty acids, and SFA, monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA) were calculated, along with the ratios of PUFA to SFA and Σn-6 PUFA to Σn-3 PUFA.
2.9.　 Quantitative real-time PCR analysis
Using qRT-PCR, we measured mRNA expression levels of genes (Table 2) involved in lipid metabolism in DSA, ASA, and visceral adipose tissues, including acetyl-CoA carboxylase α (ACCα), fatty acid synthase (FAS), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), peroxisome proliferator-activated receptor γ (PPARγ), sterol regulatory element-binding proteins 1 (SREBP1), liver X receptor α (LXRα), and ATP-binding cassette subfamily A member 1 (ABCA1) (Liu et al., 2015; Yan et al., 2020). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous control.
2.10.　 Analysis of PPARγ-pathway proteins
The protein expression levels of PPARγ, SREBP1, LXRα, ABCA1, and GAPDH in DSA tissue were determined using Western blot, as previously described (Liu et al., 2018). The primary antibodies and dilutions used are listed in Table 3. The secondary antibody (horseradish peroxidase-conjugated anti-rabbit IgG) was diluted to 1:6000. Finally, the protein bands were visualized using chemiluminescent reagents, and the density of protein bands was detected using Alpha Imager 2200 software (Alpha Innotech Corporation, San Leandro, CA, USA).
2.11.　 Statistical analysis
The treatment and control group data were compared using one-way ANOVA in SPSS (IBM Corp. Released 2011, Version 20.0. Armonk, NY, USA). Orthogonal polynomial comparisons were conducted to determine the linear and quadratic effects of increasing dietary FML supplementation on the measured traits. The pen was the experimental unit for growth performance, and the pig was used for other determinations. The results are expressed using mean values and SEM. Results with P ≤ 0.05 are considered significant, and results with 0.05 < P < 0.10 are considered trends.
3.　Results
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3.1.　 Growth performance
As reported by our previous study (Liu et al., 2022), elevating the FML level from 0.02% to 0.16% resulted in increased final body weight (linear and quadratic, P < 0.05, 74.98 ± 1.66 kg to 81.31 ± 1.91 kg) with no change in ADFI (2.34 ± 0.05 kg). Additionally, FML increased ADG (linear and quadratic, P < 0.05, 0.52 ± 0.03 kg to 0.62 ± 0.04 kg) and decreased F:G (linear and quadratic, P < 0.05, 4.54 ± 0.53 to 3.78 ± 0.37), with the highest value of ADG (0.62 ± 0.04 kg) and the lowest value of F:G (3.78 ± 0.37) in 0.16% FML group.
3.2.　 Plasma biochemical indices
Dietary FML decreased (linear and quadratic, P < 0.05) the plasma concentrations of total cholesterol and FFA in experimental pigs (Table 4). The lowest values of total cholesterol and FFA were observed in the 0.08% and 0.04% FML groups, respectively. Moreover, glucose concentration increased (quadratic, P = 0.08) with increasing FML levels. However, the triglyceride concentration decreased (quadratic, P = 0.07) with increasing FML levels.
3.3.　 Serum enzyme activities
Increasing dietary FML supplementation decreased (linear and quadratic, P < 0.01) the serum activity of HMGCR in finishing pigs and increased the activities of LPL (linear and quadratic, P < 0.05) and ATGL (linear, P < 0.05) (Table 5). The lowest activities of HMGCR and LPL were observed in the 0.08% FML and control group, respectively.
3.4.　 Adipocyte area in adipose tissues
Dietary FML decreased (linear, P = 0.05) the adipocyte area in the DSA tissue of finishing pigs and increased (quadratic, P < 0.01) the adipocyte area in the visceral adipose tissue (Figs. 1 and 2), while had no significant effect (P > 0.05) on the adipocyte area in the ASA tissue of the studied pigs.
3.5.　 Fatty acid profile in adipose tissues
As shown in Table 6, increasing dietary FML supplementation decreased (linear, P < 0.05) the C20:1 content and increased (linear, P < 0.05) the C18:3n3 and n-3PUFA contents in the DSA tissue of finishing pigs.
Increasing dietary FML supplementation decreased (linear and quadratic, P < 0.05) the C20:1 content and ∑n-6/∑n-3 ratio in the ASA tissue of finishing pigs and increased (linear, P < 0.05) the C18:2n6, C18:3n3, C20:4n6, PUFA, n-3PUFA, and n-6PUFA contents, and the PUFA/SFA ratio (Table 7). The highest values of C18:3n3, C20:4n6, and n-3PUFA contents and the PUFA/SFA ratio were all observed in the 0.08% FML group, while the lowest content of C20:1 was observed in the 0.16% FML group.
Increasing dietary FML supplementation decreased the C16:1 (quadratic, P < 0.05) and C20:1 contents (linear, P < 0.05) in the visceral adipose tissue of finishing pigs, and increased (linear and quadratic, P < 0.05) the C18:3n3, C20:4n6, n-3PUFA, and PUFA contents (Table 8). Additionally, the C18:2n6, n-6PUFA contents, and the PUFA/SFA ratio were increased (linear, P < 0.05).
3.6.　 Expression levels of lipid metabolism-related genes in adipose tissues
Dietary FML upregulated (linear and quadratic, P < 0.05) the mRNA expression levels of ABCA1 and SREBP1 in the DSA tissues of finishing pigs (Table 9). The highest values of ABCA1 and SREBP1 expression levels were observed in the 0.16% and 0.08% FML groups, respectively. And the highest values of HMGCR and PPARγ expression levels were observed in the 0.04% FML group.
Dietary FML upregulated (linear and quadratic, P < 0.01) the PPARγ expression level in the ASA tissue of finishing pigs (Table 10). The highest value of PPARγ expression level was observed in pigs fed the 0.16% FML diet.
Increasing dietary FML supplementation upregulated (linear and quadratic, P < 0.05) the mRNA expression levels of ACCα and PPARγ in the visceral adipose tissue of finishing pigs (Table 11). The highest values of ACCα and PPARγ were observed in the 0.16% and 0.08% FML groups, respectively.
3.7.　 Abundance of PPARγ-pathway proteins
The protein abundance of total or phosphorylated (P)-PPAR, PPARγ, P-SREBP1, SREBP1, LXRα, and ABCA1 in the DSA tissue of finishing pigs increased (linear or quadratic, P < 0.05) with increasing FML inclusion (Fig. 3). Additionally, increasing dietary FML supplementation increased (linear and quadratic, P < 0.05) the P-PPARγ/PPARγ ratio, with the highest value observed in the 0.16% FML group. And the highest values of P-SREBP1/SREBP1 ratio, and protein abundances of LXRα and ABCA1 were observed in the 0.08%, 0.02%, and 0.04% FML groups, respectively.
4.　Discussion
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Currently, the pork industry faces significant challenges related to sustainability and the perceived negative image of fat (Font-i-Furnols and Guerrero, 2014). Pork accounts for approximately 38% of the total amount of meat produced worldwide and is the most commonly consumed meat in European, American, and Asian countries (Szűcs and Vida, 2017). China is rich in pig breeds, mostly lard-type breeds, such as Bama miniature pigs. The Bama miniature pig (Suscrofa domestica), located in Bama County, Guangxi Province, China, is a potential animal model for studying lipid metabolism. Due to their long feeding history, genetic stability, and excellent meat quality, many studies in genetics and nutrition have used hybrid offspring of Bama mini-pigs and Berkshire pigs. Furthermore, lard is a popular table seasoning in some countries, especially China. Excessive consumption of lard can harm human health (Dunshea and D’ Souza, 2003). Obtaining healthy lard with no side effects has been the focus of research and exploration in food nutrition. FML, the main component of mulberry leaves, regulates lipid metabolism in animals. However, its ability to modulate the formation of pig fat products, such as lard, has not been studied. To our knowledge, our study is the first to demonstrate that the growth performance of Chinese hybrid barrows of Berkshire and Bama mini-pigs could be improved by including FML (0.02% to 0.16%) in their diet. We further investigated the effects of FML supplementation on the blood lipid index concentration, lipid-related enzyme activity, adipose tissue histomorphology, fatty acid composition and content in different adipose tissues, and their regulatory mechanisms. We established that FML reduced blood lipids and improved the distribution of fatty acids in the adipose tissue and could thus be an interesting source for supplying these beneficial fatty acids to humans.
The triglyceride, total cholesterol, HDL, and LDL levels are commonly used indicators of blood lipid metabolism. Studies on pigs, rats, chickens, and other animals have shown that adding mulberry leaf extract to the diet reduces blood total cholesterol and triglyceride concentrations and inhibits serum lipase activity. Song et al. (2016) found that mulberry leaf extract significantly decreased the serum concentrations of total cholesterol, triglyceride, LDL-cholesterol, and HDL-cholesterol in HFD-fed mice. Similarly, Folium Mori extract decreased body weight and triglyceride, total cholesterol, and LDL levels, improving insulin resistance in type 2 diabetes mellitus rats (Cai et al., 2016). These findings are consistent with our study, in which dietary FML decreased the plasma concentrations of total cholesterol and FFA in finishing pigs, and the triglyceride concentration decreased with increasing FML levels. Furthermore, previous studies have demonstrated the potential of mulberry leaf extract as a complementary mealtime glucose option for patients with type 2 diabetes mellitus because it significantly lowers postprandial glucose levels (Riche et al., 2017). Additionally, an ethanol extract from mulberry leaves, 1-deoxynojirimycin, lowered blood glucose levels in db/db mice (Hu et al., 2017). In our study, the glucose concentration increased with the increasing FML levels. This difference may be because FML activates the AMPK-PGC-1 pathway, improving glucose uptake and mitochondrial function (Meng et al., 2020a).
LPL plays a key role in triglyceride metabolism in animals and is vital for lipid digestion and utilization when activated. It can hydrolyze chylomicron particles, LDL, and very low-density lipoproteins in triglyceride to FFAs and monoacylglycerols, which various tissues can use. Hence, it has an evident effect in eliminating excess triglyceride from the body. HMGCR is the rate-limiting enzyme in cholesterol metabolism, which regulates the body's cholesterol levels; its inhibition can control the amount of endogenous cholesterol. In our study, increasing dietary FML supplementation decreased the serum activity of HMGCR in finishing pigs and increased the activities of LPL and ATGL. This suggests that FML increases lipolytic enzyme activity and decreases cholesterol-producing enzyme activity to reduce blood lipids.
Lipid metabolism is reflected in the lipid profile of blood and lipid accumulation in tissues. Fat deposition in the tissues primarily involves the increase in the number and volume of adipocytes. Subcutaneous and visceral adipose tissues exhibit distinct developmental and metabolic patterns, particularly regarding fatty acid composition. Typically, immature adipose tissue, such as subcutaneous fat, is located externally, and unsaturated fatty tissues, such as perirenal fat, are located internally (Jiang et al., 2013). Adipose tissue is primarily composed of fatty acids and triglyceride. The types and proportions of fatty acids in the adipose tissue are influenced by various factors, including dietary intake (Martins et al., 2012), breed, and genotype. In our study, dietary FML supplementation decreased the adipocyte area in DSA tissue, but did not affect ASA tissue. Additionally, the adipocyte area in the visceral adipose tissue increased with 0.02% and 0.04% FML administration and decreased with 0.16% FML administration. Some studies found that FML reduced the body weight and liver index of type 2 diabetes mellitus rats (Duan et al., 2022) and reduced fat deposition in obese mice (Lim et al., 2013); however, they did not investigate the effects of flavonoids on subcutaneous or visceral fat. Further research is needed to confirm these findings and determine the mechanisms underlying these effects.
Furthermore, we tested the fatty acid content in the three adipose tissues to verify the above findings. The fatty acid profiles were similar in all three adipose tissues, with oleic (C18:1n9) and palmitic acids (C16:0) accounting for approximately 70% of the total fatty acids in DSA, ASA, and visceral adipose tissues. Additionally, stearic (C18:0) and linoleic acids (C18:2n6c) accounted for approximately 25% of the total fatty acids in each tissue. This result is consistent with Lorenzo et al. (2012) and Jiang et al. (2018). Oleic acid (C18:1n9), the most abundant fatty acids, is essential for human heart health and lowers LDL (so-called bad cholesterol) levels. Its practical application recently suggests that the demand for foods with nutritional characteristics is increasing, and it has aroused interest in developing a new generation of lipids, the so-called healthy lipids. FML supplementation decreased C20:1, and increased C18:3n3 and n-3PUFA contents in DSA, ASA, and visceral adipose tissues. Additionally, FML supplementation increased the C20:4n6, n-3PUFA, n-6PUFA, and PUFA contents and the PUFA/SFA ratios in the ASA and visceral adipose tissues. PUFA is an important component of the lipid structure of biofilms, which maintains and stabilizes their structure and function. Of particular interest are the health benefits associated with the consumption of n-3PUFA. These fatty acids have shown positive effects on cardiovascular diseases, diabetes, cancer, depression, and various mental illnesses (Saini and Keum, 2018; Shahidi and Ambigaipalan, 2018). C20:4n6, C18:2n6, and C18:3n3 are the three essential PUFAs in the human body. Therefore, adding an appropriate proportion of FML to the diet can improve the composition of fatty acids in the adipose tissues of finishing pigs, specifically by increasing the content of PUFA, especially n-3PUFA in adipose tissues, and reducing the content of SFA.
Lipid metabolism is influenced by the balance between lipogenesis and lipolysis, which are critical for adipose tissue accumulation. Several genes related to lipid metabolism, including ACCα, FAS, PPARγ, SREBP1, LXRα, and ABCA1, have been identified as potential regulators. The nuclear receptor superfamily of transcription factors, including PPAR and LXR, regulates cholesterol homeostasis in the body (Li and Glass, 2004). As a member of the PPAR superfamily, PPARγ is widely expressed in animal adipose tissue with tissue specificity (Madeira et al., 2014; Chen et al., 2017). Similar to PPARγ, LXRα is a member of ligand-activated nuclear transcription factor superfamily. PPARγ and LXRα regulate adipose differentiation, transport, and lipid deposition in cells and have become new targets of drugs for hyperlipidemia. PPARγ can indirectly participate in the up-regulation of ABCA1 expression and cholesterol reverse transport by enhancing LXRα transcription. Several studies have shown that nuclear receptors PPAR and LXR, along with LPL and ABCA1, play a significant role in regulating lipid metabolism and efflux in humans and hamsters (Forcheron et al., 2002; Beaven and Tontonoz, 2006; Srivastava and He, 2010; Saurav et al., 2012). In our study, the gene expression levels of ABCA1 and PPARγ in the DSA tissues of experimental pigs were upregulated with increasing FML supplementation. Additionally, Western blot revealed that the protein abundances of LXRα and ABCA1 and the ratio of P-PPARγ/PPARγ increased significantly. Therefore, considering these findings and the decrease in adipocyte area in the DSA tissue, we inferred that FML might synergistically upregulate ABCA1 expression based on PPAR and LXR targets on the PPARγ-LXRα-ABCA1 signaling pathway, thus reducing lipid deposition in the DSA tissue. Recent in vitro research using the 3T3-L1 adipocyte IR model showed that flavonoids from mulberry leaf extract could alleviate glycolipid metabolic abnormalities (Meng et al., 2020b). However, unlike the DSA tissue, we observed increased PPARγ and LXRα expression levels and decreased ABCA1 expression levels in the ASA tissue with FML supplementation. The above phenomenon is inconsistent with the PPAR γ-LXR α-ABCA1 signaling pathway in the DSA tissue, suggesting that FML has no effect on adipocyte area in the ASA tissue. Activated LXRα can induce the transcription of SREBP-1, an important nuclear transcription factors, mainly responsible for regulating the expression of genes related to FAS, triglyceride, and glucose metabolism to maintain the dynamic balance of blood lipid (Walker et al., 2011; Peng et al., 2017). Activated SREBP-1 enhanced the expression of cholesterol and fatty acid synthesis-related genes (Hu et al., 2019). Additionally, overexpression of the SREBP-1c protein can increase the expression and activity of FAS, resulting in abnormal lipid deposition in liver tissue, thus forming a fatty liver (Zarate et al., 2017). In our study, the expression levels of SREBP1 in the visceral adipose tissues of the FML groups were lower than those of the control group; however, the expression level of ACCα increased. Combined with the fatty acid distribution in visceral adipose tissue, the adipocyte area in visceral adipose tissue first increased and decreased with an increase in the FML dose, suggesting that different FML doses may have different effects on gene expression and related signaling pathways in this tissue.
5.　Conclusions
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FML supplementation improved the growth performance, regulated the biochemical indices and enzyme activities related to lipid metabolism in the blood of finishing pigs, increased beneficial fatty acids, such as C18:3n3, n-3PUFA, and reduced the ratio of n-6PUFA to n-3PUFA in adipose tissue, thus improving the nutritional value of pig fat. Additionally, the expressions of related genes and proteins were affected by the PPARγ-LXRα-ABCA1 signaling pathway. Overall, this study suggests that FML is an effective and feasible diet/herbal therapy to improve lipid metabolism, inhibit obesity in pigs, and improve the health value of pig fat.
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doi: 10.1016/j.aninu.2023.11.003
  • Receive Date：2023-05-15
  • Online Date：2026-01-28
  • Published：2024-03-10
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  • Received：2023-05-15
  • Revised：2023-11-05
  • Accepted：2023-11-23
Affiliations
    aCollege of Animal Science and Technology, Hunan Agricultural University, Changsha, 410128, China
    bHunan Institute of Animal and Veterinary Science, Changsha, 410131, China
    cKey Laboratory of Conservation and Genetic Analysis of Local Pig Breed Germplasm Resources, Changsha, 410131, China
    dCollege of Information and Intelligence, Hunan Agricultural University, Changsha, 410128, China
    eInstitute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, 410125, China

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Corresponding authors. E-mail addresses: (Y. Yin),
(Y. Xiao).
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表12种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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