Exogenous butyrate regulates lipid metabolism through GPR41-ERK-AMPK pathway in rabbits

Abstract Diets containing higher levels of fat can lead to obesity, which is potentially harmful to health. Endogenous butyric acid is produced by intestinal microbial fermentation and participates in lipid metabolism. However, there is less butyric acid produced in the body, butyric acid has a shorter half-life in the blood. We used intraperitoneal injection of sodium butyrate to study the mechanism of butyrate involved in body lipid metabolism. Triglycerides in adipose tissue and liver were significantly reduced by butyrate. The content of triglyceride in plasma in butyrate group was also significantly decreased. In adipose tissue, butyrate up-regulated gene expression of hormone-sensitive lipase (HSL), carnitine palmitoyl transferase (CPT)1 and CPT2, and increased protein expression of G protein-coupled receptor (GPR)41, phosphorylated extracellular-signal-regulated kinase (P-ERK), adenosine 5’-monophosphate (AMP)-activated protein kinase (AMPK) and P-AMPK. Gene expression of lipoprotein lipase (LPL), differentiation-dependent factor 1 (ADD1) and fatty acid synthase (FAS) was down-regulated. In liver, butyrate up-regulated protein expression of GPR41, ERK, P-ERK, P-AMPK. Gene expression of ADD1 and FAS was down-regulated. In muscle tissue, butyrate significantly up-regulated the expression of gene LPL, fatty acid transport protein (FATP) and fatty acid-binding protein (FABP) and protein GPR41, GPR43. Thus, butyrate is involved in body lipid metabolism mainly through the GPR41-mediated ERK-AMPK pathway. Inhibits lipid synthesis in adipose tissue and the liver and promotes lipolysis, avoiding obesity caused by diets with higher fat content. Highlights Butyrate affects lipid metabolism in the body by affecting lipid synthesis/decomposition in adipose tissue and liver. Butyrate mainly through GPR41-mediated ERK-AMPK pathway, inhibit lipid synthesis, promote lipid decomposition, and avoid a large amount of fat accumulation induced by higher-fat diet.


Introduction
Lipid metabolism, including fatty acid transport, uptake, catabolism, and storage (Shen et al. 2015;. These processes are mainly carried out through coordination in the liver, muscle and adipose tissue. The liver plays a key role in lipid metabolism. Depending on species it is, more or less, the hub of fatty acid synthesis and lipid circulation through lipoprotein synthesis (Nguyen et al. 2008). Except in avian species in which de novo lipogenesis (DNL) occurs in has shown to prevent obesity, making short-chain fatty acids promising candidates for preventing energy metabolism disorders (Lin et al. 2012;Yin et al. 2016;Liu et al. 2017). Among short-chain fatty acids, especially the addition of butyrate has been found to have multiple metabolic benefits, including prevention of higher-fat diet-induced obesity, insulin resistance, and liver steatosis. Sodium butyrate is also an effective regulator of insulin homeostasis (Gao et al. 2009;Guilloteau et al. 2010;Li et al. 2018). Intravenous injection of sodium butyrate in mammals could induce fatty acid oxidation and promote cholesterol metabolism, but has no significant effect on animal appetite (Li et al. 2018;Ren et al. 2018). In the cell culture model in vitro, butyrate reduced the concentration of 22:5 (n-6) in the cellular lipids and increased the rate of lipolysis in 3T3-L1 adipocytes (Awad et al. 1991;Rumberger et al. 2014). It impaired lipid transport by inhibiting microsomal triglyceride transfer protein in Caco-2 cells (Marcil et al. 2003), reduced the secretion of triglycerides and phospholipids (Marcil et al. 2002).
G protein-coupled receptors (GPCRs) are large and diverse families of transmembrane proteins. In 2003, SCFAs were found to be ligands of orphan GPR41 and GPR43, and thus were named free fatty acids receptors 3 and 2, respectively (Brown et al. 2003). Butyrate may bind to its receptors G protein-coupled receptor (GPR41 and 43) to activate G protein-mediated second messenger signalling pathways (Nilsson et al. 2003). Among cell signalling pathways, MAPK pathway plays an important role in cell metabolism, proliferation, differentiation, apoptosis and other processes. In eukaryotic cells, there are mainly four MAPK signalling cascade circuits, namely ERK, JNK/stress-activated protein kinase, p38 MAPK and ERK5. ERK is a member of MAPK family, and ERK/MAPK pathway is the core of signal network involved in the regulation of cell metabolism, growth, development and division (Guo et al. 2020). Akt/PKB protein kinase is a serine/threonine kinase, belonging to the cAMP-dependent protein kinase A/protein kinase G/protein kinase C (AGC) superfamily, with structural homology in its catalytic domain and similar activation mechanisms (Song et al. 2005). In previous studies, Akt/PKB has become the core role of growth factor or insulin-activated signal transduction pathway and is believed to contribute to a variety of cellular functions, including nutritional metabolism, cell growth, transcriptional regulation and cell survival (Brazil and Hemmings 2001). The AMPactivated protein kinase(AMPK) is a key regulator of catabolism and anabolic processes. As an energy sensor, it can combine the energy state of the cell with the metabolic environment. These adaptations can occur not only through acute regulation of key metabolic enzymes directly phosphorylated but also through slower transcriptional adaptation responses (Cant o and Auwerx 2010). Peroxisome proliferator-activated receptor a (PPARa) is highly expressed in the liver, adipose and skeletal muscle tissues that use a lot of lipid-derived energy, where it regulates a set of enzymes crucial for fatty acid oxidation. Its primary role is to increase the cellular capacity to mobilise and catabolize fatty acids (Nguyen et al. 2008). Peroxisome proliferator-activated receptor c (PPAR c) participates in regulating energy metabolism, promoting fat formation and maintaining adipocyte differentiation (Rosen and MacDougald 2006;Sevane et al. 2013). The transcription factor sterol regulatory element binding protein-1c/differentiation-dependent factor 1 (SREBP-1c/ ADD1) has an important role in the control of fatty acid synthase (FAS) expression (Horton et al. 2002). Typically SREBP-1c and FAS genes are both expressed and correlated in tissues that synthesise fatty acids de novo (Gondret et al. 2001). FAS and carnitine palmitoyl transferase 1/2 (CPT 1/2) are involved in the regulation of fatty acid synthesis and catabolism, respectively (Thupari et al. 2001). Hormone-sensitive lipase (HSL) is a multifunctional enzyme involved in fatty acid metabolism that hydrolyses triacylglycerols (TAGs), diacylglycerols (DAGs), monoacylglycerols (MAGs), retinyl esters (REs), cholesterol esters (CEs) and other lipids in adipose tissue (Kraemer and Shen 2002;Lampidonis et al. 2011). A major step in energy metabolism is hydrolysis of triacylglycerol-rich lipoproteins (TRLs) to release fatty acids that can be used or stored. This is accomplished by lipoprotein lipase (LPL) (Olivecrona 2016). The main functions of the fatty acid transport protein (FATP) and the fatty acid-binding protein (FABP) are to regulate cellular uptake and intracellular transport of fatty acids (Chmurzy nska 2006).
Our hypothesis is that intraperitoneal injection of sodium butyrate will affect fatty acid synthesis, transport and oxidation in rabbits, and have a positive effect on rabbits lipid metabolism. The aim of this study was, therefore, to analyse the effect of sodium butyrate injection on fat deposition, fat mobilisation and to investigate which regulatory factors and signalling pathways are involved in metabolism.

Animals
Male Hyla rabbits were housed individually in cages (60 Â 40 Â 40 cm). Rabbits house temperature is 23 C, natural light, free to eat and drink. The basic feed formula was formulated according to NRC (United States National Research Council, 1977) and Nutrition of the rabbit Wiseman 1998, 2010). Additional soya oil was added to the basal diet to prepare a test diet with a higher fat content. The composition and nutrient levels of the higher-fat diet are shown in Table 1. All rabbits were fed the same diet.

Experimental protocol and sample collection
Pre-experiment: sixty male Hyla rabbits (40-day old) with similar body weight (1500 ± 10 g) were divided into 6 groups (10 rabbits per group). We selected six doses based on previous studies (Reolon et al. 2011;Khan and Jena 2014) and injected sodium butyrate intraperitoneally. The doses of sodium butyrate injected into the six groups were: 100 mg/kg/day, 300 mg/kg/day, 500 mg/kg/day, 750 mg/kg/day, 1000 mg/kg/day and 2000 mg/kg/day. After 5 days of the test, according to the rabbit's feed intake, daily gain and health status after injection, we selected 500 mg/kg/day injection dose for the formal test.
Formal experiment: forty male Hyla rabbits (40day old) with similar body weight (1500 ± 10 g) were divided into 2 groups (20 rabbits per group): control group with saline (0.9% NaCl, pH 7.4, 37 C) intraperitoneal injection for 5 days, and the butyrate treated group received a intraperitoneal injection of Sodium butyrate (200 mg/mL, pH 7.4, 37 C) for 5 days (500 mg/kg body weight per day). The control group was injected with the same volume of saline as the sodium butyrate group. The injection time is 8: 00 a.m. every day. Body weight and feed intake were recorded daily during the experiment. On the last day of the experiment, 6 rabbits were randomly selected from each group. After injection at 8 o'clock in the morning, blood samples were collected at 08:05, 08:10, 08:15, 08:20, 08:30, 09:00 and 10:00. Blood collection needles and anticoagulant vacuum blood collection tubes were used to collect rabbits plasma from ear veins. Centrifuged at 3000 xg for 10 min, aspirated the supernatant, and stored at À80 C. The samples of rabbits in each group were collected, weighed, counted and analysed separately. Rabbits were sacrificed by cervical dislocation, and the liver, skeletal muscle, shoulder fat, Subcutaneous fat, perirenal fat were collected, weighed and snap frozen in liquid nitrogen and stored at À80 C.

GC/MS
The two groups of plasma samples stored in À80 C were melted and centrifuged for 60 s at 3000 xg , and the clear liquid of the upper layer was transferred to a new centrifuge tube. The content of plasma butyrate was detected using GC/MS method described by Zhang et al. (2019).

Oil red O staining
Adipose tissue, liver, muscle oil red O staining: The tissue samples were first made into frozen sections and then stained with oil red O (Mehlem et al. 2013). Image J software (NIH Image J system, Bethesda, MD) was used for quantitative analysis.

Plasma biochemical
Plasma glucose, triglyceride and total cholesterol were detected by Japanese automatic biochemical analyser HITACHI 7020. Plasma VLDL content, hepatic lipase and lipoprotein lipase enzyme activity was determined using the method described by previous study Han et al. 2019).
Quantitative real-time PCR Total RNA extraction and qRT-PCR were carried out according to the previous research description (Zhang et al. 2011;Du et al. 2013;Liu et al. 2019aLiu et al. , 2019b. The quality and quantity of RNA were determined by agarose gel electrophoresis and biophotometer (Eppendorf, Germany), respectively. The primers were designed for exon-intron junctions using Primer 6.0 software (Primer-E Ltd., Plymouth, UK). The primer sequences are shown in Table 2. The PCR data were analysed with the 2 ÀDDCT method (Livak and Schmittgen 2001). The mRNA levels of target genes were normalised to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and b-actin (DCT) (Liu et al. 2016Wu et al. 2019). Based on the cycle  Average daily feed intake ¼ total feed intake/5; (d) average daily gain ¼ total weight gain/5; values are means ± SEM (n ¼ 6). Ã p < .05 compared with the control. threshold (CT) values, GAPDH and b-actin mRNA expression were stable across treatments in this study (p > .1).

Western blot
Six samples of adipose tissue, liver and muscle were selected, and WB was used to detect the relative expression levels of AMPK and p-AMPK in their respective tissues. The specific test procedures refer to the previous studies in Mahmood and Yang (2012).
Monoclonal mouse anti-GAPDH antibody was used as a loading control. Western blots were developed and quantified using BioSpectrum 810 with VisionWorksLS 7.1 software (UVP LLC, Upland, CA). Image J software was used for quantitative analysis (NIH Image J system, Bethesda, MD).

Statistical analysis
Use SPSS 26 software (SPSS, Chicago, IL) for data statistics. Results are presented as mean ± SEM. Statistical Figure 2. Tissue and organ weight at the end of the experiment. Reference carcase weight: weight of the chilled carcase (carcase after chilling for 24 h in a ventilated cold room (0-4 C) about 1 h after slaughter) minus the head and the above mentioned organs (liver, kidney, organs of chest and neck). Total fat weight: shoulder fat þ subcutaneous fat þ perirenal fat. Ã p < .05 compared with the control (n ¼ 6).
significance was determined at p < .05. Student's t-test was used to compare differences between two groups. GraphPad Prism 7 software (GraphPad Software, La Jolla, CA) is used for drawing.

Results
There were no significant changes (p > .05) in feed intake, average daily feed intake and average daily gain in the butyrate group injected with sodium butyrate intraperitoneally compared with the control group injected with normal saline (Figure 1(a, c, d)). From 5 to 30 min after injection of sodium butyrate, the content of plasma butyrate in the butyrate group was significantly higher (p < .05) than that in the control group (Figure 1(b)).
Compared to the control group, the butyrate group significantly reduced (p < .05) subcutaneous fat and total fat content, while having no significant effect (p > .05) on reference carcase weight, liver weight, shoulder fat weight and perirenal fat weight ( Figure 2). As shown in Figure 3, the oil red staining results of adipose tissue, liver and muscle tissue showed that sodium butyrate group significantly reduced (p < .05) the content of triglycerides in adipose tissue and liver (Figure 3(A, a, (a), B, b, (b))). However, there was no effect on triglyceride content in muscle between the two groups (p > .05; Figure 3(C, c, (c))).   It was found that the contents of triglyceride and very low density lipoprotein in plasma of butyrate group decreased significantly (Figure 4(b, d); p < .05). Plasma hepatic lipase and lipoprotein lipase activities were significantly increased (Figure 4(e, f); p < .05). There was no significant change (p > .05) in the contents of glucose and total cholesterol in plasma (Figure 4(a, c)).
As shown in Figure 5, compared with the control group, butyrate significantly up-regulated (p < .05) the protein expression of GPR41 in adipose tissue, liver and muscle. At the same time, the protein expression of GPR43 was significantly up-regulated (p < .05) in muscle, but had no significant (p > .05) effect in adipose tissue and Liver.
Compared with the control group, butyrate significantly up-regulated (p < .05) the protein expression of ERK in liver, while no significant change in adipose tissue and muscle (p > .05). AKT expression was increased in adipose tissue, liver and muscle of rabbits in the sodium butyrate group, but not significantly (p > .05). Butyrate group up-regulated (p < .05) the protein expression of P-ERK, but had no significant effect (p > .05) on the expression of protein P-AKT in adipose tissue, liver and muscle (Figure 6). In the absence of significant changes in total protein expression, sodium butyrate significantly up-regulated (p < .05) the ratio of phosphorylated protein to total protein in adipose tissue and muscle.
As shown in Figures 7 and 8, butyrate significantly upregulated (p < .05) the protein expression of AMPK and P-AMPK in adipose tissue and P-AMPK in the liver. Meanwhile, the butyrate group significantly increased (p < .05) the ratio of phosphorylated AMPK to total AMPK protein. However, there was no significant effect (p > .05) on protein expression in muscle (Figure 9).
Compared with the control group, the expression of gene LPL, ADD1 and FAS in the adipose tissue of the butyrate group was significantly down-regulated (p < .05), HSL, CPT1 and CPT2 were significantly upregulated (p < .05), and no significant effect on PPARc, PPARa (p > .05). In the liver, butyrate significantly down-regulated (p < .05) gene expression of ADD1 and FAS, and up-regulated (p < .05) CPT1 and CPT2, but had no significant effect (p > .05) on PPARa. In muscle, butyrate significantly up-regulated (p < .05) gene expression of LPL, FATP and FABP, but PPARa, CPT1 and CPT2 were not significantly altered (p > .05, Figure 10).

Discussion
Since 1980, the global prevalence of overweight and obesity has doubled, and now almost one-third of the world's population is classified as overweight or obese. Obesity has an adverse effect on almost all physiological functions of the body and poses a serious public health threat, increasing the risk of patients with many diseases (Singh et al. 2013). Obesity is a multifactorial disease caused by chronic positive energy balance, that is, when dietary energy intake exceeds energy consumption. The excess energy is converted into triglycerides and stored in adipose tissue storage, which increases the volume of the storage, which increases body fat and leads to weight gain (Rogge and Gautam 2017). In our study, it was found that plasma hepatic lipase and lipoprotein lipase enzyme activities were significantly increased after intraperitoneal administration of sodium butyrate. Plasma very low-density lipoproteins and triglycerides are significantly reduced, reducing lipid droplet deposition in adipose tissue and the liver, ultimately leading to a reduction in body fat mass. Consistent with previous studies (Mattace Raso et al. 2013;Matheus et al. 2017), butyrate alleviated obesity or lipid deposition caused by higher-fat diets.
Butyrate is the ligand for metabolite-sensing G-protein coupled receptors (GPCRs), such as GPR43, GPR41 (Coppola et al. 2021). Our study also found that the expression of GPR41 in adipose tissue, liver and muscle of rabbits in the sodium butyrate group was significantly up-regulated, and the expression of GPR43 protein in muscle was significantly increased. Therefore, after injection of sodium butyrate in rabbits, Figure 8. The expression of AMPK (A) and P-AMPK (B) in liver. (C, D) Image J software was used to make statistics on the results of the western blot (WB), and SPSS (SPSS Inc., Chicago, IL) was used for analysis. (E) Ratio of phosphorylated protein to total protein greyscale values. Values are means ± SEM (n ¼ 6). Ã p < .05 compared with the control.
G protein-coupled receptors may be activated, especially GPR41. A variety of stimulators, such as cytokines, viruses, G-protein-coupled receptor ligands and oncogenes play regulatory roles by activating the ERK/ MAPK signalling pathway (Guo et al. 2020). As a downstream effector of PI3-kinase, Akt/PKB is activated by Class 1A and Class 1B PI3-Kinase. Class 1A and Class 1B PI3-kinase are activated by tyrosine kinase and G-protein-coupled receptors, respectively (Song et al. 2005). It was found that the expression of P-ERK in the adipose tissue, liver and muscle of the sodium butyrate group and ERK in the liver were significantly increased, while the expression of AKT and P-AKT in the tissue did not change significantly. Moreover, the ratio of P-ERK to total ERK was increased in the butyrate group. Therefore, GPR41 activated by sodium butyrate could affect the body's metabolism by activating ERK.
Disorders of the phosphatidylinositol-3-kinase (PI3K)/Akt, ERK, and AMPK pathways are critical to the body's energy homeostasis and often lead to diseases such as obesity (Schultze et al. 2012). Phosphorylation of AMPK may be mediated indirectly/directly by Akt and ERK in some cellular metabolic processes (L opez-Cotarelo et al. 2015). In metabolic tissues, AMPK has a regulatory effect on factors related to lipid metabolism. When AMPK is activated, it can inhibit cell active factors such as ADD1 and FAS to inhibit lipid synthesis, and activate HSL, CPT1 and CPT2 to promote lipid decomposition (Hardie et al. 2012;Herzig and Shaw 2018). Consistent with this, butyrate significantly increased the expression of P-AMPK in adipose tissue Figure 9. The expression of AMPK (A) and P-AMPK (B) in muscle. (C, D) Image J software was used to make statistics on the results of the western blot (WB), and SPSS (SPSS Inc., Chicago, IL) was used for analysis. (E) Ratio of phosphorylated protein to total protein greyscale values. Values are means ± SEM (n ¼ 6). Ã p < .05 compared with the control. and liver, and significantly down-regulated the expressions of FAS and ADD1, and significantly up-regulated the expressions of HSL, CPT and CPT2. Therefore, butyrate regulation of lipid metabolism may be related to AMPK pathway. PPARs is a nuclear hormone receptor that acts as a ligand-activated transcription factor. Originally identified as an orphan receptor, it is now known to be activated by binding to fatty acids and to play an important role in maintaining lipid homeostasis and glucose metabolism (Darbre 2015). However, we did not find that the expression of PPARs was affected by sodium butyrate in the experiment, so we speculated that PPARs was not involved in the regulation of butyrate on lipid metabolism.

Conclusion
Butyrate mainly affects lipid metabolism in the body by affecting lipid synthesis/decomposition in adipose tissue and liver. And, mainly through GPR41-mediated ERK-AMPK pathway, inhibit lipid synthesis, promote lipid decomposition, and avoid a large amount of fat accumulation induced by higher-fat diet.

Ethical approval
All study procedures were approved by the Shandong Agriculture University Animal Care and Use Committee and were in accordance with the Guidelines for Experimental Animals established by the Ministry of Science and Technology (Beijing, China).