Epidermal growth factor activates EGFR/AMPK signalling to up-regulate the expression of SGLT1 and GLUT2 to promote intestinal glucose absorption in lipopolysaccharide challenged IPEC-J2 cells and piglets

Abstract Epidermal growth factor (EGF) plays an important role in nutrients transport. The present study was to investigate the effects of EGF on glucose absorption in a cellular injury model (IPEC-J2 cells, in vitro study) and animal injury model (weaned piglets, in vivo study) established by lipopolysaccharide (LPS). In vitro study: porcine intestinal epithelial cells (IPEC-J2) were divided into four treatments: control group (Control); 100 ng/mL EGF treatment group (EGF); 1 μg/mL LPS treatment group (LPS) and 100 ng/mL EGF plus 1 μg/mL LPS treatment group (EGF + LPS). The results showed that EGF significantly increased the alkaline phosphatase (AKP) and sodium/potassium-transporting adenosine triphosphatase (Na+/K+-ATPase) activity, and significantly improved the mRNA and protein expression of SGLT1, GLUT2, EGF receptor (EGFR) and AMP-activated protein kinase α1 (AMPK-α1) in LPS-induced injured cells. In vivo experiment: twenty-four piglets weaned at 21 days were randomly assigned into: (1) control group (basal diets); (2) EGF group (basal diet +2 mg/kg EGF); (3) LPS group (basal diet + injection with 100 μg/kg BW LPS) and (4) EGF + LPS group (basal diet + 2 mg/kg EGF + injection with 100 μg/kg BW LPS). Our results showed that EGF significantly increased the AKP and Na+/K+-ATPase activity, and significantly improved the mRNA expression of SGLT1, GLUT2, EGFR and AMPK-α1 in jejunum mucosa of piglets challenged with LPS. In conclusion, EGF can activate EGFR/AMPK signalling to up-regulate the expression of SGLT1 and GLUT2 as well as improve the AKP and Na+/K+-ATPase activity, thereby promoting intestinal glucose absorption in IPEC-J2 cells and piglets challenged by LPS. HIGHLIGHTS EGF promotes SGLT1 and GLUT2 expression and AKP and Na+/K+-ATPase activity in LPS-induced injured porcine intestinal epithelial cells. EGF promotes SGLT1 and GLUT2 expression and AKP and Na+/K+-ATPase activity in jejunum mucosa of piglets challenged by LPS. Dietary supplementation with EGF activates EGFR/AMPK signalling to up-regulate the expression of SGLT1 and GLUT2 as well as improve the AKP and Na+/K+-ATPase activity to promote intestinal glucose absorption in lipopolysaccharide challenged piglets.


Introduction
Glucose is the main carbon and energy source of eukaryotic cells, and the transport of glucose to mammalian cells is the rate-limiting step in glucose utilisation (Chaudhry et al. 2012). It has been proved that there are at least two types of glucose transporters involved in glucose transport, one is sodium/glucose cotransporter 1 (SGLT1) with high affinity and low transport capacity and the other is glucose transporter 2 (GLUT2) with low affinity and high transport capacity (Bedford et al. 2015;Xu et al. 2015;Huerzeler et al. 2019). It is believed that intracellular glucose is transported to epithelial cells by SGLT1, which is located in the brush border membrane of the intestinal mucosa, and then through GLUT2, located in basal membrane of the intestinal tract, transporting to the portal vena cava (Chaudhry et al. 2012). However, when the glucose concentration in the intestinal lumen is too high, GLUT2 can cooperate with SGLT1 to accelerate glucose absorption (Kellett et al. 2008;Chaudhry et al. 2012). Thus, the study of the function of these glucose transporters not only provides potential drug targets for human related diseases, including obesity and diabetes, but also provides ideas for controlling nutrients absorption in animals.
Epidermal growth factor (EGF), a small mitogenic polypeptide comprising 53 amino acid residues, has established as a trophic factor for the epithelial cell homeostasis (Tang et al. 2016(Tang et al. , 2018 and nutrients transport in the small intestine (Huang et al. 2007;Trapani et al. 2014;Wang L et al. 2019;. It has been established that EGF regulates the absorption of glucose in the intestinal tract via up-regulating the expression of SGLT1 (Cellini et al. 2005;Bedford et al. 2015;Xu et al. 2015;Wang CW et al. 2015;Wang L et al. 2019), but whether EGF regulates the absorption of glucose through GLUT2 is still controversial. For example, Xu et al. (2015) reported that EGF could upregulate the expression of GLUT2 in jejunum and ileum of weaned piglets, indicating that GLUT2 participates in the regulation of glucose absorption, while, Bedford et al. (2015) reported that EGF had no effect on GLUT2 mRNA expression in weaned pigs. Our previous study showed that EGF has a good repair effect on oxidative damage of pig small intestinal epithelial cells stimulated by lipopolysaccharide (LPS) (Tang et al. 2018). In theory, in the process of repairing the damaged intestinal tract, more glucose is needed to meet the energy consumption of intestinal epithelial cells. Therefore, promoting intestinal glucose absorption is of great significance for the repair of damaged intestinal tract in humans and animals. Our previous study has confirmed that EGF can promote the absorption of glucose in LPS-induced injured porcine intestinal epithelial cells (IPEC-J2) (Tang and Xiong 2021), which indirect indicated that the repair of damaged intestinal epithelial cells is related to promote the absorption of glucose. However, whether SGLT1mediated glucose transport can meet the energy needs or not in the process of intestinal repair, and is it necessary to assist glucose absorption by mobilising the expression of GLUT2 in porcine intestine have rarely been reported. Thus, we hypothesised that EGF upregulates the expression of SGLT1 and GLU2 to promote the intestinal glucose absorption in porcine intestine under damage conditions. To test this hypothesis we used LPS, a kind of endotoxin, to establish a cellular injury model (IPEC-J2 cells) and animal injury model (weaned piglets) to investigate the effects of EGF on the expression of glucose transporters SGLT1 and GLUT2 as well as to clarify the roles of SGLT1 and GLUT2 in glucose absorption during intestinal damage repair of piglets.
Determination of AKP and Na þ /K þ -ATPase activity in IPEC-J2 cells After a 24 h of EGF and/or LPS treatment, cells in 6well culture plates were gently washed with PBS (Solarbio, Beijing, China) for twice, then RIPA Lysis Buffer R2220 (Solarbio, Beijing, China) was used to lyse IPEC-J2 cells according to the instructions of the manufacturer. Cellular protein concentration was determined using the bicinchoninic acid (BCA) protein assay reagent (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) at 562 nm according to the instructions of the manufacturer. Alkaline phosphatase (AKP) assay kit (A059-2-2, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) was used to determine the AKP activity (n ¼ 6) according to the instructions of the manufacturer. Briefly, different reagents were added into blank well (5 lL double distilled water, 50 lL buffer and 50 lL matrix fluid), standard well (5 lL 0.1 mg/mL phenol standard fluid, 50 lL buffer and 50 lL matrix fluid), and measured well (5 lL sample, 50 lL buffer and 50 lL matrix fluid), fully mixed and water bath for 15 min at 37 C, then 150 lL chromogenic agent was added in each well and measured the OD at a wavelength of 520 nm used a enzyme-linked immune detector (Bio-Rad, USA). The AKP activity was calculated according to the following formulae: Sodium/potassium-transporting adenosine triphosphatase (Na þ /K þ -ATPase) activity (n ¼ 6) was measured as inorganic phosphate (Pi) released from cellular homogenates using a Na þ /K þ -ATPase assay kit (A070-2-2, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the instructions of the manufacturer. Briefly, different reagents were added into blank glass tube (0.16 mL double distilled water, 0.26 mL solution I, 0.08 mL solution II, 0.08 mL solution III) and measured glass tube (0.12 mL double distilled water, 0.1 mL sample, 0.04 mL solution X, 0.26 mL solution I, 0.08 mL solution II, 0.08 mL solution III) and incubated for 10 min at 37 C. Then 0.1 mL solution IV and 0.1 mL sample was addd in blank glass tube, and 0.1 mL solution IV was added in measured glass tube, and centrifuged at 3500 rpm for 10 min after fully mixed. Supernatant was collected for Pi measurement. Different reagents were added into blank glass tube (0.3 mL double distilled water, 1.0 mL chromogenic agent), standard glass tube (0.3 mL phosphorus standard solution, 1.0 mL chromogenic agent), control glass tube (0.3 mL supernatant, 1.0 mL chromogenic agent) and Na þ /K þ -ATPase measured glass tube (0.3 mL supernatant, 1.0 mL chromogenic agent), then reaction for 2 min, 1.0 mL solution VI was added in both glass tubes, fully mixed and reaction for 5 min at room temperature, then measured the OD value at a wavelength of 636 nm. The Na þ /K þ -ATPase activity was calculated according to the following formulae: Â 7:8g=sample protein concentration mgprot=mL ð Þ : Real-time PCR analysis of gene expression of SGLT1, GLUT2, EGFR and AMPK-a1 in IPEC-J2 cells After a 24 h of EGF and/or LPS treatment, total cell RNA was extracted and purified using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) following the protocol provided by the manufacturer. The mRNA expression of SGLT1, GLUT2, epidermal growth factor receptor (EGFR), AMP-activated protein kinase a1 (AMPK-a1) were analysed by real-time quantitative RT-PCR as described previously (Tang, Su, et al. 2019). The RT-PCR was performed using the SYBR V R Premix Ex Taq TM (Takara, Dalian, China) on an Applied Biosystems 7500 Fast Real-Time PCR System (Foster City, CA, USA). The total volume of PCR reaction system was 25 lL (12.5 lL SYBRV R Premix Ex TaqTM, 4 lLcDNA, 1 lL (10 mmol/L) forward/reverse primers and 8.5 lL dH2O). All PCRs were performed in triplicate on a 96-well RT-PCR plate under the following conditions: 95 C for 30 s followed by 39 cycles of 95 C for 5 s, 58 C for 30 s and 72 C for 60 s. The primers of genes (Sangon Biotech, Shanghai, China) were shown in Table 1. b-actin was used as a housekeeping gene to normalise target gene transcript levels. The formula 2 À(DDCt) , where DDCt ¼ (Ct Target À Ct b-actin ) treatment À (Ct Target À Ct b-actin ) control was used to calculated the relative gene expression (Peng et al. 2020).
Western blot analysis of SGLT1, GLUT2, EGFR and AMPK-a1 in IPEC-J2 cells After a 24 h of EGF and/or LPS treatment, cells in 6well culture plates were gently washed with PBS (Solarbio, Beijing, China) for twice, then RIPA Lysis Buffer R2220 (Solarbio, Beijing, China) was used to lyse IPEC-J2 cells according to the instructions of the manufacturer. Cellular protein concentration was determined using the BCA protein assay reagent (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) at 562 nm according to the instructions of the manufacturer. Western blot was performed as described previously (Tang et al. 2018). Briefly, equal amount of protein samples of cell lysate was loaded for SDS-PAGE and subsequently transferred to PVDF membrane. The membrane was blocked with PBST buffer containing 5% skim-milk for 1 h at room AKP activity ðU=g protÞ ¼ f½ðmeasured ODblank ODÞ=ðstandard OD À blank ODÞ Â standard concentration ð0:1 mg=mLÞg=sample protein concentration ðgprot=mLÞ: temperature followed by overnight hybridisation at 4 C with the indicated primary anti-bodies: anti-SGLT1, anti-GLUT2, anti-EGFR, anti-AMPKa1 and antib-actin (Proteintech, Rosemont, IL, USA). After incubation with secondary antibody HRP goat anti-rabbit IgG (Proteintech, Rosemont, IL, USA) for 1 h, signals were detected using enhanced chemiluminescence kits (ECL-Plus, Thermo, Waltham, MA, USA), and then scanned for detection of fluorescence using the BioRad gel detection system.

Animals and experimental design
Twenty-four healthy piglets with similar birth order, weaned at 21 days with a mean body weight (BW) of 5.76 ± 0.38 kg were randomly assigned into four treatments (six piglets/treatment, three males and three females, n ¼ 6). The first group fed a basal diet (Control group); the second group fed a basal diet plus 2 mg/kg EGF (ZYME FAST (Changsha) Biotechnology Co. LTD, Changsha, China) (EGF group); the third group fed a basal diet and injection with 100 lg/kg BW LPS (Sigma, Saint Louis, MO, USA) at day 8 and day 15 (LPS group); the forth group fed a basal diet plus 2 mg/kg EGF and injection with 100 lg/ kg BW LPS at day 8 and day 15 (EGF þ LPS group). The composition and nutrient levels of basal diet met the nutrient specifications for 5-10 kg BW pig according to the NRC-recommended requirements (NRC 2012) ( Table 2). The experiment lasted for 14 days. Piglets in LPS group and EGF þ LPS group were intraperitoneal injected with 100 lg/kg BW LPS at the morning of day 8 and day 15, and piglets in control group and EGF group were injected with the same amount of normal saline. All pigs were fed for 3 times per day at 8:00, 13:00 and 18:00. During the experiment, piglets were housed individually and ad libitum access to water. The room lighting was natural and the room temperature was maintained at an ambient temperature range of 25-28 C.

Sample collection
Six hours after the intraperitoneal injection of LPS, all pigs were anaesthetized with an injection of sodium pentobarbital (50 mg kg À1 BW), bled by exsanguinations to death. Middle jejunum samples from all piglets (6 piglets per treatment) were collected, one piece of jejunum segment (about 5 cm) was fixed in 10% neutral buffered formalin for examination of intestinal morphology. For another piece of jejunum segment (about 5 cm), the mucosa was scraped gently after removal of surface chime, then immediately frozen in liquid nitrogen, and stored at À80 C.
AKP and Na þ /K þ -ATPase activity in jejunum mucosa The jejunal mucosa tissues were homogenised in saline solution (1:4, weight: volume) and centrifuged at 3500 r/min for 15 min at 4 C. The supernatants protein concentration was determined using the bicinchoninic acid (BCA) protein assay reagent (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) at 562 nm according to the instructions of the manufacturer. Then the supernatants were then diluted into the optimal content for detecting the activities of AKP and Na þ /K þ -ATPase activity. The methods of determination of AKP and Na þ /K þ -ATPase activity in jejunum mucosa (n ¼ 6) were the same as the methods used in IPEC-J2 cells.

Real-time PCR analysis of gene expression of SGLT1, GLUT2, EGFR and AMPK-a1 in jejunum mucosa
Total RNA extraction from jejunal samples and the reverse transcription were conducted according to previous study (Peng et al. 2020). The methods of analysed mRNA expression of SGLT1, GLUT2, EGFR, AMPK-a1 (n ¼ 6) in jejunum mucosa were the same as the methods used in IPEC-J2 cells.

Statistical analysis
All data are expressed as mean ± standard deviation (SD, n ¼ 6). Data are statistically analysed with oneway ANOVA procedure by using the IBM SPSS statistics 21.0 software (SPSS, Inc. Chicago, IL, USA). Significant differences among the obtained means were determined by the Duncan's multiple comparison test. Probability values < 0.05 were taken to indicate statistical significance.

AKP and Na þ /K þ -ATPase activity in IPEC-J2 cells
The effects of EGF on AKP and Na þ /K þ -ATPase activity in IPEC-J2 cells challenged with LPS was presented in Figure 1. It showed that LPS induced severe damage to the AKP and Na þ /K þ -ATPase, and their activity were significantly (p < 0.05) lower than that of other groups. While EGF can promote the activity of AKP and Na þ / K þ -ATPase, and their activity were significantly (p < 0.05) higher than of other groups. EGF also promotes AKP (Figure 1(A)) and Na þ /K þ -ATPase ( Figure  1(B)) activity in LPS-induced injured cells (EGF þ LPS group), which is significantly higher (p < 0.05) than that of the LPS group and can reach a comparable level to that of the control group.

Gene expression of glucose transporters in IPEC-J2 cells
The effects of EGF on gene expression of glucose transporters in IPEC-J2 cells challenged with LPS were presented in Figure 2. Results showed that the LPS group had a lower expression of SGLT1 (Figure 2(B)), EGFR (Figure 2(C)) and AMPK-a1 (Figure 2(D)) compared with EGF and EGF þ LPS groups (p < 0.05) and Figure 1. Effects of EGF on AKP (A) and Na þ /K þ -ATPase (B) activity of IPEC-J2 cells challenged by LPS (n ¼ 6). EGF: epidermal growth factor; LPS: lipopolysaccharide; AKP: alkaline phosphatase; Na þ /K þ -ATPase: sodium/potassium-transporting adenosine triphosphatase; Ã p < 0.05. no difference compared with control group; and the EFG group had a higher expression of EGFR compared with control, LPS, and EGF þ LPS groups (p < 0.05) and a higher expression of SGLT1 and AMPK-a1 compared with control, and LPS groups (p < 0.05); EGF þ LPS group had a higher expression of GLUT2 (Figure 2(A)) compared with control, LPS and EGF groups (p < 0.05).

Protein expression of glucose transporters in IPEC-J2 cells
In accordance with above, the protein expression of GLUT2, SGLT1, EGFR and AMPK-a1 measured by western blot (Figure 3) showed that, LPS or EGF treated alone would not affect the expression of GLUT2 compared with control group (Figure 3(A)). Cells treated with EGF (EGF group) would promote the expression of SGLT1 (Figure 3(B)), EGFR (Figure 3(C)) and AMPK-a1 (Figure 3(D)) compare with control, and LPS groups. EGF plus LPS treated (EGF þ LPS group) had a higher (p < 0.05) expression of GLUT2, SGLT1, EGFR and AMPK-a1 than those cells treated with LPS (LPS group).

Intestinal histomorphology
The results of intestinal morphology are presented in Figure 4. In comparison with the other groups, the LPS group exhibited a decrease (p < 0.05) of VH, villus height to crypt depth ratio (VCR), and an increase (p < 0.05) of CD except for EGF þ LPS group; and the EGF group exhibited an increase (p < 0.05) of VH (Figure 4(A,B)), and VCR (Figure 4(A,D)) and a decrease (p < 0.05) of CD (Figure 4(A,C)) compared with other groups in the jejunum of piglets. The EGF þ LPS group had a higher (p < 0.05) VH, and VCR compared with LPS group and had no difference compared to the control group.

AKP and Na þ /K þ -ATPase activity in jejunum mucosa
The effects of EGF on AKP and Na þ /K þ -ATPase activity in jejunum mucosa of piglets challenged with LPS were presented in Figure 5. It showed that the AKP ( Figure 5(A)) and Na þ /K þ -ATPase ( Figure 5(B)) activity in jejunum mucosa decreased significantly (p < 0.05) in LPS group compared with other groups, while, EGF group remarkably increased (p < 0.05) the AKP and Na þ /K þ -ATPase activity in jejunum mucosa compared with other groups. the EGF þ LPS group had a higher AKP and Na þ /K þ -ATPase activity than the LPS group, and had no difference compared to the control group.

Gene expression of glucose transporters in jejunum mucosa
The effects of EGF on gene expression of glucose transporters in jejunum mucosa of piglets challenged with LPS were presented in Figure 6. It showed that, as the same with the cell experiment, there was no difference in the expression of GLUT2 (Figure 6(A)) among the control group, EGF group and LPS group.

Discussion
Intestinal tract is not only the main part of nutrients digestion and absorption, but also an important barrier for animals to prevent the toxins, allergens, and pathogens from the external environment into the circulation system (Arrieta et al. 2006;Tang et al. 2016;Tang, Liu, Zhong, et al. 2021). Thus, the normal integrity of epithelial cells is the structural basis for the intestinal absorption of nutrients. LPS is a major integral component of the outer membrane of gramnegative bacteria, which can induce cell injury and intestinal morphology destruction (Zhou et al. 2017;Tang et al. 2018). Intestinal morphology which often evaluated by VH, CD, and VCR can reflect the intestinal development and function, theincreased VH and reduced CD, as well as the increased VCR in animals indicated a better intestinal function (Wang M, Huang, et al. 2020). The present study also found that the intestinal morphology of piglets subjected to LPS stimulation was severely damaged, manifested as a decreased VH and increased CD. Previous studies have demonstrated that EGF plays an important role in epithelial recovery and damaged intestinal repair (Tang et al. 2018;. The present study also confirmed that EGF treatment could restore intestinal morphology to a certain extent, manifested by increased VH and decreased CD, indicating that EGF could ameliorate the jejunum damage induced by LPS stress. In theory, during the process of the damaged intestinal repairing, more energy is needed to meet the energy consumption of intestinal epithelial cells. Carbohydrates are the main source of energy for animals including piglets. Ingested carbohydrates are hydrolysed to monosaccharides by digestive enzymes in the gut, of which 80% monosaccharide is glucose. SGLT1 is the primary carrier protein responsible for the absorption of glucose from the lumen of the intestine across the brush border membrane of intestinal epithelial cells, which is dependent on Na þ /K þ -ATPase for energy supply (Drozdowski and Thomson 2006;Wang CW et al. 2015;Chen et al. 2016). Once inside the enterocytes, the glucose are either metabolised or diffuse out of the cell through the GLUT2, located on the basolateral membrane, into the blood circulation to maintain blood glucose balance (Wright et al. 1994;Chaudhry et al. 2012;Wang CW et al. 2015). Therefore, SGLT1, GLUT2 and Na þ /K þ -ATPase play important roles in glucose absorption in the small intestine. Although, studies have shown that EGF upregulated the intestinal glucose uptake by increasing the translocation of SGLT1 (Cellini et al. 2005;Bedford et al. 2015;Xu et al. 2015). Our previous study also confirmed that EGF could promote the glucose absorption in IPEC-J2 cells challenged by LPS (Tang and Xiong 2021), which can speculate that EGF repaired injured cells by increasing the absorption of glucose. However, the role of SGLT1 and GLUT2 in glucose absorption in the damaged intestine needs further confirmation.
In the present study, we used LPS to establish a cellular injury model (IPEC-J2) and animal injury model (piglets) to investigate the effects of EGF on the expression of glucose transporters SGLT1 and GLUT2 as well as to further clarify the roles of SGLT1 and GLUT2 in glucose absorption during intestinal damage repairing. The results showed that, in both IPEC-J2 cells and jejunum mucosa, LPS induced severe damage to the AKP and Na þ /K þ -ATPase activity, while EGF can promote AKP and Na þ /K þ -ATPase activity in LPSinduced injured cells and jejunum mucosa. AKP is a key enzyme in intestinal digestion and absorption, which can accelerate the uptake and transfer of nutrients, and provide energy for the body indirectly. At the same time, AKP is also an endogenous detoxification factor, which can remove LPS endotoxin secreted by pathogenic bacteria in the intestinal tract and protect intestinal health (Geddes and Philpott 2008). The increased AKP activity in EGF þ LPS group suggested that EGF could promote the absorption capacity of glucose in damaged cells and intestine. Na þ /K þ -ATPase is an integral transmembrane protein in the basolateral membrane of intestinal epithelial cells, and the main role is to maintain the electrochemical gradient and osmotic pressure balance inside and outside the cell membrane and drive the co-transport of glucose molecules to SGLT1 with Na þ (Thorsen et al. 2014;Palanikumar et al. 2015;Chen et al. 2016). SGLT1 and GLUT2 are two kinds of important glucose transporter. Previously, it was shown that EGF upregulated intestinal glucose uptake by increasing the translocation of SGLT1 (Chung et al. 2002;Cellini et al. 2005;Wang CW et al. 2015). However, recent studies indicated that GLUT2 can be trafficked to the apical membrane and contribute to glucose absorption in response to high glucose (Kellett et al. 2008;Chen et al. 2016). Results from the present study showed that LPS treatment had no effects on the expression of GLUT2 and SGLT1 in both IPEC-J2 cells and jejunum mucosa. EGF would promote the expression of SGLT in both IPEC-J2 cells and jejunum mucosa without affect the expression of GLUT2. Interestingly, EGF significantly increased the expression of GLUT2 and SGLT1 in LPS-induced damaged cells and jejunum mucosa. This indicates that in the process of intestinal repair, the glucose transported by SGLT1 is insufficient to meet the needs of the body, it is necessary to assist glucose absorption by mobilising the expression of GLUT2.
The biological function of EGF is related to its receptor, EGFR, a transmembrane glycoprotein abundantly located on the apical and basolateral aspect of villus enterocytes (Avissar et al. 2000;Tang et al. 2016). The binding of EGF with EGFR at the enterocytes surface activates a series of signalling pathways and plays a series of biological functions such as nutrients transport and intestinal repair (Wee et al. 2015;Tang et al. 2018;Wang L et al. 2019;Xue et al. 2021). AMPK is a key molecule in the regulation of biological energy metabolism and has been demonstrated to play important roles in the regulation of cellular glucose uptake (Carling 2004;Carling 2007;Dengler et al. 2017). It is widely accepted that the activation of AMPK leads to increased glucose transport in a variety of cells predominantly through the stimulation of the SGLT1 (Sopjani et al. 2010) and GLUT2 (Walker et al. 2005). The present study showed that EGF would promote the expression of EGFR and AMPK-a1 in normal or injury conditions. It indicated that under normal conditions, EGF up-regulates SGLT1 expression through the EGFR/AMPK signalling pathway to promote intestinal glucose absorption; while, in the process of intestinal repair, EGF up-regulates the expression of SGLT1 and GLUT2 through the EGFR/ AMPK signalling pathway to jointly promote intestinal glucose absorption (Figure 7). Meanwhile, due to the high similarities between pigs and humans in anatomy, physiology and nutrient metabolism, pigs can be used as an ideal animal model to study human diseases and nutrients absorption (Ferenc et al. 2014;Li et al. 2015;Hu et al. 2017). So our results may also provide some useful theoretical references for EGF in functional foods, which is beneficial for intestinal health maintain.

Conclusions
In conclusion, EGF can activate EGFR/AMPK signalling to up-regulate the expression of SGLT1 and GLUT2 as well as improve the AKP and Na þ /K þ -ATPase activity, thereby promoting intestinal glucose absorption in IPEC-J2 cells and piglets challenged by LPS.

Ethical approval
The experimental procedures involving animals were approved by the animal welfare committee of the Guizhou Normal University (Guiyang, China) with an ethic approval number GZNU-2020-0042.