Effects of electrolyte balance on intestinal barrier, amino acid metabolism, and mTORC1 signaling pathway in piglets fed low-protein diets.

A proper dietary electrolyte balance (dEB) is essential to ensure optimal growth performance of piglets. In the low-protein diet, this balance may be affected by the reduction of soybean meal and the inclusion of high levels of synthetic amino acids. The objective of this experiment was to evaluate the optimal dEB of low-protein diets and its impact on the growth performance of piglets. A total of 108 piglets (initial age of 35 d) were randomly divided into 3 groups with 6 replicates of 6 pigs each as follows: low electrolyte diet (LE group; dEB = 150 milliequivalents [mEq]/kg); medium electrolyte diet (ME group; dEB = 250 mEq/kg); high electrolyte diet (HE group; dEB = 350 mEq/kg). Results indicated that the LE and HE diet significantly decreased the average daily gain, average daily feed intake, and crude protein digestibility (P < 0.05) in piglets. Meanwhile, LE diets disrupted the structural integrity of the piglets' intestines and decreased jejunal tight junction protein (occludin and claudin-1) expression (P < 0.05). Additionally, the pH and HCO3- in the arterial blood of piglets in the LE group were lower than those in the ME and HE groups (P < 0.05). Interestingly, the LE diet significantly increased lysine content in piglet serum (P < 0.05), decreased the levels of arginine, leucine, glutamic acid, and alanine (P < 0.05), and inhibited the mammalian target of rapamycin complex 1 (mTORC1) pathway by decreasing the phosphorylation abundance of key proteins. In summary, the dietary electrolyte imbalance could inhibit the activation of the mTORC1 signaling pathway, which might be a key factor in the influence of the dEB on piglet growth performance and intestinal health. Moreover, second-order polynomial (quadratic) regression analysis showed that the optimal dEB of piglets in the low-protein diet was 250 to 265 mEq/kg.

Effects of hesperidin on mitochondrial function, mitochondria-associated endoplasmic reticulum membranes and IP3R-MCU calcium axis in the intestine of piglets exposed to deoxynivalenol.

Deoxynivalenol (DON) pollution is prevalent in crops, and can induce oxidative stress and intestinal injury. Hesperidin is one of the major flavonoids in citrus fruits that has various biological activities such as antioxidant and anti-inflammatory activities. However, whether hesperidin could alleviate DON-induced intestinal injury and the mechanism remain unclear. Mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) have attracted attention for their crucial signaling points to regulate ER-mitochondria calcium transfer. This study aims to evaluate the effects of hesperidin on the intestinal barrier, mitochondrial function, MAMs, and inositol 1,4,5-triphosphate receptor (IP3R)-mitochondrial calcium uniporter (MCU) calcium axis in the intestine of piglets exposed to DON. Twenty-four piglets were randomly divided into four groups in a 2 × 2 factorial arrangement for a 21-d experiment: Control: basal diet; hesperidin group: basal diet + 300 mg kg-1 hesperidin; DON: basal diet + 1.5 mg kg-1 DON; DON + hesperidin group: basal diet + 1.5 mg kg-1 DON + 300 mg kg-1 hesperidin. The data showed that when compared with the DON group, hesperidin improved growth performance and the intestinal barrier, alleviated intestinal oxidative stress and ER stress, and decreased the serum alanine aminotransferase (ALT) level (P < 0.05). Hesperidin also alleviated mitochondrial dysfunction and ferroptosis in the intestine of piglets exposed to DON (P < 0.05). Importantly, hesperidin prevented excessive MAM formation by downregulating the protein levels of Mitofusin 2 (Mfn2) and glucose-regulated protein 75 (GRP75), decreasing the ratio of the mitochondria with MAMs/total mitochondria and the ratio of MAM length/mitochondrial perimeter and lengthening the mitochondria-ER distance in MAMs (P < 0.05). Furthermore, hesperidin regulated the IP3R-glucose-regulated protein 75 (GRP75)-voltage-dependent anion channel 1 (VDAC1)-MCU calcium axis by decreasing the protein levels of GRP75 and MCU and the calcium level of the mitochondria compared with the DON group (P < 0.05). An in vitro experiment was conducted to further explore whether IP3R-mediated ER-mitochondria calcium transfer was involved in the protective effects of hesperidin on the intestinal epithelium barrier and mitochondria. Data showed that hesperidin may exert protective effects on the intestinal epithelium barrier and mitochondria via inhibiting ER-mitochondrial calcium transfer mediated by IP3Rs. These data suggested that hesperidin could alleviate MAM-mediated mitochondrial calcium overload, thereby improving mitochondrial function and alleviating oxidative stress and intestinal injury in DON-challenged piglets.

Mitochondria-associated endoplasmic reticulum membranes involve in oxidative stress-induced intestinal barrier injury and mitochondrial dysfunction under diquat exposing.

Many factors induced by environmental toxicants have made oxidative stress a risk factor for the intestinal barrier injury and growth restriction, which is serious health threat for human and livestock and induces significant economic loss. It is well-known that diquat-induced oxidative stress is implicated in the intestinal barrier injury. Although some studies have shown that mitochondria are the primary target organelle of diquat, the underlying mechanism remains incompletely understood. Recently, mitochondria-associated endoplasmic reticulum membranes (MAMs) have aroused increasing concerns among scholars, which participate in mitochondrial dynamics and signal transduction. In this study, we investigated whether MAMs involved in intestinal barrier injury and mitochondrial dysfunction induced by diquat-induced oxidative stress in piglets and porcine intestinal epithelial cells (IPEC-J2 cells). The results showed that diquat induced growth restriction and impaired intestinal barrier. The mitochondrial reactive oxygen species (ROS) was increased and mitochondrial membrane potential was decreased following diquat exposure. The ultrastructure of mitochondria and MAMs was also disturbed. Meanwhile, diquat upregulated endoplasmic reticulum stress marker protein and activated PERK pathway. Furthermore, loosening MAMs alleviated intestinal barrier injury, decrease of antioxidant enzyme activity and mitochondrial dysfunction induced by diquat in IPEC-J2 cells, while tightening MAMs exacerbated diquat-induced mitochondrial dysfunction. These results suggested that MAMs may be associated with the intestinal barrier injury and mitochondrial dysfunction induced by diquat in the jejunum of piglets.

Effects of low protein diets on acid-base balance, electrolyte balance, intestinal structure, and amino acid transport in piglets.

Reducing the dietary crude protein (CP) could effectively reduce pressure on protein ingredient supplies. However, few data have been reported about the extent to which CP can be reduced and whether limiting the use of soybean meal leads to electrolyte imbalance. In this experiment, using the low protein (LP) diet [2% lower than NRC (2012)], seventy-two piglets (35 days old) were randomly divided into 2 groups with 6 replicates of 6 piglets each: CON group (CP = 18.5%) and LP group (CP = 16.5%), to investigate the effect of the LP diet on electrolyte balance, acid-base balance, intestinal structure and amino acid transport in piglets. The results revealed that the LP diet decreased the average daily gain and dietary CP digestibility, and damaged the villi structure of the small intestine. Compared with the CON diet, the potassium content decreased and the chlorine content increased in the LP diet, and similar trends were shown in piglet serum. The arterial pH, pCO2, HCO3 -, and base excess of piglets in the LP group were lower than those in the CON group, while pO2 was higher than those in the CON group. Interestingly, the LP diet significantly increased the lysine content in piglet serum and significantly decreased the levels of arginine, leucine, and glutamic acid. Furthermore, the LP diet significantly affected the expression of some amino acid transport vectors (B0AT1, EAAC1, and y+LAT1). In summary, these findings suggested that the LP diet leads to acid-base imbalance, amino acid transport disorder and amino acids imbalance in piglets, and the dietary electrolyte may be a key factor in the impact of the LP diet on piglet growth performance and intestinal health.

Deoxynivalenol induced intestinal barrier injury, mitochondrial dysfunction and calcium overload by inositol 1,4,5-triphosphate receptors (IP3Rs)-mitochondrial calcium uniporter (MCU) calcium axis.

Deoxynivalenol (DON) contamination is widespread in crops and could easily cause intestinal injury, which brings hazards to animals. Mitochondria are considered as an important target of DON, nevertheless, the mechanism is still unclear. Mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) have gained arousing interest and are recognized as critical signaling hubs that control calcium signaling transduction between ER and mitochondria. This study aims to investigate the effects of DON on intestinal barrier, mitochondria, MAMs and inositol 1,4,5-triphosphate receptors (IP3Rs)-mitochondrial calcium uniporter (MCU) calcium axis in piglets and porcine intestinal epithelial cells (IPEC-J2). Furthermore, inhibition of IP3Rs or MCU was used to explore whether IP3Rs-MCU axis of MAMs was involved in the mitochondria dysfunction and intestinal epithelium barrier injury induced by DON in IPEC-J2. The data showed that DON induced intestinal barrier injury, mitochondrial dysfunction and ERS in piglets' jejunum and IPEC-J2. Moreover, DON increased MAMs by upregulating the protein level of Mitofusin 2 (Mfn2), increasing the percentage of mitochondria with MAMs/total mitochondria and the ratio of MAMs length/mitochondrial perimeter and shortening the distance between mitochondria and ER of MAMs. Importantly, DON influenced IP3Rs-glucose-regulated protein 75 (GRP75)-voltage-dependent anion channel 1 (VDAC1)-MCU calcium axis by increasing the protein levels of GRP75 and MCU and the interaction of VDAC1-GRP75-IP3Rs complex, which in turn induced mitochondrial calcium overload. Furthermore, inhibition of IP3Rs or MCU alleviated DON-induced intestinal epithelium barrier injury, mitochondrial dysfunction and mitochondrial calcium overload of IPEC-J2. The current investigation proposed that DON induced intestinal injury, mitochondrial dysfunction and calcium overload via IP3Rs-GRP75-VDAC1-MCU calcium axis.

Effects of DON on Mitochondrial Function, Endoplasmic Reticulum Stress, and Endoplasmic Reticulum Mitochondria Contact Sites in the Jejunum of Piglets.

Recent research has emphasized the significance of investigating the interplay between organelles, with endoplasmic reticulum mitochondria contact sites (ERMCSs) being recognized as critical signaling hubs between organelles. The objective of the current study was to assess the impact of deoxynivalenol (DON) on jejunal mitochondria, ER, and ERMCSs. Twelve piglets (35 d, 10.22 ± 0.35 kg) were randomized into two groups: control group, basal diet; the DON group, basal diet + 1.5 mg/kg DON. The findings revealed that DON decreased growth performance, induced jejunal oxidative stress, and impaired jejunal barrier function. DON was also found to induce mitochondrial dysfunction, trigger endoplasmic reticulum stress (ERS) in the piglets' jejunum, and activate mitochondrial and ER apoptosis pathways by upregulating apoptosis-related proteins (Caspase-8, Caspase-12, Bax, and CHOP). To investigate the involvement of ERMCSs in DON-induced intestinal injury, we measured the protein levels of ERMCS proteins, such as mitofusin 1 (Mfn1), mitofusin 2 (Mfn2), and glucose-regulated protein 75 (GRP75) and Pearson's correlation coefficient of ERMCS proteins and ERMCS ultrastructure. Our finding showed that DON upregulated the protein level of Mfn2 and GRP75 and increased the percentage of mitochondria with ERMCSs/total mitochondria, the length of ERMCSs compared to the perimeter of mitochondria, and the Pearson's correlation coefficient of voltage-dependent anion-selective channel protein 1 (VDAC1) and inositol 1,4,5-triphosphate receptors (IP3Rs) in piglets' jejunum. Furthermore, DON shortened the distance between mitochondria and ER at ERMCSs. These findings suggested that DON impaired mitochondrial function, triggered ERS, and increased ERMCSs, indicating that the increased ERMCSs could be related to mitochondrial dysfunction and ERS involved in the intestinal injury of piglets induced by DON.