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Fatty liver hemorrhagic syndrome (FLHS) is a prevalent metabolic disorder observed in egg-laying hens, characterized by fatty deposits and cellular steatosis in the liver. Our preliminary investigations have revealed a marked decrease in the concentration of butyric acid in the FLHS strain of laying hens. It has been established that sodium butyrate (NaB) protects against metabolic disorders. However, the underlying mechanism by which butyrate modulates hepato-lipid metabolism to a great extent remains unexplored. In this study, we constructed an isolated in vitro model of chicken primary hepatocytes to induce hepatic steatosis by free fatty acids (FFA). Our results demonstrate that treatment with NaB effectively mitigated FFA-induced hepatic steatosis in chicken hepatocytes by inhibiting lipid accumulation, downregulating the mRNA expression of lipo-synthesis-related genes (sterol regulatory element binding transcription factor 1 (SREBF1), acetyl-CoA carboxylase 1(ACC1), fatty acid synthase (FASN), stearoyl-CoA desaturase 1 (SCD1), liver X receptor α (LXRα), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR)) (P < 0.05), and upregulating the mRNA and protein expression of AMP-activated protein kinase α1 (AMPKα1), peroxisome proliferator-activated receptor α (PPARα), and carnitine palmitoyl-transferase 1A (CPT1A) (P < 0.05). Moreover, AMPK and PPARα inhibitors (Compound C (Comp C) and GW6471, respectively) reversed the protective effects of NaB against FFA-induced hepatic steatosis by blocking the AMPK/PPARα pathway, leading to lipid droplet accumulation and triglyceride (TG) contents in chicken primary hepatocytes. With these findings, NaB can alleviate hepatocyte lipoatrophy injury by activating the AMPK/PPARα pathway, promoting fatty acid oxidation, and reducing lipid synthesis in chicken hepatocytes, potentially being able to provide new ideas for the treatment of FLHS., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Berberine (BBR) is a natural alkaloid with multiple biotical effects that has potential as a treatment for fatty liver hemorrhagic syndrome (FLHS). However, the mechanism underlying the protective effect of BBR against FLHS remains unclear. The present study aimed to investigate the effect of BBR on FLHS induced by a high-energy, low-protein (HELP) diet and explore the involvement of the gut microbiota and bile acid metabolism in the protective effects. A total of 90 healthy 140-day-old Hy-line laying hens were randomly divided into three groups, including a control group (fed a basic diet), a HELP group (fed a HELP diet), and a HELP+BBR group (high-energy, high-protein diet supplemented with BBR instead of maize). Our results show that BBR supplementation alleviated liver injury and hepatic steatosis in laying hens. Moreover, BBR supplementation could significantly regulate the gut's microbial composition, increasing the abundance of Actinobacteria and Romboutsia. In addition, the BBR supplement altered the profile of bile acid. Furthermore, the gut microbiota participates in bile acid metabolism, especially taurochenodeoxycholic acid and α-muricholic acid. BBR supplementation could regulate the expression of genes and proteins related to glucose metabolism, lipid synthesis (FAS, SREBP-1c), and bile acid synthesis (FXR, CYP27a1). Collectively, our findings demonstrate that BBR might be a potential feed additive for preventing FLHS by regulating the gut microbiota and bile acid metabolism.

Tumor necrosis factor-α-induced protein eight like 1 (TIPE1) plays important role in autophagy, immunity, and lipid metabolism. The potential role of TIPE1 in fatty liver hemorrhage syndrome (FLHS) is elusory. In the present study, the full-length coding sequence of TIPE1 was cloned, and the polyclonal antibody of TIPE1 was produced by the recombinant TIPE1 protein. The bioinformatic analysis showed that the chicken TIPE1 protein, which was predicted to be a hydrophobic and non-transmembrane protein without signal peptide was highly different from that of mammals. Furthermore, proceeded by using TIPE1 polyclonal antibody, the tissue distribution analysis showed that TIPE1 protein is ubiquitously expressed in various tissues in adult hens and chicks, with its level being higher in the liver and, spleen, moderate in intestinal, brain, and heart. Besides, immunohistochemistry and immunofluorescence observation demonstrated that TIPE1 mainly existed in the cytoplasm in liver, duodenum, and cecum cell. Notably, the TIPE1 expressions were significantly decreased in laying hens suffering from FLHS. Collectively, these results showed that the chicken TIPE1 polyclonal antibody was successfully prepared and further used to analyze the expression profiles of chicken. And the expression of TIPE1 was reduced in FLHS which provided the foundation for further investigation in FLHS., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Peroxisome proliferator-activated receptor α (PPARα) play a key role in the regulation of metabolic homeostasis, inflammation, cellular growth, and differentiation. To further explore the potential role of PPARα in the energy homeostasis of fatty liver hemorrhagic syndrome (FLHS), we reported the prokaryotic expression and purification of chicken PPARα subunit protein, and successfully prepared a polyclonal antibody against PPARα recombinant protein. The 987 bp PPARα subunit genes were cloned into the pEASY-T3 clone vector. Then the plasmid PCR products encoding 329 amino acids were ligated to pEASY-Blunt E2 vector and transformed into BL21 to induce expression. The recombinant PPARα subunit protein, containing His-tag, was purified by affinity column chromatography using Ni-NTA affinity column. Rabbit antiserum was generated by using the concentration of recombinant PPARα subunit protein as the antigen. The results of western blotting showed that the antiserum can specifically recognize chicken endogenous PPARα protein. Immunohistochemistry and immunofluorescence showed that the PPARα mainly existed in the nucleus of hepatocytes, renal epithelial cells and hypothalamic endocrine nerve cells. More importantly, western blotting and real-time quantitative PCR indicated that FLHS significantly decreased the expression of PPARα., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Fatty liver haemorrhagic syndrome (FLHS) is a widespread metabolic disease in laying hens that causes a decrease in egg production and even death. Insulin resistance is a major contributor to the pathogenesis of nonalcoholic fatty liver disease. However, the relationship between FLHS and the insulin resistance mechanisms underlying FLHS is not well elucidated. Therefore, we established an FLHS model induced by feeding a high-energy low-protein diet. In the current study, we found that the fasting glucose and insulin concentrations were elevated in the FLHS group compared with the control group during the experimental period. The results of the oral glucose tolerance test (OGTT) and insulin sensitivity test (IST) showed a high level of insulin resistance in the FLHS model. InsR, 4EBP-1, Glut-1 and Glut-3 mRNA expression were decreased, and TOR, S6K1, and FOXO1 were elevated (P < 0.05). Metabolomic analysis with GC/MS identified 46 differentially expressed metabolites between these two groups, and of these, 14 kinds of metabolism molecules and 32 kinds of small metabolism molecules were decreased (P < 0.05). Further investigation showed that glucose, lipid and amino acid metabolism blocks in the progression of FLHS by GO functional and pathway analysis. Overall, these results suggest that insulin resistance participated in FLHS; comprehensively, metabolites participated in the dysregulated biological process.

In mammals, the AMP-activated protein kinase (AMPK) pathways in the central and peripheral tissues coordinately integrate inputs from multiple sources to regulate energy balance. To investigate the effects of the fatty liver hemorrhagic syndrome (FLHS) caused by high-energy, low-protein diets and to explore the potential role of AMPK in the energy homeostasis of FLHS, 60 laying hens were equally divided into 2 groups: control group (basal diet) and experimental group (high-energy, low-protein diet). Liver tissues were subjected to histopathological analysis. Liver tissues were also collected on the 100th day to determine the levels of total cholesterol, triglyceride (TG), high-density lipoprotein cholesterol (HDL-Ch), low-density lipoprotein cholesterol (LDL-Ch), aspartate aminotransferase, and alanine aminotransferase in plasma. Additionally, the mRNA expression levels of AMPK signaling pathway related genes in liver were determined by quantitative RT-PCR. The results showed that histopathological lesions presented different degrees of lipid vacuolization in hepatocytes. In combination with hematoxylin and eosin and oil red O staining, the experimental group was divided into mild group and severe group. In the severe group, contents of TG and LDL-Ch were extremely significantly increased (P < 0.01) compared to the control group, and HDL-Ch content was extremely significantly decreased (P < 0.01). The serine-threonine kinase 11 and AMPKα1 mRNA expression levels were downregulated, while acetyl-CoA carboxylase, fatty acid synthase, hepatocyte nuclear factor-4α, 3-hydroxy-3-methyl glutaryl coenzyme A reductase and carnitine palmitoyltransferase-I mRNA expression levels were upregulated by a high-energy and low-protein diet. Taken together, these findings suggest that a functional AMPK signaling pathway exists in chickens and AMPK may alter the energy balance in the FLHS induced by high-energy, low-protein diets., (© 2019 Poultry Science Association Inc.)

Metabolism-associated fatty liver disease (MAFLD) is one of the most common causes of liver disease; however, the underlying processes remain unknown. This study aimed to investigate the changes of free fatty acids (FFA) on the expression of genes related to the AMP-activated protein kinase (AMPK) signaling pathway in the primary hepatocytes of laying hens. The primary hepatocytes of laying hens were treated with FFA (containing a 2:1 ratio of oleic and palmitic acids) for 24 h. FFA significantly increased lipid droplet accumulation, decreased glycogen synthesis, increased the levels of triglycerides (TG), total cholesterol (TC), reactive oxygen species (ROS), malondialdehyde (MDA), and glucose content in the supernatant (GLU) in the primary hepatocytes of laying hens, and decreased the levels of total antioxidant capacity (T-AOC) and superoxide dismutase (SOD), as well as mitochondrial membrane potential (MMP). The results of the PCR array combined with Western blotting experiments showed that the activity of AMPK was inhibited. Inhibition of AMPK signaling pathway decreases the expression of genes involved in fatty acid oxidation, increases the expression of genes involved in lipid synthesis, decreases the expression of genes involved in glycogen synthesis, increases the expression of genes involved in glycolysis, increases the expression of genes involved in oxidative stress, and increases the expression of genes involved in cell proliferation and apoptosis. Taken together, our results suggest that FFA can affect the homeostasis of the AMPK signaling pathway by altering energy metabolic homeostasis, inducing oxidative stress, and adjusting the onset of cell proliferation and apoptosis. [ABSTRACT FROM AUTHOR]

Fatty liver hemorrhage syndrome (FLHS), a nutritional and metabolic disease that frequently occurs in laying hens, causes serious losses to the poultry industry. Nowadays, the traditional clinical diagnosis of FLHS still has its limitations. Therefore, searching for some metabolic biomarkers and elucidating the metabolic pathway in vivo are useful for the diagnosis and prevention of FLHS. In the present study, a model of FLHS in laying hens induced by feeding a high-energy, low-protein diet was established. Gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) was used to analyze the metabolites in serum at days 40 and 80. The result showed that, in total, 40 differential metabolites closely related to the occurrence and development of FLHS were screened and identified, which were mainly associated with lipid metabolism, amino acid metabolism, and energy metabolism pathway disorders. Further investigation of differential metabolites showed 10 potential biomarkers such as 3-hydroxybutyric acid, oleic acid, palmitoleic acid, and glutamate were possessed of high diagnostic values by analyzing receiver operating characteristic (ROC) curves. In conclusion, this study showed that the metabolomic method based on GC-TOF-MS can be used in the clinical diagnosis of FLHS in laying hens and provide potential biomarkers for early risk evaluation of FLHS and further insights into FLHS development. [ABSTRACT FROM AUTHOR]

Berberine (BBR), a well-known quaternary ammonium alkaloid, is recognized for its ability to prevent and alleviate metabolic disorders because of its anti-oxidative and anti-inflammatory properties. However, the underlying mechanisms of BBR to mitigate fatty liver hemorrhagic syndrome (FLHS) through the modulation of gut microbiota and their metabolism remained unclear. The results revealed that BBR ameliorates lipid metabolism disorder in high-energy and low-protein (HELP) diet-induced FLHS laying hens, as evidenced by improved liver function and lipid deposition of the liver, reduced blood lipids, and the expression of liver lipid synthesis-related factors. Moreover, BBR alleviated HELP diet-induced barrier dysfunction, increased microbial population, and dysregulated lipid metabolism in the ileum. BBR reshaped the HELP-perturbed gut microbiota, particularly declining the abundance of Desulfovibrio_piger and elevating the abundance of Bacteroides_salanitronis_DSM_18170. Meanwhile, metabolomic profiling analysis revealed that BBR reshaped microbial metabolism and function, particularly by reducing the levels of hydrocinnamic acid, dehydroanonaine, and leucinic acid. Furthermore, fecal microbiota transplantation (FMT) experiments revealed that BBR-enriched gut microbiota alleviated hepatic lipid deposition and intestinal inflammation compared with those chicks that received a gut microbiota by HELP. Collectively, our study provided evidence that BBR effectively alleviated FLHS induced by HELP by reshaping the microbial and metabolic homeostasis within the liver-gut axis. [ABSTRACT FROM AUTHOR]