Serum macroelements and microelements levels in periparturient dairy cows in relation to fatty liver diseases.

BACKGROUND: Fatty liver in dairy cows is a common metabolic disease defined by triglyceride (TG) buildup in the hepatocyte. Clinical diagnosis of fatty liver is usually done by liver biopsy, causing considerable economic losses in the dairy industry owing to the lack of more effective diagnostic methods. Therefore, this study aimed to investigate the potential utility of blood biomarkers for the diagnosis and early warning of fatty liver in dairy cows. RESULTS: A total of twenty-four lactating cows within 28 days after parturition were randomly selected as experimental animals and divided into healthy cows (liver biopsy tested, n = 12) and cows with fatty liver (liver biopsy tested, n = 12). Inductively coupled plasma mass spectrometry (ICP-MS) was used to determine the macroelements and microelements in the serum of two groups of cows. Compared to healthy cows (C), concentrations of calcium (Ca), potassium (K), magnesium (Mg), strontium (Sr), selenium (Se), manganese (Mn), boron (B) and molybdenum (Mo) were lower and copper (Cu) was higher in fatty liver cows (F). Meanwhile, the observed differences in macroelements and microelements were related to delivery time, with the greatest major disparity between C and F occurring 7 days after delivery. Multivariable analysis was used to test the correlation between nine serum macroelements, microelements and fatty liver. Based on variable importance projection and receiver operating characteristic (ROC) curve analysis, minerals Ca, Se, K, B and Mo were screened as the best diagnostic indicators of fatty liver in postpartum cows. CONCLUSIONS: Our data suggested that serum levels of Ca, K, Mg, Se, B, Mo, Mn, and Sr were lower in F than in C. The most suitable period for an early-warning identification of fatty liver in cows was 7 days after delivery, and Ca, Se, K, B and Mo were the best diagnostic indicators of fatty liver in postpartum cows.

Mechanisms of hepatic steatosis in chickens: integrated analysis of the host genome, molecular phenomics and gut microbiome.

Hepatic steatosis is the initial manifestation of abnormal liver functions and often leads to liver diseases such as nonalcoholic fatty liver disease in humans and fatty liver syndrome in animals. In this study, we conducted a comprehensive analysis of a large chicken population consisting of 705 adult hens by combining host genome resequencing; liver transcriptome, proteome, and metabolome analysis; and microbial 16S ribosomal RNA gene sequencing of each gut segment. The results showed the heritability (h2 = 0.25) and duodenal microbiability (m2 = 0.26) of hepatic steatosis were relatively high, indicating a large effect of host genetics and duodenal microbiota on chicken hepatic steatosis. Individuals with hepatic steatosis had low microbiota diversity and a decreased genetic potential to process triglyceride output from hepatocytes, fatty acid ß-oxidation activity, and resistance to fatty acid peroxidation. Furthermore, we revealed a molecular network linking host genomic variants (GGA6: 5.59-5.69 Mb), hepatic gene/protein expression (PEMT, phosphatidyl-ethanolamine N-methyltransferase), metabolite abundances (folate, S-adenosylmethionine, homocysteine, phosphatidyl-ethanolamine, and phosphatidylcholine), and duodenal microbes (genus Lactobacillus) to hepatic steatosis, which could provide new insights into the regulatory mechanism of fatty liver development.

Mechanism of fibroblast growth factor 1 regulating fatty liver disorder in mule ducks.

Mule ducks tend to accumulate abundant fat in their livers via feeding, which leads to the formation of a fatty liver that is several times larger than a normal liver. However, the mechanism underlying fatty liver formation has not yet been elucidated. Fibroblast growth factor 1 (FGF1), a member of the FGF superfamily, is involved in cellular lipid metabolism and mitosis. This study aims to investigate the regulatory effect of FGF1 on lipid metabolism disorders induced by complex fatty acids in primary mule duck liver cells and elucidate the underlying molecular mechanism. Hepatocytes were induced by adding 1,500:750 µmol/L oleic and palmitic acid concentrations for 36 h, which were stimulated with FGF1 concentrations of 0, 10, 100, and 1000 ng/mL for 12 h. The results showed that FGF1 significantly reduced the hepatic lipid droplet deposition and triglyceride content induced by complex fatty acids; it also reduced oxidative stress; decreased reactive oxygen species fluorescence intensity and malondialdehyde content; upregulated the expression of antioxidant factors nuclear factor erythroid 2 related factor 2 (Nrf2), HO-1, and NQO-1; significantly enhanced liver cell activity; promoted cell cycle progression; inhibited cell apoptosis; upregulated cyclin-dependent kinase 1 (CDK1) and BCL-2 mRNA expression; and downregulated Bax and Caspase-3 expression. In addition, FGF1 promoted AMPK phosphorylation, activated the AMPK pathway, upregulated AMPK gene expression, and downregulated the expression of SREBP1 and ACC1 genes, thereby alleviating excessive fat accumulation in liver cells induced by complex fatty acids. In summary, FGF1 may alleviate lipid metabolism disorders induced by complex fatty acids in primary mule duck liver cells by activating the AMPK signaling pathway.

Hepatic lipid accumulation is associated with multiple metabolic pathway alterations but not dyslipidemia and insulin resistance in central bearded dragons (Pogona vitticeps).

OBJECTIVE: To investigate associations between hepatic fat accumulation, fibrosis, and plasma values of primary metabolites, biochemical measurands, insulin, and lipoproteins in bearded dragons. ANIMALS: 48 adult central bearded dragons (Pogona vitticeps). METHODS: Dragons were sedated with alfaxalone, and a blood sample was collected. Plasma was submitted for untargeted primary metabolomics using gas chromatography time-of-flight mass spectrometry, a biochemistry panel, and a lipoprotein panel determined by PAGE. Hepatic lipid content was quantified by liver attenuation measurements from CT images and digital image analysis of standardized histologic sections of the liver. Fibrosis was quantified by digital image analysis on Masson's trichrome-stained histologic sections. Severity was determined from pathologic review of liver sections according to a standardized grading system. Statistical associations were investigated using serial linear models adjusted for false discovery rate and multivariate statistics. RESULTS: Both hepatic fat and fibrosis had a significant effect on CT liver attenuation values. Several oligosaccharides (maltotriose, maltose, ribose, trehalose) and alkaline phosphatase were significantly and linearly increased with hepatic lipid content (all q < .05). On partial least square-discriminant analysis, ß-hydroxybutyric acid was the most important discriminatory variable between fatty liver severity grades on histology. No significant associations were found with insulin, lipoproteins, and succinic acid. CLINICAL RELEVANCE: Bearded dragons with hepatic lipid accumulation experienced multiple metabolic pathway disruptions, some being compatible with mitochondrial dysfunction. No evidence of insulin resistance or dyslipidemia was found. Hepatic biopsy and histopathology remain recommended for reliably diagnosing and staging fatty liver disease in bearded dragons.

FAdV-4-induced ferroptosis affects fat metabolism in LMH cells.

Ferroptosis is a form of controlled cell death that was first described relatively recently and that is dependent on the formation and accumulation of lipid free radicals through an iron-mediated mechanism. A growing body of evidence supports the close relationship between pathogenic infections and ferroptotic cell death, particularly for viral infections. Ferroptosis is also closely tied to the pathogenic development of hepatic steatosis and other forms of liver disease. Fowl adenovirus serotype 4 (FAdV-4) is a hepatotropic aviadenovirus causing hydropericardium syndrome (HPS) that is capable of impacting fat metabolism. However, it remains uncertain as to what role, if any, ferroptotic death plays in the context of FAdV-4 infection. Here, FAdV-4 was found to promote ferroptosis via the p53-SLC7A11-GPX4 axis, while ferrostain-1 was capable of inhibiting this FAdV-4-mediated ferroptotic death through marked reductions in lipid peroxidation. The incidence of FAdV-4-induced fatty liver was also found to be associated with the activation of ferroptotic activity. Together, these results offer novel insights regarding potential approaches to treating HPS.

Epigenetic regulation of H3K27me3 in laying hens with fatty liver hemorrhagic syndrome induced by high-energy and low-protein diets.

BACKGROUND: Fatty liver hemorrhagic syndrome (FLHS) in the modern poultry industry is primarily caused by nutrition. Despite encouraging progress on FLHS, the mechanism through which nutrition influences susceptibility to FLHS is still lacking in terms of epigenetics. RESULTS: In this study, we analyzed the genome-wide patterns of trimethylated lysine residue 27 of histone H3 (H3K27me3) enrichment by chromatin immunoprecipitation-sequencing (ChIP-seq), and examined its association with transcriptomes in healthy and FLHS hens. The study results indicated that H3K27me3 levels were increased in the FLHS hens on a genome-wide scale. Additionally, H3K27me3 was found to occupy the entire gene and the distant intergenic region, which may function as silencer-like regulatory elements. The analysis of transcription factor (TF) motifs in hypermethylated peaks has demonstrated that 23 TFs are involved in the regulation of liver metabolism and development. Transcriptomic analysis indicated that differentially expressed genes (DEGs) were enriched in fatty acid metabolism, amino acid, and carbohydrate metabolism. The hub gene identified from PPI network is fatty acid synthase (FASN). Combined ChIP-seq and transcriptome analysis revealed that the increased H3K27me3 and down-regulated genes have significant enrichment in the ECM-receptor interaction, tight junction, cell adhesion molecules, adherens junction, and TGF-beta signaling pathways. CONCLUSIONS: Overall, the trimethylation modification of H3K27 has been shown to have significant regulatory function in FLHS, mediating the expression of crucial genes associated with the ECM-receptor interaction pathway. This highlights the epigenetic mechanisms of H3K27me3 and provides insights into exploring core regulatory targets and nutritional regulation strategies in FLHS.

Quantitative lipidomics reveals the changes of lipids and antioxidant capacity in egg yolk from laying hens with fatty liver hemorrhagic syndrome.

In laying hens, fatty liver hemorrhagic syndrome (FLHS) is a common metabolic disorder, which can affect egg production and nutritional value. However, the impact of FLHS on the lipid content in egg yolks was not clear. In this study, FLHS model was induced by using high-energy low-protein diet, and the egg quality was evaluated. Egg yolk lipids were quantitatively analyzed by using ultra-performance liquid chromatography-mass spectrometry combined with multivariate statistical analysis. Gene expressions of the lipoprotein were determined by qRT-PCR and antioxidant capacity of the egg yolk were determined by kits. The elevated blood lipids and extensive lipid droplets observed indicated successful establishment of the FLHS model in laying hens. Measurements of egg quality showed that egg yolk weight was increased in the FLHS group. Lipidomics revealed that 1,401 lipids, comprising 27 lipid subclasses in the egg yolk. According to score plots of principal component analysis and orthogonal partial least squares discriminant analysis, different lipid profile was observed between the control and FLHS groups. A total of 97 different lipid species were screen out. Sphingolipid and glycerophospholipid metabolism were identified as key pathways. Free polyunsaturated fatty acids (PUFA) exhibited an increase in the FLHS group (P < 0.05). Notably, the form of PUFAs was changed that the FLHS group showed an increase in triacylglycerol-docosahexenoic acid and triacylglycerol-arachidonic acid in the egg yolk, while triacylglycerol-α-linolenic acid was decreased (P < 0.05). Total superoxide dismutase was decreased in the egg yolks affected by FLHS. Gene expressions of vitellogenin 2 (VTG2), VTG3, very low-density apolipoprotein II and apolipoprotein B were increased in the liver of laying hens with FLHS (P < 0.05). In conclusion, FLHS promoted the lipid transport from the liver to the yolk by upregulating lipoprotein expression, which altered lipid profile, and reduced antioxidant capacity in the yolk. This study provided a foundation for understanding the changes in lipids, lipid transport and lipid antioxidation capacity in egg yolk from laying hens with FLHS.

Sodium butyrate alleviates free fatty acid-induced steatosis in primary chicken hepatocytes via the AMPK/PPARα pathway.

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.

Dietary supplementation of magnolol alleviates fatty liver hemorrhage syndrome in postpeak Xinhua laying hens via regulation of liver lipid metabolism.

As a metabolic disease, fatty liver hemorrhagic syndrome (FLHS) has emerged as a major cause of noninfectious mortality in laying hens, resulting in substantial economic losses to the poultry industry. This study aimed to investigate the therapeutic effects of magnolol on FLHS in postpeak laying hen model, focusing on lipid metabolism, antioxidative capacity, and potential molecular mechanisms of action. We selected 150 Xinhua laying hens aged 50 wk and divided them into normal diet group (ND), high-fat diet group (HFD), 100 mg/kg magnolol group (MG100), 300 mg/kg magnolol group (MG300), 500 mg/kg magnolol group (MG500) on average. The experiment lasted for 6 wk, and liver samples were collected from the hens at the end of the experiment. The results demonstrated that the inclusion of magnolol in the diet had a significant impact on various factors. It led to a reduction in weight, an increase in egg production rate, a decrease in blood lipid levels, and an improvement in abnormal liver function, liver steatosis, and oxidative stress. These effects were particularly prominent in the MG500 group. The RNA-Seq analysis demonstrated that in the MG500 group, there was a down-regulation of genes associated with fatty acid synthesis (Acc, Fasn, Scd, Srebf1, Elovl6) compared to the HFD group. Moreover, genes related to fatty acid oxidation (CPT1A and PGC1α) were found to be up-regulated. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of these differentially expressed genes indicated their enrichment in the PPAR signaling pathway. These findings demonstrate that magnolol can mitigate FLHS by inhibiting fatty acid synthesis and promoting fatty acid oxidation. This discovery offers a novel approach for treating FLHS in laying hens, reducing the economic losses associate with FLHS.

Insights into the influence of three types of sugar on goose fatty liver formation from endoplasmic reticulum stress (ERS).

This study analyzed the formation of goose fatty liver due to endoplasmic reticulum stress (ERS) caused by 3 types of sugar. Transcriptome analysis was performed for liver tissues from geese fed a traditional diet (maize flour), geese overfed with traditional diet, and geese overfed with diet supplemented with glucose, fructose, or sucrose. Correlation analysis of the liver tissue transcriptomes showed that differentially expressed genes (DEGs) involved in ERS were significantly negatively correlated with DEGs involved in inflammation response in the sucrose overfeeding group, and significantly positively correlated with the DEGs involved in lipid metabolism in fructose overfeeding group. Goose primary hepatocytes were isolated in vitro and then treated with glucose or fructose. Some were also treated with ERS inhibitor 4-phenylbutyric acid (4-PBA). In the hepatocytes, mRNA expression of X-Box Binding Protein 1 (XBP1), activating transcription factor 6 (AFT6) and glucose-regulated protein 78 (GRP78) genes increased in the two sugar groups (glucose and fructose), but were suppressed by adding 4-PBA. The mRNA expression data, protein kinase contents, and triglyceride (TG) and very low-density lipoprotein (VLDL) concentrations all suggest that ERS regulates lipid deposition induced by glucose and fructose via elevating lipid synthesis, inhibiting fatty acid oxidation, and decreasing lipid transportation. In conclusion, glucose, or fructose cause ERS and then ERS causes lipid deposition in goose primary hepatocytes. Three types of sugar cause lipid accumulation and then lipid accumulation prevents ERS during goose fatty liver formation, which suggests a potential mechanism protects goose livers from ERS. The different sugars may induce lipid deposition in different ways.

Medium-Chain Fatty Acid Feeding Reduces Oxidation and Causes Panacinar Steatosis in Livers of Neonatal Pigs.

BACKGROUND: Medium-chain fatty acids (MCFAs) are commonly used to enhance the caloric content of infant formulas. We previously reported that pigs fed MCFA developed hepatic steatosis when compared to those fed isocaloric long-chain fatty acid (LCFA) rich formula. OBJECTIVES: The objectives of this study were to investigate: 1) whether MCFA and LCFA feeding affect hepatic fatty acid oxidation, and 2) how fat type alters the expression of hepatic fatty acid metabolic genes. METHODS: Twenty-six, 7-d-old pigs were fed a low-energy control (CONT) formula, or 2 isocaloric high-energy formulas rich in LCFA or MCFA for 22 days. Livers were collected for examining ex vivo fatty acid oxidation, fatty acid content, and mRNA expression of fatty acid metabolic genes. RESULTS: Liver fat was 20% for pigs in the MCFA compared with 2.9% and 4.6% for those in the CONT and LCFA groups (P < 0.05). MCFA-fed pigs had greater amounts of hepatic laurate, myristate, palmitate, and palmitoleate (14, 34, 49, and 9.3 mg · g-1) than those fed LCFA and CONT (1.8, 1.9, 19, 1.5 mg · g-1) formulas (P ≤ 0.05). Hepatic laurate and palmitate oxidation was reduced for pigs fed MCFA (29 mmol · mg-1 · h-1) compared with those fed CONT (54 mmol · mg-1 · h-1) and LCFA (51 mmol · mg-1 · h-1) formulas (P < 0.05). Expression of fatty acid synthase 3 (FASN-3), fatty acid binding protein 1 (FABP-1), and acetyl-CoA carboxylase 1 (ACACA-1) were 8-, 6-, and 2-fold greater for pigs in the MCFA than those in the LCFA and CONT groups (P < 0.05). CONCLUSIONS: Feeding MCFA resulted in hepatic steatosis compared with an isocaloric formula rich in LCFA. Steatosis occurred concomitantly with reduced fatty acid oxidation but greater mRNA expression of fatty acid synthetic and catabolic genes.

Transcriptome and lipidome integration unveils mechanisms of fatty liver formation in Shitou geese.

Geese evolved from migratory birds, and when they consume excessive high-energy feed, glucose is converted into triglycerides. A large amount of triglyceride deposition can induce incomplete oxidation of fatty acids, leading to lipid accumulation in the liver and the subsequent formation of fatty liver. In the Chaoshan region of Guangdong, China, Shitou geese develop a unique form of fatty liver through 24 h overfeeding of brown rice. To investigate the mechanisms underlying the formation of fatty liver in Shitou geese, we collected liver samples from normally fed and overfed geese. The results showed that the liver size in the treatment group was significantly larger, weighing 3.5 times more than that in the control group. Extensive infiltration of lipid droplets was observed in the liver upon staining of tissue sections. Biochemical analysis revealed that compared to the control group, the treatment group showed significantly elevated levels of total cholesterol (T-CHO), triglycerides (TG), and glycogen in the liver. However, no significant differences were observed in the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which are common indicators of liver damage. Furthermore, we performed a combined transcriptomic and lipidomic analysis of the liver samples and identified 1,510 differentially expressed genes (DEGs) and 1,559 significantly differentially abundant metabolites (SDMs). The enrichment analysis of the DEGs revealed their enrichment in metabolic pathways, cellular process-related signaling pathways, and specific lipid metabolism pathways. We also conducted KEGG enrichment analysis of the SDMs and compared them with the enriched signaling pathways obtained from the DEGs. In this study, we identified 3 key signaling pathways involved in the formation of fatty liver in Shitou geese, namely, the biosynthesis of unsaturated fatty acids, glycerol lipid metabolism, and glycerophospholipid metabolism. In these pathways, genes such as glycerol-3-phosphate acyltransferase, mitochondrial (GPAM), 1-acylglycerol-3-phosphate O-acyltransferase 2 (AGPAT2), diacylglycerol O-acyltransferase 2 (DGAT2), lipase, endothelial (LIPG), lipoprotein lipase (LPL), phospholipase D family member 4 (PLD4), and phospholipase A2 group IVF (PLA2G4F) may regulate the synthesis of metabolites, including triacylglycerol (TG), phosphatidate (PA), 1,2-diglyceride (DG), phosphatidylethanolamine (PE), and phosphatidylcholine (PC). These genes and metabolites may play a predominant role in the development of fatty liver, ultimately promoting the accumulation of TG in the liver and leading to the progression of fatty liver.

Enrichment efficiency of lutein in eggs and its function in improving fatty liver hemorrhagic syndrome in aged laying hens.

In this study, we evaluated the enrichment efficiency of lutein in eggs and its function in preventing fatty liver hemorrhagic syndrome (FLHS) in aged laying hens. Five groups of laying hens (65 wk old) were fed basal diets supplemented with 0, 30, 60, 90, or 120 mg/kg of lutein. The supplementation period lasted 12 wk followed by 2 wk of lutein depletion in feed. The results revealed that lutein efficiently enriched the egg yolks and improved their color with a significant increase in relative redness (P < 0.001). Lutein accumulation increased in the egg yolk until day 10, then depletion reached a minimum level after 14 d. Overall, zeaxanthin content in all the groups was similar throughout the experimental period. However, triglycerides and total cholesterol were significantly decreased in the liver (P < 0.05) but not significantly different in the serum (P > 0.05). In the serum, the lipid metabolism enzyme acetyl-CoA synthetase was significantly reduced (P < 0.05), whereas dipeptidyl-peptidase 4 was not significantly different (P > 0.05), and there was no statistical difference of either enzyme in the liver (P > 0.05). Regarding oxidation and inflammation-related indexes, malondialdehyde, tumor necrosis factors alpha, interleukin-6, and interleukin-1 beta were decreased, whereas superoxide dismutase and total antioxidant capacity increased in the liver (P < 0.001). The function of lutein for the same indexes in serum was limited. It was concluded that lutein efficiently enriched the egg yolk of old laying hens to improve their color and reached the highest level on day 10 without being subject to a significant conversion into zeaxanthin. At the same time, lutein prevented liver steatosis in aged laying hens by exerting strong antioxidant and anti-inflammatory functions, but also through the modulation of lipid metabolism, which may contribute to reducing the incidence of FLHS in poultry.

Ultrasonographic image of fatty infiltration of the liver correlates with selected biochemical parameters and back fat thickness of periparturient Holstein-Friesian cows.

During the transition period, the cow's body activates adaptive mechanisms aimed at adjusting to the changing demand for energy and nutrients, which are necessary for the growing fetus and the subsequent start of milk production. This time is also associated with an increased risk of metabolic diseases and reproductive disorders. Our study aimed to identify prepartum and postpartum biochemical markers and weight loss patterns that could differentiate cows that would exhibit ultrasonographic signs of liver fatty infiltration during the latter half of the transition period. The study was performed in a single herd of Holstein-Friesian cows and the animals were divided into two groups: CON (n=13) - cows without ultrasonographic signs of fatty liver, and FL (n=16) - cows with ultrasonographic signs of fatty liver. Backfat thickness and specific biochemical parameters were measured weekly from one week before parturition to 9 weeks postpartum. Our study highlights the importance of using a combination of monitoring methods to assess the metabolic status of transition dairy cattle. The results showed that ultrasound measurements of backfat thickness, blood NEFA levels, glucose concentration, and AST activity were all different (p<0.05) between the control and FL groups, indicating the usefulness of these parameters in monitoring the health status of transition cows. Additionally, the results suggest that high prepartum glucose levels (4.99 mmol/l) could serve as a potential marker for future FL, while the elevated NEFA levels (0.51 mmol/l) and decreased AST activity (80.56 u/l) in FL animals indicate their potential as indicators of lipid mobilization and liver structural damage, respectively.

Saikosaponin a ameliorates diet-induced fatty liver via regulating intestinal microbiota and bile acid profile in laying hens.

Fatty liver hemorrhagic syndrome is a widespread metabolic disease in laying hens that decreases egg production and even causes death in severe cases. Many traditional Chinese medicine ingredients, such as saikosaponin a (SSa), have been shown to alleviate fatty liver, but the underlying mechanisms remain unclear. In this study, we aimed to explore the alleviation of dietary SSa on excessive hepatic lipid deposition and the interactions between intestinal microbiota and bile acid (BA) in laying hens. Fifty-four 35-wk-old laying hens were randomly allocated into 3 treatment groups with 6 replicates (3 birds per replicate) and fed with a basal diet (CON), high-energy and low-protein diet (HELP), and HELP diet with 30 mg/kg SSa (HELP + SSa). SSa reversed diet-induced egg production rate decrease (P < 0.05). SSa could potently ameliorate HELP-induced accumulation of hepatic cholesterol and liver injury via the increase (P < 0.05) of mRNA expression of BA synthesis gene, such as cholesterol 7 alpha-hydroxylase 1. SSa treatment alleviated gut dysbiosis, especially reducing (P < 0.05) the relative abundance of bile salt hydrolase (BSH)-producing bacteria such as Lactobacillus, Bifidobacterium, and Turicibacter. Ileal BA metabolomic analysis revealed that SSa increased (P < 0.05) the content of tauro-conjugated BAs, mainly taurochenodeoxycholic acid and tauro-α-muricholic acid. The mRNA expression of farnesoid X receptor (FXR) and fibroblast growth factor 19 were decreased (P < 0.05) in intestine, which was associated with increased gene expression of enzymes in the BA synthesis that reduced the levels of cholesterol. Moreover, SSa treatment inhibited intestinal BA reabsorption via decreasing (P < 0.05) the mRNA expression of apical sodium-dependent bile acid transporter. Our findings indicated that SSa reduced liver cholesterol accumulation and alleviated fatty liver in laying hens through microbiota-BA-intestinal FXR crosstalk.

Effect of dietary glycine supplementation on productive performance, egg quality, stress response, and fatty liver incidence in laying hens raised under heat stress conditions.

The current experiment aimed to investigate the effect of dietary glycine (Gly) supplementation on productive performance, egg quality, stress response, and fatty liver incidence in laying hens raised under heat stress (HS) conditions. A total of two hundred eighty 24-wk-old Lohmann Brown-Lite laying hens were randomly allotted to 1 of 4 dietary treatments with 7 replicates. The negative control (NC) diet was prepared to meet or exceed the nutrient and energy requirement for Lohmann Brown laying hens, whereas the positive control (PC) diet was formulated to increase AMEn by 100 kcal/kg compared with the NC diet. Two additional diets were prepared by supplementing 0.341% and 0.683% Gly to the NC diet. All hens were exposed to cyclic HS at 31.4 ± 1.17°C for 8 h/d and 26.7 ± 1.10°C for the remaining time for a 12-wk trial. Results indicated that increasing supplementation of Gly in diets tended (linear, P = 0.088) to decrease the FCR of laying hens. Increasing supplementation of Gly in diets increased (linear, P < 0.05) eggshell lightness and decreased (linear, P < 0.05) egg yolk color. Moreover, a tendency for a quadratic association (P < 0.10) of serum aspartate aminotransferase and alanine aminotransferase concentrations with increasing supplementation of Gly was observed. Increasing supplementation of Gly in diets decreased (linear, P < 0.05) blood heterophil:lymphocyte ratio of laying hens. Hens fed the NC diet showed higher fatty liver incidence (P < 0.05) than those fed the PC diet, but increasing supplementation of Gly decreased (linear, P < 0.05) fatty liver incidence of laying hens. In conclusion, increasing supplementation of Gly up to 0.683% in diets decreases FCR, stress response, and fatty liver incidence in laying hens raised under HS conditions.

Regulation of cholesterol metabolism during high fatty acid-induced lipid deposition in calf hepatocytes.

Cholesterol in the circulation is partly driven by changes in feed intake, but aspects of cholesterol metabolism during development of fatty liver are not well known. The objective of this study was to investigate mechanisms of cholesterol metabolism in calf hepatocytes challenged with high concentrations of fatty acids (FA). To address mechanistic insights regarding cholesterol metabolism, liver samples were collected from healthy control dairy cows (n = 6; 7-13 d in milk) and cows with fatty liver (n = 6; 7-11 d in milk). In vitro, hepatocytes isolated from 3 healthy female calves (1 d old) were challenged with or without a mix of 1.2 mM FA to induce metabolic stress. In addition, hepatocytes were processed with 10 µmol/L of the cholesterol synthesis inhibitor simvastatin or 6 µmol/L of the cholesterol intracellular transport inhibitor U18666A with or without the 1.2 mM FA mix. To evaluate the role of cholesterol addition, hepatocytes were treated with 0.147 mg/mL methyl-ß-cyclodextrin (MßCD + FA) or 0.147 mg/mL MßCD with or without 10 and 100 µmol/L cholesterol before incubation with FA (CHO10 + FA and CHO100 + FA). In vivo data from liver biopsies were analyzed by 2-tailed unpaired Student's t-test. Data from in vitro calf hepatocytes were analyzed by one-way ANOVA. Compared with healthy cows, blood plasma total cholesterol and plasma low-density lipoprotein cholesterol content in cows with fatty liver was markedly lower, whereas the hepatic total cholesterol content did not differ. In contrast, compared with healthy controls, the triacylglycerol content in the liver and the content of FA, ß-hydroxybutyrate, and aspartate aminotransferase in the plasma of cows with fatty liver were greater. The results revealed that both fatty liver in vivo and challenge of calf hepatocytes with 1.2 mM FA in vitro led to greater mRNA and protein abundance of sterol regulatory element binding transcription factor 1 (SREBF1) and fatty acid synthase (FASN). In contrast, mRNA and protein abundance of sterol regulatory element binding transcription factor 2 (SREBF2), acyl coenzyme A-cholesterol acyltransferase, and ATP-binding cassette subfamily A member 1 (ABCA1) were lower. Compared with the FA group, the cholesterol synthesis inhibitor simvastatin led to greater protein abundance of microsomal triglyceride transfer protein and mRNA abundance of SREBF2, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), ACAT2, and lower ABCA1 and FASN protein abundance. In contrast, compared with the FA group, the cholesterol intracellular transport inhibitor U18666A + FA led to greater total cholesterol concentration and greater protein and mRNA abundance of FASN. Compared with the MßCD + FA group, the addition of 10 µmol/L cholesterol led to greater concentration of cholesteryl ester and excretion of apolipoprotein B100, and greater protein and mRNA abundance of ABCA1 and microsomal triglyceride transfer protein, and lower concentration of malondialdehyde. Overall, a reduction in cholesterol synthesis promoted FA metabolism in hepatocytes likely to relieve the oxidative stress caused by the high FA load. The data suggest that maintenance of normal cholesterol synthesis promotes very low-density lipoprotein excretion and can reduce lipid accumulation and oxidative stress in dairy cows that experience fatty liver.

Proteomic analysis of fatty liver induced by starvation of medaka fish larvae.

When medaka fish (Oryzias latipes) larvae are grown in the absence of exogenous nutrition, the liver becomes dark and positive to Oil Red O staining from 7 days post-hatch (dph). We determined the mechanism of this starvation-induced development of fatty liver by proteomic analysis using livers obtained from larvae grown in the presence or absence of 2% glucose at 5 dph. Results showed that changes in the expression levels of enzymes involved in glycolysis or the tricarboxylic acid cycle were modest, whereas the expression levels of enzymes involved in amino acid catabolism or ß-oxidation of fatty acids were significantly elevated, suggesting that they become major energy sources under starvation conditions. Expression levels of enzymes for the uptake and ß-oxidation of fatty acids as well as synthesis of triacylglycerol were elevated, whereas those for the synthesis of cholesterol as well as export of cholesterol and triacylglycerol were decreased under starvation conditions, which explains the accumulation of triacylglycerol in the liver. Our results provide the basis for future research to understand how gene malfunction(s) affects the development of fatty liver, which can lead to nonalcoholic steatohepatitis and then to liver cirrhosis.Key words: amino acid catabolism, ß-oxidation, triacylglycerol, cholesterol, export.

AdipoRon alleviates fatty acid-induced lipid accumulation and mitochondrial dysfunction in bovine hepatocytes by promoting autophagy.

During the transition period in dairy cows, high circulating concentrations of nonesterified fatty acids (NEFA) increase hepatic lipid deposits and are considered a major pathological factor for liver damage. We investigated whether AdipoRon, a synthetic small-molecule agonist of adiponectin receptors 1 and 2 shown to prevent liver lipid accumulation in nonruminants, could alleviate NEFA-induced lipid accumulation and mitochondrial dysfunction. Bovine hepatocytes were isolated from 5 healthy Holstein female newborn calves (1 d of age, 30-40 kg, fasting), and independently isolated hepatocytes from at least 3 different calves were used for each subsequent experiment. The composition and concentration of NEFA used in this study were selected according to hematological criteria of dairy cows with fatty liver or ketosis. First, hepatocytes were cultured with various concentrations of NEFA (0, 0.6, 1.2, or 2.4 mM) for 12 h. In a second experiment, hepatocytes were treated with AdipoRon at different concentrations (0, 5, 25, or 50 µM for 12 h) and times (25 µM for 0, 6, 12, or 24 h) with or without NEFA (1.2 mM) treatment. In the last experiment, hepatocytes were treated with AdipoRon (25 µM), NEFA (1.2 mM), or both for 12 h after treatment with or without the autophagy inhibitor chloroquine. Hepatocytes treated with NEFA had increased protein abundance of sterol regulatory element-binding protein 1c (SREBP-1c) and mRNA abundance of acetyl-CoA carboxylase 1 (ACACA), and decreased protein abundance of peroxisome proliferator-activated receptor α (PPARA), proliferator-activated receptor gamma coactivator-1 α (PGC-1α), mitofusin 2 (MFN2), cytochrome c oxidase subunit IV (COX IV), and mRNA abundance of carnitine palmitoyltransferase 1A (CPT1A), along with lower ATP concentrations. AdipoRon treatment reversed these effects, suggesting this compound had a positive effect on lipid metabolism and mitochondrial dysfunction during the NEFA challenge. In addition, upregulated expression of microtubule-associated protein 1 light chain 3-II (LC3-II, encoded by MAP1LC3) and downregulated expression of sequestosome-1 (SQSTM1, also called p62) indicated that AdipoRon enhanced autophagic activity in hepatocytes. The fact that chloroquine impeded the beneficial effects of AdipoRon on lipid accumulation and mitochondrial dysfunction suggested a direct role for autophagy during NEFA challenge. Our results suggest that autophagy is an important cellular mechanism to prevent NEFA-induced lipid accumulation and mitochondrial dysfunction in bovine hepatocytes, which is consistent with other studies. Overall, AdipoRon may represent a promising therapeutic agent to maintain hepatic lipid homeostasis and mitochondrial function in dairy cows during the transition period.

Protective effects of genistein on the production performance and lipid metabolism disorders in laying hens with fatty liver hemorrhagic syndrome by activation of the GPER-AMPK signaling pathways.

The aim of this study was to evaluate the beneficial effects and potential mechanisms of genistein (GEN) on production performance impairments and lipid metabolism disorders in laying hens fed a high-energy and low-protein (HELP) diet. A total of 120 Hy-line Brown laying hens were fed with the standard diet and HELP diet supplemented with 0, 50, 100, and 200 mg/kg GEN for 80 d. The results showed that the declines in laying rate (P < 0.01), average egg weight (P < 0.01), and egg yield (P < 0.01), and the increase of the ratio of feed to egg (P < 0.01) induced by HELP diet were markedly improved by 100 and 200 mg/kg of GEN treatment in laying hens (P < 0.05). Moreover, the hepatic steatosis and increases of lipid contents (P < 0.01) in serum and liver caused by HELP diet were significantly alleviated by treatment with 100 and 200 mg/kg of GEN in laying hens (P < 0.05). The liver index and abdominal fat index of laying hens in the HELP group were higher than subjects in the control group (P < 0.01), which were evidently attenuated by dietary 50 to 200 mg/kg of GEN supplementation (P < 0.05). Dietary 100 and 200 mg/kg of GEN supplementation significantly reduced the upregulations of genes related to fatty acid transport and synthesis (P < 0.01) but enhanced the downregulations of genes associated with fatty acid oxidation (P < 0.01) caused by HELP in the liver of laying hens (P < 0.05). Importantly, 100 and 200 mg/kg of GEN supplementation markedly increased G protein-coupled estrogen receptor (GPER) mRNA and protein expression levels and activated the AMP-activated protein kinase (AMPK) signaling pathway in the liver of laying hens fed a HELP diet (P < 0.05). These data indicated that the protective effects of GEN against the decline of production performance and lipid metabolism disorders caused by HELP diet in laying hens may be related to the activation of the GPER-AMPK signaling pathways. These data not only provide compelling evidence for the protective effect of GEN against fatty liver hemorrhagic syndrome in laying hens but also provide the theoretical basis for GEN as an additive to alleviate metabolic disorders in poultry.

Fatty liver hemorrhagic syndrome (FLHS) is a nutritional and metabolic disease that seriously threatens the health and performance of laying hens, which is characterized by hepatic steatosis and lipid metabolism disorders. As an isoflavone phytoestrogen, genistein (GEN) exerts many beneficial functions, including alleviating lipid metabolism disorders and anti-inflammatory properties. However, further research is needed on the protective effect and potential mechanism of GEN on the FLHS in laying hens. Here, we found that GEN treatment improved liver injury and decline of production performance in laying hens with FLHS. Moreover, GEN treatment alleviated hepatic steatosis and lipid metabolism disorders through reducing the expression levels of mRNA related to fatty acid transport and synthesis and enhancing the mRNA expression levels of factors associated with fatty acid oxidation in FLHS layers, which may be achieved by activation of the G protein-coupled estrogen receptor­adenosine 5'-monophosphate (AMP)-activated protein kinase signaling pathways. These data not only provide compelling evidence for the protective effects and mechanisms of GEN against FLHS in laying hens but also provide the theoretical basis for GEN to alleviate other metabolic disorders in poultry.