G protein-coupled estrogen receptor 1 ameliorates nonalcoholic steatohepatitis through targeting AMPK-dependent signaling.

Nonalcoholic fatty liver disease (NAFLD), especially nonalcoholic steatohepatitis (NASH), has emerged as a prevalent cause of liver cirrhosis and hepatocellular carcinoma, posing severe public health challenges worldwide. The incidence of NASH is highly correlated with an increased prevalence of obesity, insulin resistance, diabetes, and other metabolic diseases. Currently, no approved drugs specifically targeted for the therapies of NASH partially due to the unclear pathophysiological mechanisms. G protein-coupled estrogen receptor 1 (GPER1) is a membrane estrogen receptor involved in the development of metabolic diseases such as obesity and diabetes. However, the function of GPER1 in NAFLD/NASH progression remains unknown. Here, we show that GPER1 exerts a beneficial role in insulin resistance, hepatic lipid accumulation, oxidative stress, or inflammation in vivo and in vitro. In particular, we observed that the lipid accumulation, inflammatory response, fibrosis, or insulin resistance in mouse NAFLD/NASH models were exacerbated by hepatocyte-specific GPER1 knockout but obviously mitigated by hepatic GPER1 activation in female and male mice. Mechanistically, hepatic GPER1 activates AMP-activated protein kinase signaling by inducing cyclic AMP release, thereby exerting its protective effect. These data suggest that GPER1 may be a promising therapeutic target for NASH.

Dehydroepiandrosterone protects against hepatic glycolipid metabolic disorder and insulin resistance induced by high fat via activation of AMPK-PGC-1α-NRF-1 and IRS1-AKT-GLUT2 signaling pathways.

BACKGROUND/OBJECTIVES: Mitochondrial dysfunction, oxidative stress, or fatty liver are the key pathophysiological features for insulin resistance and obesity. Dehydroepiandrosterone (DHEA) can ameliorate obesity and insulin resistance; however, the mechanisms of these actions are poorly understood. The present study aimed to investigate the effect and possible mechanism of DHEA against glycolipid metabolic disorder and insulin resistance. SUBJECTS/METHODS: Rats fed a high-fat diet (HFD) and palmitic acid (PA)-induced BRL-3A cells were employed to analyze the effect of DHEA on factors related to metabolic disorder and insulin resistance in vivo and in vitro. RESULTS: DHEA prevented lipid metabolism disorders by enhancing phospho (p)-protein kinase AMP-activated catalytic subunit alpha (AMPKα) (Thr172) protein level and its downstream lipid metabolism-related factors in liver of rats fed an HFD or in PA-induced BRL-3A cells. Meanwhile, DHEA ameliorated mitochondrial dysfunction through activation of the AMPK-peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α)-nuclear respiratory factor-1 (NRF-1) pathway, which represented as the enhancing of the mtDNA copy number, ATP level, and membrane potential, and decreasing of reactive oxygen species production. Moreover, DHEA alleviated insulin resistance via increasing the phosphorylated insulin receptor substrate 1 (p-IRS1) (Tyr612) level and decreasing that of p-IRS1 (Ser307) level in liver of rats fed an HFD or in PA-induced BRL-3A cells, which subsequently enhanced p-protein kinase B (AKT) (Ser473) and membrane glucose transporter type 2 (GLUT2) expression levels. CONCLUSIONS: The protective effect of DHEA on high-fat-induced hepatic glycolipid metabolic disorder and insulin resistance are achieved through activation of the AMPK-PGC-1α-NRF-1 and IRS1-AKT-GLUT2 signaling pathways. The results provide compelling evidence for the mechanism by which DHEA prevents glycolipid metabolic disorder, and suggest its potential applications for controlling diabetes and obesity in animals and humans.

Dehydroepiandrosterone resisted E. Coli O157:H7-induced inflammation via blocking the activation of p38 MAPK and NF-κB pathways in mice.

Dehydroepiandrosterone (DHEA), a critical metabolite in cholesterol metabolism, can regulate the inflammatory responses in humans or animals. However, the precise mechanisms of these beneficial actions remains poorly understood. Present study aims to clarify the anti-inflammatory function of DHEA and its possible mechanisms in the E. coli O157:H7-stimulated mice. The results indicated that DHEA reduced the mortality of mice and bacterial concentration in the peritoneal fluid in the E. coli-stimulated mice. DHEA increased the spleen index, the activity of lactate dehydrogenase and acid phosphatase; while it decreased the nitric oxide (NO) content and inducible nitric oxide synthase (iNOS) activity in mice. The mRNA levels of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and interferon gamma (IFN-γ) were decreased, whereas the interleukin-4 (IL-4) and interleukin-10 (IL-10) mRNA levels were increased in the E. coli-stimulated mice treated with DHEA. Moreover, DHEA treatment reversed the increasing of IFN-γ/IL-4 ratio in mice caused by E. coli infection. Importantly, DHEA blocked the nuclear translocation of p65 through down-regulation the IκB-α protein phosphorylation level in the mice stimulated with E. coli O157:H7. No statically changes were showed on the phospho (p)-ERK1/2 and p-JNK1/2 protein level, while DHEA significantly suppressed the p-p38 protein level in mice. The above results indicated that DHEA alleviated inflammatory responses by suppressing NO secretion and promoting Th2-associated anti-inflammatory cytokines production in mice; and this action might relate to the blocking of p38 MAPK and NF-κB signaling pathways activation. All the above results provide substantial information for understanding the anti-inflammatory function of DHEA and further support it as a potential immunomodulatory in prevention inflammatory and bacterial infection diseases.

Integrated analysis of proteomics-delineated and metabolomics-delineated hepatic metabolic responses to (-)-hydroxycitric acid in chick embryos.

(-)-Hydroxycitric acid [(-)-HCA] is widely used as a nutritional supplement to control body weight and fat accumulation in animals and humans, whereas the underlying biochemical mechanism is unclear. Broiler chicken was used as a model for studies of obesity due to its natural hyperglycemia and being insulin resistant. The current study aimed to obtain a systematic view of serum metabolites and hepatic proteins and well understand the mechanism of hepatic metabolic response to (-)-HCA treatment in chick embryos. The results showed that 22, 90, and 82 of differentially expressed proteins were identified at E14d, E19d, and H1d in chick embryos treated with (-)-HCA, respectively. Meanwhile, 5, 83, and 88 of serum metabolites significantly changed at E14d, E19d, and H1d in chick embryos after (-)-HCA treatment. Bioinformatics analysis showed that the key proteins and metabolites, which were significantly altered in chick embryos treated with (-)-HCA, were mainly involved in the citrate cycle, glycolysis/gluconeogenesis, fatty acid metabolism, and pyruvate metabolism. Our data indicated that (-)-HCA treatment might promote fat metabolism via regulating the key protein expression levels and metabolite contents in the citrate cycle, glycolysis/gluconeogenesis, and oxidative phosphorylation during chicken embryonic development. These results will deepen our understanding of the mechanism of fat reduction by (-)-HCA and provide substantial information for (-)-HCA as a nutritional supplement to control body weight gain and curb obesity-related diseases.

Dehydroepiandrosterone rehabilitate BRL-3A cells oxidative stress damage induced by hydrogen peroxide.

This study aimed to investigate the molecular mechanism of dehydroepiandrosterone (DHEA) rehabilitated BRL-3A cells oxidative stress damage induced by hydrogen peroxide (H2 O2 ). Results showed that DHEA reversed the decrease of cell viability and ameliorated nuclear chromatin damage in H2 O2 -induced BRL-3A cells. DHEA increased the activities of superoxide dismutase, catalase, peroxidase, and glutathione peroxidase, and decreased the reactive oxygen species (ROS) production in H2 O2 -induced BRL-3A cells. DHEA attenuated the protein damage and lipid peroxidation, and reduced the apoptosis in H2 O2 -induced BRL-3A cells. The mRNA levels of Bax, Caspase-9, and Caspase-3 were decreased, while the Bcl-2 mRNA level was increased in H2 O2 -induced BRL-3A cells treated with DHEA. Our results showed that DHEA treatment increased the PI3K and p-Akt protein levels, while decreased the Bax and capase-3 protein levels in H2 O2 -induced BRL-3A cells. However, the rise in PI3K and p-Akt protein levels, and the decrease in Bax and capase-3 protein levels induced by DHEA treatment were reversed when the cells pretreated with LY294002 (PI3K inhibitor). These results indicated that DHEA ameliorated H2 O2 -induced oxidative damage by increasing anti-oxidative enzyme activities and ameliorating the protein damage and lipid peroxidation in BRL-3A cells. In addition, DHEA decreased the apoptosis by inhibiting caspase-3 and Bax protein levels and this action mainly achieved via the activation of PI3K/Akt signaling pathways in H2 O2 -induced BRL-3A cells. These results provided substantial information for DHEA as a nutritional supplement to treat oxidative stress and it related diseases in animals and humans.

Proteomics reveals changes in hepatic proteins during chicken embryonic development: an alternative model to study human obesity.

BACKGROUND: Chicken embryos are widely used as a model for studies of obesity; however, no detailed information is available about the dynamic changes of proteins during the regulation of adipose biology and metabolism. Thus, the present study used an isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic approach to identify the changes in protein abundance at different stages of chicken embryonic development. RESULTS: In this study, the abundances of 293 hepatic proteins in 19-day old of chicken embryos compared with 14-day old and 160 hepatic proteins at hatching compared with 19-day old embryos were significantly changed. Pathway analysis showed that fatty acid degradation (upregulated ACAA2, CPT1A, and ACOX1), protein folding (upregulated PDIs, CALR3, LMAN1, and UBQLN1) and gluconeogenesis (upregulated ACSS1, AKR1A1, ALDH3A2, ALDH7A1, and FBP2) were enhanced from embryonic day 14 (E14) to E19 of chicken embryo development. Analysis of the differentially abundant proteins indicated that glycolysis was not the main way to produce energy from E19 to hatching day during chicken embryo development. In addition, purine metabolism was enhanced, as deduced from increased IMPDH2, NT5C, PGM2, and XDH abundances, and the decrease of growth rate could be overcome by increasing the abundance of ribosomal proteins from E19 to the hatching day. CONCLUSION: The levels of certain proteins were coordinated with each other to regulate the changes in metabolic pathways to satisfy the requirement for growth and development at different stages of chicken embryo development. Importantly, ACAA2, CPT1A, and ACOX1 might be key factors to control fat deposition during chicken embryonic development. These results provided information showing that chicken is a useful model to further investigate the mechanism of obesity and insulin resistance in humans.

Dehydroepiandrosterone reduces accumulation of lipid droplets in primary chicken hepatocytes by biotransformation mediated via the cAMP/PKA-ERK1/2 signaling pathway.

Dehydroepiandrosterone (DHEA) is commonly used as a nutritional supplement to control fat deposition, but the mechanism of this action is poorly understood. In this study, we demonstrated that DHEA increased phosphorylation of AMP-activated protein kinase (p-AMPK). Elevated p-AMPK levels resulted in reduced expression of sterol regulatory element binding protein-1c, acetyl CoA carboxylase, fatty acid synthase and enhanced expression of peroxisome proliferators-activated receptor α and carnitine palmitoyl transferase-I, ultimately leading to the reduction of lipid droplet accumulation in primary chicken hepatocytes. We found that DHEA activates the cyclic adenosine 3', 5'-monophosphate/protein kinase A - extracellular signal-regulated kinase 1/2 (cAMP/PKA-ERK1/2) signaling pathway, which regulates the conversion of DHEA into testosterone and estradiol by increasing the 17ß-hydroxysteroid dehydrogenase and aromatase protein expression. Importantly, the fat-reducing effects of DHEA are more closely associated with the conversion of DHEA into estradiol than with the action of DHEA itself as an active biomolecule, or to its alternative metabolite, testosterone. Taken together, our results indicate that DHEA is converted into active hormones through activation of the cAMP/PKA-ERK1/2 signaling pathway; the fat-reducing effects of DHEA are achieved through its conversion into estradiol, not testosterone, and not through direct action of DHEA itself, which led to the activation of the p-AMPK in primary chicken hepatocytes. These data provide novel insight into the mechanisms underlying the action of DHEA in preventing fat deposition, and suggest potential applications for DHEA treatment to control fat deposition or as an agent to treat disorders related to lipid metabolism in animals and humans.

(-)-Hydroxycitric Acid Reduced Lipid Droplets Accumulation Via Decreasing Acetyl-Coa Supply and Accelerating Energy Metabolism in Cultured Primary Chicken Hepatocytes.

BACKGROUND/AIMS: (-)-Hydroxycitric acid (HCA) had been shown to suppress fat accumulation in animals and humans, while the underlying biochemical mechanism is not fully understood, especially little information is available on whether (-)-HCA regulates energy metabolism and consequently affects fat deposition. METHODS: Hepatocytes were cultured for 24 h and then exposed to (-)-HCA (0, 1, 10, 50 µM), enzyme protein content was determined by ELISA; lipid metabolism gene mRNA levels were detected by RT-PCR. RESULTS: (-)-HCA significantly decreased the number and total area of lipid droplets. ATP-citrate lyase, fatty acid synthase and sterol regulatory element binding protein-1c mRNA level were significantly decreased after (-)-HCA treatment, whereas peroxisome proliferator-activated receptor α mRNA level was significantly increased. (-)-HCA significantly decreased ATP-citrate lyase activity and acetyl-CoA content in cytosol, but significantly increased glucose consumption and mitochondrial oxygen consumption rate. (-)-HCA promoted the activity/content of glucokinase, phosphofructokinase-1, pyruvate kinase, pyruvate dehydrogenase, citrate synthase, aconitase, succinate dehydrogenase, malate dehydrogenase, NADH dehydrogenase and ATP synthase remarkably. CONCLUSIONS: (-)-HCA decreased lipid droplets accumulation by reducing acetyl-CoA supply, which mainly achieved via inhibition of ATP-citrate lyase, and accelerating energy metabolism in chicken hepatocytes. These results proposed a biochemical mechanism of fat reduction by (-)-HCA in broiler chickens in term of energy metabolism.

(-)-Hydroxycitric acid reduced fat deposition via regulating lipid metabolism-related gene expression in broiler chickens.

BACKGROUND: Chicken as a delicious food for a long history, and it is well known that excess fat deposition in broiler chickens will not only induced metabolic diseases, but also lead to adverse effect in the consumer's health. (-)-Hydroxycitric acid (HCA), a major active ingredient of Garcinia Cambogia extracts, had shown to suppress fat accumulation in animals and humans. While, the precise physiological mechanism of HCA has not yet been full clarified, especially its action in broiler chickens. Thus, this study aimed to assess the effect of (-)-HCA on lipid metabolism in broiler chickens. METHODS: A total of 120 1-day-old broiler chickens were randomly allocated to four groups, with each group was repeated three times with 10 birds. Birds received a commercial diet supplemented with (-)-HCA at 0, 1000, 2000 or 3000 mg/kg, respectively, for a period of 4 weeks ad libitum. RESULTS: Body weight (BW) in the 2000 and 3000 mg/kg (-)-HCA groups was significantly decreased (P < 0.05) than that in control group. A significantly decreased of serum triglyceride (TG) and density lipoprotein-cholesterol (LDL-C) content were observed in 3000 mg/kg (-)-HCA group (P < 0.05). Broiler chickens supplmented with 2000 and 3000 mg/kg (-)-HCA had pronouncedly higher hepatic lipase (HL) activity, hepatic glycogen and non-esterified fatty acid (NEFA) contents in liver (P < 0.05). Serum free triiodothyronine (FT3) and thyroxin (T4) contents were significantly higher in 3000 mg/kg (-)-HCA group (P < 0.05) compared with the control group. Supplemental (-)-HCA markedly decreased fatty acid synthase (FAS) and sterol regulatory element binding protein-1c (SREBP-1c) (P < 0.05) mRNA levels, while the mRNA abundance of adenosine 5'-monophosphate-activated protein kinaseß2 (AMPKß2) (P < 0.05) was significantly increased. In addition, ATP-citrate lyase (ACLY) mRNA level (P < 0.05) was significantly decreased in broiler chickens supplemented with 3000 mg/kg (-)-HCA. No differences was observed on carnitine palmitoyl transferase-I(CPT-I), while peroxisome proliferators-activated receptor α (PPARα) mRNA level (P < 0.05) was significantly increased in broiler chickens supplemented with 2000 and 3000 mg/kg (-)-HCA. CONCLUSIONS: Supplemental (-)-HCA inhibited lipogenesis by inhibiting ACLY, SREBP-1c and FAS expression, and accelerated lipolysis through enhancing HL activity and PPARα expression, which eventually led to the reduced abdominal fat deposition in broiler chickens. Graphical abstract Mechanism of (-)-HCA effect on hepatic lipids metabolism.

(-)-Hydroxycitric Acid Nourishes Protein Synthesis via Altering Metabolic Directions of Amino Acids in Male Rats.

(-)-Hydroxycitric acid (HCA), a major active ingredient of Garcinia Cambogia extracts, had shown to suppress body weight gain and fat accumulation in animals and humans. While, the underlying mechanism of (-)-HCA has not fully understood. Thus, this study was aimed to investigate the effects of long-term supplement with (-)-HCA on body weight gain and variances of amino acid content in rats. Results showed that (-)-HCA treatment reduced body weight gain and increased feed conversion ratio in rats. The content of hepatic glycogen, muscle glycogen, and serum T4 , T3 , insulin, and Leptin were increased in (-)-HCA treatment groups. Protein content in liver and muscle were significantly increased in (-)-HCA treatment groups. Amino acid profile analysis indicated that most of amino acid contents in serum and liver, especially aromatic amino acid and branched amino acid, were higher in (-)-HCA treatment groups. However, most of the amino acid contents in muscle, especially aromatic amino acid and branched amino acid, were reduced in (-)-HCA treatment groups. These results indicated that (-)-HCA treatment could reduce body weight gain through promoting energy expenditure via regulation of thyroid hormone levels. In addition, (-)-HCA treatment could promote protein synthesis by altering the metabolic directions of amino acids. Copyright © 2016 John Wiley & Sons, Ltd.

Dehydroepiandrosterone-Regulated Testosterone Biosynthesis via Activation of the ERK1/2 Signaling Pathway in Primary Rat Leydig Cells.

BACKGROUND: Dehydroepiandrosterone decreases with age and this reduction has been shown to be associated with physical health in human. Some studies have suggested that the effects of DHEA are exerted after it is biotransformed into more biologically-active hormones in peripheral target cells. This study investigated the effects of DHEA on the testosterone biosynthesis and possible signaling pathway mechanism underlying these DHEA effects were also explored in primary rat Leydig cells. METHODS: Primary Leydig cells were treated with DHEA and then detected testosterone content by RIA and steroidogenic enzymes, ERK1/2 signal pathway factors protein expression level by Western blot. RESULTS: Incubation of primary Leydig cells with DHEA significantly increased testosterone content and 3ß-HSD and 17ß-HSD protein expression levels, while aromatase protein expression levels were decreased. Compared with the control group, p-ERK1/2 and p-CREB protein levels were significantly increased in DHEA-treated groups. Testosterone content was significantly decreased in the DHEA-treated group pre-incubated with U0126 (p-ERK1/2 inhibitor). Additionally, the rise in p-ERK1/2, 3ß-HSD and 17ß-HSD protein levels induced by DHEA was reversed when cells were pre-incubated with U0126. Interestingly, no significant difference was found in aromatase protein expression level in cells pretreated with U0126. CONCLUSION: These findings demonstrate that (a) exogenous DHEA might preferentially convert to testosterone rather than estradiol due to the up-regulation of 3ß-HSD and 17ß-HSD protein levels and the down-regulation of aromatase protein level in primary Leydig cells, and (b) the action of DHEA is at least partly associated with the elevation of p-ERK1/2 and p-CREB protein levels.

The influence of dietary taurine and reduced housing density on hepatic functions in laying hens.

To investigate the influence of dietary taurine and reduced housing density on hepatic functions in laying hens, green-shell laying hens were randomly assigned to 3 groups: a free-range group, a caged group with low-density, and a caged group with high-density. Each group was further divided into the control (C) and taurine-treatment (T) groups. All the test birds were fed the same basic diet, except that the T groups were supplemented with 0.1% taurine. After 15 d, sera and liver were aseptically collected. The results show that dietary taurine supplementation and reduced housing density significantly attenuated physiopathological changes in the liver. When compared with the free-range group, serum alanine aminotransterase and aspartate aminotransterase in the caged hens were significantly higher and were deceased by taurine (P < 0.05). Serum inducible nitric oxide synthase activity in caged hens was higher than that in free-range hens, and taurine reduced serum inducible nitric oxide synthase activities in the low-density group (P < 0.05). Nuclear factor-κB DNA-binding activity increased significantly in the high-density housing group when compared with the other 2 housing patterns and was decreased by taurine (P < 0.05). Taurine reduced the expression of tumor necrosis factor-α mRNA in all 3 rearing patterns, IL-4 mRNA expression in the high-density group, and IL-10 in the low-density group (P < 0.05). Malondialdehyde levels decreased in serum and liver from T groups and serum total antioxidation capability levels increased significantly (P < 0.05) in the low-density group. Dietary taurine supplementation decreased acetyl-CoA and sterol regulatory element-binding protein-1c mRNA expression in the high-density groups (P < 0.05). Taurine significantly increased lipoprotein lipase mRNA expression in the high-density group and peroxisome proliferator receptor mRNA expression both in the low- and high-density groups (P < 0.05). Taurine supplementation reduced total cholesterol levels in the low- and high-density groups, decreased triglyceride and low-density lipoprotein cholesterol levels in high-density groups, and increased high-density lipoprotein cholesterol levels in all 3 rearing patterns (P < 0.05). Our data demonstrate that dietary taurine and reduced housing density offer significant protection from hepatic damage in laying hens.

Genistein alleviates chronic heat stress-induced lipid metabolism disorder and mitochondrial energetic dysfunction by activating the GPR30-AMPK-PGC-1α signaling pathways in the livers of broiler chickens.

The objective of this study was to investigate the preventive effects and mechanisms of genistein (GEN) on production performance and metabolic disorders in broilers under chronic heat stress (HS). A total of 120 male 3-wk-old Ross broilers were randomly assigned to 5 groups: a thermoneutral zone (TN) group maintained at normal temperature (21°C ± 1°C daily), an HS group subjected to cyclic high temperature (32°C ± 1°C for 8 h daily), and 3 groups exposed to HS with varying doses of GEN (50, 100, or 150 mg/kg diet). The experimental period lasted for 3 wk. Here, HS led to a decline in growth performance parameters and hormone secretion disorders (P < 0.05), which were improved by 100 and 150 mg/kg GEN treatment (P < 0.05). Moreover, the HS-induced increases in the liver index (P < 0.01) and abdominal fat rate (P < 0.05) were attenuated by 150 mg/kg GEN (P < 0.05). The HS-induced excessive lipid accumulation in the liver and serum (P < 0.01) was ameliorated after 100 and 150 mg/kg GEN treatment (P < 0.05). Furthermore, the HS-induced decreases in lipolysis-related mRNA levels and increases in lipid synthesis-related mRNA levels in the liver (P < 0.01) were effectively blunted after 100 and 150 mg/kg GEN treatment (P < 0.05). Importantly, the HS-stimulated hepatic mitochondrial energetic dysfunction and decreases in the mRNA or protein levels of peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α), nuclear respiratory factor 1, and mitochondrial transcription factor A in the liver were ameliorated by 150 mg/kg GEN (P < 0.05). Moreover, 50 to 150 mg/kg GEN treatment resulted in a significant increase in the mRNA or protein levels of G protein-coupled estrogen receptor (GPR30), AMP-activated protein kinase (AMPK) α1, phosphorylated AMPKα, and phosphorylated acetyl-CoA carboxylase α. Collectively, GEN alleviated metabolic disorders and hepatic mitochondrial energetic dysfunction under HS, possibly through the activation of GPR30-AMPM-PGC-1α pathways. These data provide a sufficient basis for GEN as an additive to alleviate HS in broilers.

Dioscin improves fatty liver hemorrhagic syndrome by promoting ERα-AMPK mediated mitophagy in laying hens.

BACKGROUND: Mitochondria play a crucial role in upholding metabolic homeostasis. Mitochondrial damage closely associated with the pathogenesis of fatty liver hemorrhagic syndrome (FLHS), while mitophagy being among the most effective methods for eliminating the damaged mitochondria. Dioscin, a natural extract, can activate autophagy; however, its effects on FLHS regarding mitophagy regulation remain unelucidated. PURPOSE: We explored the impact of dioscin on FLHS induced by a high-energy and low-protein (HELP) diet in laying hens, mainly focused the protective effects of dioscin on mitochondrial injury. METHOD: To investigate the impact of dioscin on fatty liver syndrome in laying hens, we first induced the condition by feeding them a high-energy and low-protein diet. Then, we assessed lipid metabolism-related markers using oil red staining and a commercial detection kit. In addition, the role of dioscin on fatty liver syndrome in laying hens was confirmed by assessing the activation of hepatocyte fat deposition and hepatocyte apoptosis; and the mechanism of dioscin in FLHS was investigated through LMH cell experiment in vitro. Furthermore, CETSA and molecular docking were conducted for additional confirmation. RESULT: The results showed that dioscin alleviated mitochondrial damage, relieved the excessive deposition of hepatic lipid droplets and oxidative stress induced by HELP diet in laying hens. Furthermore, dioscin regulated the mitophagy by activating the estrogen receptor α (ERα)/adenosine 5'-monophosphate-activated protein kinase (AMPK) signaling pathway, thus mitigating mitochondria injury and apoptosis in hepatocytes. In addition, we found that dioscin promoted the translocation of nuclear transcription factor into nucleus by activating ERα-AMPK signaling, facilitating autophagic flux in the liver of laying hens and LMH cells. Furthermore, cells pretreated with the lysosomal acidification inhibitor bafilomycin A1 blocked the inhibitory effect of dioscin on the apoptosis induced by palmitic acid (PA)-stimulation in LMH cells, suggesting that dioscin reduces PA-induced apoptosis by activating mitophagy. Moreover, dioscin-induced lysosomal acidification and mitochondrial biogenesis were reversed in PA-induced LMH cells pretreated with ERα-specific inhibitor methylpiperidino pyrazole. CONCLUSION: This study firstly demonstrated that dioscin alleviates fatty liver syndrome induced by HELP diet in laying hens. The findings from this study illustrated that dioscin plays a significant role in reducing mitochondrial damage and apoptosis, and these beneficial effects mainly achieve through promotion of ERα-AMPK signaling, which mediates autophagy within the liver of laying hens fed a HELP-diets. These findings provide a theoretical basis for considering dioscin as a possible treatment option for mitigating FLHS in egg-laying hens.

Synergistic effect of genistein and adiponectin reduces fat deposition in chicken hepatocytes by activating the ERß-mediated SIRT1-AMPK signaling pathway.

Dietary supplementation with bioactive substances that can regulate lipid metabolism is an effective approach for reducing excessive fat deposition in chickens. Genistein (GEN) has the potential to alleviate fat deposition; however, the underlying mechanism of GEN's fat-reduction action in chickens remains unclear. Therefore, the present study aimed to explore the underlying mechanism of GEN on the reduction of fat deposition from a novel perspective: intercellular transmission of adipokine between adipocytes and hepatocytes. The findings showed that GEN enhanced the secretion of adiponectin (APN) in chicken adipocytes, and the enhancement effect of GEN was completely blocked when the cells were pretreated with inhibitors targeting estrogen receptor ß (ERß) or proliferator-activated receptor γ (PPARγ) signals, respectively. Furthermore, the results demonstrated that both co-treatment with GEN and APN or treatment with the medium supernatant (Med SUP) derived from chicken adipocytes treated with GEN significantly decreased the content of triglyceride and increased the protein levels of ERß, Sirtuin 1 (SIRT1) and phosphor-AMP-activated protein kinase (p-AMPK) in chicken hepatocytes compared to the cells treated with GEN or APN alone. Moreover, the increase in the protein levels of SIRT1 and p-AMPK induced by GEN and APN co-treatment or Med SUP treatment were blocked in chicken hepatocytes pretreated with the inhibitor of ERß signals. Importantly, the up-regulatory effect of GEN and APN co-treatment or Med SUP treatment on the protein level of p-AMPK was also blocked in chicken hepatocytes pretreated with a SIRT1 inhibitor; however, the increase in the protein level of SIRT1 induced by GEN and APN co-treatment or Med SUP treatment was not reversed when the hepatocytes were pretreated with an AMPK inhibitor. In conclusion, the present study demonstrated that GEN enhanced APN secretion by activating the ERß-Erk-PPARγ signaling pathway in chicken adipocytes. Subsequently, adipocyte-derived APN synergized with GEN to activate the ERß-mediated SIRT1-AMPK signaling pathway in chicken hepatocytes, ultimately reducing fat deposition. These findings provide substantial evidence from a novel perspective, supporting the potential use of GEN as a dietary supplement to prevent excessive fat deposition in poultry.

Dimethyl fumarate restores Ca2+ dyshomeostasis through activation of the SIRT1 signal to treat nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is characterized by an excessive lipid accumulation in the liver, with a global prevalence of approximately 25 %. While early-stage steatosis is reversible and can be intervened upon, it has the potential to progress to some serious complications, including cirrhosis and even liver cancer. Dimethyl fumarate (DMF), a derivative of fumaric acid shows promise in intervening in certain diseases. However, the precise effect and underlying mechanism of DMF on hepatic steatosis remain unclear. In this study, we demonstrated that DMF mitigates hepatic steatosis in mice subjected to high-fat/high-cholesterol (HFHC) diets. Meanwhile, our in vivo and in vitro results showed that DMF relieves lipid accumulation, oxidative stress, and endoplasmic reticulum (ER) stress. Mechanically, our findings revealed that the effect of DMF on reducing lipid accumulation is linked to the restoration of Ca2+ homeostasis. Furthermore, we found that activation of the SIRT1 signal by DMF plays an important role in correcting the mishandling of the Ca2+ signal, and knockdown of SIRT1 expression reverses the beneficial role of DMF PA-incubated AML12 cells. In conclusion, our results suggested DMF's amelioration of hepatic steatosis is related to the activation of SIRT1-mediated Ca2+ signaling.

Synthesis and antioxidant activity of novel Mannich base of 1,3,4-oxadiazole derivatives possessing 1,4-benzodioxan.

A new series of Mannich base of 1,3,4-oxadiazole derivatives possessing 1,4-benzodioxan (6a-6ae) were synthesized and characterized by (1)H NMR, ESI-MS and elemental analysis. The structure of 6b was further confirmed by single crystal X-ray diffraction. All these novel compounds were screened for their in vitro antioxidant activity employing 2,2'-diphenyl-1-picrylhydrazyl radical (DPPH), 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonate) cationic radical (ABTS(+)) and ferric reducing antioxidant power (FRAP) scavenging assays. Due to the combination of 1,4-benzodioxan, 1,3,4-oxadiazoles and substituted phenyl ring, most of them exhibited nice antioxidant activities. In all of these three assays mentioned above, compounds 6f and 6e showed significant radical scavenging ability comparable to the commonly used antioxidants, BHT and Trolox. Seven compounds with representative substituents or activities were selected for further assays in chemical simulation biological systems-inhibition of microsomal lipid peroxidation (LPO) and protection against 2,2'-azobis (2-amidinopropane hydrochloride) (AAPH) induced DNA strand breakage, in which, 6f and 6e were demonstrated to be of the most potent antioxidant activities.

Flavipin in Chaetomium globosum CDW7, an endophytic fungus from Ginkgo biloba, contributes to antioxidant activity.

1,2-Benzenedicarboxaldehyde-3,4,5-trihydroxy-6-methyl (flavipin) was found to be antagonistic against nematodes and fungi. Here we demonstrated that flavipin is a potent antioxidant in vitro and in vivo, which has great potential in the therapy for free radical-associated diseases. Therefore, flavipin-producing bio-source was screened from 80 endophytes in Ginkgo biloba. Seven endophytic fungi were able to synthesize antioxidant substances and identified by ITS rDNA sequences. Among them, Chaetomium globosum CDW7 was a remarkable producer of flavipin. The fermentation parameters of CDW7 were then optimized for high flavipin production. Cultured under the optimal condition (25 °C, 100/250 mL flask, 12 discs/flask, 150 rpm, pH 6.5) for 14 days, CDW7 was able to synthesize flavipin at a production of 315.5 mg/L. In addition, flavipin output was positively correlated to antioxidant activities of crude extracts with a correlation coefficient of 0.8235, indicating that flavipin was the major antioxidant component of CDW7's metabolites. These data demonstrated that CDW7 was a highly yielded bio-source of antioxidant flavipin.

Dehydroepiandrosterone alleviates oleic acid-induced lipid metabolism disorders through activation of AMPK-mTOR signal pathway in primary chicken hepatocytes.

The incident of lipid metabolism disorders has obviously increased under the undue pursuit of efficiency, which had seriously threatened to the health development of poultry industry. As an important cholesterol-derived intermediate, though dehydroepiandrosterone (DHEA) has the fat-reduction effect in animals and humans, but the underlying mechanism still poorly understood. Herein, the present study aimed to investigate the regulatory effects and its molecular mechanism of DHEA on disturbance of lipid metabolism induced by oleic acid (OA) in primary chicken hepatocytes. The hepatocytes were treated with 0, 0.1, 1, 10 µM DHEA for 4 h, and then supplemented with 0 or 0.5 mM OA stimulation for another 24 h. Our findings demonstrated that DHEA treatment effectively reduced TG content and alleviated lipid droplet deposition in OA-induced hepatocytes. DHEA inhibited the lipogenesis related factors (ACC, FAS, SREBP-1c, and ACLY) mRNA level and increased the lipolysis key factors (CPT-1 and PPARα) mRNA levels. In addition, DHEA obviously elevated the protein levels of CPT-1A, p-ACC, and ECHS1; whereas decreased the protein levels of FAS and SREBP-1 in hepatocytes stimulated by OA. Furthermore, DHEA promoted the phosphorylation of AMP-activated protein kinase (AMPK) and inhibited the phosphorylation of mammalian target of rapamycin (mTOR). Mechanistically, the hepatocytes were pre-treated with AMPK inhibitor compound C or AMPK activator AICAR before addition of DHEA treatment, and the results certified that DHEA activated cAMP/AMPK pathway and which subsequently led the inhibition of mTOR signal, which finally reduced the fat excessive accumulation in OA-stimulated hepatocytes. Collectively, our study unveiled that DHEA protects against the lipid metabolism disorders triggered by OA stimulation through activation of AMPK-mTOR signaling pathway, which prompts the value of DHEA as a potential nutritional supplement in regulating the lipid metabolism and its related disease in poultry.

Genistein activated SIRT1-AMPK signaling pathway mediated by ERß-FOXO1-Nampt to reduce fat accumulation in chicken hepatocytes.

Excessive fat accumulation in broiler chickens would seriously threaten the poultry industry. It leads to lower feed conversion rate and worse meat quality. Even worse, it harms the consumers' health due to the intake of high-fat chicken products. Dietary supplements with bioactive ingredients have been considered an effective way to solve this problem. Genistein is the primary phytoestrogen in soybean. Its fat-reduction effect has been reported, but the molecular mechanism is unclear. The present study found that genistein reduced lipid droplets accumulation by regulating lipid metabolism-related factors expression in chicken hepatocytes. The research showed that genistein significantly increased phosphor (p)-AMP-activated protein kinase (p-AMPK) and Sirtuin 1 (SIRT1) protein expressions. The effect of genistein on reducing lipid droplets accumulation and upregulating p-AMPK protein level was blocked entirely when pretreated with SIRT1 inhibitor. These results implied that SIRT1 is required to activate AMPK. Furthermore, genistein treatment significantly upregulated the SIRT1 protein level when pretreated with AMPK inhibitor. We demonstrated that the activation of estrogen receptor ß-Forkhead box O1-Nicotinamide phosphoribosyl transferase (ERß-FOXO1-Nampt) signaling pathway upregulated the NAD+ concentration in hepatocytes, and activated SIRT1 ultimately. In summary, we demonstrated that genistein suppressed lipid droplets accumulation in chicken hepatocytes by activating SIRT1-AMPK. The SIRT1-AMPK signaling pathway was mediated by ERß-FOXO1-Nampt. These findings increase our understanding of the mechanisms of genistein on fat reduction, and provide compelling evidence for it as a nutritional supplement to prevent excessive fat deposition and lipid metabolism-related diseases in animals and even humans.