6-PPD triggered lipid metabolism disorder and inflammatory response in larval zebrafish (Danio rerio) by regulating PPARγ/NF-κB pathway.

The fatty liver is one of the main problems in aquaculture. In addition to the nutritional factors, endocrine disrupter chemicals (EDCs) are one of the causes of fatty liver in fish. Bisphenol A (BPA) is a plasticizer widely used in the production of various plastic products and exhibits certain endocrine estrogen effects. Our previous study found that BPA could increase the accumulation of triglyceride (TG) in fish liver by disturbing the expression of lipid metabolism-related genes. How to recover the lipid metabolism disorder caused by BPA and other environmental estrogens remains to be explored. In the present study, Gobiocypris rarus was used as a research model, and 0.01% resveratrol, 0.05% bile acid, 0.01% allicin, 0.1% betaine, and 0.01% inositol were added to the feed of the G. rarus that exposed to 15 μg/L BPA. At the same time, a BPA exposure group without feed additives (BPA group) and a blank group with neither BPA exposure nor feed additives (Con group) were setted. The liver morphology, hepatosomatic index (HSI), hepatic lipid deposition, TG level, and expression of lipid metabolism-related genes were analyzed after 5 weeks of feeding. The HSI in bile acid and allicin groups was significantly lower than that in Con group. The TG in resveratrol, bile acid, allicin, and inositol groups returned to Con level. Principal component analysis of TG synthesis, decomposition, and transport related genes showed that dietary bile acid and inositol supplementation had the best effect on the recovery of BPA-induced lipid metabolism disorder, followed by allicin and resveratrol. In terms of lipid metabolism-related enzyme activity, bile acid and inositol were the most effective in recovering BPA-induced lipid metabolism disorders. The addition of these additives had a restorative effect on the antioxidant capacity of G. rarus livers, but bile acids and inositol were relatively the most effective. The results of the present study demonstrated that under the present dosage, bile acids and inositol had the best improvement effect on the fatty liver of G. rarus caused by BPA. The present study will provide important reference for solving the problem of fatty liver caused by environmental estrogen in aquaculture.

The adiponectin receptor agonist AdipoRon alleviates lipotoxic injury in LMH cells

Sulfated Polysaccharides from Enteromorpha prolifera Attenuate Lipid Metabolism Disorders in Mice with Obesity Induced by a High-Fat Diet via a Pathway Dependent on AMP-Activated Protein Kinase

Puerarin attenuates cadmium-induced hepatic lipid metabolism disorder by inhibiting oxidative stress and inflammation in mice

Obesity is characterized by excessive lipid accumulation and chronic inflammation that disrupt metabolic homeostasis. This study investigated the effects of Lactiplantibacillus plantarum GBCC_F0227 on lipid metabolism using differentiated 3T3-L1 adipocytes and a high-fat diet (HFD)-induced obese mouse model. In 3T3-L1 adipocytes, exposure to GBCC_F0227 culture supernatant was associated with reduced intracellular triglyceride accumulation and lower expression of lipid metabolism-related genes. Consistent with the in vitro observations, oral administration of GBCC_F0227 attenuated body weight gain and adipose tissue expansion in HFD-fed mice, together with lower expression of HFD-induced lipid metabolism-related genes (Pparg, Fabp4, Dgat2, and Cs) in white adipose tissue (WAT). GBCC_F0227 treatment also reduced the expression of inflammatory markers (Il-6, Il-1b) as well as Leptin, in epididymal WAT. In the liver, GBCC_F0227 administration lowered the expression of Pparg and Cd36 and reduced hepatic lipid accumulation. Collectively, these findings indicate that GBCC_F0227 administration is associated with coordinated changes in lipid accumulation and lipid metabolism-related gene expression in WAT and liver under HFD conditions. These results highlight the potential of GBCC_F0227 as a probiotic candidate for modulating obesity-associated lipid metabolic alterations.

Tributyltin triggers lipogenesis in macrophages via modifying PPARγ pathway.

Flower of Citrus aurantium L. var. amara Engl. (CAVA) has been confirmed to have promising anti-obesity effects. However, the regulation of alkaloid extracts from flower of CAVA (Al) on lipid metabolism remain unknown. In this study, Al was optimized by ultrasound-assisted extraction using response surface methodology. The optimal conditions were ultrasonic time 72 min, ethanol concentration 78% and liquid/solid ratio 30 ml/g with the maximum alkaloid yield 5.66%. LC-MS assay indicated that the alkaloid compounds were enriched in Al after optimization. Nine alkaloid compounds were identified in Al by LC-MS assay and stachydrine, caffeine and cathine appeared as the major alkaloid compounds. Bioactivity assay showed that Al treatment significantly increased superoxide dismutase (SOD) activity, and reduced malonaldehyde (MDA) and reactive oxygen species (ROS) levels. Al administration also reversed oleic acid-induced hepatic steatosis in Hep G2 cells by inhibiting the expression of lipogenesis-signaling genes including fatty acid synthase (FAS), peroxisome proliferator-activated receptor subtype γ (PPARγ), uncoupling protein 2 (UCP2), and retinol binding protein (RBP4). However, OA-induced reduction of lipolysis-related gene carnitine palmitoyl transferase 1A (CPT1A) in Hep G2 cells was not improved by Al supplementation. Moreover, the increased SOD activity and decreased MDA and ROS contents were also observed in Caenorhabditis elegans by Al addition. Al intervention exhibited the ability to inhibit lipid accumulation in C. elegans by suppressing expression of lipid metabolism-related genes. These results suggested that the alkaloid extracts from the flower of CAVA showed great potential to regulate lipid metabolism. PRACTICAL APPLICATIONS: The extraction of alkaloid extracts from the flower of CAVA was optimized with a maximum yield of 5.66%. The regulatory effects and mechanisms of Al on lipid metabolism of Hep G2 cells and Caenorhabditis elegans were also investigated. More clinical studies are required to evaluate the potential of using alkaloids from the flower of CAVA as therapeutic agents against lipid metabolic disorders.

Protective effects of AdipoRon on the liver of Huoyan goose fed a high-fat diet

Ginsenoside CK regulates non-alcoholic fatty liver disease by regulating liver fat metabolism and gut microbiota.

Short-term developmental effects and potential mechanisms of azoxystrobin in larval and adult zebrafish (Danio rerio)

Arsenic is a widespread environmental toxic agent that has been shown to cause diverse tissue and cell damage and at the same time to be an effective anti-cancer therapeutic agent. The objective of this study is to explore the signaling mechanisms involved in arsenic toxicity. We show that the IkappaB kinase beta (IKKbeta) plays a crucial role in protecting cells from arsenic toxicity. Ikkbeta(-)(/)(-) mouse 3T3 fibroblasts have decreased expression of antioxidant genes, such as metallothionein 1 (Mt1). In contrast to wild type and IKKbeta-reconstituted Ikkbeta(-)(/)(-) cells, IKKbeta-null cells display a marked increase in arsenic-induced reactive oxygen species (ROS) accumulation, which leads to activation of the MKK4-c-Jun NH(2)-terminal kinase (JNK) pathway, c-Jun phosphorylation, and apoptosis. Pretreatment with the antioxidant N-acetylcysteine (NAC) and expression of MT1 in the Ikkbeta(-)(/)(-) cells prevented JNK activation; moreover, NAC pretreatment, MT1 expression, MKK4 ablation, and JNK inhibition all protected cells from death induced by arsenic. Our data show that two signaling pathways appear to be important for modulating arsenic toxicity. First, the IKK-NF-kappaB pathway is crucial for maintaining cellular metallothionein-1 levels to counteract ROS accumulation, and second, when this pathway fails, excessive ROS leads to activation of the MKK4-JNK pathway, resulting in apoptosis.

To investigate the effect of Hepatitis B Virus X Protein (HBx) on the expression of lipid metabolism-related genes and its role in pathogenesis of hepatocyte fatty degeneration. Hepatitis B Virus X gene eukaryon expression vector pIRES2-eGFP-HBx was transfected into HepG2 cells to establish HepG2/HBx cell model for HBx expression. HepG2 cells transfected with pIRES2-eGFP (HepG2/pIRES2 cell) and non-transfected were used as controls. At 24, 48 and 72 hours after transfection, the expression of green fluorescent protein (GFP) was observed by fluorescence microscope and the triglyceride(TG) content was detected. RT-PCR and Western blot were applied to detect the levels of sterol regulatory element binding protein-1 (SREBP-1), liver x receptor alpha (LXRalpha) mRNA and the levels of HBx, LXRalpha and fatty acid synthase (FAS) protein. At 24, 48 and 72 hours after transfection, the expression of GFP was found in HepG2/HBx and HepG2/pIRES2 cells, and increased gradually. The expression of HBx was detected only in HepG2/HBx cells, and was increased with time after transfection (F = 32.21, P less than 0.01). These suggested successful obtaining of HepG2-HBx cell model for HBx expression. At 24h, 48h and 72h after transfection, the expression levels of LXRalpha mRNA (0.386+/-0.055, 0.505+/-0.071, 0.649+/-0.058 ) and SREBP-1 mRNA (0.395+/-0.055, 0.548+/-0.047, 0.795+/-0.058), as well as the levels of LXRalpha protein(0.178+/-0.036, 0.263+/-0.047, 0.347+/-0.058) and FAS protein(0.436+/-0.055, 0.608+/-0.053, 0.827+/-0.046) in HepG2-HBx group were dramatically higher than those in the controls at the same time points (all P less than 0.05/0.01), and were gradually increased with time (all P less than 0.05/0.01). A positive correlationship was observed between HBX protein level and the LXRalpha, SREbP-1 mRNA and LXRalpha, FAS protein levels. The difference of TG content between HepG2/HBx group and control groups was not statistically significant (P more than 0.05). HBx-LXRalpha-SREBP-1/FAS pathway suggested regulating transcription and expression of lipid metabolism-related genes, which might be one of the important molecular mechanism causing hepatocyte fatty degeneration.

To explore the effects of feeding mode on biological clock and circadian expression of lipids metabolism-related genes in mice. Ninety healthy male ICR mice were divided into 3 groups with 30 in each: ad libitum-feeding, daytime-feeding and nighttime-feeding groups, in a 12 h to 12 h light-dark cycle. After two weeks of feeding the animals was sacrificed in batches (5 in each batch) at 4, 8, 12, 16, 20 and 24 h, the circadian expression of lipids metabolism-related genes in the liver and brain was detected by real time quantitative RT PCR at 6 time points. The circadian oscillator in the brain was more sensitive to alteration of feeding mode than that in the liver, nighttime feeding decreased peak mRNA levels of Cry2, Per1, and Per2 (5.5, 4.3 and 7.1 folds, respectively) in the brain. However, there was no difference in the expression rhythm of hepatic clock genes between nighttime-feeding and ad libitum group. In addition, changed feeding mode significantly decreased the peak value of Rev erbα (2 folds for daytime feeding, 3.4 folds for nighttime feeding) and Dbp (10.6 folds for daytime feeding, 2.8 folds for nighttime feeding), which two had opposite expression mode in different feeding modes. Different expression rhythm of lipid metabolism related genes SREBP1-c, PPARα, FAS, and CPT was shown with decreased mRNA expression levels of SREBP1-c and PPARα in daytime feeding (5.5 folds, 4 folds) and nighttime feeding (4.4 folds, 4.8 folds). Changing the feeding mode could entrain circadian oscillators both in the brain and liver. What is more, hepatic circadian oscillators couple with the feeding time.

Accumulation of lipid in the liver is the first hallmark of both alcohol-related liver disease (ALD) and non-alcohol-related fatty liver disease (NAFLD). Recent studies indicate that specific mutations in lipid genes confer risk and might influence disease progression to irreversible liver cirrhosis. This study aimed to understand the function/s of lipid risk genes driving disease development in zebrafish genetic models of alcohol-related and non-alcohol-related fatty liver. We used zebrafish larvae to investigate the effect of alcohol and high fat to model fatty liver and tested the utility of this model to study lipid risk gene functions. CRISPR/Cas9 gene editing was used to create knockdowns in 5 days post-fertilisation zebrafish larvae for the available orthologs of human cirrhosis risk genes (pnpla3, faf2, tm6sf2). To establish fatty liver models, larvae were exposed to ethanol and a high-fat diet (HFD) consisting of chicken egg yolk. Changes in morphology (imaging), survival, liver injury (biochemical tests, histopathology), gene expression (qPCR) and lipid accumulation (dye-specific live imaging) were analysed across treatment groups to test the functions of these genes. Exposure of 5-day post-fertilisation (dpf) WT larvae to 2% ethanol or HFD for 48 h developed measurable hepatic steatosis. CRISPR-Cas9 genome editing depleted pnpla3, faf2 and tm6sf2 gene expression in these CRISPR knockdown larvae (crispants). Depletion significantly increased the effects of ethanol and HFD toxicity by increasing hepatic steatosis and hepatic neutrophil recruitment ≥2-fold in all three crispants. Furthermore, ethanol or HFD exposure significantly altered the expression of genes associated with ethanol metabolism (cyp2y3) and lipid metabolism-related gene expression, including atgl (triglyceride hydrolysis), axox1, echs1 (fatty acid β-oxidation), fabp10a (transport), hmgcra (metabolism), notch1 (signalling) and srebp1 (lipid synthesis), in all three pnpla3, faf2 and tm6sf2 crispants. Nile Red staining in all three crispants revealed significantly increased lipid droplet size and triglyceride accumulation in the livers following exposure to ethanol or HFD. We identified roles for pnpla3, faf2 and tm6sf2 genes in triglyceride accumulation and fatty acid oxidation pathways in a zebrafish larvae model of fatty liver.

The Hypothalamic-Pituitary-Adrenal (HPA) axis and its final effector, glucocorticoids (GCs), are important players in maintaining homeostasis of an organism upon stress exposure. However, overexposure to GCs during early life is involved in developmental programming of the HPA axis and is linked to detrimental effects in health. The hypothalamus is a key target for developmental programming due to its pivotal role as an integrator of input signals coming from sensory systems and other brain regions and as a translator of neuronal signals into endocrine signals. Yet, little is known about the molecular mechanisms involved in hypothalamic programming mediated by stressful experiences during early life. Elucidation of these mechanisms is essential for understanding the link between early life stress, dysregulation of the stress response, and detrimental health in later stages.
\nHere, I used the zebrafish model to elucidate the molecular correlates of early adverse experience in hypothalamic cells. First, I developed a stimulation protocol using vortex flows to activate the hypothalamic-pituitary-interrenal (HPI) axis, the homolog of the HPA axis in teleost, and characterized the stress response at early stages by measuring cortisol (the main GC in zebrafish) and behavioral correlates. I then identified a critical time window in which HPI axis activity matures. Subsequently, I established an early life stress protocol to induce hypercortisolic states and alter stress response maturation. Endocrinological, behavioral, and cellular characterization of the early life stress paradigm showed an overall downregulation of the stress response with attenuated locomotor and cortisol response to subsequent stressful events as well as reduced calcium activity and expression of stress related peptides (AVP, CRH, and OXT) in hypothalamic cells. To dissect the molecular correlates of early adverse experience, I then performed transcriptomic analysis of hypothalamus-specific cell populations after exposure to the early life stress paradigm. Candidate molecules involved in the adaptive process occurring in hypothalamic cells were identified. Moreover, gene ontology and pathway analysis showed that lipid metabolism and molecular transport pathways were downregulated after zebrafish larvae were subjected to the early life stress protocol. In contrast, cellular movement and inflammatory response pathways were upregulated. Finally, I characterized the cortisol profiles of optogenetic and targeted transgenic tools which have been generated to manipulate the HPI axis activity in freely swimming larvae. Here, I show evidence of altered levels of endogenous cortisol in larvae that were manipulated at any of the three levels of the HPI axis. 
\nAltogether, the main contributions of this thesis are: 1) establishment of a novel stress protocol to activate the HPI axis in zebrafish larvae in a highly controlled and strength-dependent manner; 2) characterization of the cortisol response of developing zebrafish and identification of a critical time window of stress response maturation; 3) development of an early life stress paradigm and elucidation of the effects of early adverse experience at the cellular, behavioral, and endocrinological level; 4) identification of candidate molecules and metabolic pathways in hypothalamic cells involved in adaptive processes after early adverse experience, and 5) characterization of the cortisol profiles of optogenetic and genetic tools to manipulate the HPI axis activity at any of its three levels (hypothalamus, pituitary, and interrenal gland).