Sirt3-Sod2-mROS-Mediated Manganese Triggered Hepatic Mitochondrial Dysfunction and Lipotoxicity in a Freshwater Teleost.

Exposure to excessive manganese (Mn) is toxic to humans and animals. However, the toxic effects and mechanisms of excessive Mn influencing the vertebrates have been highly overlooked. In the present study, dietary Mn overload significantly increased hepatic lipid and Mn contents, decreased superoxide dismutase 2 (Sod2) activity, increased the Sod2 acetylation level, and induced mitochondrial dysfunction; Mn induced mitochondrial dysfunction through Mtf1/sirtuin 3 (Sirt3)-mediated acetylation of Sod2 at the sites K55 and K70. Meanwhile, mitochondrial oxidative stress was involved in Mn-induced lipotoxicity. Mechanistically, Mn-induced lipotoxicity was via oxidative stress-induced Hsf1 nucleus translocation and its DNA binding capacity to the regions of a peroxisome proliferator-activated receptor g (pparg) promoter, which in turn induced the transcription of lipogenic-related target genes. For the first time, our study demonstrated that Mn-induced hepatic lipotoxicity via a mitochondrial oxidative stress-dependent Hsf1/Pparg pathway and Mtf1/sirt3-mediated Sod2 acetylation participated in mitochondrial dysfunction. Considering that lipid metabolism and lipotoxicity are widely used as the biomarkers for environmental assessments of pollutants, our study provided innovative and important insights into Mn toxicological and environmental evaluation in aquatic environments.

Selenoprotein F (SELENOF)-mediated AKT1-FOXO3a-PYGL axis contributes to selenium supranutrition-induced glycogenolysis and lipogenesis.

Mounting evidence showed that excess selenium (10.0-15.0-fold of adequate Se) intake caused severe hepatic lipid deposition in the vertebrate. However, the underlying mechanism remains unclear. The study was performed to elucidate the mechanism of Se supranutrition mediated-changes of lipid deposition and metabolism. We found that dietary excessive Se addition increased hepatic TGs and glucose contents, up-regulated lipogenic enzyme activities and reduced hepatic glycogen contents. Transcriptomic and immunoblotting analysis showed that Se supranutrition significantly influenced serine/threonine kinase 1 (AKT1)-forkhead box O3a (FOXO3a)-PYGL signaling and protein levels of SELENOF. Knockdown of SELENOF and PYGL by RNA interference revealed that the AKT1-FOXO3a-PYGL axis was critical for Se supranutrition-induced lipid accumulation. Moreover, Se supranutrition-induced lipid accumulation was via the increased DNA binding capacity of FOXO3a to PYGL promoter, which increased glycogenolysis, and accordingly promoted lipogenesis and lipid accumulation. Our finding provides new insight into the mechanism of Se supranutrition-induced lipid accumulation and suggests that SELENOF may be a therapeutic target for Se supranutrition induced-lipid disorders in the vertebrates.

Lipophagy mediated glucose-induced changes of lipid deposition and metabolism via ROS dependent AKT-Beclin1 activation.

High dietary carbohydrate intake leads to lipid accumulation in the intestinal tract, but the molecular mechanism remains unknown. In the present study, using yellow catfish (Pelteobagrus fulvidraco) as a model, we found that (1) high carbohydrate diets (HCD) and high glucose (HG) increased lipid deposition, up-regulated lipogenesis and fatty acid ß-oxidation, activated autophagy and induced oxidative stress in the intestinal tissues and intestinal epithelial cells (IECs); (2) lipophagy alleviated HG-induced lipid accumulation via the up-regulation of fatty acid ß-oxidation; (3) Akt interacted directly with Beclin1; (4) HG suppressed Akt1 phosphorylation, downregulated Akt1-mediated phosphorylation of Beclin1, activated lipophagy and alleviated the increment of TG deposition induced by HG with S87 and S292 being the key phosphorylation residues of Beclin1 in response to HG; (5) ROS generation mediated HG-induced activation of lipophagy and HG-induced suppression of AKT phosphorylation, activated AMPK and alleviated HG-induced increase of TG deposition. Our study provides mechanistic evidence that high carbohydrate- and glucose-induced lipophagy in intestine and IECs is associated with ROS-AKT-Beclin1-dependent activation of autophagy, which alleviates glucose-induced lipid accumulation. Our findings are important since the regulation of autophagy can be used as potential molecular targets for the prevention and treatment of lipotoxicity in the intestine of vertebrates, including humans.

Physiological and transcriptomic analyses reveal the toxicological mechanism and risk assessment of environmentally-relevant waterborne tetracycline exposure on the gills of tilapia (Oreochromis niloticus).

With the increasing application of tetracycline (TC) in medical treatment, animal husbandry and aquaculture in recent decades, high quantities of TC have been frequently detected in the aquatic environment, and accordingly TC-related toxicity and environmental pollution have become a global concern. The present study was performed to explore the toxicological influences of TC exposure at its environmentally relevant concentrations on the gills of tilapia Oreochromis niloticus, based on the alteration in histopathology, oxidative stress, inflammatory response, cell cycle, mitochondrial function, apoptosis, and transcriptomic analysis. Our findings revealed that TC exposure damaged the structure and function, induced oxidative stress, affected inflammatory responses, and reduced Na+/K+-ATPase (NKA) activity in the gills. TC also caused the inhibition in cell cycle, resulted in mitochondrial dysfunction and activated apoptosis. Further transcriptomic analysis indicated the extensive influences of TC exposure on the gill function, and immune system was the main target to waterborne TC exposure. These results elucidated that environmental TC had more complex toxicological effects on gills of fish than previously assessed, and provided novel insight into molecular toxicology of TC on fish and good basis for assessing the environmental risk of TC.

Dietary selenium sources differentially regulate selenium concentration, mRNA and protein expression of representative selenoproteins in various tissues of yellow catfish Pelteobagrus fulvidraco.

The study was conducted to determine the effects of three dietary Se sources, such as sodium-selenite (S-S), seleno-yeast (S-Y) and seleno-methionine (S-M), on Se concentration, glutathione peroxidase (GPX) and TXNRD activities, and mRNA expression of fifteen representative selenoproteins, and protein expression of four endoplasmic reticulum-resided selenoproteins in a wide range of tissues of yellow catfish. Compared with S-S and S-M groups, dietary S-Y significantly decreased growth performance and feed utilisation of yellow catfish. Dietary Se sources significantly influenced Se contents in the spleen, dorsal muscle and the kidney, GPX activities in spleen, kidney, intestine, muscle and mesenteric fat, and TXNRD activities in the heart, intestine and mesenteric fat. Among ten tested tissues, dietary Se sources influenced mRNA expression of GPX4 and SELENOK in three tissues; GPX3, SELENOS and TXNRD2 in four tissues; SELENOF, SELENON and DIO2 in five tissues; SELENOM, GPX1/2 and TXNRD3 in six tissues; SELENOW in seven tissue and SELENOP and SELENOT in eight tissues. Based on these observations above, S-S and S-M seem to be suitable Se sources for improving growth performance and feed utilisation of yellow catfish. Dietary Se sources differentially influence the expression of selenoproteins in various tissues of yellow catfish. For the first time, we determined the expression of selenoproteins in fish in responses to dietary Se sources, which contributes to a better understanding of the functions and regulatory mechanisms of selenoporteins.

Environmentally relevant concentrations of oxytetracycline and copper increased liver lipid deposition through inducing oxidative stress and mitochondria dysfunction in grass carp Ctenopharyngodon idella.

Oxytetracycline (OTC) and Cu are prevalent in aquatic ecosystems and their pollution are issues of serious concern. The present working hypothesis is that the toxicity of Cu and OTC mixture on physiological activity of fish was different from single OTC and Cu alone. The present study indicated that, compared to single OTC or Cu alone, Cu+OTC mixture reduced growth performance and feed utilization of grass carp, escalated the contents of Cu, OTC and TG, increased lipogenesis, induced oxidative stress, damaged the mitochondrial structure and functions and inhibited the lipolysis in the liver tissues and hepatocytes of grass carp. Cu+OTC co-treatment significantly increased the mRNA abundances and protein expression of Nrf2. Moreover, we found that Cu+OTC mixture-induced oxidative stress promoted Nrf2 recruitment to the SREBP-1 promoter and increased SREBP-1-mediated lipogenesis; Nrf2 sited at the crossroads of oxidative stress and lipid metabolism, and mediated the regulation of oxidative stress and lipid metabolism. Our findings clearly indicated that OTC and Cu mixture differed in environmental risks from single antibiotic or metal element itself, and thus posed different toxicological responses to aquatic animals. Moreover, our findings suggested that Nrf2 functioned as an important antioxidant regulator linking oxidative stress to lipogenic metabolism, and thus elucidated a novel regulatory mechanism for lipid metabolism.

Phospholipase C signal mediated the glucose-induced changes of glucose absorption and lipid accumulation in the intestinal epithelial cells of yellow catfish Pelteobagrus fulvidraco.

In present study, we explored the effects and the underlying mechanisms of phospholipase C (PLC) mediating glucose-induced changes in intestinal glucose transport and lipid metabolism by using U-73122 (a PLC inhibitor). We found that glucose incubation activated the PLC signal and U-73122 pre-incubation alleviated the glucose-induced increase in plcb2, plce1 and plcg1 mRNA expression. Meanwhile, U-73122 pre-treatment blunted the glucose-induced increase in sodium/glucose co-transporters 1/2 mRNA and protein expressions. U-73122 pre-treatment alleviated the glucose-induced increase in TAG content, BODIPY 493/503 fluorescence intensity, lipogenic enzymes (glucose 6-phospate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), malic enzyme and fatty acid synthase (FAS)) activity and the mRNA expressions of lipogenic genes and related transcription factors (6pgd, g6pd, fas, acca, srebp1 and carbohydrate response element-binding protein (chrebp)) in intestinal epithelial cells of yellow catfish. Further research found that U-73122 pre-incubation mitigated the glucose-induced increase in the ChREBP protein expression and the acetylation level of ChREBP in HEK293T cells. Taken together, these data demonstrated that the PLC played a major role in the glucose-induced changes of glucose transport and lipid metabolism and provide a new perspective for revealing the molecular mechanism of glucose-induced changes of intestinal glucose absorption, lipid deposition and metabolism.

Functional Analysis of Steroidogenic Factor 1 (sf-1) and 17α-Hydroxylase/Lyase (cyp17α) Promoters in Yellow Catfish Pelteobagrus fulvidraco.

The present study was performed to clone and characterize the structures and functions of steroidogenic factor 1 (sf-1) and 17α-hydroxylase/lyase (cyp17α) promoters in yellow catfish Pelteobagrus fulvidraco, a widely distributed freshwater teleost. We successfully obtained 1981 and 2034 bp sequences of sf-1 and cyp17α promoters, and predicted the putative binding sites of several transcription factors, such as Peroxisome proliferator-activated receptor alpha (PPARα), Peroxisome proliferator-activated receptor gamma (PPARγ) and Signal transducer and activator of transcription 3 (STAT3), on sf-1 and cyp17α promoter regions, respectively. Overexpression of PPARγ significantly increased the activities of sf-1 and cyp17α promoters, but overexpression of PPARα significantly decreased the promoter activities of sf-1 and cyp17α. Overexpression of STAT3 reduced the activity of the sf-1 promoter but increased the activity of the cyp17α promoter. The analysis of site-mutation and electrophoretic mobility shift assay suggested that the sf-1 promoter possessed the STAT3 binding site, but did not the PPARα or PPARγ binding sites. In contrast, only the PPARγ site, not PPARα or STAT3 sites, was functional with the cyp17α promoter. Leptin significantly increased sf-1 promoter activity, but the mutation of STAT3 and PPARγ sites decreased leptin-induced activation of sf-1 promoter. Our findings offered the novel insights into the transcriptional regulation of sf-1 and cyp17α and suggested leptin regulated sf-1 promoter activity through STAT3 site in yellow catfish.

Isolation and Characterization of Three Sodium-Phosphate Cotransporter Genes and Their Transcriptional Regulation in the Grass Carp Ctenopharyngodon idella.

It is important to explore the regulatory mechanism of phosphorus homeostasis in fish, which help avoid the risk of P toxicity and prevent P pollution in aquatic environment. The present study obtained the full-length cDNA sequences and the promoters of three SLC20 members (slc20a1a, slc20a1b and slc20a2) from grass carp Ctenopharyngodon idella, and explored their responses to inorganic phosphorus (Pi). Grass carp SLC20s proteins possessed conservative domains and amino acid sites relevant with phosphorus transport. The mRNAs of three slc20s appeared in the nine tissues, but their expression levels were tissue-dependent. The binding sites of three transcription factors (SREBP1, NRF2 and VDR) were predicted on the slc20s promoters. The mutation and EMSA analysis indicated that: (1) SREBP1 binding site (-783/-771 bp) negatively but VDR (-260/-253 bp) binding site positively regulated the activities of slc20a1a promoter; (2) SREBP1 (-1187/-1178 bp), NRF2 (-572/-561 bp) and VDR(615/-609 bp) binding sites positively regulated the activities of slc20a1b promoter; (3) SREBP1 (-987/-977 bp), NRF2 (-1469/-1459 bp) and VDR (-1124/-1117 bp) binding sites positively regulated the activities of the slc20a2 promoter. Moreover, Pi incubation significantly reduced the activities of three slc20s promoters, and Pi-induced transcriptional inactivation of slc20s promoters abolished after the mutation of the VDR element but not SREBP1 and NRF2 elements. Pi incubation down-regulated the mRNA levels of three slc20s. For the first time, our study elucidated the transcriptional regulatory mechanisms of SLC20s and their responses to Pi, which offered new insights into the Pi homeostatic regulation and provided the basis for reducing phosphorus discharge into the waters.

Waterborne copper exposure up-regulated lipid deposition through the methylation of GRP78 and PGC1α of grass carp Ctenopharyngodon idella.

Early molecular events after the exposure of heavy metals, such as aberrant DNA methylation, suggest that DNA methylation was important in regulating physiological processes for animals and accordingly could be used as environmental biomarkers. In the present study, we found that copper (Cu) exposure increased lipid content and induced the DNA hypermethylation at the whole genome level. Especially, Cu induced hypermethylation of glucose-regulated protein 78 (grp78) and peroxisome proliferator-activated receptor gamma coactivator-1α (pgc1α). CCAAT/enhancer binding protein α (C/EBPα) could bind to the methylated sequence of grp78, whereas C/EBPß could not bind to the methylated sequence of grp78. These synergistically influenced grp78 expression and increased lipogenesis. In contrast, DNA methylation of PGC1α blocked the specific protein 1 (SP1) binding and interfered mitochondrial function. Moreover, Cu increased reactive oxygen species (ROS) production, activated endoplasmic reticulum (ER) stress and damaged mitochondrial function, and accordingly increased lipid deposition. Notably, we found a new toxicological mechanism for Cu-induced lipid deposition at DNA methylation level. The measurement of DNA methylation facilitated the use of these epigenetic biomarkers for the evaluation of environmental risk.

Characterization and expression analysis of seven selenoprotein genes in yellow catfish Pelteobagrus fulvidraco to dietary selenium levels.

BACKGROUND: Selenium (Se) appears in the selenoproteins in the form of selenocysteine (Sec) and is important for the growth and development of vertebrates. The present study characterized seven selenoproteins, consisting of the GPX1, GPX3, GPX4, SELENOW, SELENOP, TXNRD2 and TXNRD3 cDNAs in various tissues of yellow catfish, explored their regulation to dietary Se addition. METHODS: 3' and 5' RACE PCR were used to clone full-length cDNA sequences of seven selenoprotein genes (GPX1, GPX3, GPX4, SELENOW, SELENOP, TXNRD2 and TXNRD3). Their molecular characterizations were analyzed, including conservative motifs and the SECIS elements. The phylogenetic trees were generated through neighbor-joining (NJ) method with MEGA 6.0 with 1000 bootstrap replications. Quantitative real-time PCR was used to explore their mRNA tissue distribution in the heart, anterior intestine, dorsal muscle, head kidney, gill, liver, brain, spleen and mesenteric fat. Yellow catfish (mixed sex) were fed diets with dietary Se contents at 0.03 (low Se), 0.25 (adequate Se) and 6.39 (high Se) mg Se/kg, respectively, for 12 weeks, and their spleen, kidney, testis and brain were used for the determination of the mRNA levels of the seven selenoproteins. RESULTS: The seven selenoproteins had similar domains to their corresponding members of other vertebrates. They were widely expressed in nine tissues, including heart, liver, brain, spleen, head kidney, dorsal muscle, mesenteric fat, anterior intestine and gill, but showed tissue-dependent expression patterns. Dietary Se addition affected the expression of the seven genes in spleen, kidney, testis and brain tissues of yellow catfish. CONCLUSION: Taken together, our study demonstrated the characterization, expression and regulation of seven selenoproteins, which increased our understanding of the biological functions of Se and selenoproteins in fish.

Dietary Glucose Increases Glucose Absorption and Lipid Deposition via SGLT1/2 Signaling and Acetylated ChREBP in the Intestine and Isolated Intestinal Epithelial Cells of Yellow Catfish.

BACKGROUND: Dietary carbohydrate affects intestinal glucose absorption and lipid deposition, but the underlying mechanisms are unknown. OBJECTIVES: We used yellow catfish and their isolated intestinal epithelial cells (IECs) to test the hypothesis that sodium/glucose cotransporters (SGLTs) 1/2 and acetylated carbohydrate response element binding protein (ChREBP) mediated glucose-induced changes in glucose absorption and lipid metabolism. METHODS: Yellow catfish (mean ± SEM weight: 4.68 ± 0.02 g, 3 mo old, mixed sex) were fed diets containing 250 g carbohydrates/kg from glucose (G, control), corn starch (CS), sucrose (S), potato starch (PS), or dextrin (D) for 10 wk. IECs were isolated from different yellow catfish and incubated for 24 h in a control or glucose (15 mM) solution with or without a 2-h pretreatment with an inhibitor [sotagliflozin (LX-4211) or tubastatin A (TBSA)]. Human embryonic kidney cells (HEK293T cells) were transfected with a Flag-ChREBP plasmid to explore ChREBP acetylation. Triglyceride (TG) and glucose concentrations and enzymatic activities were measured in the intestine and IECs of yellow catfish. They also were subjected to immunofluorescence, immunoprecipitation, qPCR, and immunoblotting. Immunoblotting and immunoprecipitation were performed with HEK293T cells. RESULTS: The G group had greater intestine TGs (0.99- to 2.30-fold); activities of glucose 6-phospate dehydrogenase, 6-phosphogluconate dehydrogenase, and isocitrate dehydrogenase (0.12- to 2.10-fold); and expression of lipogenic genes (0.32- to 2.34-fold) than the CS, PS, and D groups. The G group had greater intestine sglt1/2 mRNA and protein expression than the CS, S and D groups (0.35- to 1.12-fold and 0.40- to 4.67-fold, respectively), but lower mRNA amounts of lipolytic genes (48.6%-65.8%) than the CS and PS groups. LX-4211 alleviated the glucose-induced increase in sglt1/2 mRNA (38.2%-47.4%) and SGLT1 protein (48.0%) expression, TGs (29.3%), and lipogenic enzyme activities (27.7%-42.1%) and gene expression (38.0%-55.5%) in the IECs. TBSA promoted the glucose-induced increase in TGs (11.3%), fatty acid synthase activity (32.6%), and lipogenic gene expression (21.6%-34.4%) in the IECs and acetylated ChREBP (10.5%) in HEK293T cells. CONCLUSIONS: SGLT1/2 signaling and acetylated ChREBP mediated glucose-induced changes in glucose absorption and lipid metabolism in the intestine and IECs of yellow catfish.

Waterborne Cu exposure increased lipid deposition and lipogenesis by affecting Wnt/ß-catenin pathway and the ß-catenin acetylation levels of grass carp Ctenopharyngodon idella.

Lipid metabolism could be used as a biomarker for environmental monitoring of metal pollution, including Cu. Given the potential role of the Wnt/ß-catenin signaling pathway and acetylation in lipid metabolism, the aim of this study was to investigate the mechanism of Wnt signaling and acetylation mediating Cu-induced lipogenesis. Grass carp Ctenopharyngodon idella, widely distributed freshwater teleost, were used as the model. We found that waterborne Cu exposure increased the accumulation of Cu and lipid, up-regulated lipogenesis, suppressed Wnt signaling, reduced ß-catenin protein level and its nuclear location, reduced the sirt1 mRNA levels and up-regulated the ß-catenin acetylation level. Further investigation found that Cu up-regulated lipogenesis through Wnt/ß-catenin pathway; Cu regulated the ß-catenin acetylation, and K311 was the key acetylated residue after Cu incubation. SIRT1 mediated Cu-induced changes of acetylated ß-catenin and played an essential role in nuclear accumulation of ß-catenin and Cu-induced lipogenesis. Cu facilitated lipid accumulation via the regulation of Wnt pathway by SIRT1. For the first time, our study uncovered the novel mechanism for Wnt/ß-catenin pathway and ß-catenin acetylation levels mediating Cu-induced lipid deposition, which provided insights into the association between Cu exposure and lipid metabolism in fish and had important environmental implications for monitoring metal pollution in the water by using new biomarkers involved in lipid metabolism.

Functional analysis of MTF-1 and MT promoters and their transcriptional response to zinc (Zn) and copper (Cu) in yellow catfish Pelteobagrus fulvidraco.

Metal-responsive transcription factor-1 (MTF-1) and metallothionein (MT) expression are involved in metal homeostasis and detoxification. Here, we characterized the structure and functions of mtf-1 and mt promoters in yellow catfish Pelteobagrus fulvidraco. Many important binding sites of transcriptional factors, such as heat shock promoter element (HSE) and metal responsive element (MRE), were predicted on their promoter regions. Cu did not significantly influence the activity of mtf-1 promoter, but Zn increased its promoter activity. Cu and Zn induced the increase of mt promoter activity. HSE site of mtf-1 promoter was the functional binding locus responsible for Zn-induced mtf-1 transcriptional activation. Zn and Cu induced transcriptional activation of mt gene through the MTF-1- and MRE-dependent pathway. Using primary hepatocytes of yellow catfish, we found that Cu and Zn induced the mt expression; Cu did not significantly influence the mRNA and total protein levels of MTF-1, but Zn up-regulated its mRNA and total protein expression. Both Zn and Cu treatment also up-regulated MTF-1 nuclear protein expression, which in turn increased the mt expression. Taken together, these findings delineated the transcriptional regulation of MT and MTF-1 under Zn or Cu treatments, and provided some mechanisms for the regulation of Cu and Zn homeostasis in vertebrates.

Lipophagy mediated carbohydrate-induced changes of lipid metabolism via oxidative stress, endoplasmic reticulum (ER) stress and ChREBP/PPARγ pathways.

High-carbohydrate diets (HCD) can induce the occurrence of nonalcoholic fatty liver disease (NAFLD), characterized by dramatic accumulation of hepatic lipid droplets (LDs). However, the potential molecular mechanisms are still largely unknown. In this study, we investigated the role of autophagy in the process of HCD-induced changes of hepatic lipid metabolism, and to examine the process of underlying mechanisms during these molecular contexts. We found that HCD significantly increased hepatic lipid accumulation and activated autophagy. Using primary hepatocytes, we found that HG increased lipid accumulation and stimulated the release of NEFA by autophagy-mediated lipophagy, and that lipophagy significantly alleviated high glucose (HG)-induced lipid accumulation. Oxidative and endoplasmic reticulum (ER) stress pathways played crucial regulatory roles in HG-induced lipophagy activation and HG-induced changes of lipid metabolism. Further investigation found that HG-activated lipophagy and HG-induced changes of lipid metabolism were via enhancing carbohydrate response element-binding protein (ChREBP) DNA binding capacity at PPARγ promoter region, which in turn induced transcriptional activation of the key genes related to lipogenesis and autophagy. The present study, for the first time, revealed the novel mechanism for lipophagy mediating HCD-induced changes of lipid metabolism by oxidative stress and ER stress, and ChREBP/PPARγ pathways. Our study provided innovative evidence for the direct relationship between carbohydrate and lipid metabolism via ChREBP/PPARγ pathway.

Novel insights for SREBP-1 as a key transcription factor in regulating lipogenesis in a freshwater teleost, grass carp Ctenopharyngodon idella.

Disturbances in lipid metabolism are at the core of several health issues facing modern society, including fatty liver and obesity. The sterol regulatory element-binding protein 1 (SREBP-1) is one important transcription factor regulating lipid metabolism, but the relevant mechanism still remains unknown. The present study determined the transcriptional regulation of SREBP-1 and its target genes (including acetyl-CoA carboxylase α (accα), fatty acid synthase (fas) and stearoyl-CoA desaturase 1 (scd1)) in a freshwater teleost, grass carp Ctenopharyngodon idella. We cloned and characterised the 1988 bp, 2043 bp, 1632 bp and 1889 bp sequences of srebp-1, accα, scd1 and fas promoters, respectively. A cluster of putative binding sites of transcription factors, such as specific protein, yin yang 1, nuclear factor Y, sterol response elements (SRE) and enhancer box (E-box) element, were predicted on their promoter regions. Overexpression of nSREBP-1 reduced srebp-1 promoter activity, increased scd1 and fas promoter activity but did not influence accα promoter activity. The site-mutation and electrophoretic mobility shift assay analysis indicated that srebp-1, fas and scd1 promoters, but not accα promoter, possessed SRE. In Ctenopharyngodon idella kidney (CIK) cells of grass carp, nSREBP-1 overexpression significantly reduced srebp-1 mRNA expression and up-regulated miR-29 mRNA expression. The 3'UTR of srebp-1 possessed the potential miR-29 binding site and miR-29 up-regulated the luciferase activity of srebp-1 3'UTR and srebp-1 mRNA expression, implying a self-activating loop of SREBP-1 and miR-29 in grass carp. Based on the above-mentioned results, we found two novel transcriptional mechanisms for SREBP-1 in grass carp: (1) the auto-regulation sited on the SREBP-1 promoter regions was suppressive and (2) there was a self-activating loop of SREBP-1 and miR-29.

SREBP-1 and LXRα pathways mediated Cu-induced hepatic lipid metabolism in zebrafish Danio rerio.

The present study was performed to explore the underlying molecular mechanism of Cu-induced disorder of lipid metabolism in fish. To this end, adult zebrafish were exposed to three waterborne Cu concentrations (0 (control), 8 and 16 µg Cu/L, respectively) for 60 days. Hepatic Cu content and hepatosomatic index increased after waterborne Cu exposure. H&E and oil red O stainings showed extensive steatosis in the liver of Cu-exposed fish. Cu exposure up-regulated lipogenic enzymes activities of ME, ICDH, 6PGD, G6PD and FAS, but down-regulated CPTI activities. Transcriptomic analysis indicated that lipid metabolism related pathways were significantly enriched in both low-dose and high-dose Cu exposure group. Genes involved in lipogenic process from fatty acid biosynthesis, fatty acid elongation, fatty acid desaturation to glycerolipid biosynthesis were up-regulated by Cu. To elucidate the mechanism, LXRα inhibitor SR9243 and SREBP1 inhibitor fatostatin were used to verify the role of LXRα and SREBP1 in Cu-induced disorder of lipid metabolism. Both SR9243 and fatostatin significantly attenuated the Cu-induced increase of TG accumulation of hepatocytes. Meanwhile, SR9243 significantly attenuated the Cu-induced up-regulation of expression of lipogenic genes (acaca, fas, icdh, dgat1, moat2 and moat3), and fatostatin significantly attenuated the up-regulation of expression of acaca, fas, g6pd, dgat1 and moat2. Enzymes analysis showed both SR9243 and fatostatin blocked the Cu-induced increase of lipogenic enzymes activities. Taken together, our findings highlight the importance of LXRα and SREBP1 in Cu-induced hepatic lipid deposition, which proposed a novel mechanism for elucidating metal element exposure inducing the disorder of lipid metabolism in aquatic vertebrates.

CREB element is essential for unfolded protein response (UPR) mediating the Cu-induced changes of hepatic lipogenic metabolism in Chinese yellow catfish (Pelteobagrus fulvidraco).

The present study was conducted to explore the underlying mechanism of unfolded protein response (UPR) mediating the Cu-induced changes of hepatic lipogenic metabolism in a low vertebrate, freshwater teleost yellow catfish Pelteobagrus fulvidraco. To this end, three experiments were conducted. In Exp. 1, we cloned the regions of grp78, perk, ire-1α and atf-6α promoters, and found that multiple cAMP-response element binding protein (CREB) binding sites were identified in their promoter regions. Furthermore, these CREB binding sites played crucial role in transcriptional regulation of UPR. In Exp. 2, the involvement of perk, ire-1α and atf-6α in Cu-induced changes of hepatic lipid metabolism was confirmed by specific miRNA. In Exp. 3, the regulatory mechanism of CREB underlying UPR mediating Cu-induced hepatic lipogenic metabolism were investigated. Cu induced UPR via the activation of CREB binding sites in the promoter regions of grp78, perk, ire-1α and atf-6α. In addition, the inhibition of CREB markedly attenuated the Cu-induced up-regulation of hepatic lipogenic metabolism in hepatocytes. This conclusion was further supported by the results from the trial of CREB over-expression. Taken together, the present study indicated that CREB was essential for UPR mediating Cu-induced lipogenic metabolism, supporting a mechanistic link among CREB, UPR and Cu-induced changes of lipid metabolism.

Functional Analysis of Promoters of Genes in Lipid Metabolism and Their Transcriptional Response to STAT3 under Leptin Signals.

We characterized the promoters of target genes of the signal transducer and activator of transcription 3, STAT3 (carnitine palmitoyltransferase I, CPT Iα1b, acetyl-CoA carboxylase alpha, ACCα; fatty acid synthase, FAS; and peroxisome proliferator-activated receptor gamma, PPARγ) in a teleost Pelteobagrus fulvidraco. Binding sites of STAT3 were predicted on these promoters, indicating that STAT3 probably mediated their transcriptional activities. Leptin had no effect on the activity of ACCα and PPARγ promoters, but increased CPT Iα1b promoter activity and decreased FAS promoter activity. The −979/−997 STAT3 binding site of CPT Iα1b and the −794/−812 STAT3 binding site of FAS were functional binding loci responsible for leptin-induced transcriptional activation. The study provided direct evidence that STAT3 regulated the expression of CPT Iα1b and FAS at the transcription level, and determined the STAT3 response element on promoters of CPT Iα1b and FAS under leptin signal.

Zinc reduces hepatic lipid deposition and activates lipophagy via Zn2+/MTF-1/PPARα and Ca2+/CaMKKß/AMPK pathways.

Zinc (Zn) deficiency is the most consistently discovered nutritional manifestations of fatty liver disease. Although Zn is known to stimulate hepatic lipid oxidation, little is known about its underlying mechanism of action in lipolysis. Given the potential role of lipophagy in lipid metabolism, the purpose of this study was to test the hypothesis that Zn attenuates hepatic lipid accumulation by modulating lipophagy. The present study indicated that Zn is a potent promoter of lipophagy. Zn administration significantly alleviated hepatocellular lipid accumulation and increased the release of free fatty acids in association with enhanced fatty acid oxidation and inhibited lipogenesis, which was accompanied by activation of autophagy. Moreover, Zn reduced lipid accumulation and stimulated lipolysis by autophagy-mediated lipophagy. Zn-induced up-regulation of autophagy and lipid depletion is free Zn2+-dependent in the cytosols. Zn-induced autophagy and lipid turnover involved up-regulation of the calcium/calmodulin-dependent protein kinase kinase-ß (Ca2+/CaMKKß)/AMPK pathway. Meanwhile, Zn2+-activated autophagy and lipid depletion were via enhancing metal response element-binding transcription factor (MTF)-1 DNA binding at PPARα promoter region, which in turn induced transcriptional activation of the key genes related to autophagy and lipolysis. Zn activated the pathways of Zn2+/MTF-1/ Peroxisome proliferator-activated receptor (PPAR)α and Ca2+/CaMKKß/AMPK, resulting in the up-regulation of lipophagy and accordingly reduced hepatic lipid accumulation. Our study, for the first time, provided innovative evidence of the direct relationship between metal elements (Zn) and lipid metabolism. The present study also indicated the novel mechanism for Zn-induced lipolysis by the activation of Zn2+/MTF-1/PPARα and Ca2+/CaMKKß/AMPK pathways, which induced the occurrence of lipophagy. These results provide new insight into Zn nutrition and its potential beneficial effects on the prevention of fatty liver disease in vertebrates.-Wei, C.-C., Luo, Z., Hogstrand, C., Xu, Y.-H., Wu, L.-X., Chen, G.-H., Pan, Y.-X., Song, Y.-F. Zinc reduces hepatic lipid deposition and activates lipophagy via Zn2+/MTF-1/PPARα and Ca2+/CaMKKß/AMPK pathways.