Androgen signaling inhibits de novo lipogenesis to alleviate lipid deposition in zebrafish

Testosterone is closely associated with lipid metabolism and known to affect body fat composition and muscle mass in males.However,the mechanisms by which testosterone acts on lipid metabolism are not yet fully understood,especially in teleosts.In this study,cyp17a1-/-zebrafish(Danio rerio)exhibited excessive visceral adipose tissue(VAT),lipid content,and up-regulated expression and activity of hepatic de novo lipogenesis(DNL)enzymes.The assay for transposase accessible chromatinwithsequencing(ATAC-seq)results demonstrated that chromatin accessibility of DNL genes was increased in cyp17a1-/-fish compared to cyp17a1+/+male fish,including stearoyl-CoA desaturase(scd)and fatty acid synthase(fasn).Androgen response element(ARE)motifs in the androgen signaling pathway were significantly enriched in cyp17a1+/+male fish but not in cyp17a1-/-fish.Both androgen receptor(ar)-/-and wildtype(WT)zebrafish administered with Ar antagonist flutamide displayed excessive visceral adipose tissue,lipid content,and up-regulated expression and activity of hepatic de novo lipogenesis enzymes.The Ar agonist BMS-564929 reduced the content of VAT and lipid content,and down-regulated acetyl-CoA carboxylase a(acaca),fasn,and scd expression.Mechanistically,the rescue effect of testosterone on cyp17a1-/-fish in terms of phenotypes was abolished when ar was additionally depleted.Collectively,these findings reveal that testosterone inhibits lipid deposition by down-regulating DNL genes via Ar in zebrafish,thus expanding our understanding of the relationship between testosterone and lipid metabolism in teleosts.

Successful Production of an All-Female Common Carp (Cyprinus carpio L.) Population Using cyp17a1-Deficient Neomale Carp

Due to sexual dimorphism in the growth of certain cultured fish species,the production of monosex fishes is desirable for the aquaculture industry.Nowadays,the most widely practiced technique available for the mass production of monosex fish populations is sex steroid-induced sex reversal.Here,a novel strategy for the successful production of all-female(AF)common carp(Cyprinus carpio L.),to take advantage of the sexual dimorphism in growth documented in this species,has been developed using genetic engineering via single gene-targeting manipulation without any exogenous hormone treatments.Male and female heterozygous cyp17a1-deficient common carp were first obtained using the clustered regularly interspaced short palindromic repeats/CRISPR-associated endonuclease 9(CRISPR/Cas9)technique.An all-male phenotype for homozygous cyp17a1-deficient carp,regardless of the individuals’sexdetermination genotypes(XY or XX),has been observed.A male-specific DNA marker newly identified in our laboratory was used to screen the neomale carp population with the XX genotype from the cyp17a1-deficient carp.These neomale carp develop a normal testis structure with normal spermatogenesis and sperm capacity.The neomale common carp were then mated with wild-type(WT)females(cyp17a1^(+/+)XX genotype)using artificial fertilization.All the AF offspring sample fish from the neomale-WT female mating were confirmed as having the cyp17a^(1+/-)XX genotype,and normal development of gonads to ovaries was observed in 100.00%of this group at eight months post-fertilization(mpf).A total of 1000 carp fingerlings,500 from the WT male and female and 500 from the neomale and WT female mating,were mixed and reared in the same pond.The average body weight of cyp17a1^(+/-)XX females was higher by 6.60%(8 mpf)and 32.66%(12 mpf)than that of the control common carp.Our study demonstrates the first successful production of a monosex teleost population with the advantages of sexual dimorphism in growth using genetic manipulation targeting a single locus.

Prevalence of and risk factors for diabetic macular edema in a northeastern Chinese population

AIM:To estimate the prevalence of diabetic macular edema(DME) and clinically significant macular edema(CSME),and to assess their risk factors in a population with type 2 diabetic mellitus(T2DM) located in northeast China.METHODS:Patients were included from the Fushun Diabetic Retinopathy Cohort Study(FS-DIRECT),a community-based study conducted in northeast China.The presence of DME and CSME was determined by the Early Treatment Diabetic Retinopathy Study(ETDRS) retinopathy scale of fundus photographs.The age-standardized prevalence of DME and CSME was estimated.The association between DME/CSME and risk factors was analyzed in a multivariate Logistical analysis.RESULTS:A total of 292(15.4%) and 166(8.8%) patients were diagnosed as DME and CSME,yielding the age and sex standardized prevalence of 13.5%(95%CI:11.9%-15.0%),and 7.1%(95%CI:5.9%-8.3%),respectively.Female patients had a higher prevalence of DME compared to their male counterparts(15.7% vs 10.4%,P=0.03).Multivariable Logistic regression analysis showed that younger age,insulin use,proteinuria,longer duration of diabetes,and higher glycosylated hemoglobin A1c,were associated with the prevalence of DME and CSME.Patients with higher fasting plasma glucose,systolic blood pressure,and blood urea nitrogen were also found to be associated with DME.CONCLUSION:Early fundus screening in diabetic patients is invaluable and given the relatively high prevalence of DME and CSME in this study cohort,those with a high risk of sight threatening maculopathy would invariably benefit from earlier detection.

糖尿病患者视网膜血管直径的影响因素

目的:了解2型糖尿病患者视网膜血管直径及其影响因素。方法:横断面研究。将2012年7月至2013年5月辽宁省抚顺市将军街道15个社区所有已确诊为2型糖尿病的居民1913例纳入研究。采用计算机辅助软件定量测量视网膜中央动脉直径(CRAE)、视网膜中央静脉直径(CRVE),并计算视网膜动静脉比值(AVR)。不同特征的组间CRAE、CRVE和AVR差异比较采用Student-t检验或者方差分析。采用广义线性回归分别探讨CRAE、CRVE和AVR的影响因素。采用LOWESS下的广义估计方程探讨糖尿病视网膜病变(DR)严重程度与CRAE、CRVE和AVR之间可能存在的非线性关系。结果:身体质量指数(BMI)较大、高血压、高密度脂蛋白较低、糖化血红蛋白较低患者的CRAE较低,差异有统计学意义(均P<0.05);年龄较大、女性、糖尿病病程较长、不吸烟、糖化血红蛋白<7%患者的CRVE较低,差异有统计学意义(均P<0.05);年龄较大、女性、不吸烟、BMI较小、无高血压、高密度脂蛋白较高、甘油三酯较低、糖化血红蛋白<7%患者的AVR较高,差异有统计学意义(均P<0.05)。多因素回归显示男性、高血压、高糖化血红蛋白、较低的高密度脂蛋白和高BMI是较小的CRAE的影响因素(均P<0.05);年龄较大、吸烟、糖尿病病程增加、较高的高密度脂蛋白是较大的CRVE的影响因素(均P<0.05);年龄较小、吸烟、高血压是较小的AVR的影响因素(均P<0.05)。随着DR严重程度增加,CRAE、CRVE、AVR的改变并不呈线性关系,CRAE和CRVE存在先逐渐增加再逐渐减小的趋势;AVR存在先减小再增加的趋势。结论:DR患者的CRAE、CRVE显著降低,AVR显著升高,这种关系在中度非增生型DR和增生性DR患者中更明显。

Hyperandrogenism in POMCa-deficient zebrafish enhances somatic growth without increasing adiposity

The endocrine regulatory roles of the hypothalamic-pituitary-adrenocortical axis on anxiety-like behavior and metabolic status have been found throughout animal taxa.However,the precise effects of the balancing adrenal corticosteroid biosynthesis under the influence of adrenocorticotrophic hormone(ACTH),a pro-opiomelanocortin(POMC)-derived peptide,on animal energy expenditure and somatic growth remain unknown.POMC has also been identified as one of the candidate loci for polycystic ovary syndrome,which features hyperandrogenism and some prevalence of obesity in patients.Here we show that zebrafish lacking functional POMCa exhibit similar phenotypes of stress response and body weight gain but not obesity as observed in mammalian models.In contrast with the impaired anorexigenic signaling cascade of melanocyte-stimulating hormones and leptin,which are responsible for their obesity-prone weight gain observed in various pome mutant mammals,analyses with our pomca mutant series indicate that ACTH is the key regulator for the phenotype with enhanced somatic growth without obesity in pomca-deficient zebrafish.Hypocortisolism associated with hyperandrogenism has been observed in the pomca-deficient zebrafish,with enhanced activation of mammalian target of rapamycin complex 1;reutilization of amino acids and fatty acid^-oxidation are observed in the muscle tissue of the pomca-deficient fish.After reducing hyperandrogenism by crossing our pomca mutant fish with a cy p l 7a 1-deficient background,the phenotype of enhanced somatic growth in pomca-deficient fish was no longer observed.Thus,our work also demonstrated that the role of POMCa in stress response seems to be conserved in vertebrates,whereas its effect on adipostasis is unique to teleosts.

近视与糖尿病视网膜病变的相关性

目的:利用双眼糖尿病视网膜病变(DR)程度的不对称性探讨近视与DR的相关性。方法:横断面研究。根据糖尿病早期治疗研究(ETDRS)标准,将抚顺糖尿病视网膜病变队列研究的患者进行DR与糖尿病性黄斑水肿(DME)分级。DR分级包括无DR、轻度非增殖性DR(NPDR)、中度NPDR、重度NPDR与增殖性DR(PDR)。纳入双眼DR程度至少相差1级(314例,628眼)或仅单眼DME(74例,148眼)的患者共388例(776眼)。近视与高度近视分别定义为等效球镜度(SE)<-1 D与<-5 D。采用配对t检验比较较好眼与较差眼间SE的差异,采用McNemar配对检验比较较好眼与较差眼间近视及高度近视比例的差异。结果:388例患者年龄(60.6±8.5)岁,其中男147例(37.9%)。双眼DR程度大多数相差1级(297例,76.5%)。较好眼的SE较对侧眼偏负[(-0.22±2.24)D与(0.00±1.95)D,t=3.01,P=0.003],较好眼高度近视比例高于对侧眼(4.4%与2.1%,χ^2=6.23,P=0.01)。按较好眼DR程度分组后,NPDR患者较好眼的SE较对侧眼偏负[(-0.37±2.76)D与(0.14±1.89)D,t=2.57,P=0.01],高度近视比例高于对侧眼(7.8%与1.1%,χ^2=6.00,P=0.01)。无DR患者以及DME患者中,较好眼与较差眼间各屈光参数差异均无统计学意义。结论:本研究利用双眼DR程度的不对称性,证实了高度近视与DR的负相关关系。

Depletion of insulin receptors leads to β-cell hyperplasia in zebrafish

Hyperglycemia in type 2 diabetes results from an inability of insulin to regulate gluconeogenesis.To characterize the role of the insulin/insulin receptor pathway in glycometabolism and type 2 diabetes,we created a zebrafish model in which insulin receptors a and b(insra and insrb) have been ablated.We first observed that insra and insrb were both expressed abundantly during embryonic development and in various adult tissues.Increased expression of insulin and number of b-cells were observed in insra-/-/-insrb-/-fish together with higher glucose in insra-/-,insrb-/-,or insra-/-/-insrb-/-fish,indicating that insra and insrb were knocked out effectively.However,compared to the wild-type fish,insra-/-/-insrb-/-fish died between 5 and 16 days post-fertilization(dpf) with severe pericardial edema and increased level of cell apoptosis,which was not induced by increased total body glucose content.Increased gluconeogenesis and decreased glycolysis were also observed in both single and double knockout fish,but no mortality or malformation was observed in single knockout fish.Given the importance of insulin receptors in glucose homeostasis and embryonic development,transcriptome analysis was used to provide an important model of defective insulin signaling and to study its developmental consequences in zebrafish.The results indicated that both insra and insrb played a pivotal role in glucose metabolism and embryonic development,and insra was more critical than insrb in the insulin signaling pathway.

Genomic polymorphisms at the crhr2 locus improve feed conversion efficiency through alleviation of hypothalamus-pituitary-interrenal axis activity in gibel carp(Carassius gibelio)

Improvement in fish feed conversion efficiency(FCE)is beneficial for sustaining global food fish supplies.Here,we show that a set of polymorphisms at locus of the corticotropin releasing hormone receptor 2(crhr2),which is involved in hypothalamuspituitary-interrenal(HPI)axis signaling,is associated with improved FCE in farmed allogynogenetic gibel carp strain CAS Ⅲ compared with that in the wild gibel carp strain Dongting(DT).This set of polymorphisms downregulates the expression levels of crhr2 mRNA in the brain and pituitary tissues in gibel carp strain CAS Ⅲ compared with those in strain DT.Furthermore,compromised HPI axis signaling is observed in gibel carp strain CAS Ⅲ,such as decreased α-melanocyte stimulating hormone protein levels,plasma cortisol content,and stress responses.Moreover,enhanced activation of protein kinase B/mammalian target of rapamycin complex 1 signaling observed in the muscle tissue of strain CAS Ⅲ in comparison to that in strain DT indicated elevated anabolic metabolism in strain CAS Ⅲ.Thus,these studies demonstrate that the genetic markers associated with compromised HPI axis signaling,such as crhr2,are potentially useful for genetic selection toward improvement in farmed fish growth and FCE,which would reduce fishmeal consumption and thereby indirectly facilitate sustainable fisheries.

Androgen signaling regulates the transcription of anti-Müllerian hormone via synergy with SRY-related protein SOX9A

Anti-Mullerian hormone （amh） is one of the earliest functional genes expressed during testicular differentiation. It has been suggested that androgen signaling regulates critical genes for the differentiation and development of the testis. To elucidate the exact regulatory mechanisms involved in arnh transcription mediated by androgen signaling, androgen signaling was manipulated in zebrafish by cytochrome P450 17al （cyplTal） knockout and Flutamide treatment. In cyp17a1-deficient and Flutamide-treated testes, up-regulated sry-box9a （soxga） and down-regulated amh were observed. Moreover, a physical association of the zebrafish androgen receptor （AR） and SOX9A was found. The interaction between AR and SOX9A was mediated via the DNA binding domain （DBD） of AR and the transactivation domain （TA） of SOX9A, and was further enhanced by 5-alpha dihydrotestosterone （DHT）, one of the most potent androgens. Intriguingly, together with SOX9A, androgen signaling synergistically promoted amh transcription, mainly through the proximal 1 kb of the amh promoter region. Taken together, our data demonstrate a critical mechanism underlying the direct synergy of androgen signaling and SOX9A in the regulation of amh transcription.

LRH-1 senses signaling from phosphatidylcholine to regulate the expansion growth of digestive organs via synergy with Wnt/β-catenin signaling in zebrafish

Liver receptor homolog-1(LRH-1)is an orphan nuclear receptor that is critical for the growth and proliferation of cancer cells and other biological processes,including lipid transportation and metabolism,sexual determination and steroidogenesis.However,because homozygous lrh-1mice die in utero,the regulatory mechanisms involved in embryonic development mediated by this receptor are poorly understood.In the present study,we performed transcription activator-like effector nuclease(TALEN)-mediated loss-of-function assays,taking advantage of zebrafish external fertilization,to investigate the function of lrh-1.The digestive organs were affected by lrh-1 depletion as a result of cellcycle arrest(at the checkpoint of G1 to S phase),but not cell apoptosis.Biochemical analysis revealed that LRH-1 augments the transcriptional activity of b-catenin 1 and 2 via physical interactions.Screening the specific ligand(s)sensed by LRH-1 during organogenesis revealed that phosphatidylcholine(PC),a potential ligand,is the upstream target of LRH-1 during endoderm development.These data provide evidence for the crosstalk between the PC/LRH-1 and Wnt/β-catenin signaling pathways during the expansion growth of endoderm organs.