👤 Zhiping Guan

MUFAs are unsaturated FAs with one double bond and are derived from endogenous synthesis and dietary intake. Accumulating evidence has suggested that plasma and erythrocyte MUFA levels are associated with cardiometabolic disorders, including CVD, T2D, and metabolic syndrome (MS). Previous genome-wide association studies (GWASs) have identified seven loci for plasma and erythrocyte palmitoleic and oleic acid levels in populations of European origin. To identify additional MUFA-associated loci and the potential functional variant at each locus, we performed ethnic-specific GWAS meta-analyses and trans-ethnic meta-analyses in more than 15,000 participants of Chinese and European ancestry. We identified novel genome-wide significant associations for vaccenic acid at Show less

Angiotensin II (Ang II) is a major contributor to the development of heart failure. However, the molecular and cellular mechanisms that underlie this process remain elusive. Inadequate angiogenesis in the myocardium leads to a transition from cardiac hypertrophy to dysfunction, and our previous study showed that Ang II significantly impaired the angiogenesis response. The current study was designed to examine the role of Jagged1-Notch signaling in the effect of Ang II during impaired angiogenesis and cardiac hypertrophy. Ang II was subcutaneously infused into 8-week-old male C57BL/6 mice at a dose of 200 ng·kg-1·min-1 for 2 weeks using Alzet micro-osmotic pumps. N-[N-(3, 5-difluorophenacetyl)-L-alanyl]-S-phenylglycine tert-butyl ester (DAPT), a γ-secretase inhibitor, was injected subcutaneously during Ang II infusion at a dose of 10.0 mg·kg-1·d-1. Forty mice were divided into four groups (n = 10 per group): control group; Ang II group, treated with Ang II; DAPT group, treated with DAPT; and Ang II + DAPT group, treated with both Ang II and DAPT. At the end of experiments, myocardial (left ventricle [LV]) tissue from each experimental group was evaluated using immunohistochemistry, Western blotting, and real-time polymerase chain reaction. Data were analyzed using one-way analysis of variance test followed by the least significant difference method or independent samples t-test. Ang II treatment significantly induced cardiac hypertrophy and impaired the angiogenesis response compared to controls, as shown by hematoxylin and eosin (HE) staining and immunohistochemistry for CD31, a vascular marker (P < 0.05 for both). Meanwhile, Jagged1 protein was significantly increased, but gene expression for both Jag1 and Hey1 was decreased in the LV following Ang II treatment, compared to that in controls (relative ratio for Jag1 gene: 0.45 ± 0.13 vs. 0.84 ± 0.15; relative ratio for Hey1 gene: 0.51 ± 0.08 vs. 0.91 ± 0.09; P < 0.05). All these cellular and molecular effects induced by Ang II in the hearts of mice were reduced by DAPT treatment. Interestingly, Ang II stimulated Hey1, a known Notch target, but did not affect the expression of Hey2, another Notch target gene. A Jagged1-Hey1 signal might mediate the impairment of angiogenesis induced by Ang II during cardiac hypertrophy. Show less

NRBF2/Atg38 has been identified as the fifth subunit of the macroautophagic/autophagic class III phosphatidylinositol 3-kinase (PtdIns3K) complex, along with ATG14/Barkor, BECN1/Vps30, PIK3R4/p150/Vps15 and PIK3C3/Vps34. However, its functional mechanism and regulation are not fully understood. Here, we report that NRBF2 is a fine tuning regulator of PtdIns3K controlled by phosphorylation. Human NRBF2 is phosphorylated by MTORC1 at S113 and S120. Upon nutrient starvation or MTORC1 inhibition, NRBF2 phosphorylation is diminished. Phosphorylated NRBF2 preferentially interacts with PIK3C3/PIK3R4. Suppression of NRBF2 phosphorylation by MTORC1 inhibition alters its binding preference from PIK3C3/PIK3R4 to ATG14/BECN1, leading to increased autophagic PtdIns3K complex assembly, as well as enhancement of ULK1 protein complex association. Consequently, NRBF2 in its unphosphorylated form promotes PtdIns3K lipid kinase activity and autophagy flux, whereas its phosphorylated form blocks them. This study reveals NRBF2 as a critical molecular switch of PtdIns3K and autophagy activation, and its on/off state is precisely controlled by MTORC1 through phosphorylation. Show less

TNKS1BP1 is a member of the poly(ADP-ribose) polymerase (PARP) superfamily. Our previous studies have demonstrated that TNKS1BP1 plays an important role in DNA damage response. But whether and how TNKS1BP1 associates with cancer is still not clear. Here, we found that TNKS1BP1 was upregulated in human lung adenocarcinoma (LAC) tissues, and was associated with poor overall survival (OS) in LAC patients. Dysregulation of TNKS1BP1 affected the sensitivity of A549 cells to several DNA damage agents including cisplatin, bleomycin, and ionizing radiation. Mechanically, overexpression of TNKS1BP1 increased the accumulation of S phase cells, which was accompanied by a decrease in M phase cells. More importantly, we found TNKS1BP1 regulated genome stability, mainly through affecting the homologous recombination pathway of DNA double-strand breaks by inhibiting the RAD51 foci formation. Overall, our study indicates that, in LAC, aberrant expressions of TNKS1BP1 are common events, and overexpression of TNKS1BP1 might affect outcomes of cancer patients to chemotherapy and radiotherapy. Show less

The Goto-Kakizaki (GK) rat is an animal model of non-obese type 2 diabetes (T2D). The GK rat was generated through the introduction of various genetic mutations from continuous inbreeding; these rats develop diabetes spontaneously. The mutated genes in GK rats may play key roles in the regulation of diabetes. The hypothalamus plays a central role in systematic energy homeostasis. Here, the hypothalamic transcriptomes in GK and Wistar rats at 4, 8 and 12 weeks were investigated by RNA-seq, and multiple variants and gene expression profiles were obtained. The number of variants identified from GK rats was significantly greater than that of Wistar rats, indicating that many variants were fixed and heritable in GK rats after selective inbreeding. The differential gene expression analysis indicated that GK rats had a dysfunctional hypothalamic melanocortin system and attenuation of the hypothalamic glucose-sensing pathway. In addition, we generated integrated gene network modules by combining the protein-protein interaction (PPI) network, co-expression network and mutations in GK and Wistar rats. In the modules, GK-specific genes, such as Bad, Map2k2, Adcy3, Adcy2 and Gstm6, may play key roles in hypothalamic regulation in GK rats. Our research provides a comprehensive map of the abnormalities in the GK rat hypothalamus, which reveals the new mechanisms of pathogenesis of T2D. Show less

Leukemia inhibitory factor (LIF) has many physiological roles. However, its effects on Leydig cell development are still unclear. Rat immature and adult Leydig cells were cultured with different concentrations of LIF alone or in combination with luteinizing hormone (LH) for 24 h. LIF (1 and 10 ng/ml) significantly increased androgen production in immature Leydig cells, but had no effects on testosterone production in adult Leydig cells. Further studies revealed that LIF dose-dependently increased Star and Hsd17b3 expression levels in immature Leydig cells. Gene microarray revealed that the upregulation of anti-oxidative genes and Star might contribute to LIF-induced androgen production. In conclusion, LIF has stimulatory effects on androgen production in rat immature Leydig cells. Show less

The SNP of rs964184 in ZPR1 has recently been associated with type 2 diabetes mellitus (T2DM) in Japanese individuals. To comprehensively investigate the association of common variants in ZPR1 with T2DM in Han Chinese individuals, we designed a two-stage case-control study of 3,505 T2DM patients and 6,911 unrelated healthy Han Chinese individuals. A total of 24 single nucleotide polymorphisms (SNPs) were genotyped, and single-SNP association, imputation and gender-specific association analyses were performed. To increase the coverage of genetic markers, we implemented imputation techniques to extend the number of tested makers to 280. A novel SNP, rs2075290, and the previously reported SNP, rs964184, were significantly associated with T2DM in the two independent datasets, and individuals harboring the CC genotype of rs2075290 and GG genotype of rs964184 exhibited higher levels of fasting plasma glucose (FPG) and blood hemoglobin A1c (HbA1c) than individuals of other genotypes. Additionally, haplotype analyses indicated that two haplotype blocks containing rs2075290 or rs964184 were also significantly associated with T2DM. In summary, these results suggest that ZPR1 plays an important role in the etiology of T2DM, and this gene might be involved in abnormal glucose metabolism. Show less

Polyunsaturated fatty acids (PUFAs) have a major impact on human health. Recent genome-wide association studies (GWAS) have identified several genetic loci that are associated with plasma levels of n-3 and n-6 PUFAs in primarily subjects of European ancestry. However, the relevance of these findings has not been evaluated extensively in other ethnic groups. The primary aim of this study was to evaluate for genetic loci associated with n-3 and n-6 PUFAs and to validate the role of recently identified index loci using data from a Singaporean Chinese population. Using a GWAS approach, we evaluated associations with plasma concentrations of three n-3 PUFAs [alphalinolenic acid (ALA), eicosapentaenoic acid and docosahexaenoic acid], four n-6 PUFAs [linoleic acid (LA), gammalinolenic acid, dihomogammalinolenic acid (DGLA) and arachidonic acid], and estimates of delta-5 desaturase and delta-6 desaturase activities among the participants (N = 1361) of the Singaporean Chinese Health Study. Our results reveal robust genome-wide associations (p value <5 × 10(-8)) with ALA, all four n-6 PUFAs, and delta-6 desaturase activity at the FADS1/FADS2 locus. We further replicated the associations between common index variants at the NTAN1/PDXDC1 locus and n-6 PUFAs LA and DGLA, and between the JMJD1C locus and n-6 PUFA LA (p value between 0.0490 and 9.88 × 10(-4)). These associations were independent of dietary intake of PUFAs. In aggregate, we show that genetic loci that influence plasma concentrations of n-3 and n-6 PUFAs are shared across different ethnic groups. Show less

Mammalian milk is a key source of lipids, providing not only important calories but also essential fatty acids. Veterinary medicine and omics systems sciences intersection, termed as "veterinomics" here, has received little attention to date but stands to offer much promise for building bridges between human and animal health. We determined the changes in porcine mammary genes and proteomics expression associated with milk triacylglycerol (TAG) synthesis and secretion from late pregnancy to lactation. TAG content and fatty acid (FA) composition were determined in porcine colostrum (the 1st day of lactation) and milk (the 17th day of lactation). The mammary transcriptome for 70 genes and 13 proteins involved in TAG synthesis and secretion from six sows, each at d -17(late pregnancy), d 1(early lactation), and d 17 (peak lactation) relative to parturition were analyzed using quantitative real-time PCR and Western blot analyses. The TAG content and the concentrations of de novo synthesized FAs, saturated FAs, and monounsaturated FAs were higher in milk than in colostrum (p<0.05). Robust upregulation with high relative mRNA abundance was evident during lactation for genes associated with FA uptake (VLDLR, LPL, CD36), FA activation (ACSS2, ACSL3), and intracellar transport (FABP3), de novo FA synthesis (ACACA, FASN), FA elongation (ELOVL1), FA desaturation (SCD, FADS1), TAG synthesis (GPAM, AGPAT1, LPIN1, DGAT1), lipid droplet formation (BTN2A1, XDH, PLIN2), and transcription factors and nuclear receptors (SREBP1, SCAP, INSIG1/2). In conclusion, a wide variety of lipogenic genes and proteins regulate the channeling of FAs towards milk TAG synthesis and secretion in porcine mammary gland tissue. These findings inform future omics strategies to increase milk fat production and lipid profile and attest to the rise of both veterinomics and lipidomics in postgenomics life sciences. Show less

Etomidate is a rapid hypnotic intravenous anesthetic agent. The major side effect of etomidate is the reduced plasma concentration of corticosteroids, leading to the abnormal reaction of adrenals. Cortisol and testosterone biosynthesis has similar biosynthetic pathway, and shares several common steroidogenic enzymes, such as P450 side chain cleavage enzyme (CYP11A1) and 3β-hydroxysteroid dehydrogenase 1 (HSD3B1). The effect of etomidate on Leydig cell steroidogenesis during the cell maturation process is not well established. Immature Leydig cells isolated from 35 day-old rats were cultured with 30 μM etomidate for 3 hours in combination with LH, 8Br-cAMP, 25R-OH-cholesterol, pregnenolone, progesterone, androstenedione, testosterone and dihydrotestosterone, respectively. The concentrations of 5α-androstanediol and testosterone in the media were measured by radioimmunoassay. Leydig cells were cultured with various concentrations of etomidate (0.3-30 μM) for 3 hours, and total RNAs were extracted. Q-PCR was used to measure the mRNA levels of following genes: Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Srd5a1, and Akr1c14. The testis mitochondria and microsomes from 35-day-old rat testes were prepared and used to detect the direct action of etomidate on CYP11A1 and HSD3B1 activity. In intact Leydig cells, 30 μM etomidate significantly inhibited androgen synthesis. Further studies showed that etomidate also inhibited the LH- stimulated androgen production. On purified testicular mitochondria and ER fractions, etomidate competitively inhibited both CYP11A1 and HSD3B1 activities, with the half maximal inhibitory concentration (IC50) values of 12.62 and 2.75 μM, respectively. In addition, etomidate inhibited steroidogenesis-related gene expression. At about 0.3 μM, etomidate significantly inhibited the expression of Akr1C14. At the higher concentration (30 μM), it also reduced the expression levels of Cyp11a1, Hsd17b3 and Srd5a1. In conclusion, etomidate directly inhibits the activities of CYP11A1 and HSD3B1, and the expression levels of Cyp11a1 and Hsd17b3, leading to the lower production of androgen by Leydig cells. Show less

Cholesterol efflux has been thought to be the main and basic mechanism by which free cholesterol is transferred from extra hepatic cells to the liver or intestine for excretion. Salvianolic acid B (Sal B) has been widely used for the prevention and treatment of atherosclerotic diseases. Here, we sought to investigate the effects of Sal B on the cholesterol efflux in THP-1 macrophages. After PMA-stimulated THP-1 cells were exposed to 50 mg/L of oxLDL and [(3)H] cholesterol (1.0 μCi/mL) for another 24 h, the effect of Sal B on cholesterol efflux was evaluated in the presence of apoA-1, HDL2 or HDL3. The expression of ATP binding cassette transporter A1 (ABCA1), peroxisome proliferator-activated receptor-gamma (PPAR-γ), and liver X receptor-alpha (LXRα) was detected both at protein and mRNA levels in THP-1 cells after the stimulation of Sal B. Meanwhile, specific inhibition of PPAR-γ and LXRα were performed to investigate the mechanism. The results showed that Sal B significantly accelerated apoA-I- and HDL-mediated cholesterol efflux in both dose- and time-dependent manners. Meanwhile, Sal B treatment also enhanced the expression of ABCA1 at both mRNA and protein levels. Then the data demonstrated that Sal B increased the expression of PPAR-γ and LXRα. And the application of specific agonists and inhibitors of further confirmed that Sal exert the function through PPAR-γ and LXRα. These results demonstrate that Sal B promotes cholesterol efflux in THP-1 macrophages through ABCA1/PPAR-γ/LXRα pathway. Show less

TNKS1BP1 was originally identified as an interaction protein of tankyrase 1, which belongs to the poly(ADP-ribose) polymerase (PARP) superfamily. PARP members play important roles for example in DNA repair, telomere stability and mitosis regulation. Although the TNKS1BP1 protein was considered to be a poly(ADP-ribosyl)ation acceptor of tankyrase 1, its function is still unknown. Here we firstly identified that TNKS1BP1 was up-regulated by ionizing radiation (IR) and the depletion of TNKS1BP1 significantly sensitized cancer cells to IR. Neutral comet assay, pulsed-field gel electrophoresis, and γH2AX foci analysis indicated that TNKS1BP1 is required for the efficient repair of DNA double-strand breaks (DSB). The TNKS1BP1 protein was demonstrated to interact with DNA-dependent protein kinase (DNA-PKcs) and poly(ADP-ribose) polymerase 1 (PARP-1), by co-immunoprecipitation analysis. Moreover, TNKS1BP1 was shown to promote the association of PARP-1 and DNA-PKcs. Overexpression of TNKS1BP1 induced the autophosphorylation of DNA-PKcs/Ser2056 in a PARP-1 dependent manner, which contributed to an increased capability of DNA DSB repair. Inhibition of PARP-1 blocked the TNKS1BP1-mediated DNA-PKcs autophosphorylation and attenuated the PARylation of DNA-PKcs. TNKS1BP1 is a newly described component of the DNA DSB repair machinery, which provides much more mechanistic evidence for the rationale of developing effective anticancer measures by targeting PARP-1 and DNA-PKcs. Show less

Fat deposition influences both meat quality and animal productivity. However, it is not clear how fat development is regulated in growing and fattening beef cattle. This study characterized proteomic changes in subcutaneous adipose tissue from steers fed a high-grain diet in an effort to understand the molecular mechanisms of fat development during feedlot production. Eight British-Continental crossbred steers had two subcutaneous adipose tissue biopsies at 12 and 15 mo of age. Protein expression in fat samples was profiled using liquid chromatography-tandem mass spectrometry (LC-MS/MS). During the finishing period, steers increased subcutaneous adipose tissue mass with concomitant changes in the proteome profile, but the nature of these changes varied among steers. The expression of 123 out of 627 identified proteins differed (P <: 0.05) between 2 ages. Functional analyses on differentially expressed proteins revealed that 20.2% of them were associated with cellular growth and proliferation of adipose tissue. There were 17 out of 108 differentially expressed proteins associated with lipid metabolism, which were acyl-CoA synthetase medium-chain family member 1 (ACSM1), annexin A1 (ANXA1), apolipoprotein C-III (APOC3), apolipoprotein H (beta-2-glycoprotein I; APOH), EH-domain containing 1 (EHD1), coagulation factor II (thrombin; F2), gelsolin (GSN), lamin A/C (LMNA), mitogen-activated protein kinase kinase 1 (MAP2K1), myosin, heavy chain 9, non-muscle (MYH9), orosomucoid 1 (ORM1), protein disulfide isomerase family A, member 3 (PDIA3), retinol binding protein 4, plasma (RBP4), renin binding protein (RENBP), succinate dehydrogenase complex, subunit A, flavoprotein (Fp; SDHA), serpin peptidase inhibitor, clade C (antithrombin), member 1 (SERPINC1), and serpin peptidase inhibitor, clade G (C1 inhibitor), member 1 (SERPING1). Further analysis of the expression levels of proteins associated with lipid metabolism indicated a downregulation in the synthesis of fatty acids at the cellular level at 15 compared to 12 mo of age. These results suggest that even though adipose tissue expanded, fat anabolism was reduced in adipocytes during growth, revealing a coordinated balance between subcutaneous fat mass and the cellular abundance of lipogenic proteins to control the rate of fat deposition in growing beef cattle. The findings observed in this study expand our understanding on how proteome of bovine adipose tissue is regulated during growth, which might help the development in the future of new strategies to manipulate adiposity in beef cattle in a manner that improves meat quality and animal productivity. Show less

On the basis of their self-renewal capacity and their ability to differentiate into derivatives of all three germ layers, germ line-derived multipotent adult stem cells (maGSCs) from mouse testis might serve as one of preferable sources for pluripotent stem cells in regenerative medicine. In our study, we aimed for an efficient hepatic differentiation protocol that is applicable for both maGSCs and embryonic stem cells (ESCs). We attempted to accomplish this goal by using a new established co-culture system with OP9 stroma cells for direct differentiation of maGSCs and ESCs into hepatic cells. We found that the hepatic differentiation of maGSCs was induced by the OP9 co-culture system in comparison to the gelatin culture. Furthermore, we showed that the combination of OP9 co-culture with activin A resulted in the increased expression of endodermal and early hepatic markers Gata4, Sox17, Foxa2, Hnf4, Afp, and Ttr compared to differentiated cells on gelatin or on OP9 alone. Moreover, the hepatic progenitors were capable of differentiating further into mature hepatic cells, demonstrated by the expression of liver-specific markers Aat, Alb, Tdo2, Krt18, Krt8, Krt19, Cps1, Sek, Cyp7a1, Otc, and Pah. A high percentage of maGSC-derived hepatic progenitors (51% AFP- and 61% DLK1-positive) and mature hepatic-like cells (26% ALB-positive) were achieved using this OP9 co-culture system. These generated hepatic cells successfully demonstrated in vitro functions associated with mature hepatocytes, including albumin and urea secretion, glycogen storage, and uptake of low-density lipoprotein. The established co-culture system for maGSCs into functional hepatic cells might serve as a suitable model to delineate the differentiation process for the generation of high numbers of mature hepatocytes in humans without genetic manipulations and make germ line-derived stem cells a potential autologous and alternative cell source for hepatic transplants in metabolic liver disorders. Show less

High altitude environments are of particular interest in the studies of local adaptation as well as their implications in physiology and clinical medicine in human. Some Chinese pig breeds, such as Tibetan pig (TBP) that is well adapted to the high altitude and Dahe pig (DHP) that dwells at the moderate altitude, provide ideal materials to study local adaptation to altitudes. Yet, it is still short of in-depth analysis and understanding of the genetic adaptation to high altitude in the two pig populations. In this study we conducted a genomic scan for selective sweeps using FST to identify genes showing evidence of local adaptations in TBP and DHP, with Wuzhishan pig (WZSP) as the low-altitude reference. Totally, we identified 12 specific selective genes (CCBE1, F2RL1, AGGF1, ZFPM2, IL2, FGF5, PLA2G4A, ADAMTS9, NRBF2, JMJD1C, VEGFC and ADAM19) for TBP and six (OGG1, FOXM, FLT3, RTEL1, CRELD1 and RHOG) for DHP. In addition, six selective genes (VPS13A, GNA14, GDAP1, PARP8, FGF10 and ADAMTS16) were shared by the two pig breeds. Among these selective genes, three (VEGFC, FGF10 and ADAMTS9) were previously reported to be linked to the local adaptation to high altitudes in pigs, while many others were newly identified by this study. Further bioinformatics analysis demonstrated that majority of these selective signatures have some biological functions relevant to the altitude adaptation, for examples, response to hypoxia, development of blood vessels, DNA repair and several hematological involvements. These results suggest that the local adaptation to high altitude environments is sophisticated, involving numerous genes and multiple biological processes, and the shared selective signatures by the two pig breeds may provide an effective avenue to identify the common adaptive mechanisms to different altitudes. Show less

Liver X receptors (LXRs) including LXRα and LXRβ are members of the nuclear hormone receptor superfamily of ligand activated transcription factors, which serve as lipid sensors to regulate expression of genes controlling many aspects of cholesterol and fatty acid metabolism. The liver is the central organ in controlling lipid metabolism. In the present study, we aimed at elucidating the role of LXR activation in hepatic fatty acid homeostasis. We treated C57BL/6 mice with a synthetic non-selective LXR agonist TO901317. Fatty acid profile of lipid esters in the livers was analyzed by gas-liquid chromatography. Real-time polymerase chain reaction (PCR) and western blot were used to determine the expression of SREBP1c and SCD1 in TO901317-treated livers and HepG2 cells. Oral administration of TO901317 resulted in increased fatty acid desaturation in the liver, with concomitant increase in hepatic stearoyl CoA desaturase-1 (SCD1) expression. TO901317-induced SCD1 expression was observed in LXRβ-/- mice, but not in LXRα-/- mice. Furthermore, TO901317 significantly increased expression of sterol regulatory element-binding protein 1c (SREBP1c), the deficiency of which almost completely abolished the induction of SCD1 by TO901317. This drug induced both SREBP1c and SCD1 expression in HepG2 cells. Overexpression of SREBP1c resulted in a significant increase in SCD1 promoter activity and expression. Taken together, the present studies demonstrate that pan-LXR activation increases hepatic fatty acid desaturation via the induction of SCD1 expression in an LXRα-dependent and SREBP1c-mediated manner. Show less

Autophagy, a highly conserved process conferring cytoprotection against stress, contributes to the progression of cerebral ischemia. β-arrestins are multifunctional proteins that mediate receptor desensitization and serve as important signaling scaffolds involved in numerous physiopathological processes. Here, we show that both ARRB1 (arrestin, β 1) and ARRB2 (arrestin, β 2) were upregulated by cerebral ischemic stress. Knockout of Arrb1, but not Arrb2, aggravated the mortality, brain infarction, and neurological deficit in a mouse model of cerebral ischemia. Accordingly, Arrb1-deficient neurons exhibited enhanced cell injury upon oxygen-glucose deprivation (OGD), an in vitro model of ischemia. Deletion of Arrb1 did not affect the cerebral ischemia-induced inflammation, oxidative stress, and nicotinamide phosphoribosyltransferase upregulation, but markedly suppressed autophagy and induced neuronal apoptosis/necrosis in vivo and in vitro. Additionally, we found that ARRB1 interacted with BECN1/Beclin 1 and PIK3C3/Vps34, 2 major components of the BECN1 autophagic core complex, under the OGD condition but not normal conditions in neurons. Finally, deletion of Arrb1 impaired the interaction between BECN1 and PIK3C3, which is a critical event for autophagosome formation upon ischemic stress, and markedly reduced the kinase activity of PIK3C3. These findings reveal a neuroprotective role for ARRB1, in the context of cerebral ischemia, centered on the regulation of BECN1-dependent autophagosome formation. Show less

The Notch3 signaling pathway is thought to play a critical role in cancer development, as evidenced by the Notch3 amplification and rearrangement observed in human cancers. However, the molecular mechanism by which Notch3 signaling contributes to tumorigenesis is largely unknown. In an effort to identify the molecular modulators of the Notch3 signaling pathway, we screened for Notch3-intracellular domain (N3-ICD) interacting proteins using a human proteome microarray. Pathway analysis of the Notch3 interactome demonstrated that ubiquitin C was the molecular hub of the top functional network, suggesting the involvement of ubiquitination in modulating Notch3 signaling. Thereby, we focused on functional characterization of an E3 ubiquitin-protein ligase, WWP2, a top candidate in the Notch3 interactome list. Co-immunoprecipitation experiments showed that WWP2 interacted with N3-ICD but not with intracellular domains from other Notch receptors. Wild-type WWP2 but not ligase-deficient mutant WWP2 increases mono-ubiquitination of the membrane-tethered Notch3 fragment, therefore attenuating Notch3 pathway activity in cancer cells and leading to cell cycle arrest. The mono-ubiquitination by WWP2 may target an endosomal/lysosomal degradation fate for Notch3 as suggested by the fact that the process could be suppressed by the endosomal/lysosomal inhibitor. Analysis of The Cancer Genome Atlas dataset showed that the majority of ovarian carcinomas harbored homozygous or heterozygous deletions in WWP2 locus, and there was an inverse correlation in the expression levels between WWP2 and Notch3 in ovarian carcinomas. Furthermore, ectopic expression of WWP2 decreased tumor development in a mouse xenograft model and suppressed the Notch3-induced phenotypes including increase in cancer stem cell-like cell population and platinum resistance. Taken together, our results provide evidence that WWP2 serves as a tumor suppressor by negatively regulating Notch3 signaling in ovarian cancer. Show less

BACKGROUND- Palmitic acid (16:0), stearic acid (18:0), palmitoleic acid (16:1n-7), and oleic acid (18:1n-9) are major saturated and monounsaturated fatty acids that affect cellular signaling and metabolic pathways. They are synthesized via de novo lipogenesis and are the main saturated and monounsaturated fatty acids in the diet. Levels of these fatty acids have been linked to diseases including type 2 diabetes mellitus and coronary heart disease. METHODS AND RESULTS- Genome-wide association studies were conducted in 5 population-based cohorts comprising 8961 participants of European ancestry to investigate the association of common genetic variation with plasma levels of these 4 fatty acids. We identified polymorphisms in 7 novel loci associated with circulating levels of ≥1 of these fatty acids. ALG14 (asparagine-linked glycosylation 14 homolog) polymorphisms were associated with higher 16:0 (P=2.7×10(-11)) and lower 18:0 (P=2.2×10(-18)). FADS1 and FADS2 (desaturases) polymorphisms were associated with higher 16:1n-7 (P=6.6×10(-13)) and 18:1n-9 (P=2.2×10(-32)) and lower 18:0 (P=1.3×10(-20)). LPGAT1 (lysophosphatidylglycerol acyltransferase) polymorphisms were associated with lower 18:0 (P=2.8×10(-9)). GCKR (glucokinase regulator; P=9.8×10(-10)) and HIF1AN (factor inhibiting hypoxia-inducible factor-1; P=5.7×10(-9)) polymorphisms were associated with higher 16:1n-7, whereas PKD2L1 (polycystic kidney disease 2-like 1; P=5.7×10(-15)) and a locus on chromosome 2 (not near known genes) were associated with lower 16:1n-7 (P=4.1×10(-8)). CONCLUSIONS- Our findings provide novel evidence that common variations in genes with diverse functions, including protein-glycosylation, polyunsaturated fatty acid metabolism, phospholipid modeling, and glucose- and oxygen-sensing pathways, are associated with circulating levels of 4 fatty acids in the de novo lipogenesis pathway. These results expand our knowledge of genetic factors relevant to de novo lipogenesis and fatty acid biology. Show less

The protein, thyroid hormone-responsive SPOT 14 homolog (Thrsp), has been reported to be a lipogenic gene in cultured hepatocytes, implicating an important role of Thrsp in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Thrsp expression is known to be regulated by a variety of transcription factors, including thyroid hormone receptor, pregnane X receptor, and constitutive androstane receptor. Emerging in vitro evidence also points to a critical role of liver X receptor (LXR) in regulating Thrsp transcription in hepatocytes. In the present study, we showed that Thrsp was up-regulated in livers of db/db mice and high-fat-diet-fed mice, two models of murine NAFLD. Hepatic overexpression of Thrsp increased triglyceride accumulation with enhanced lipogenesis in livers of C57Bl/6 mice, whereas hepatic Thrsp gene silencing attenuated the fatty liver phenotype in db/db mice. LXR activator TO901317 induced Thrsp expression in livers of wild-type (WT) and LXR-β gene-deficient mice, but not in LXR-α or LXR-α/β double-knockout mice. TO901317 treatment significantly enhanced hepatic sterol regulatory element-binding protein 1c (SREBP-1c) expression and activity in WT mice, but failed to induce Thrsp expression in SREBP-1c gene-deficient mice. Sequence analysis revealed four LXR response-element-like elements and one sterol regulatory element (SRE)-binding site within a -2,468 ∼+1-base-pair region of the Thrsp promoter. TO901317 treatment and LXR-α overexpression failed to induce, whereas overexpression of SREBP-1c significantly increased Thrsp promoter activity. Moreover, deletion of the SRE site completely abolished SREBP-1c-induced Thrsp transcription. Thrsp is a lipogenic gene in the liver that is induced by the LXR agonist through an LXR-α-mediated, SREBP-1c-dependent mechanism. Therefore, Thrsp may represent a potential therapeutic target for the treatment of NAFLD. Show less

Autophagy is a cellular defense response to stress conditions, such as nutrient starvation. The type III phosphatidylinositol (PtdIns) 3-kinase, whose catalytic subunit is PIK3C3/VPS34, plays a critical role in intracellular membrane trafficking and autophagy induction. PIK3C3 forms multiple complexes and the ATG14-containing PIK3C3 is specifically involved in autophagy induction. Mechanistic target of rapamycin (MTOR) complex 1, MTORC1, is a key cellular nutrient sensor and integrator to stimulate anabolism and inhibit catabolism. Inactivation of TORC1 by nutrient starvation plays a critical role in autophagy induction. In this report we demonstrated that MTORC1 inactivation is critical for the activation of the autophagy-specific (ATG14-containing) PIK3C3 kinase, whereas it has no effect on ATG14-free PIK3C3 complexes. MTORC1 inhibits the PtdIns 3-kinase activity of ATG14-containing PIK3C3 by phosphorylating ATG14, which is required for PIK3C3 inhibition by MTORC1 both in vitro and in vivo. Our data suggest a mechanistic link between amino acid starvation and autophagy induction via the direct activation of the autophagy-specific PIK3C3 kinase. Show less

The class III phosphatidylinositol (PtdIns)-3 kinase, PIK3C3/VPS34, forms multiple complexes and regulates a variety of cellular functions, especially in intracellular vesicle trafficking and autophagy. Even though PtdIns3P, the product of PIK3C3, is thought to be a critical membrane marker for the autophagosome, it is unclear how PIK3C3 is regulated in response to autophagy-inducing stimuli. A complexity of PIK3C3 biology is due in part to the existence of multiple complexes, of which the ATG14- or UVRAG-containing complexes play important roles in autophagy. We recently discovered differential regulation of distinct PIK3C3 complexes in response to energy starvation and showed a mechanism by which AMPK directly phosphorylates PIK3C3 and BECN1 to regulate non- and pro-autophagic PIK3C3 complexes, respectively. Show less

The aim of the present research was to investigate clinicopathologic correlations of immunohistochemically- demonstrated axin (axis inhibition) and β-catenin expression in primary hepatocellular carcinomas (HCCs), in comparison with paraneoplastic, cirrhotic and normal liver tissues. Variation in Axin expression across groups were significant (P < 0.01), correlating with alpha fetoprotein (AFP), HBsAg, cancer plugs in the portal vein, and clinical stage of HCCs(P < 0.05); however, there were no links with sex, age, and tumour size (P > 0.05). Differences in cell membrane β-catenin expression were also statistically significant (P < 0.01), again correlated with AFP, HBsAg, cancer plugs in the portal vein, and clinical stage in HCCs (P < 0.05) but not with sex, age, and tumour size (P > 0.05). Axin expression levels in tissues with reduced membrane β-catenin were low (P < 0.05), also being low with nuclear β-catenin expression (P < 0.05). Axin and β-catenin may play an important role in the genesis and progression of HCC via the Wnt signal transmission pathway. Simultaneous determination of axin, β-catenin, AFP, and HBsAg may be useful for early diagnosis, and metastatic and clinical staging of HCCs. Show less

Endothelin-1 (ET-1), predominantly produced by vascular endothelial cells (VECs), plays an important role in the pathogenesis of inflammatory diseases. Liver X receptor (LXR), a typical nuclear receptor, is known for inhibiting expression of inflammatory molecules. However, it remains unclear whether LXR suppresses ET-1 expression. In the present study, we showed that pretreatment with GW3965, a specific ligand of LXR, significantly attenuated lipopolysaccharide (LPS)-induced ET-1 in mice plasma. The in vitro experiments showed that both LXRα and β were expressed in human VECs, and they are functional as demonstrated by induction of the target gene ABCA1 after treatment with GW3965. Moreover, activation of LXR with GW3965 in human VECs dramatically attenuated the basal and LPS-stimulated ET-1 production at both transcriptional and translational levels. Luciferase reporter assays indicated that LXR activation suppressed the transcriptional activity of the human ET-1 gene promoter, and repressed the activity of a heterologous promoter driven by the response elements of activator-1 (AP-1) or nuclear factor-κB (NF-κB). Electrophoretic mobility shift and chromatin immunoprecipitation assays showed that activation of LXR reduced the binding of the transcriptional factors AP-1 and NF-κB to the ET-1 gene promoter region. In conclusion, activation of LXR represses ET-1 expression in vivo and in vitro, which may be involved in the negatively interfering with AP-1/NF-κB signaling. These results suggest that LXRs may serve as a novel molecular target for modulating ET-1 expression in VECs, and even for the treatment of ET-1-associated inflammatory diseases. Show less

The present study demonstrates a key role for the oxysterol receptor liver X receptor β (LXRβ) in the etiology of diabetes insipidus (DI). Given free access to water, LXRβ(-/-) but not LXRα(-/-) mice exhibited polyuria (abnormal daily excretion of highly diluted urine) and polydipsia (increased water intake), both features of diabetes insipidus. LXRβ(-/-) mice responded to 24-h dehydration with a decreased urine volume and increased urine osmolality. To determine whether the DI was of central or nephrogenic origin, we examined the responsiveness of the kidney to arginine vasopressin (AVP). An i.p. injection of AVP to LXRβ(-/-) mice revealed a partial kidney response: There was no effect on urine volume, but there was a significant increase of urine osmolality, suggesting that DI may be caused by a defect in central production of AVP. In the brain of WT mice LXRβ was expressed in the nuclei of magnocellular neurons in the supraoptic and paraventricular nuclei of the hypothalamus. In LXRβ(-/-) mice the expression of AVP was markedly decreased in the magnocellular neurons as well as in urine collected over a 24-h period. The persistent high urine volume after AVP administration was traced to a reduction in aquaporin-1 expression in the kidney of LXRβ(-/-) mice. The LXR agonist (GW3965) in WT mice elicited an increase in urine osmolality, suggesting that LXRβ is a key receptor in controlling water balance with targets in both the brain and kidney, and it could be a therapeutic target in disorders of water balance. Show less

We studied serum proteomic profiling in patients with graft versus host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT) by two-dimensional gel electrophoresis (2-DE) and mass spectrometry analysis. The expression of a group of proteins, haptoglobin (Hp), alpha-1-antitrypsin, apolipoprotein A-IV, serum paraoxonase and Zn-alpha-glycoprotein were increased and the proteins, clusterin precursor, alpha-2-macroglobulin, serum amyloid protein precursor, sex hormone-binding globulin, serotransferrin and complement C4 were decreased in patients with extensive chronic GVHD (cGVHD). Serum haptoglobin (Hp) levels in patients with cGVHD were demonstrated to be statistically higher than in patients without cGVHD and normal controls (p < 0.01). We used immunoblotting and PCR in combination with 2-DE gel image analysis to determine Hp polymorphisms in 25 allo-HCT patients and 16 normal donors. The results demonstrate that patients with cGVHD had a higher incidence of HP 2-2 phenotype (43.8%), in comparison to the patients without cGVHD (0%) and normal donors (18.7%), suggesting the possibility that specific Hp polymorphism may play a role in the development of cGVHD after allo-HCT. In this study, quantitative serum Hp levels were shown to be related to cGVHD development. Further, the data suggest the possibility that specific Hp polymorphisms may be associated with cGVHD development and warrant further investigation. Show less

In the title compound, {[Ag(2)Pr(C(6)H(4)NO(2))(4)(H(2)O)(4)]ClO(4)·H(2)O}(n), the Pr(III) atom, lying on a twofold rotation axis, has a distorted square-anti-prismatic coordination geometry, defined by four O atoms from four nicotinate (nic) ligands and four water mol-ecules. The Ag(I) atom is coordinated in an almost linear fashion by two pyridyl N atoms from two nicotinate ligands. The linear coordination is augmented by weak inter-actions with three O atoms from one perchlorate anion, one uncoordinated water mol-ecule and one carboxyl-ate group. Two Pr atoms link two {Ag(nic)(2)}(+) units into a ring, which is further extended into an infinite zigzag chain by sharing the Pr atoms. These chains are further connected into a three-dimensional network via weak Ag⋯O inter-actions, O-H⋯O hydrogen bonds, Ag⋯Ag inter-actions [3.357 (2) Å] and π-π inter-actions between the pyridyl rings [centroid-centroid distance = 3.685 (4) Å]. Show less

To study the role of the carbohydrate response element binding protein (ChREBP) in excessive lipid deposition in the liver of db/db mouse. The deposition of neutral lipids in the liver was evaluated by Oil Red O staining. Immunohistochemical assay was utilized to determine the localization of ChREBP protein expression in mouse liver. The expressions of ChREBP and its target genes including acetyl-coenzyme A carboxylase 1 (Acc-1), fatty acid synthase (Fas), glycerol-3-phosphate acyltransferase (Gpat) were analyzed by Real-time PCR and Western blot. Significant lipid droplet deposition was detected in the livers of db/db mice. ChREBP was diffusely expressed in heptocytes with relative higher expression levels around portal and central veins. ChREBP was predominantly located in the cytosol in non-diabetic db/m mice, but was translocated to the nucleus in db/db mice. Nuclear ChREBP protein levels were 8.2-fold higher in db/db mice than in db/m mice(P<0.01). In contrast, another lipogenic transcription factor, sterol regulatory element binding protein-1(SREBP-1), remained unchanged. Consistent with increased nuclear ChREBP levels, expressions of ChREBP target genes involved in lipogenesis including Acc-1, Fas and Gpat were upregulated by 2-fold(P<0.05),1.7-fold (P<0.05) and 4.2-fold(P<0.05), respectively, in db/db mice. The db/db mouse exhibits significantly higher liver ChREBP activity, which may be associated with the development of hepatic steatosis frequently occurring in type 2 diabetes. Targeting ChREBP might represent a new intervention strategy for fatty liver. Show less

The liver X receptors (LXRs) regulate a set of genes involved in lipid metabolism and reverse cholesterol transport. We investigated the mechanism by which shear stress regulates LXR in vascular endothelial cells (ECs). Western blot showed that the protein level of LXRalpha and its target ABCA1 in the mouse thoracic aorta was higher than that in the aortic arch. As well, the mRNA level of LXR and its target genes ABCA1, ABCG1, ApoE, and LPL in the thoracic aorta was higher. In vitro, bovine aortic ECs were subjected to a steady laminar flow (12 dyne/cm2). The expressions of LXR and the LXR-mediated transcription were increased by laminar shear stress. Laminar flow increased LXR-ligand binding and the gene expression of sterol 27-hydroxylase (CYP27), which suggests an increased level of LXR ligand in ECs. This effect was attenuated by LXRalpha and CYP27 RNAi. The decrease of LXR in the aorta of PPARgamma+/- mice and that of C57 mice fed with PPARgamma antagonist suggest the involvement of PPARgamma in the LXR induction by flow. Laminar flow increases LXR function via a PPARgamma-CYP27 dependent mechanism, which reveals an atheroprotective role for laminar flow exerting on endothelium. Show less