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Saturated fatty acids (SFAs) induce reactive oxygen species (ROS) production in neurons. Extracellular signal regulated kinase (ERK)/nuclear factor erythroid-2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) is a ROS response pathway. Therefore, high ROS is always accompanied by increase of HO-1, an anti-oxidative enzyme; but it remains unknown why there is no significant reduction of ROS with the increase of HO-1 in SFAs-treated neurons. We hypothesized that the up-regulation of HO-1 is compensatory for response to fatty acid-induced oxidative stress but not enough to reduce ROS levels. We evaluated the anti-ROS effect of HO-1 and the involved pathway in palmitic acid (PA)-treated human neuroblastoma BE(2)-M17 cells. As expected, PA-induced ROS increase was accompanied by activation of the ERK-Nrf2-HO-1 pathway, as demonstrated by an increase in ERK phosphorylation, Nrf2 phosphorylation and nuclear accumulation, and HO-1 expression at the mRNA and protein levels, in a PA-dose-dependent manner. In contrast, administration of the ROS scavenger NAC significantly reduced the levels of PA-regulated ROS and HO-1 protein. However, the ERK inhibitor U0126 not only reversed the activating effect of PA on the ERK-Nrf2-HO-1 pathway but also aggravated PA-induced ROS. Furthermore, the Nrf2-specific activator NK-252 significantly increased PA-up-regulated HO-1 protein and alleviated PA-induced ROS. Therefore, our results suggest that up-regulation of HO-1 in PA-treated neurons is a compensatory response to ROS increase and that increasing HO-1 expression by Nrf2 activation can prevent the process of ROS production in PA-treated neurons.

Rationale: Abdominal aortic aneurysms (AAAs) are characterized by pathological remodeling of the aortic wall. Although both increased Krüppel-like factor 5 (KLF5) expression and macrophage infiltration have been implicated in vascular remodeling, the role of KLF5 in macrophage infiltration and AAA formation remains unclear. Objective: To determine the role of KLF5 in AAA formation and macrophage infiltration into AAAs. Methods and Results: KLF5 expression was significantly increased in human AAA tissues and in 2 mouse models of experimental AAA. Moreover, in myeloid-specific Klf5 knockout mice (myeKlf5 −/− mice), macrophage infiltration, medial smooth muscle cell loss, elastin degradation, and AAA formation were markedly decreased. In cell migration and time-lapse imaging analyses, the migration of murine myeKlf5 −/− macrophages was impaired, and in luciferase reporter assays, KLF5 activated Myo9b (myosin IXB) transcription by direct binding to the Myo9b promoter. In subsequent coimmunostaining studies, Myo9b was colocalized with filamentous actin, cortactin, vinculin, and Tks5 in the podosomes of phorbol 12,13-dibutyrate–treated macrophages, indicating that Myo9b participates in podosome formation. Gain- and loss-of-function experiments showed that KLF5 promoted podosome formation in macrophages by upregulating Myo9b expression. Furthermore, RhoA-GTP levels increased after KLF5 knockdown in macrophages, suggesting that KLF5 lies upstream of RhoA signaling. Finally, Myo9b expression was increased in human AAA tissues, located in macrophages, and positively correlated with AAA size. Conclusions: These data are the first to indicate that KLF5-dependent regulation of Myo9b/RhoA is required for podosome formation and macrophage migration during AAA formation, warranting consideration of the KLF5–Myo9b–RhoA pathway as a therapeutic target for AAA treatment.

Epithelial to mesenchymal transition (EMT) is an important prerequisite for metastasis to secondary organs. Interleukin-like EMT inducer (ILEI) protein has been shown to translationally upregulated during EMT and metastatic progression as a consequence of aberrant TGF-β signaling. Our initial evaluation of FAM3C (encoding ILEI) and ILEI expression in normal prostate (PCS-440-010) and prostate cancer cell lines (DU145, LNCaP, and PC3) revealed detectable protein expression in only LNCaP cell line even though all cell lines tested had comparable FAM3C expression. Given that PC3 and DU145 cell lines did not have detectable ILEI expression hinted at additional level of regulation of ILEI expression. Treatment with MG-132 resulted in robust detection of ILEI in the PCS-440-010, PC3 and DU145 cell lines, suggesting that at least in these cell lines, ILEI is actively degraded by the proteasome. Mass spectrometric analysis of FLAG immunoprecipitates of untreated and MG-132 treated FLAG-ILEI transfected cells indicated that UBE4A and UBE3C ubiquitin ligases were interacting with ILEI. Ectopic overexpression of UBE4A, but not UBE3C, resulted in destabilization of ILEI in LNCaP cells, whereas RNAi-mediated silencing of UBE4A in PCS-440-010, PC3 and DU145 cell lines resulted in robust accumulation of ILEI, indicating UBE4A as the cognate ubiquitin ligase for ILEI. Co-immunoprecipitation experiments established direct interaction of endogenous ILEI and UBE4A. Furthermore, co-immunoprecipitation of FLAG-tagged ILEI in cells co-transfected with either HA-UBE4A or HA-UBE3C revealed robust polyubiquitinated smear of ILEI in cells transfected with UBE4A, but not UBE3C, thus confirming UBE4A as the ubiquitin ligase for ILEI degradation. Ectopic overexpression of UBE4A, but not UBE3C, in cells was downregulated in vitro migration and invasion in these cells. Cumulatively, our data reveals a novel post-translational regulatory mechanism of regulating ILEI1 expression, a protein required for metastatic progression in prostate cancer cells. © 2016 IUBMB Life, 69(1):16-21, 2017.

We evaluated the effects of administration of 1,25-dihydroxyvitamin D (1,25(OH)2D) during pregnancy on relieving adverse outcomes of preeclampsia and the pathologic and biochemical changes in reduction in uteroplacental perfusion (RUPP) model of rats. On day 1, 7, and 14 of pregnancy, rats in pregnant RUPP plus 1,25(OH)2D (RUPP+VD) group (n = 15) received 120 ng/100 g body weight/week of 1,25(OH)2D by subcutaneous injection, while rats in normal pregnant (n = 12) and the RUPP group (n = 14) received 1,25(OH)2D vehicle (saline solution). On day 19 of pregnancy, after measure of blood pressure and cardiac function and urine collection, rats were euthanized, and fetal and maternal serum, placenta, and heart and kidney were collected. Fetal mortality, urinary protein, glucose, and parameters for kidney function in serum were measured. We evaluated vitamin D receptor expression and pathological and ultrastructural changes in rat heart, kidney, and placenta. Levels of oxidative stress, endoplasmic reticulum (ER) stress, apoptosis, and autophagy were measured in placenta. Compared to RUPP rats, 1,25(OH)2D decreased fetal mortality, mean blood pressure, 24-h urinary protein, urine microalbumin, and hyperglycemia in RUPP+VD rats. These were consistent with the improvements of structure impairment in heart, kidney, and placenta of RUPP rat by 1,25(OH)2D. In placenta of RUPP rat, the decrease in oxidative stress and ER stress by 1,25(OH)2D treatment was accompanied by autophagy activation and apoptosis attenuation. 1,25(OH)2D plays a beneficial effect on preeclampsia at the early gestation and might be used as a potential protective agent for preeclampsia. However, the RUPP model only recapitulated the hypoxic origin of preeclampsia; further randomized controlled trial is expected to be performed for validation and evaluation.

Background Lipotoxicity contributes to diabetic myocardial disease. In this study, we investigated the lipid species contributing to lipotoxicity and the relationship with peroxisomal β-oxidation in the heart of diabetic mice. Methods Male C57BL/6 mice were randomly divided into a Diabetic group (intraperitoneal injection of STZ) and a Control group (saline). Cardiac function indexes [ejection fraction (EF%) and fractional shortening (FS%)] were evaluated by echocardiography. Morphological changes in the myocardial tissues and mitochondria were assessed by electron microscopy following hematoxylin and eosin staining. Blood myocardial injury indexes and lipids were measured using an automatic biochemical analyzer. Cardiac ATP levels were analyzed using a commercially available kit. mRNA levels of glucose transporter 4 (GLUT4), fatty acid binding protein 3 (FABP3), palmitoyl transferase 1α (CPT-1α), acyl-CoA oxidase 1 (AOX1), D-bifunctional protein (DBP), 3-ketoacyl-CoA thiolase A (THLA), uncoupling protein (UCP) 2 and UCP3 were investigated by quantitative reverse-transcription polymerase chain reaction. FABP3 protein expression was analyzed by Western blotting. Non-targeted metabolomics by LC-MS/MS was applied to evaluate profile of lipid metabolism in heart. Results Compared with controls, EF% and FS% were significantly reduced in diabetic mice. Furthermore, blood myocardial injury indexes and lipids, as well as myocardial mitochondrial cristae fusion were significantly increased. In the diabetic heart, GLUT4 expression was decreased, while expression of FABP3, CPT-1α, AOX1, DBP, THLA, UCP2 and UCP3 was increased, and ATP levels were reduced. In total, 113 lipids exhibited significant differential expression (FC > 2, P

Docosahexaenoic acid (DHA) biosynthesis in the liver is the most important source of extra-hepatic tissue DHA. However, the levels of DHA and DHA biosynthesis in the livers of animals with type 2 diabetes mellitus remain unknown. In the current study, we used type 2 diabetic rats induced by high-fat diets and low-dose streptozotocin (STZ, 25 mg/kg) to investigate DHA biosynthesis in the liver during type 2 diabetes mellitus. The serum and livers were collected. Biochemical characteristics, fatty acid profiles, the activity of peroxisomal β-oxidation, and the expressions of enzymes involved in the DHA biosynthesis pathway were assayed in the liver of diabetic rats. The data showed that DHA levels were unexpectedly increased despite of the lower expressions of key enzymes, Δ5- and Δ6-desaturases (Δ5D and Δ6D) in the endoplasmic reticulum (ER). The increased DHA was accompanied by an increase in the expression of straight-chain acyl-CoA oxidase (SCOX), D-bifunctional protein (DBP), and 3-oxoacyl-CoA thiolase (THL), as well as elevated activity of peroxisomal β-oxidation and an increased capacity for DHA biosynthesis from C24:6n-3. These findings indicate that DHA biosynthesis is increased in the livers of diabetic rats induced by high-fat diets and STZ, and that this effect is related to increased activity of peroxisomal β-oxidation, but not of ER desaturases. 1 Practical applications The results support the hypothesis that the dietary supplementation in diabetes with DHA and EPA is desirable. The pathway of DHA biosynthesis in the liver of rats induced by high-fat diets and STZ is shown. DHA biosynthesis is increased in the livers of diabetic rats induced by high-fat diets and STZ, and this effect is related to increased activity of peroxisomal β-oxidation, but is not related to the activity of ER desaturases.

Hepatocellular carcinoma (HCC) arises in a setting of chronic inflammation induced by inflammatory cytokines, such as nuclear factor-kappaB (NF-κB). HCC is a male-predominant cancer that can be attenuated by estradiol (E2) in vitro and in vivo. Although 17β-hydroxysteroid dehydrogenase 4 (HSD17B4) has been implicated as an estradiol-inactivating enzyme, and its promoter sequence contains two putative NF-κB elements: it is currently unknown whether HSD17B4 is the link between inflammation, estradiol and proliferation in hepatoma cells. In this study, HepG2 cells were used to investigate the role of HSD17B4 in the proliferation of liver cancer cells treated with the NF-κB activator, tumor necrosis factor-alpha (TNF-α), with the inhibitor of NF-κB activation, pyrrolidinedithiocarbamate (PDTC), or with a related specific siRNA. We demonstrated that the human HSD17B4 gene is a target for NF-κB activation in inflammation-stimulated HepG2 cells. HSD17B4 is up-regulated via the binding of activated NF-κB to the distal NF-κB-responsive element via TNF-α stimulation, which then promotes cell proliferation by decreasing the levels of E2 and enhancing the expression of interleukin 6 (IL-6), cyclin D1 and proliferating cell nuclear antigen (PCAN). These results from HepG2 cells are consistent with the observation that HSD17B4 is highly expressed and activated NF-κB is highly co-localized with the NF-κB-responsive element of HSD17B4 in liver tumor tissues from HCC patients. Our findings indicate for the first time that HSD17B4 plays an important role in aggravated HCC progression and provides a novel therapeutic target for HCC.

Background Present study aimed to better understand the potential apoptotic pathways that involved in docosahexaenoic acid (DHA)-induced apoptosis of prostate cancer cells. Methods Human prostate cancer DU145 cells were treated with different concentrations of fish oil, omega-3 PUFA (DHA, and Eicosapentaenoic acid, EPA), or omega-6 PUFA (Arachidonic acid, AA). Cell viability and apoptosis were evaluated by MTT assay and Hoechst staining. Pathway-focused gene expression profiling of DU145 cells was analyzed with the RT2 Profile PCR Array System. The results were verified by real time quantitative polymerase chain reaction (RT-qPCR). Results AA exposure showed no obvious effect on viability of DU145 cells. However, exposure with fish oil, EPA, or DHA for 24 h significantly affected cell viability. The growth inhibition of DHA was more pronounced than that of EPA and showed a time-dependent increase. DHA exposure caused typical apoptotic characteristics. Ten genes were more expressed, while 5 genes were less expressed following DHA exposure. RT-qPCR confirmed the time dependent effect of DHA on the expression of these differentially expressed genes. KEGG pathway analysis showed that DHA may induce the apoptosis of cancer cells preferentially through mediating P53, MAPK, TNF, PI3K/AKT, and NF-κB signaling pathways. Conclusion Our study demonstrated the beneficial action of DHA on human prostate carcinoma cell line DU145. The pro-apoptotic effect of DHA on DU145 cells may involve mediation various pathways, especially P53, MAPK, TNF, PI3K/AKT, and NF-κB signaling pathways. Molecular mechanisms of DHA on apoptosis of cancer cells still need to be further clarified. Electronic supplementary material The online version of this article (doi:10.1186/s12944-017-0442-5) contains supplementary material, which is available to authorized users.

Apoptosis pathway-focused gene expression profiling of DU145 cells following DHA exposure. (DOCX 21Â kb)

Epithelial to mesenchymal transition (EMT) is an important prerequisite for metastasis to secondary organs. Interleukin-like EMT inducer (ILEI) protein has been shown to translationally upregulated during EMT and metastatic progression as a consequence of aberrant TGF-β signaling. Our initial evaluation of FAM3C (encoding ILEI) and ILEI expression in normal prostate (PCS-440-010) and prostate cancer cell lines (DU145, LNCaP, and PC3) revealed detectable protein expression in only LNCaP cell line even though all cell lines tested had comparable FAM3C expression. Given that PC3 and DU145 cell lines did not have detectable ILEI expression hinted at additional level of regulation of ILEI expression. Treatment with MG-132 resulted in robust detection of ILEI in the PCS-440-010, PC3 and DU145 cell lines, suggesting that at least in these cell lines, ILEI is actively degraded by the proteasome. Mass spectrometric analysis of FLAG immunoprecipitates of untreated and MG-132 treated FLAG-ILEI transfected cells indicated that UBE4A and UBE3C ubiquitin ligases were interacting with ILEI. Ectopic overexpression of UBE4A, but not UBE3C, resulted in destabilization of ILEI in LNCaP cells, whereas RNAi-mediated silencing of UBE4A in PCS-440-010, PC3 and DU145 cell lines resulted in robust accumulation of ILEI, indicating UBE4A as the cognate ubiquitin ligase for ILEI. Co-immunoprecipitation experiments established direct interaction of endogenous ILEI and UBE4A. Furthermore, co-immunoprecipitation of FLAG-tagged ILEI in cells co-transfected with either HA-UBE4A or HA-UBE3C revealed robust polyubiquitinated smear of ILEI in cells transfected with UBE4A, but not UBE3C, thus confirming UBE4A as the ubiquitin ligase for ILEI degradation. Ectopic overexpression of UBE4A, but not UBE3C, in cells was downregulated in vitro migration and invasion in these cells. Cumulatively, our data reveals a novel post-translational regulatory mechanism of regulating ILEI1 expression, a protein required for metastatic progression in prostate cancer cells. © 2016 IUBMB Life, 69(1):16-21, 2017.

The aim of the present study was to determine the effects of fluvastatin, a relatively hydrophilic 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) inhibitor, on endogenous amyloid beta (Aβ) production and if such effects would be associated with changes in brain total cholesterol in rats. Wistar male rats were treated with fluvastatin at a dose of 20 mg/kg/day or vehicle (controls) by oral gavage for 28 days. We examined serum and brain cholesterol levels by CHOD-PAP method, brain Aβ levels by radioimmunoassay, mRNA levels of HMGR and cholesterol 24S-hydroxylase (CYP46) involving in cholesterol balance, β-secretase (BACE1) and α-secretase (ADAM10) involving in amyloid beta precursor protein (AβPP) processing by RT-PCR and protein levels of AβPP by immunohistochemistry. Serum total cholesterol and brain Aβ levels were significantly reduced in fluvastatin-treated rats. There was no change in total cholesterol levels, HMGR and CYP46 mRNA levels in the brain of fluvastatin-treated rats. Fluvastatin reduced AβPP protein levels and up-regulated ADAM10 but down-regulated BACE1 mRNA expression in rat brain under used condition. These results suggest that reduction of brain Aβ levels by fluvastatin is associated with changes in level of AβPP and AβPP cleavage-related enzyme mRNA, and is independent of brain total cholesterol. It may contribute to one of neuroprotective effects of fluvastatin and reveal that administration of fluvastatin could be beneficial in the preventation of AD. Key words: Alzheimer’s disease, cholesterol, amyloid β, amyloid-β protein precursor, fluvastatin, statin.

Diabetes mellitus correlates with subsequent development of Alzheimer's disease (AD). An accumulation of very long chain fatty acids (VLCFAs) was observed in AD brains. We found previously that inhibiting peroxisomal β-oxidation by an inhibitor caused increases in VLCFA and β-amyloid peptide (Aβ) in the cortex and primary cultured neurons of rats. Therefore, we investigated whether there was an impaired peroxisomal β-oxidation and elevated VLCFA related to the increased Aβ in the diabetic brain. This study was conducted in a type 2 diabetic rat model induced by a high-fat diet and low-dose streptozotocin. A decrease in peroxisomal β-oxidation activity caused by down-regulated thiolase expression and a consequent increase in C26:0 were observed. Meanwhile, decreases in eicosapentenoic acid (EPA) and increases in oxidative stress [indicated by levels of malondialdehyde (MDA), and the protein expression of NOX4, p47(phox) and HO-1], Aβ, and the expression of AβPP and BACE1, two proteins involved in Aβ production, were observed. C26:0 levels were positively correlated with Aβ and MDA. This work suggests that in addition to decreases in EPA, increases in C26:0 by impaired peroxisomal β-oxidation can be a potential risk factor contributing to the progression of AD in diabetic brains via inducing oxidative stress.

Aims Several studies have shown associations between the composition of polyunsaturated fatty acids (PUFAs) in various tissues and type 2 diabetes mellitus (T2DM) development in European populations. Genetic variants of fatty acid desaturase (FADS) contribute to the variations of PUFA composition. Here we have explored whether similar correlations are also true among Chinese Han people. Methods A case–control study was employed to examine this correlation in Han Chinese people. The study included 421 healthy adults and 331 T2DM patients. Results The ratio of arachidonic acid/linoleic acid (AA/LA), which reflects Δ6 desaturase activity, was significantly increased in T2DM patients. Furthermore, the ratio of eicosapentaenoic acid/α-linolenic acid (EPA/ALA), which reflects Δ5 desaturase activity, was markedly decreased in T2DM patients. Importantly, among four single nucleotide polymorphisms (rs174545, rs2072114, rs174602 and rs174616) in the FADS1-FADS2 gene cluster, only minor allele (T) of rs174616 was associated with decreased risk of T2DM in both codominant and dominant models after adjustment for age, gender and BMI. Furthermore, the ratio of AA/LA in both controls and T2DM was reduced in T carriers while an increased proportion of LA was seen in T2DM patients compared with control patients. Conclusion These data suggest that in northern Han Chinese people, the minor allele (T) of rs174616 in the FADS1-FADS2 gene cluster is associated with a decreased conversion rate of LA to AA, which may contribute to decreased reduced risk of developing T2DM.

Ligand-activated Liver X Receptor (LXR) is known to increase cholesterol efflux from cells and reduce the production of amyloid β (Aβ) from amyloid-beta precursor protein (APP). However, little is known about the effects of LXRβ, one subtype of LXR, on endogenous Aβ. In this study, we show that LXRβ inactivation with specific antisense oligodeoxynucleotides (As-ODN) significantly reduced secreted Aβ and decreased mRNA levels of APP(751+770), and α-, β-secretase (ADAM10, BACE1) in primary rat neurons. We also show that As-ODN down-regulated the LXR responsive genes ABCA1 and HMG-CoA reductase (HMGCR). These changes are associated with decreased cellular cholesterol levels. The effect of LXRβ inactivation on Aβ levels is likely due to the alteration of cholesterol production and APP processing. Thus, our data suggest that LXRβ has an important function in cholesterol homeostasis and endogenous Aβ maintenance in neurons.

Objective The purpose of this study was to screen aberrantly expressed miRNAs and genes in prostate cancer (PCA), and further uncover the underlying mechanisms for the development of PCA. Materials and Methods We searched the Gene Expression Omnibus database for miRNA and gene expression datasets of PCA, and then separately integrated miRNA and gene expression datasets to identify miRNA and gene expression profiles in PCA. Target genes of differentially expressed miRNAs were predicted through miRWalk database. We matched these target genes with the list of differentially expressed genes to identify miRNA-target gene pairs whose expression was inversely correlated. The function of these target genes was annotated. Results Twenty-nine differentially expressed miRNAs and 946 differentially expressed genes were identified between PCA and normal control. Seven hundred fifty-one miRNA-target gene pairs that showed inverse expression in PCA were obtained to establish a regulatory network. In this regulatory network, 10 genes ( BCL2 , BNC2 , CCND2 , EPM2A , MRAS , NAV2 , RASL12 , STK33 , TCEAL1 , WWC2 ) were co-regulated by 5 miRNAs (hsa-miR-106b, hsa-miR-130b, hsa-miR-93, hsa-miR-153, hsa-miR-182). The expression of hsa-miR-182 was significantly associated with PCA survival through the online validation tool of SurvMicro, suggesting the potential use as a diagnostic or prognostic biomarker in PCA. Conclusion This integrated analysis was performed to infer new miRNA regulation activities, which provides insights into the understanding of underlying molecular mechanisms of PCA, and guides for exploration of novel therapeutic targets.

Background There is overwhelming evidence that dietary supplementation with n-3 polyunsaturated fatty acids (PUFAs), mainly EPA (C20:5n-3) and DHA (C22:6n-3), has cardiovascular protective effects on patients with type 2 diabetes mellitus (T2DM) but not on healthy people. Because the T2DM heart increases fatty acid oxidation (FAO) to compensate for the diminished utilization of glucose, we hypothesize that T2DM hearts consume more n-3 PUFAs and, therefore, need more n-3 PUFAs. In the present study, we investigated the changes in cardiac n-3 PUFAs and peroxisomal beta-oxidation, which are responsible for the degradation of PUFAs in a high-fat diet (HFD) and low-dose streptozotocin- (STZ) induced type 2 diabetic rat model. Methods and results The capillary gas chromatography results showed that all the n-3 (or omega-3) PUFAs, especially DHA (~50%) and EPA (~100%), were significantly decreased, and the n-6/n-3 ratio (~115%) was significantly increased in the hearts of diabetic rats. The activity of peroxisomal beta-oxidation, which is crucial to very-long-chain and unsaturated FA metabolism (including DHA), was significantly elevated in DM hearts. Additionally, the real-time PCR results showed that the mRNA expression of most peroxisomal beta-oxidation key enzymes were up-regulated in T2DM rat hearts, which might contribute to the reduction of n-3 (or omega-3) PUFAs. Conclusion In conclusion, our results indicate that T2DM hearts consume more n-3 PUFAs, especially DHA and EPA, due to exaggerated peroxisomal beta-oxidation.