SEPP1 Expression Research Articles

Single-cell transcriptome in the examination of the molecular characteristics of macrophages in lung cancer brain metastasis.

e20546 Background: Brain metastasis is one of the most common distant metastasis types in lung cancer patients. Its mechanism involves multiple factors, including cell-cell interactions, immune evasion, and the influence of the microenvironment. Single-cell RNA sequencing technology has significantly advanced our understanding of tumors at a single-cell resolution. It enables an in-depth understanding of the expression profiles of metastatic cells in brain tissue, providing potential predictive biomarkers for future monitoring, diagnosis, and effective treatment. Methods: We collected 50 samples from the GEO database, including 17 normal lung samples, 17 lung cancer samples, and 16 brain metastasis samples. We used the MNN method for batch correction and performed differential analysis using findAllMarkers and FindMarkers. Additionally, we conducted enrichment analysis using ClusterProfiler. Results: After batch correction and re-clustering of the macrophages, we identified 9 clusters. Based on their biological functions, they were classified into 5 macrophage subtypes: RTM_macro, Angio_macro, SEPP1_macro, Prolif_macro, and Mono_like. The angiogenic macrophages (Angio_macro) were most prominent in lung cancer brain metastasis samples (52%), followed by lung cancer (25%), and least present in normal samples (17%). Enrichment analysis revealed that iron death, antigen processing and presentation, and endoplasmic reticulum protein processing were more enriched in brain metastasis. This correlates with the high proportion of angiogenic macrophages present in the brain. The SEPP1_macro subtype showed high expression of SEPP1 and VCAN, genes associated with angiogenesis. Studies suggest that SEPP1 is associated with poor prognosis as it promotes neovascularization and tumor invasion. This subtype was highly expressed in brain metastasis (40%) and lung cancer (45%), but rare in normal samples (4%). In the SEPP1_macro subtype, brain metastasis samples were enriched in signaling pathways such as tumor necrosis factor, PI3K-AKT, NF-kB, and Mitogen-activated protein kinase (MAPK), focusing mainly on the interaction of cytokines and cytokine receptors. Conclusions: In lung cancer and lung cancer brain metastasis, there is a significant presence of macrophages that promote angiogenesis and subpopulations of tumor-promoting cells associated with poor prognosis. The existence of these two cell subsets may indicate their activation during tumor metastasis and their regulatory role in the formation of new blood vessels, thereby promoting tumor growth.

#2716 Selenoprotein-P1 (SEPP-1) in human proximal tubule cells after ischemic-reperfusion injury: an in-vitro model

Abstract Background and Aims Novel biomarkers, anticipating serum creatinine rise, are eagerly needed to have a speedy diagnosis of acute kidney injury (AKI) and thus to mitigate and to preserve kidney function with well-timed therapeutic plans. Recently, in a pilot study on patients undergoing elective major cardiac surgery with cardiopulmonary bypass (CPB), selenoprotein-p1 (SEPP-1) has emerged as a promising candidate plasmatic biomarker for an early AKI risk stratification. SEPP-1 increased particularly between 4th to 8th hour after CPB and with a strict relation with ischemia duration (Bolignano et Al. Rev. Cardiovasc. Med. 2022). SEPP1 is primarily secreted from liver and acts as a selenium transporter supplying tissues and organs with this trace mineral which elicits the activity of specific glutathione peroxidase selenoenzymes. Additionally, renal tubular cells are able to synthetize this protein but remain unknown if, during AKI, SEPP1 increases as defeat answer to renal cells damage or if it's hepatic SEPP1 that accumulates for a reduced renal glomerular filtration rate. The primary aim of this study was to investigate the release of SEPP-1 by renal tubular cells in an experimental model of AKI induced by ischemia-reperfusion and secondarily evaluate the effect of selenium supplying on renal cells injury. Method Human proximal tubular cells (PTC), HK2 cells and human embryonic kidney 293 cells (HEK293) were cultured in Keratinocyte-SFM a complete serum-free medium supplemented with human recombinant Epidermal Growth Factor (rEGF) and Bovine Pituitary Extract (BPE) (LGC Standards s.r.l., Milan, Italy) and in Eagle's Minimum Essential Medium (E-MEM) (LGC Standards s.r.l., Milan, Italy) containing 10% fetal bovine serum, respectively. The cells were pre-incubated with medium containing Sodium Selenite 100 nM for 24 h and then were treated for 24 hours with CoCl2 (Sigma Aldrich S.r.l., Milan, Italy) a chemical hypoxia inducer (500 µM for HK-2 and 250 µM for HEK-293). After that cell viability was evaluated by MTT and trypan bleu dye exclusion assay, while, protein expression of HIF (hypoxia marker) and SEPP1 was analyzed by western blot analysis. Results Ischemia-reperfusion was simulated by the exposure of HK-2 and HEK-293 to CoCl2 and confirmed by HIF expression. The expression of SEPP-1 increased significantly in HK-2 and HEK-293 after hypoxia stress (Fig. 1). Furthermore, the treatment with CoCl2 determined a decrease of cell viability (about 50% less) and Sodium selenite alone increased cell proliferation (about 15% more) in both cell lines. Interestingly cell growth after hypoxia augmented significantly if cell lines were pre-treated with sodium-selenite, highly in HK-2 cells (p < 0.001). Conclusion These preliminary data evidenced, in vitro, that AKI is able to induce renal tubular expression of SEPP-1 and probably SEPP-1 release in human after renal injury is not only a result of a reduced filtration of the hepatic released protein. Furthermore, SEPP-1 showed a possible protective role in AKI. In fact, AKI induced by ischemia-reperfusion, in our in vitro cellular model, was able to induce an increased expression of SEPP-1 in renal tubular cells suggesting a shielding mechanism against injury related to selenium transport. This hypothesis was supported by an exalted SEPP-1 expression after Sodium-Selenite adding to cells’ culture. Supplementary experiments, including functional molecular assays in vitro and in vivo studies will allow to better characterize the role of SEPP-1 in AKI.

Abstract 3032: Selenoprotein Sepp1 determines cysteine dependence in pancreatic cancer

Abstract Pancreatic cancer (PaCa) represents the fourth leading cause of overall cancer death, despite its relatively low incidence compared to other malignancies (Lung, prostate, breast, etc.). Approximately 57,600 people were estimated to be diagnosed with pancreatic cancer in the USA, and 47,050 were estimated to die of the disease in 2020 reported by The American Cancer Society. Novel effective therapeutic strategies are urgently needed. Metabolic changes are typically associated with tumor development to support fast cell proliferation and malignant transition. Such metabolic deregulations differentiate tumor cells from normal cells and act as potential targets of anticancer therapies. Previous studies have shown cysteine dependence often presents in various cancers, since cysteine metabolism supplies glutathione - an important substrate for the cellular reactive oxygen species (ROS) cleanup cycle. Studies in breast and kidney cancers have shown that such cysteine-dependence is a prominent feature in the cancer subtypes with a mesenchymal cell phenotype, instead of in the epithelial subtype. We found that cysteine dependence exists in pancreatic cancer as well. Mesenchymal PaCa cells, in contrast to the epithelial tumor cells, appear to be more sensitive to cysteine depletion with extensive induction of ferroptosis, a programmed cell death pathway involving iron and lipid peroxidation. Gene expression analysis revealed the selenium transporter protein Sepp1 to be absent in the mesenchymal PaCa cells. Knockdown of Sepp1 expression in cysteine-independent cells renders cells dependent on cysteine. Selenium is a micronutrient required for the synthesis of selenoproteins including GPX4 and Sepp1. The addition of selenium alleviated cell reliance on cysteine and caused resistance to GPX4 inhibitor-induced ferroptosis. We observed that Sepp1 regulates the thioredoxin pathway to rescue the cells under the glutathione deficiency. Interestingly, the conditioned media assay also revealed that Sepp1 protects cells from ROS stress by a symbiotic mechanism. Our findings suggest that Sepp1 could serve as a biomarker of tumor cysteine-dependency and a potential molecular target for therapeutic strategies to overcome drug resistance in pancreatic cancer. Citation Format: Arslan Amer, Tahiyat Alothaim, Xiaohu Tang. Selenoprotein Sepp1 determines cysteine dependence in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3032.

Lack of Association between Common Polymorphisms in Selenoprotein P Gene and Susceptibility to Colorectal Cancer, Breast Cancer, and Prostate Cancer: A Meta-Analysis.

Method We search the PubMed, Embase, Google Scholar, and Wanfang (China) databases (up to December 1, 2020) to identify all eligible publications. The pooled odds ratio (OR) correspondence with 95% confidence interval (CI) was calculated to evaluate the associations. Results Finally, nine eligible studies with 7,157 cases and 6,440 controls and five studies with 2,278 cases and 2,821 controls were enrolled in rs3877899 and rs7579 polymorphisms, individually. However, a null significant association was detected between the two polymorphisms in SEPP1 and susceptibility to colorectal, breast, and prostate cancer in all comparison models. Subsequently, subgroup analysis based on tumor type, no significant association was identified for prostate, breast, and colorectal cancer. In addition, when the stratification analyses were conducted by the source of control, HWE status, and ethnicity, yet no significant association was found. Conclusions The current meta-analysis shows that SEPP1 rs3877899 and rs7579 polymorphisms may not be associated with susceptibility to colon cancer, breast cancer, and prostate cancer, and further well-designed studies with a larger sample size are warranted to validate our findings.

Low-Se Diet Can Affect Sperm Quality and Testicular Glutathione Peroxidase-4 activity in Rats.

This study aimed to observe the influence of selenium (Se) deficiency on sperm quality and selenoprotein expression in rats. Four-week male Wista rats were randomly divided into three groups: Se-A, Se-L, and Se-D (respectively for Se- adequate, low, and deficient group). After 9 weeks, the rats were sacrificed by anesthesia, with the cauda epididymidis quickly fetched for sperm count, motility, and deformity. Meanwhile the blood, liver, brain, heart, and testis were collected for Se and biochemical analysis. It was found that the rats in Se-D had poor growth, while the Se concentrations in blood, liver, and heart for Se-D decreased significantly, compared with Se-A and Se-L (p < 0.01). But no significant difference was observed in testis and brain and also no statistical significance for sperm count. The sperm motility for Se-A (63.07%) was significantly higher than Se-L (53.91%) and Se-D (54.15%). Deformities were observed in both Se-L and Se-D. Both glutathione peroxidases (GPxs) and selenoprotein-P (SEPP1) levels in plasma and tissues of Se-D were significantly lower than those of Se-A and Se-L (p < 0.01). The SEPP1 levels in heart and brain of Se-L were lower than Se-A (p < 0.01). There was no statistical difference for GPx1 between Se-A and Se-L. The GPx4 level in testis of Se-L was lower than Se-A (p < 0.05). However, the SEPP1 in plasma, liver, testis, and the GPx3 level in plasma of Se-L were higher than those of Se-A (p < 0.05 or p < 0.01). Our results show that dietary Se deficiency could reduce GPx4 and SEPP1 expression in testis, which further influence sperm motility and may cause sperm deformity. Selenoprotein expression in some tissues of Se-L was higher than that of Se-A, but sperm quality and GPx4 expression in testis were not improved for Se-L. Low active pseudoselenoproteins might be synthesized in low-Se condition. The underlying mechanism needs to be further investigated.

LncRNA DLEU2 acts as a miR-181a sponge to regulate SEPP1 and inhibit skeletal muscle differentiation and regeneration.

Sarcopenia is a serious public health problem associated with the loss of muscle mass and function. The purpose of this study was to identify molecular markers and construct a ceRNA pathway as a significant predictor of sarcopenia. We designed a prediction model to select important differentially expressed mRNAs (DEMs), and constructed a sarcopenia associated ceRNA network. After correlation analysis of each element in the ceRNA network based on clinical samples and GTEX database, C2C12 mouse myoblasts were used as a model to verify the identified ceRNA pathways. A new model for predicting sarcopenia based on four molecular markers SEPP1, SV2A, GOT1, and GFOD1 was developed. The model was used to construct a ceRNA network and showed high accuracy. Correlation analysis showed that the expression levels of lncDLEU2, SEPP1, and miR-181a were closely associated with a high risk of sarcopenia. lncDLEU2 inhibits muscle differentiation and regeneration by acting as a miR-181a sponge regulating SEPP1 expression. In this study, a highly accurate prediction tool was developed to improve the prediction outcomes of sarcopenia. These findings suggest that the lncDLEU2-miR-181a-SEPP1 pathway inhibits muscle differentiation and regeneration. This pathway may be a new therapeutic target for the treatment of sarcopenia.

Selenoprotein P inhibits cell proliferation and ROX production in HCC cells.

Selenoprotein P (SEPP1) is a kind of secretory glycoproteins with an antioxidant effect during the development of some diseases. In this study, we attempted to observe the expression of SEPP1 in livers from the patients with hepatocellular carcinoma (HCC) and explore its effect on HCC cells. All the tissues from patients with HCC were obtained from Affiliated Hospital of Nantong University. Western blot and immunohistochemical results showed that SEPP1 was reduced in HCC liver tissues. Its expression was negatively correlated with Ki67 expression in tissues. The expression of SEPP1 in normal liver cell line was significantly higher than those in the liver cancer cell lines. Serum starvation and release experiment demonstrated that SEPP1 expression was reduced and PCNA expression was increased, when the serum was re-added into cell culture system and the cells were on a proliferation state. After SEPP1 over-expression plasmid was transfected into HepG2 cells, cell proliferation of HepG2 cells and PCNA expression level were all inhibited by SEPP1. Results obtained via 8-isoprostane ELISA further indicated that inhibited ROS level was found in HepG2 cells transfected with SEPP1 over-expression plasmid. In addition, RT-qPCR results demonstrated that GPX1 expression levels increased in HepG2 cells transfected with SEPP1 over-expression plasmid. In conclusion, SEPP1 may inhibit the proliferation of HCC cells, accompanied by the reduction of ROS production and the increasing of GPX1 expression.

Antioxidant capacity, lipid oxidation status and expression of specific selenoprotein mRNA in Longissimus dorsi muscle of lambs (Ovies aries) supplemented with supranutritional selenium

Selenium is known to play a key role in maintenance of redox status of tissues, immunity, reproduction, thyroid and muscle functions. The present investigation was carried out to understand the effect of supranutritional dietary Se supplementation on antioxidant capacity, lipid oxidation and expression of specific selenoprotein mRNA in Longissimus dorsi muscles of growing lambs. Twenty male lambs of 5–6 months of age were fed basal diet supplemented with 0.5, 1.5 or 4.5 ppm Se-yeast (organic Se) or without Se (control) for 90 days. The antioxidant capacity, lipid oxidation of meat during different days of storage and the mRNA expression of GPX1, GPX2, GPX3, TXNRD1, TXNRD3, DIO1, DIO2, DIO3, SEPP1, SEP15 and SEPW1 were studied in Longissimus dorsi muscles of sheep. The results indicated improvement in antioxidant status by supplementation of 1.5 and 4.5 ppm Se, and reduction in meat lipid oxidation status on day 0 without any further reduction after 3 and 7 days of storage in all the Se supplemented lambs. A selective change in expression of GPX2, GPX3, TXNRD1, DIO2, DIO3, SEPP1, SEP15 and SEPW1 mRNA was observed by supranutritional Se while GPX1, TXNRD3, DIO1 and expressions remained unaffected by supplementation. In conclusion, supranutritional Se supplementation in lambs increased antioxidant status, reduced lipid oxidation status with limited effect on oxidative stability of meat during storage and regulated Longissimus dorsi muscle selenoprotein mRNA expression differentially depending on the Se feeding levels. Our results thus provided new insights into the regulation of selenoprotein gene expression by supranutritional levels of dietary Se.

Evaluation of SEPP1 and Selenoprotein S Gene Polymorphisms (rs7579 and rs34713741) in Relation to Colorectal Cancer Susceptibility in Subset of Iranian Population: A Case-control Study.

Background:Colorectal cancer (CRC) is rated as the second cause of cancer death worldwide. Selenium (Se) has antioxidant activity and antitumor effect, especially in colon cancer. This important role occurs through selenoproteins. Low Se intake or low plasma Se and selenoproteins concentrations are associated with higher risk of CRC. rs7579 polymorphism in 3’ untranslated region of the SEPP1 gene can effect on selenocysteine incorporation during protein synthesis and also effect on microRNA –messengerRNA interaction and sequentially change in SEPP1 expression. rs34713741 polymorphism as a promoter variant in selenoprotein S (SELS) gene can effect on SElS expression and finally lead to increased CRC risk.Methods:A case–control study using 60 CRC patients and 74 noncancerous counterparts were undertaken in order to determine rs7579 and rs34713741 genotypes using real-time polymerase chain reaction high-resolution melting method.Results:We found an association of borderline statistical significance between allele A for rs7579 in SEPP1 and CRC risk (adjusted odds ratio = 1.63; confidential interval = 0.99–2.07; P = 0.05). The frequency of genotypes rs34713741 of the mentioned polymorphisms was not significantly different between case and control groups (P = 0.23 and P = 0.93, respectively).Conclusions:The results suggest that these polymorphisms probably has not a substantial role in Iranian CRC risk and is not a serious potential factor in risk assessment of mentioned disease among Iranians.

PO037 Effects of Selenium Supplementation On Expression of SEPP1 In mRNA and Protein Levels In Subjects With and Without Metabolic Syndrome Suffering From Coronary Artery Disease: Selenegene Study A Double-Blind Randomized Controlled Trial

PO037 Effects of Selenium Supplementation On Expression of SEPP1 In mRNA and Protein Levels In Subjects With and Without Metabolic Syndrome Suffering From Coronary Artery Disease: Selenegene Study A Double-Blind Randomized Controlled Trial

MiR-200a-5p augments cardiomyocyte hypertrophy induced by glucose metabolism disorder via the regulation of selenoproteins.

Selenium and selenoproteins are identified as potential determinants in pathological cellular hypertrophy. Cardiomyocytes hypertrophy is a compensatory form of heart disease characterized by increased size of cardiomyocytes. However, the link between cardiac hypertrophy and Se-specific microRNA (miRNA) remains to be characterized. In the current study, we established a miR-200a-5p mimic and an inhibitor cardiomyocytes model. Cardiomyocytes hypertrophy was induced in the miR-200a-5p mimic group. Hence, we detected the glucose level of cardiomyocytes to estimate the cellular glucose uptake. The effect of miR-200a-5p overexpression and the low expression on 25 selenoproteins mRNA levels was further explored using reverse transcription polymerase chain reaction. Overexpression of miR-200a-5p elevated glucose uptake and Txnrd2, 3 expression and reduced Sepp1, Seln, Selt, and Sep15 expression in cardiomyocytes. Contrary results were observed in cardiomyocytes with the knockdown of miR-200a-5p. We next assessed glucose metabolism-related genes in cardiomyocytes. The results showed that miR-200a-5p had a negative correlation with insulin-like growth factor gene-1, insulin-like growth factor binding protein (IGFBP)1, IGFBP2, IGFBP3, IGFBP4, and IGFBP5 and had a positive correlation with Akt, glucose transporter family (GLUT)2, GLUT3, and GLUT4. These results support the involvement of selenoproteins and glucose metabolism in the control of cardiomyocytes hypertrophy by Se-specific miRNA, suggesting that miR-200a-5p inhibited the expression of stress-related selenoproteins to alter glucose transport leading to glucose metabolism disorder, eventually inducing cardiomyocytes hypertrophy. Our finding highlights a pivotal role of Se-specific miRNA and selenoproteins in cardiac hypertrophy.

Effects of selenium supplementation on expression of SEPP1 in mRNA and protein levels in subjects with and without metabolic syndrome suffering from coronary artery disease: Selenegene study a double-blind randomized controlled trial.

Selenoprotein P (SePP) is involved in the protection against diseases. The present study is the first investigation of the effect of selenium supplementation on plasma selenium and expression of SEPP1 in mRNA and protein levels based on metabolic syndrome (MetS), in individuals suffering from coronary artery diseases. In this clinical trial, 160 patients with angiographically documented stenosis of more than 75% in each vessel were enrolled. Patients received either 200-mg selenium yeast tablets or placebo tablets orally after a meal, once daily for 60 days. The mRNA and protein levels of the selenium and SePP1 products were determined before and after the study. From the initial 160 participants, 145 subjects (71 MetS-affected individuals, 74 MetS-unaffected individuals) enrolled in this study. Comparing the selenium and placebo groups, no significant percentage changes of plasma selenium, △Ct SEPP1, or SePP were shown (P > 0.05). Moreover, beyond a significant difference for the expression of SePP in the selenium group compared to its baseline level (P < 0.05), no other significant differences were revealed for plasma selenium and △Ct SEPP1 after the intervention in either group (P > 0.05). Selenium supplementation did not affect plasma selenium or the mRNA or protein level of SePP in either groups after a 2-months intervention beyond a significant increase of SePP in the MetS group. This trial suggests that further studies should investigate the long-term use of selenium supplementation and the effect of a SePP increase on MetS as a potential therapeutic effect.

Effects of Selenium Supplementation on Expression of SEPP1 in Subjects with T Metabolic Syndrome and History of CVD: a Double-blind Randomized Controlled Trial

Effects of Selenium Supplementation on Expression of SEPP1 in Subjects with T Metabolic Syndrome and History of CVD: a Double-blind Randomized Controlled Trial

Selenium (Se) Homeostasis in Chicken Central Nervous System Involves Selenoprotein Expression from Insufficient to Excess Se

Selenium (Se) incorporated in selenoproteins as selenocysteine and supports various important cellular and organismal functions. When Se deficient, brain present the first priority grade to get it. It becomes a homeostasis of Se content. We recently reported that O‐Phosphoseryl‐tRNA: selenocysteinyl‐tRNA synthase (SepSecS) plays an important role in Se homeostasis regulation in the avian brain (PloS One 7:e35761 (2012)). To better understand the relationship of Se homeostasis and selenoprotein expression in chicken central nervous system (CNS), day‐old HyLine White chickens were fed a low Se diet (Se‐L, 0.028 mg/g) supplemented with 4 levels of dietary Se (0 to 5.0 mg Se/kg) as Na2SeO3 for 8 wk. Then chickens were dissected for getting the CNS, which included cerebral contex, cerebellum, thalamus, bulbus cinereus and marrow. Se content in CNS was measured. The expression of selenoproteome which have 24 selenoproteins were detected by the qRT‐PCR array. Se homeostasis was observed during changed the content of Se in chicken CNS from insufficient to excess Se, especially in cerebral contex and bulbus cinereus. In cerebellum, the expression of SePP1 had augmented when Se content was abundant. It implied that SePP1 was the main Se transporters. In bulbus cinereus, the selenoprotein had increased obviously as the content of Se was 3.0 mg/kg. Furthermore, at the 5.0 mg/kg Se, there was a special increase about the selenoprotein expression in the marrow. The distribution of Se in chicken CNS is very notable. The expression of selenoproteins has a hierarch while changing Se content, and Selt, Selk, Selw, Selu, Gpx3, Gpx4, Sepp1, Selo, Sel15, Seln, Gpx2 and Sepp2 take more necessary function in the chicken CNS. In summary, hierarchy of regulated selenoprotein expression makes an important role of CNS Se metabolism and transport in avian.Support or Funding InformationChina New Century Excellent Talents in University (NECT‐1207‐02), China Program for New Century Excellent Talents In Heilongjiang Provincial University (1252‐NCET‐009), Doctor Initial Funding of Northeast Agricultural University (2012RBC52), Research Special funds for Scientific Innovation Talents of Haerbin (2012RFQXN006) and Science Foundation of the Education Department of Heilongjiang Province (12511031).

Diet-induced obesity in the selenocysteine lyase knockout mouse.

Selenocysteine lyase (Scly) mediates selenocysteine decomposition. It was previously demonstrated that, upon adequate caloric intake (12% kcal fat) and selenium deficiency, disruption of Scly in mice leads to development of metabolic syndrome. In this study, we investigate the effect of a high-fat (45% kcal) selenium-adequate diet in Scly knockout (KO) mice on development of metabolic syndrome. Involvement of selenoproteins in energy metabolism after Scly disruption was also examined in vitro in the murine hepatoma cell line, Hepa1-6, following palmitate treatment. Scly KO mice were more susceptible to diet-induced obesity than their wild-type counterparts after feeding a high-fat selenium-adequate diet. Scly KO mice had aggravated hyperinsulinemia, hypercholesterolemia, glucose, and insulin intolerance, but unchanged inflammatory cytokines and expression of most selenoproteins, except increased serum selenoprotein P (Sepp1). Scly KO mice also exhibited enhanced hepatic levels of pyruvate and enzymes involved in the regulation of pyruvate cycling, such as pyruvate carboxylase (Pcx) and pyruvate dehydrogenase (Pdh). However, in vitro silencing of Scly in Hepa1-6 cells led to diminished Sepp1 expression, and concomitant palmitate treatment decreased Pdh expression. The role of selenium in lipid metabolism is recognized, but specific selenium-dependent mechanisms leading to obesity are unclear. This study uncovers that Scly has a remarkable effect on obesity and metabolic syndrome development triggered by high-fat exposure, independent of the expression of most selenoproteins. Diet-induced obesity in Scly KO mice is aggravated, with effects on pyruvate levels and consequent activation of energy metabolism independent of selenoprotein levels.

Exendin-4 Inhibits the Expression of SEPP1 and Fetuin-A via Improvement of Palmitic Acid-Induced Endoplasmic Reticulum Stress by AMPK.

BackgroundSelenoprotein P (SEPP1) and fetuin-A, both circulating liver-derived glycoproteins, are novel biomarkers for insulin resistance and nonalcoholic fatty liver disease. However, the effect of exendin-4 (Ex-4), a glucagon-like peptide-1 receptor agonist, on the expression of hepatokines, SEPP1, and fetuin-A, is unknown.MethodsThe human hepatoma cell line HepG2 was treated with palmitic acid (PA; 0.4 mM) and tunicamycin (tuni; 2ug/ml) with or without exendin-4 (100 nM) for 24 hours. The change in expression of PA-induced SEPP1, fetuin-A, and endoplasmic reticulum (ER) stress markers by exendin-4 treatment were evaluated using quantitative real-time reverse transcription polymerase chain reaction and Western blotting. Transfection of cells with AMP-activated protein kinase (AMPK) small interfering RNA (siRNA) was performed to establish the effect of exendin-4-mediated AMPK in the regulation of SEPP1 and fetuin-A expression.ResultsExendin-4 reduced the expression of SEPP1, fetuin-A, and ER stress markers including PKR-like ER kinase, inositol-requiring kinase 1α, activating transcription factor 6, and C/EBP homologous protein in HepG2 cells. Exendin-4 also reduced the expression of SEPP1 and fetuin-A in cells treated with tunicamycin, an ER stress inducer. In cells treated with the AMPK activator 5-aminoidazole-4-carboxamide ribonucleotide (AICAR), the expression of hepatic SEPP1 and fetuin-A were negatively related by AMPK, which is the target of exendin-4. In addition, exendin-4 treatment did not decrease SEPP1 and fetuin-A expression in cells transfected with AMPK siRNA.ConclusionThese data suggest that exendin-4 can attenuate the expression of hepatic SEPP1 and fetuin-A via improvement of PA-induced ER stress by AMPK.

Selenoprotein P as a diabetes-associated hepatokine that impairs angiogenesis by inducing VEGF resistance in vascular endothelial cells

Impaired angiogenesis induced by vascular endothelial growth factor (VEGF) resistance is a hallmark of vascular complications in type 2 diabetes; however, its molecular mechanism is not fully understood. We have previously identified selenoprotein P (SeP, encoded by the SEPP1 gene in humans) as a liver-derived secretory protein that induces insulin resistance. Levels of serum SeP and hepatic expression of SEPP1 are elevated in type 2 diabetes. Here, we investigated the effects of SeP on VEGF signalling and angiogenesis. We assessed the action of glucose on Sepp1 expression in cultured hepatocytes. We examined the actions of SeP on VEGF signalling and VEGF-induced angiogenesis in HUVECs. We assessed wound healing in mice with hepatic SeP overexpression or SeP deletion. The blood flow recovery after ischaemia was also examined by using hindlimb ischaemia model with Sepp1-heterozygous-knockout mice. Treatment with glucose increased gene expression and transcriptional activity for Sepp1 in H4IIEC hepatocytes. Physiological concentrations of SeP inhibited VEGF-stimulated cell proliferation, tubule formation and migration in HUVECs. SeP suppressed VEGF-induced reactive oxygen species (ROS) generation and phosphorylation of VEGF receptor 2 (VEGFR2) and extracellular signal-regulated kinase 1/2 (ERK1/2) in HUVECs. Wound closure was impaired in the mice overexpressing Sepp1, whereas it was improved in SeP (-/-)mice. SeP (+/-)mice showed an increase in blood flow recovery and vascular endothelial cells after hindlimb ischaemia. The hepatokine SeP may be a novel therapeutic target for impaired angiogenesis in type 2 diabetes.

Metformin Suppresses Expression of the Selenoprotein P Gene via an AMP-activated Kinase (AMPK)/FoxO3a Pathway in H4IIEC3 Hepatocytes

Selenoprotein P (SeP; encoded by SEPP1 in humans) is a liver-derived secretory protein that induces insulin resistance in type 2 diabetes. Suppression of SeP might provide a novel therapeutic approach to treating type 2 diabetes, but few drugs that inhibit SEPP1 expression in hepatocytes have been identified to date. The present findings demonstrate that metformin suppresses SEPP1 expression by activating AMP-activated kinase (AMPK) and subsequently inactivating FoxO3a in H4IIEC3 hepatocytes. Treatment with metformin reduced SEPP1 promoter activity in a concentration- and time-dependent manner; this effect was cancelled by co-administration of an AMPK inhibitor. Metformin also suppressed Sepp1 gene expression in the liver of mice. Computational analysis of transcription factor binding sites conserved among the species resulted in identification of the FoxO-binding site in the metformin-response element of the SEPP1 promoter. A luciferase reporter assay showed that metformin suppresses Forkhead-response element activity, and a ChIP assay revealed that metformin decreases binding of FoxO3a, a direct target of AMPK, to the SEPP1 promoter. Transfection with siRNAs for Foxo3a, but not for Foxo1, cancelled metformin-induced luciferase activity suppression of the metformin-response element of the SEPP1 promoter. The overexpression of FoxO3a stimulated SEPP1 promoter activity and rescued the suppressive effect of metformin. Metformin did not affect FoxO3a expression, but it increased its phosphorylation and decreased its nuclear localization. These data provide a novel mechanism of action for metformin involving improvement of systemic insulin sensitivity through the regulation of SeP production and suggest an additional approach to the development of anti-diabetic drugs.

Selenoprotein P regulates 1-(4-Chlorophenyl)-benzo-2,5-quinone-induced oxidative stress and toxicity in human keratinocytes

Polychlorinated biphenyls and their metabolites are environmental pollutants that are believed to have adverse health effects presumably by inducing oxidative stress. To determine if 1-(4-Chlorophenyl)-benzo-2,5-quinone (4-ClBQ; metabolite of 4-monochlorobiphenyl, PCB3)-induced oxidative stress is associated with changes in the expression of specific antioxidant genes, mRNA levels of 92 oxidative stress-response genes were analyzed using TaqMan Array Human Antioxidant Mechanisms (Life Technologies), and results were verified by performing quantitative RT-PCR assays. The expression of selenoprotein P (sepp1) was significantly downregulated (8- to 10-fold) in 4-ClBQ-treated HaCaT human skin keratinocytes, which correlated with a significant increase in MitoSOX oxidation. Overexpression of Mn-superoxide dismutase or catalase or treatment with N-acetyl-l-cysteine suppressed 4-ClBQ-induced toxicity. Sodium selenite supplementation also suppressed 4-ClBQ-induced decrease in sepp1 expression, which was associated with a significant inhibition in cell death. Furthermore, HaCaT cells overexpressing sepp1 were resistant to 4-ClBQ-induced oxidative stress and toxicity. These results demonstrate that SEPP1 represents a previously unrecognized regulator of PCB-induced biological effects. These results support the speculation that selenoproteins can be an attractive countermeasure for PCB-induced adverse biological effects.

The Relationship between Selenoprotein P and Glucose Metabolism in Experimental Studies

Selenium is an essential trace element in the diet of mammals which is important for many physiological functions. However, a number of epidemiological studies have suggested that high selenium status is a possible risk factor for the development of type 2 diabetes, although they cannot distinguish between cause and effect. Selenoprotein P (Sepp1) is central to selenium homeostasis and widely expressed in the organism. Here we review the interaction between Sepp1 and glucose metabolism with an emphasis on experimental evidence. In models with or without gene modification, glucose and insulin can regulate Sepp1 expression in the pancreas and liver, and vice versa. Especially in the liver, Sepp1 is regulated virtually like a gluconeogenic enzyme. Combining these data suggests that there could be a feedback regulation between hepatic Sepp1 and pancreatic insulin and that increasing circulating Sepp1 might be the result rather than the cause of abnormal glucose metabolism. Future studies specifically designed to overexpress Sepp1 are needed in order to provide a more robust link between Sepp1 and type 2 diabetes.