Selenoprotein P Expression Research Articles

Ccdc152 is not necessary for male fertility, but contributes to maintaining sperm morphology.

Selenoprotein P (SeP) is synthesized in the liver and plays a vital role in maintaining selenium homeostasis via transport throughout the body. Previous studies have shown that SeP-deficient mice have severely reduced expression of selenoproteins essential for testicular function, leading to male infertility. We previously reported that the high expression of Ccdc152 in hepatocytes acts as a lncRNA, suppressing SeP expression in the liver. Ccdc152 reduces SeP translation by binding to SeP mRNA and decreasing its interaction with SECIS-binding protein 2. Although Ccdc152 is highly expressed in testes, its function remains unclear. Therefore, this study aimed to elucidate the role of Ccdc152 in the testes. Using the CRISPR/Cas9 system, we generated mice lacking all exons of Ccdc152 and found that SeP expression levels in the liver and plasma, as well as overall selenium homeostasis, remained unchanged. No significant differences were observed in the expression of glutathione peroxidase 1/4 or level of selenium in the testes. Subsequent investigation of the impact on male reproductive function revealed no abnormalities in sperm motility or Mendelian ratios of the offspring. However, a slight decrease in testicular weight and an increased rate of sperm malformations in the epididymis were observed. RNA-seq and pathway analyses identified the reduced expression of multiple genes related to kinesin and reproductive pathways. Based on these findings, Ccdc152 may not be essential for male reproductive function, but it may enhance reproductive capabilities by maintaining the expression of genes necessary for reproduction.

Hepatokine selenoprotein P has an important role in cardiac remodeling

Abstract Background In heart failure, the failure of the pump function results in reduced blood flow and dysfunction of energy metabolism in tissues and organs throughout the body. The liver is the largest organ in the human body and plays a central role in lipid and glucose metabolism. Hepatokine selenoprotein P (SeP) contributes to insulin resistance and hyperglycemia in patients with type 2 diabetes. Inhibition of SeP protects the heart from ischemia reperfusion injury and serum levels of SeP are elevated in patients with heart failure with reduced ejection fraction. Objective We investigated the role of SeP in the regulation of cardiac remodeling in response to pressure overload. Methods and Results To examine the role of SeP in cardiac remodeling, transverse aortic constriction (TAC) was subjected to SeP knockout (KO) and wild-type (WT) mice for 2 weeks. LV weight/tibial length (TL) was significantly smaller in SeP KO mice than in WT mice. Lung weight/TL was significantly smaller in SeP KO than in WT mice. TAC-induced cardiac upregulation of the fetal type genes, including atrial and brain natriuretic factors, was significantly attenuated in SeP KO compared to WT. Furthermore, azan staining revealed that there was significantly less interstitial fibrosis in hearts after TAC in SeP KO than in WT mice. Expression of SeP in the liver of WT mice was significantly increased by TAC, while expression in the heart was unchanged. Hepatocyte-specific SeP KO mice were generated using the albumin-Cre-LoxP system. LV weight/TL was significantly smaller in hepatocyte-specific SeP KO than in WT mice. To determine whether hepatic overexpression of SeP affects TAC-induced cardiac hypertrophy, a hydrodynamic injection method was used to generate mice that overexpress SeP mRNA in the liver. Hepatic overexpression of SeP in SeP KO mice lead to a significant increase in LV weight/TL after TAC compared to that in other SeP KO mice. Conclusions These results suggest that cardiac pressure overload induced hepatic expression of SeP and the absence of endogenous SeP attenuated cardiac hypertrophy, dysfunction and fibrosis in response to pressure overload in mice. SeP possibly plays a maladaptive role against progression of heart failure through the cardiohepatic interaction.

Sulforaphane decreases serum selenoprotein P levels through enhancement of lysosomal degradation independent of Nrf2

Selenoprotein P (SeP) is a major selenoprotein in serum predominantly produced in the liver. Excess SeP impairs insulin secretion from the pancreas and insulin sensitivity in skeletal muscle, thus inhibition of SeP could be a therapeutic strategy for type 2 diabetes. In this study, we examine the effect of sulforaphane (SFN), a phytochemical of broccoli sprouts and an Nrf2 activator, on SeP expression in vitro and in vivo. Treatment of HepG2 cells with SFN decreases inter- and intra-cellular SeP levels. SFN enhances lysosomal acidification and expression of V-ATPase, and inhibition of this process cancels the decrease of SeP by SFN. SFN activates Nrf2 in the cells, while Nrf2 siRNA does not affect the decrease of SeP by SFN or lysosomal acidification. These results indicate that SFN decreases SeP by enhancing lysosomal degradation, independent of Nrf2. Injection of SFN to mice results in induction of cathepsin and a decrease of SeP in serum. The findings from this study are expected to contribute to developing SeP inhibitors in the future, thereby contributing to treating and preventing diseases related to increased SeP.

Study of selenomethionine on the selenoproteins expression in hepatocyte L02 and the synergistic effect of serine

To observe the effect of selenomethionine(SeMet)on the selenoproteins expression in hepatocyte L02 and the synergistic effect of serine. The L02 cells were cultured and divided into SeMet group and Serine+SeMet group. SeMet dose was set as 0. 001, 0. 01, 0. 1, 1 and 10 μmol/L. Serine and SeMet were mixed according to 2∶1 molar ratio(Serine∶SeMet=2∶1). The L02 cells were cultured for 48 h after SeMet and Serine added. Finally, the cell culture supernatants and homogenates were collected for the selenoprotein P(SEPP)and glutathione peroxidase 1(GPx1)concentrations detection by a double-antibody sandwich enzyme-linked immuno-sorbent assay(ELISA). The expression of SEPP and GPx1 in cell homogenates was detected by western blot(WB). ELISA and WB results showed that GPx1 and SEPP expressions were dose dependent in a low SeMet concentration range, reached their inflection points when SeMet concentration was 1 μmol/L and 0. 1 μmol/L respectively, and began to decrease when SeMet concentration was further increased to 10 μmol/L. WB result showed that serine had a synergistic effect on GPx1 and SEPP expressions as SeMet concentration was 0. 001 μmol/L to 10 μmol/L. There was an inflection point in selenium protein synthesis when SeMet acted on normal hepatocyte(L02), the specific mechanism needs to be further explored. Secondly, serine had a synergistic effect on GPx1 and SEPP expressions of SeMet in hepatocyte(L02).

SeP is elevated in NAFLD and participates in NAFLD pathogenesis through AMPK/ACC pathway.

Nonalcoholic fatty liver disease (NAFLD) is prevalent chronic liver diseases with unknown mechanism and no curative treatment. Hepatokines have demonstrated importance in NAFLD but, role of selenoprotein P (SeP) in NAFLD is unknown. A total of 79 patients with NAFLD and 79 healthy controls were included in this case-control study. SeP is elevated in patients with NAFLD. With elevating level of SeP, NAFLD prevalence, and detecting rate increases. As NAFLD aggravated, serum SeP increases. Correlation analysis demonstrates that SeP is positively associated with NAFLD risk factors including body mass index, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, and serum uric acid. Both NAFLD in vivo and in vitro models, SeP protein level is higher in liver. Small interfering RNA of SEPP1 inhibited TG accumulation by activating adenosine monophosphate activated protein kinase/acetyl-CoA carboxylase (AMPK/ACC), and overexpression of SEPP1 aggravated lipid accumulation and inhibited AMPK/ACC phosphorylation. SeP expression is activated in NAFLD and exacerbated NAFLD through AMPK/ACC, providing insight into new diagnostic, therapeutic target in NAFLD.

Abstract 354: The Role of the Liver Heart Axis During Cardiac Remodeling

Background: Hepatokine selenoprotein P (SeP) contributes to insulin resistance and hyperglycemia in patients with type 2 diabetes. Although clinical studies suggest the insulin resistance is an independent risk factor of heart failure and inhibition of SeP protects the heart from ischemia reperfusion injury, the role of SeP in pathogenesis of chronic heart failure is not well understood. Objective: We examined the role of SeP in the regulation of cardiac remodeling in response to pressure overload. Methods and Results: We measured serum SeP levels in 22 patients for heart failure with reduced ejection fraction (HFrEF; LVEF<50%) and 22 normal subjects. Serum levels of SeP were significantly elevated in patients with HFrEF compared to in normal subjects (3.55 ± 0.43 vs 2.98 ± 0.43, p<0.01). To examine the role of SeP in cardiac remodeling, SeP knockout (KO) and wild-type (WT) mice were subjected to pressure overload (transverse aortic constriction (TAC)) for 2 weeks. The mortality rate following TAC was significantly decreased in SeP KO mice compared to WT mice (22.5 % in KO mice (n=40) vs 52.3 % in WT mice (n=39) p<0.01). LV weight/tibial length (TL) was significantly smaller in SeP KO mice than in WT mice (6.75 ± 0.24 vs 8.33 ± 0.32, p<0.01). Lung weight/TL was significantly smaller in SeP KO than in WT mice (10.46 ± 0.44 vs 16.38 ± 1.12, p<0.05). Interestingly, hepatic expression of SeP in WT was significantly increased by TAC. To determine whether hepatic overexpression of SeP affects TAC-induced cardiac hypertrophy, a hydrodynamic injection method was used to generate mice that overexpress SeP mRNA in the liver. Hepatic overexpression of SeP in SeP KO mice lead to a significant increase in LV weight/TL and Lung weight/TL after TAC compared to that in other SeP KO mice. Conclusions: These results suggest that serum levels of SeP were elevated in patients with heart failure with reduced ejection fraction and cardiac pressure overload induced hepatic expression of SeP in mice model. Gene deletion of SeP attenuated cardiac hypertrophy and dysfunction in response to pressure overload in mice. SeP possibly plays a pivotal role in promoting cardiac remodeling through the liver-heart axis.

SeP Is Elevated in NAFLD and Participates in NAFLD Pathogenesis Through AMPK/ACC Pathway

Background: Non-alcoholic fatty liver disease (NAFLD) is prevalent chronic liver diseases with unknown mechanism and no curative treatment. Hepatokines have demonstrated important role in NAFLD pathogenesis but, role of selenoprotein P (SeP) in NAFLD is unknown. Methods: We included NAFLD patients as well as equivalent healthy controls and collected demographic data, medical history and general information. Serum sample was sent for blood routine, biochemical study and also SeP by ELISA. NAFLD in vivo and in vitro models were generated with high fat diet and palmitic acid, respectively. siRNA, overexpression plasmid and purified protein of SeP were used to manipulate SeP level in hepatocyte. qRT-PCR, Western Blot and quantative triglyceride measurement were used to evaluated corresponding effect. Findings: A total of 79 NAFLD patients and 79 healthy controls were included in this case-control study. SeP is elevated in NAFLD patients (P < 0.05). With elevating level of SeP, NAFLD prevalence and detecting rate increases (P < 0.05). As NAFLD aggravated, serum SeP increases. Correlation analysis demonstrates that SeP is positively associated with NAFLD risk factors BMI, ALT, AST, GGT and serum uric acid (P < 0.05). Both NAFLD in vivo and in vitro models, SeP protein level is higher in liver. siRNA of SEPP1 inhibited TG accumulation by activating AMPK/ACC, and overexpression of SEPP1 with plasmid or recombinant protein both aggravated lipid accumulation and inhibited AMPK/ACC phosphorylation. Interpretation: SeP expression is activated in NAFLD and excerbated NAFLD through AMPK/ACC pathway, providing insight into new diagnostic, therapeutic target in NAFLD. Funding Statement: The authors stated: None. Declaration of Interests: The authors declare there are no conflicting financial interests. Ethics Approval Statement: The study protocol was approved by the Hospital Ethics Committee and performed in accordance with Declaration of Helsinki. All persons gave their informed consent prior to their inclusion in the study and study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki. Study protocol has been approved by ethical committee of our hospital and conforms to Animal Research: Reporting of in vivo Experiments (ARRIVE) guidelines.

P6479Diagnostic and prognostic significance of serum levels of selenoprotein P in patients with pulmonary arterial hypertension

Abstract Background Despite the recent progress in upfront combination therapy for pulmonary arterial hypertension (PAH), a useful biomarker for the disorder still remains to be developed. Selenoprotein P (SeP) is a glycoprotein secreted mainly from hepatocytes but also from other various kinds of cells, including pulmonary artery smooth muscle cells (PASMCs), to maintain selenium homeostasis and cellular energy metabolism. We have recently demonstrated that SeP expression in PASMCs is markedly up-regulated in PAH patients and plays crucial roles in the pathogenesis of the disorder. In this study, we thus examined whether serum levels of SeP could be a useful biomarker for the disorder. Methods In the experimental study, we performed gene expression microarray and in silico analyses to identify a novel therapeutic target for PAH. We also used the lung, serum, and cultured PAMSCs derived from patients with PAH for mechanistic experiments. In the clinical study, we enrolled a total of 65 consecutive patients with PAH who underwent right heart catheterization for hemodynamic assessment. We measured serum SeP levels and evaluated their prognostic impacts during follow-up (mean 1,520 days, IQR: 1,393–1,804 days). Serum SeP level was measured using a newly developed sol particle homogeneous immunoassay. As controls, we collected serum samples from 20 controls without any known cardiac disorders evaluated by hematological examination, echocardiography, and coronary angiography. In PAH patients, we examined the relationship between baseline SeP levels and composite endpoint of all-cause death and lung transplantation. The correlation between the absolute changes in SeP and those in hemodynamic parameters during follow-up were also examined. Results In the experimental study, SeP promoted PASMC proliferation through increased oxidative stress and mitochondrial metabolic dysfunction, which were associated with activated HIF-1α and dysregulated glutathione metabolism. In the clinical study, PAH patients showed significantly higher levels of serum SeP compared with controls (3.07±0.57 vs. 2.43±0.25 mg/L, P&lt;0.0001). Higher SeP levels (cut-off point, 3.47 mg/L) were significantly associated with the composite endpoint of all-cause death and lung transplantation in PAH patients [HR: 4.85 (1.42 to 16.6), P&lt;0.01]. Importantly, we found that absolute changes in SeP levels in PAH patients significantly correlated with those in mean pulmonary artery pressure, pulmonary vascular resistance, and cardiac index in response to PAH-specific therapy (R=0.78, 0.76, and −0.71, respectively, all P&lt;0.0001). Furthermore, the increases in SeP levels during follow-up predicted the poor outcome in PAH patients [Figure, HR: 4.29 (1.27 to 14.4), P&lt;0.05]. Figure 1 Conclusions These results indicate that SeP is a novel therapeutic target of PAH and that serum SeP levels are a novel biomarker for diagnosis and assessment of treatment efficacy and long-term prognosis in PAH patients. Acknowledgement/Funding Grants-in-aid for Scientific Research from the Japan Agency for Medical Research and Development, Tokyo, Japan (16ek0109176h0001, 17ek0109227h0001).

P2590A liver-derived secretory protein, selenoprotein P causes pressure overload-induced cardiac hypertrophys

Abstract Background Hepatokine selenoprotein P (SeP) contributes to insulin resistance and hyperglycemia in patients with type 2 diabetes. Inhibition of SeP protects the heart from ischemia reperfusion injury and serum levels of SeP are elevated in patients with heart failure with reduced ejection fraction. Objective We investigated the role of SeP in the regulation of cardiac remodeling in response to pressure overload. Methods and results To examine the role of SeP in cardiac remodeling, transverse aortic constriction (TAC) was subjected to SeP knockout (KO) and wild-type (WT) mice for 2 weeks. Hepatic expression of SeP in WT was significantly increased by TAC. LV weight/tibial length (TL) was significantly smaller in SeP KO mice than in WT mice (6.75±0.24 vs 8.33±0.32, p&lt;0.01). Lung weight/TL was significantly smaller in SeP KO than in WT mice (10.46±0.44 vs 16.38±1.12, p&lt;0.05). TAC-induced cardiac upregulation of the fetal type genes, including atrial and brain natriuretic factors, was significantly attenuated in SeP KO compared to WT. Furthermore, azan staining revealed that there was significantly less interstitial fibrosis in hearts after TAC in SeP KO than in WT mice. To determine whether hepatic overexpression of SeP affects TAC-induced cardiac hypertrophy, a hydrodynamic injection method was used to generate mice that overexpress SeP mRNA in the liver. Hepatic overexpression of SeP in SeP KO mice lead to a significant increase in LV weight/TL and Lung weight/TL after TAC compared to that in other SeP KO mice. Conclusions These results suggest that cardiac pressure overload induced hepatic expression of SeP and the absence of endogenous SeP attenuated cardiac hypertrophy, dysfunction and fibrosis in response to pressure overload in mice. SeP possibly plays a maladaptive role against progression of heart failure through the liver-heart axis.

Protectin DX ameliorates palmitate‐induced hepatic insulin resistance through AMPK/SIRT1‐mediated modulation of fetuin‐A and SeP expression

The role as well as the molecular mechanisms of protectin DX (PDX) in the prevention of hepatic insulin resistance, a hallmark of type 2 diabetes, remains unknown. Therefore, the present study was designed to explore the direct impact of PDX on insulin resistance and to investigate the expression of fetuin-A and selenoprotein P (SeP), hepatokines that are involved in insulin signalling, in hepatocytes. Human serum levels of PDX as well as fetuin-A and SeP were determined by high-performance liquid chromatography (HPLC). Human primary hepatocytes were treated with palmitate and PDX. NF-κB phosphorylation as well as expression of insulin signalling associated genes and hepatokines were determined by Western blotting analysis. FOXO1 binding levels were measured by quantitative real-time PCR. Selected genes from candidate pathways were evaluated by small interfering (si) RNA-mediated gene suppression. Serum PDX levels were significantly (P<0.05) downregulated, whereas serum fetuin-A and SeP levels were increased (P<0.05) in obese subjects compared with healthy subjects. In in vitro experiments, PDX treatment increased AMP-activated protein kinase (AMPK) phosphorylation and SIRT1 expression and attenuated palmitate-induced fetuin-A and SeP expression and insulin resistance in hepatocytes. AMPK or SIRT1 siRNA mitigated the suppressive effects of PDX on palmitate-induced fetuin-A through NF-κB and SeP expression linked to FOXO1 and insulin resistance. Recombinant fetuin-A and SeP reversed the suppressive effects of fetuin-A and SeP expression on palmitate-mediated impairment of insulin signalling. The current finding provides novel insight into the underlying mechanism linking hepatokines to the pathogenesis of hepatic insulin resistance.

Selenoprotein P Promotes the Development of Pulmonary Arterial Hypertension: Possible Novel Therapeutic Target.

Excessive proliferation and apoptosis resistance of pulmonary artery smooth muscle cells (PASMCs) are key mechanisms of pulmonary arterial hypertension (PAH). Despite the multiple combination therapy, a considerable number of patients develop severe pulmonary hypertension (PH) because of the lack of diagnostic biomarker and antiproliferative therapies for PASMCs. Microarray analyses were used to identify a novel therapeutic target for PAH. In vitro experiments, including lung and serum samples from patients with PAH, cultured PAH-PASMCs, and high-throughput screening of 3336 low-molecular-weight compounds, were used for mechanistic study and exploring a novel therapeutic agent. Five genetically modified mouse strains, including PASMC-specific selenoprotein P (SeP) knockout mice and PH model rats, were used to study the role of SeP and therapeutic capacity of the compounds for the development of PH in vivo. Microarray analysis revealed a 32-fold increase in SeP in PAH-PASMCs compared with control PASMCs. SeP is a widely expressed extracellular protein maintaining cellular metabolism. Immunoreactivity of SeP was enhanced in the thickened media of pulmonary arteries in PAH. Serum SeP levels were also elevated in patients with PH compared with controls, and high serum SeP predicted poor outcome. SeP-knockout mice ( SeP-/-) exposed to chronic hypoxia showed significantly reduced right ventricular systolic pressure, right ventricular hypertrophy, and pulmonary artery remodeling compared with controls. In contrast, systemic SeP-overexpressing mice showed exacerbation of hypoxia-induced PH. Furthermore, PASMC-specific SeP-/- mice showed reduced hypoxia-induced PH compared with controls, whereas neither liver-specific SeP knockout nor liver-specific SeP-overexpressing mice showed significant differences with controls. Altogether, protein levels of SeP in the lungs were associated with the development of PH. Mechanistic experiments demonstrated that SeP promotes PASMC proliferation and resistance to apoptosis through increased oxidative stress and mitochondrial dysfunction, which were associated with activated hypoxia-inducible factor-1α and dysregulated glutathione metabolism. It is important to note that the high-throughput screening of 3336 compounds identified that sanguinarine, a plant alkaloid with antiproliferative effects, reduced SeP expression and proliferation in PASMCs and ameliorated PH in mice and rats. These results indicate that SeP promotes the development of PH, suggesting that it is a novel biomarker and therapeutic target of the disorder.

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.

Abstract 62: Selenoprotein P Promotes Vascular Smooth Muscle Cell Proliferation and Pulmonary Hypertension -A Possible Novel Therapeutic Target-

Background: Excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) is the main characteristics in pulmonary arterial hypertension (PAH); however, its mechanism still remains unclear. Methods &amp; Results: To explore a novel therapeutic target in PAH, we performed a gene expression microarray screening using PASMCs from patients with PAH (PAH-PASMCs) and those from controls and found up-regulation of selenoprotein P (SeP) in PAH-PASMCs (&gt;5-fold, P&lt;0.01). SeP is a widely expressed extracellular protein, which transports selenium to whole body. Immunostaining showed strong expression of SeP in thickened pulmonary arteries (PA) in PAH. Chronic exposure of mice to hypoxia (O 2 10%) increased SeP expression in the lung. In PAH-PASMCs, hypoxia (O 2 2%, 24 hours) increased SeP expression compared with normoxic controls (O 2 21%) (1.6-fold, P&lt;0.05). Treatment with human recombinant SeP (10μg/mL) up-regulated pro-proliferative genes, activated signals associated with cell-cycle, and increased PAH-PASMCs proliferation (P&lt;0.05, n=8 each). SeP siRNA suppressed pro-proliferative signals with a resultant reduction in PASMCs proliferation. Next, SeP-deficient mice ( SeP -/- ) were exposed to hypoxia (O 2 10%) for 4 weeks, which resulted in suppression of right ventricular systolic pressure (RVSP), RV hypertrophy and PA remodeling compared with controls (all P&lt;0.05, n=11 each). PASMCs proliferation was reduced in SeP -/- PASMCs compared with SeP +/+ PASMCs (P&lt;0.05). SeP -/- PASMCs showed ERK1/2 inactivation, AMPK activation, and reduced secretion of pro-inflammatory cytokines. SMC-targeted deletion of SeP suppressed the development of hypoxia-induced PH compared with controls (all P&lt;0.05, n=10). In contrast, liver-specific deletion of SeP did not affect the development of hypoxia-induced PH (all P&gt;0.05, n=8-9). In the clinical study, serum SeP levels were significantly elevated in PAH patients (n=203) compared with controls (n=20) (P&lt;0.001). Event-free curve revealed that high plasma SeP (≥27μg/ml) predicted poor outcome in PH patients (death or lung transplantation, P&lt;0.01). Conclusions: These results indicate that SeP, especially in PASMCs, promotes PA remodeling, suggesting that it is a novel therapeutic target for PAH.

Control of serum copper (Cu) and selenium (Se) status by thyroid hormones

Copper and selenium are trace elements essentially needed for the expression and activity of a number of important and rate-limiting human enzymes. Among the Cu-dependent proteins are the bilirubin, lysyl and catechol oxidases, the endocrine relevant enzymes dopamine-beta and peptidyl-glycine mono-oxygenases and the antioxidant enzyme superoxide dismutase. In serum, Cu is mainly transported by hepatically-derived ceruloplasmin (CP). Se is needed for the expression of selenoproteins including the deiodinases implicated in thyroid hormone (TH) activation and inactivation, the glutathione peroxidases needed for the physiologically controlled degradation of peroxides and the thioredoxin reductases controlling the intracellular redox status. Se is transported by selenoprotein P (SePP), a tightly controlled Se-containing serum protein and Se biomarker. In rodents, we have previously observed a stringent regulation of hepatic CP and SePP expression by TH. Now, we tested whether TH also affect serum Se and Cu concentrations in healthy humans.

AMPK activator-mediated inhibition of endoplasmic reticulum stress ameliorates carrageenan-induced insulin resistance through the suppression of selenoprotein P in HepG2 hepatocytes

Carrageenan (CGN) has been shown to cause inflammation through toll-like receptor 4, which may play an important role in insulin resistance and type 2 diabetes mellitus. Selenoprotein P (SeP) has recently been identified as a novel hepatokine that causes insulin resistance. Here, we report that treatment of HepG2 cells with CGN increased both CCAAT enhancer binding protein homologous protein (CHOP) and SeP expression. Pretreatment with 4-phenylbutyrate (4-PBA), an endoplasmic reticulum stress inhibitor, and PD98059, a c-Jun N-terminal kinase (JNK) inhibitor, reversed CGN-induced SeP expression. Moreover, both 4-PBA and knock-down of SeP improved CGN-induced insulin resistance. In addition, we found that adenosine monophosphate-activated protein kinase (AMPK) activators ameliorated CGN-induced insulin resistance in addition to suppressing CHOP and SeP expression. In conclusion, CGN-induced ER stress increased the expression of SeP through the JNK pathway, while AMPK activators ameliorated CGN-induced insulin resistance via SeP inhibition through the AMPK-mediated alleviation of ER stress in hepatocytes.

Salsalate and Adiponectin Improve Palmitate-Induced Insulin Resistance via Inhibition of Selenoprotein P through the AMPK-FOXO1α Pathway

Selenoprotein P (SeP) was recently identified as a hepatokine that induces insulin resistance (IR) in rodents and humans. Recent clinical trials have shown that salsalate, a prodrug of salicylate, significantly lowers blood glucose levels and increases adiponectin concentrations. We examined the effects of salsalate and full length-adiponectin (fAd) on the expression of SeP under hyperlipidemic conditions and explored their regulatory mechanism on SeP. In palmitate-treated HepG2 cells as well as high fat diet (HFD)-fed male Spraque Dawley (SD) rats and male db/db mice, SeP expression and its regulatory pathway, including AMPK-FOXO1α, were evaluated after administration of salsalate and salicylate. Palmitate treatment significantly increased SeP expression and aggravated IR, while knock-down of SeP by siRNA restored these changes in HepG2 cells. Palmitate-induced SeP expression was inhibited by both salsalate and salicylate, which was mediated by AMPK activation, and was blocked by AMPK siRNA or an inhibitor of AMPK. Chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift (EMSA) assay showed that salsalate suppressed SeP expression by AMPK-mediated phosphorylation of FOXO1α. Moreover, fAd also reduced palmitate-induced SeP expression through the activation of AMPK, which results in improved IR. Both salsalate and salicylate treatment significantly improved glucose intolerance and insulin sensitivity, accompanied by reduced SeP mRNA and protein expression in HFD-fed rats and db/db mice, respectively. Taken together, we found that salsalate and adiponectin ameliorated palmitate-induced IR in hepatocytes via SeP inhibition through the AMPK-FOXO1α pathway. The regulation of SeP might be a novel mechanism mediating the anti-diabetic effects of salsalate and adiponectin.

Reducing selenoprotein P expression suppresses adipocyte differentiation as a result of increased preadipocyte inflammation.

Oxidative stress and low-grade inflammation have been implicated in obesity and insulin resistance. As a selenium transporter, ubiquitously expressed selenoprotein P (SeP) is known to play a role in the regulation of antioxidant enzyme activity. However, SeP expression and regulation in adipose tissue in obesity and its role in inflammation and adipocyte biology remain unexplored. In this study, we examined Sepp1 gene expression and regulation in adipose tissue of obese rodents and characterized the role of Sepp1 in adipose inflammation and adipogenesis in 3T3-L1 adipocytes. We found that Sepp1 gene expression was significantly reduced in adipose tissue of ob/ob and high-fat diet-induced obese mice as well as in primary adipose cells isolated from Zucker obese rats. Rosiglitazone administration increased SeP protein expression in adipose tissue of obese mice. Treatment of either TNFα or H(2)O(2) significantly reduced Sepp1 gene expression in a time- and dose-dependent manner in 3T3-L1 adipocytes. Interestingly, Sepp1 gene silencing resulted in the reduction in glutathione peroxidase activity and the upregulation of inflammatory cytokines MCP-1 and IL-6 in preadipocytes, leading to the inhibition of adipogenesis and adipokine and lipogenic gene expression. Most strikingly, coculturing Sepp1 KD cells resulted in a marked inhibition of normal 3T3-L1 adipocyte differentiation. We conclude that SeP has an important role in adipocyte differentiation via modulating oxidative stress and inflammatory response.

Selenoprotein P expression is controlled through interaction of the coactivator PGC-1α with FoxO1a and hepatocyte nuclear factor 4α transcription factors

Selenoprotein P (SeP), the major selenoprotein in plasma, is produced mainly by the liver, although SeP expression is detected in many organs. Recently, we reported stimulation of SeP promoter activity by the forkhead box transcription factor FoxO1a in hepatoma cells and its attenuation by insulin. Here, we demonstrate that this translates into fine-tuning of SeP production and secretion by insulin. Overexpression of peroxisomal proliferator activated receptor-gamma coactivator 1alpha (PGC-1alpha) enhanced the stimulatory effect of FoxO1a on SeP promoter activity. We identified a novel functional binding site for hepatocyte nuclear factor (HNF)-4alpha, termed hepatocyte nuclear factor binding element 1, in the human SeP promoter directly upstream of the FoxO-responsive element daf16-binding element 2 (DBE2). Point mutations in hepatocyte nuclear factor binding element 1 alone or together with DBE2 decreased basal activity and responsiveness of the SeP promoter to PGC-1alpha. Moreover, the PGC-1alpha-inducing glucocorticoid dexamethasone strongly enhanced SeP messenger RNA levels and protein secretion in cultured rat hepatocytes, whereas insulin suppressed the stimulation of both PGC-1alpha and SeP caused by dexamethasone treatment. In a brain-derived neuroblastoma cell line with low basal SeP expression, SeP transcription was stimulated by PGC-1alpha together with FoxO1a, and overexpression of HNF-4alpha potentiated this effect. High-level expression of SeP in liver is ensured by concerted action of the coactivator PGC-1alpha and the transcription factors FoxO1a and HNF-4alpha. Hence, the production of SeP is regulated similarly to that of the gluconeogenic enzyme glucose-6-phosphatase. As hepatic SeP production is crucial for selenium distribution throughout the body, the present study establishes PGC-1alpha as a key regulator of selenium homeostasis.

Aberrant expression of selenoproteins in the progression of colorectal cancer

Since damage to DNA and other cellular molecules by reactive oxygen species ranks high as a major culprit in the onset and development of colorectal cancer, the aim of the present study is to clarify the role of antioxidant seleonoproteins including glutathione peroxidase (GPx), thioredoxin reductase (TXR) and selenoprotein P (SePP), and the effect of oxidative stress on the progression of colorectal cancer. Expression of 14 oxidative stress-related molecules in both tumorous and non-tumorous tissues in 41 patients was examined by immunohistochemistry and Western blot analysis. Expression levels of proteins modified by 4-hydroxy-2-nonenal (4-HNE), malonyldialdehyde (MDA) and 4-hydroxy-2-hexenal (4-HHE), and the positive rate of 8-hydroxy-2’-deoxyguanosine (8-OHdG) in tumorous tissues were much higher than those in non-tumorous tissues. Glutathione (GSH) content in tumor tissues was much lower than that in non-tumorous tissues. Expression level of selenoproteins such as GPx-1, GPx-3, and SePP, which are rapidly degraded during selenium deprivation, was significantly decreased in tumorous tissues, whereas that of GPx-2, which is resistant to selenium deprivation, was increased. Expression of SePP was decreased at stage III and IV, compared to that of stage II. These data suggest that contrasting expression pattern of the antioxidant selenoproteins plays an important role in the progression of colorectal cancer.

Stimulation of selenoprotein P promoter activity in hepatoma cells by FoxO1a transcription factor

Selenoprotein P (SeP) is the major selenoprotein in human plasma, acting as an antioxidant and serving the transport of selenium from the liver to extrahepatic tissues. We here demonstrate that the human SeP promoter responds to overexpression of FoxO1a as well as of a constitutively active form of FoxO1a. Two FoxO-responsive elements were identified and characterized by generation of point mutation and deletion constructs. Similarly, SeP mRNA was upregulated in response to activation of FoxO1a in rat hepatoma cells stably transfected with a hydroxytamoxifen-regulatable form of FoxO1a. Insulin, stimulating the phosphorylation and inactivation of FoxO1a via phosphoinositide 3-kinase (PI3K) and Akt, suppressed SeP promoter activity and mRNA synthesis. This suppressive effect of insulin on SeP expression was attenuated by inhibitors of PI3K. In conclusion, the selenoprotein P promoter is a target of the Akt/FoxO signal transduction cascade and SeP expression is regulated at the level of transcription by the forkhead box protein FoxO1a in human and rat hepatoma cells.