Amy K. Motley

Selenoprotein Metabolism and Function: Evidence for More than One Function for Selenoprotein P

Biological functions of selenium are exerted by selenoproteins that contain selenocysteine in their primary structure. Selenocysteine is synthesized and inserted into proteins cotranslationally by a complex process. Families of selenoproteins include the glutathione peroxidases, the iodothyronine deiodinases and the thioredoxin reductases. These are redox enzymes that take advantage of the chemical properties of selenium to catalyze, respectively, removal of hydroperoxides by glutathione, deiodination of thyroid hormones and support of cellular processes requiring reduction of disulfides. Approximately 10 additional selenoproteins have been identified. One of them, selenoprotein P, is an extracellular protein that contains most of the selenium in plasma. It associates with endothelial cells, probably through its heparin-binding properties. Selenoprotein P has been postulated to protect against oxidative injury and to transport selenium from the liver to peripheral tissues. Selenium-dependent protection against diquat-induced liver necrosis and lipid peroxidation in the rat correlates with the presence of selenoprotein P. Recent results support a transport function. When (75)SeO(3)(2-) was administered intravenously to rats, liver tissue took up (75)Se within minutes, associated with a rapid decline in plasma (75)Se. Brain tissue did not begin accumulating (75)Se until (75)Se-labeled selenoprotein P had begun appearing in the plasma after 30 min. These results suggest that tissues like liver can take up small-molecule forms of selenium whereas presence of the element in selenoprotein P facilitates uptake by tissues like brain. Thus, there is evidence for both antioxidant and selenium transport functions of selenoprotein P.

Effects of Chemical Form of Selenium on Plasma Biomarkers in a High-Dose Human Supplementation Trial

Intervention trials with different forms of selenium are under way to assess the effects of selenium supplements on the incidence of cancer and other diseases. Plasma selenium biomarkers respond to selenium administration and might be useful for assessing compliance and safety in these trials. The present study characterized the effects of selenium supplementation on plasma selenium biomarkers and urinary selenium excretion in selenium-replete subjects. Moderate (∼200 μg/d) to large (∼600 μg/d) selenium supplements in the forms sodium selenite, high-selenium yeast (yeast), and l-selenomethionine (selenomethionine) were administered. Subjects were randomized into 10 groups (placebo and three dose levels of each form of selenium). Plasma biomarkers (selenium concentration, selenoprotein P concentration, and glutathione peroxidase activity) were determined before supplementation and every 4 weeks for 16 weeks. Urinary selenium excretion was determined at 16 weeks. Supplementation with selenomethionine and yeast raised the plasma selenium concentration in a dose-dependent manner. Selenite did not. The increased selenium concentration correlated with the amount of selenomethionine administered. Neither glutathione peroxidase activity nor selenoprotein P concentration responded to selenium supplementation. Urinary selenium excretion was greater after selenomethionine than after selenite, with excretion after yeast being intermediate and not significantly different from either of the other two. We conclude that plasma selenium concentration is useful in monitoring compliance and safety of selenium supplementation as selenomethionine but not as selenite. Plasma selenium seems to reflect the selenomethionine content of yeast but not the other yeast selenium forms. As judged by urinary selenium excretion, selenium in the form of selenomethionine is better absorbed than selenite. (Cancer Epidemiol Biomarkers Prev 2006;15(4):804–10)

Establishing optimal selenium status: results of a randomized, double-blind, placebo-controlled trial

BACKGROUND Dietary recommendations for selenium differ between countries, mainly because of uncertainties over the definition of optimal selenium status. OBJECTIVE The objective was to examine the dose-response relations for different forms of selenium. DESIGN A randomized, double-blind, placebo-controlled dietary intervention was carried out in 119 healthy men and women aged 50-64 y living in the United Kingdom. Daily placebo or selenium-enriched yeast tablets containing 50, 100, or 200 microg Se ( approximately 60% selenomethionine), selenium-enriched onion meals ( approximately 66% gamma-glutamyl-methylselenocysteine, providing the equivalent of 50 microg Se/d), or unenriched onion meals were consumed for 12 wk. Changes in platelet glutathione peroxidase activity and in plasma selenium and selenoprotein P concentrations were measured. RESULTS The mean baseline plasma selenium concentration for all subjects was 95.7 +/- 11.5 ng/mL, which increased significantly by 10 wk to steady state concentrations of 118.3 +/- 13.1, 152.0 +/- 24.3, and 177.4 +/- 26.3 ng/mL in those who consumed 50, 100, or 200 microg Se-yeast/d, respectively. Platelet glutathione peroxidase activity did not change significantly in response to either dose or form of selenium. Selenoprotein P increased significantly in all selenium intervention groups from an overall baseline mean of 4.99 +/- 0.80 microg/mL to 6.17 +/- 0.85, 6.73 +/- 1.01, 6.59 +/- 0.64, and 5.72 +/- 0.75 microg/mL in those who consumed 50, 100, or 200 microg Se-yeast/d and 50 microg Se-enriched onions/d, respectively. CONCLUSIONS Plasma selenoprotein P is a useful biomarker of status in populations with relatively low selenium intakes because it responds to different dietary forms of selenium. To optimize the plasma selenoprotein P concentration in this study, 50 microg Se/d was required in addition to the habitual intake of approximately 55 microg/d. In the context of established relations between plasma selenium and risk of cancer and mortality, and recognizing the important functions of selenoprotein P, these results provide important evidence for deriving estimated average requirements for selenium in adults. This trial was registered at clinicaltrials.gov as NCT00279812.

Deletion of Apolipoprotein E Receptor-2 in Mice Lowers Brain Selenium and Causes Severe Neurological Dysfunction and Death When a Low-Selenium Diet Is Fed

Selenoprotein P (Sepp1) is a plasma and extracellular protein that is rich in selenium. Deletion of Sepp1 results in sharp decreases of selenium levels in the brain and testis with dysfunction of those organs. Deletion of Sepp1 also causes increased urinary selenium excretion, leading to moderate depletion of whole-body selenium. The lipoprotein receptor apolipoprotein E receptor-2 (apoER2) binds Sepp1 and facilitates its uptake by Sertoli cells, thus providing selenium for spermatogenesis. Experiments were performed to assess the effect of apoER2 on the concentration and function of selenium in the brain and on whole-body selenium. ApoER2−/− and apoER2+/+ male mice were fed a semipurified diet with selenite added as the source of selenium. ApoER2−/− mice had depressed brain and testis selenium, but normal levels in liver, kidney, muscle, and the whole body. Feeding a selenium-deficient diet to apoER2−/− mice led to neurological dysfunction and death, with some of the characteristics exhibited by Sepp1−/− mice fed the same diet. Thus, although it does not affect whole-body selenium, apoER2 is necessary for maintenance of brain selenium and for prevention of neurological dysfunction and death under conditions of selenium deficiency, suggesting an interaction of apoER2 with Sepp1 in the brain.

Optimization of selenoprotein P and other plasma selenium biomarkers for the assessment of the selenium nutritional requirement: a placebo-controlled double-blind study of selenomethionine supplementation in selenium-deficient Chinese subjects

BACKGROUND The intake of selenium needed for optimal health has not been established. Selenoproteins perform the functions of selenium, and the selenium intake needed for their full expression is not known. OBJECTIVE This study sought to determine the intake of selenium required to optimize plasma selenoprotein P (SEPP1) and to compare SEPP1 with other plasma selenium biomarkers. DESIGN A 40-wk placebo-controlled, double-blind study of selenium repletion was carried out in 98 healthy Chinese subjects who had a daily dietary selenium intake of 14 micro g. Fourteen subjects each were assigned randomly to daily dose groups of 0, 21, 35, 55, 79, 102, and 125 micro g Se as l-selenomethionine. Plasma glutathione peroxidase (GPX) activity, SEPP1, and selenium were measured. A biomarker was considered to be optimized when its value was not different from the mean value of the subjects receiving larger supplements. RESULTS The SEPP1 concentration was optimized at 40 wk by the 35- micro g supplement, which indicated that 49 micro g/d could optimize it. GPX activity was optimized by 21 micro g (total ingestion: 35 micro g/d). The selenium concentration showed no tendency to become optimized. CONCLUSIONS The present results indicate that SEPP1 concentration is the best plasma biomarker studied for assessing optimal expression of all selenoproteins, because its optimization required a larger intake of selenium than did GPX activity. On the basis of the selenium intake needed for SEPP1 optimization with adjustments for body weight and individual variation, ap 75 micro g Se/d as selenomethionine is postulated to allow full expression of selenoproteins in US residents. This trial was registered at clinicaltrials.gov as NCT00428649.

The Selenium-rich C-terminal Domain of Mouse Selenoprotein P Is Necessary for the Supply of Selenium to Brain and Testis but Not for the Maintenance of Whole Body Selenium

Selenoprotein P (Sepp1) has two domains with respect to selenium content: the N-terminal, selenium-poor domain and the C-terminal, selenium-rich domain. To assess domain function, mice with deletion of the C-terminal domain have been produced and compared with Sepp1–/– and Sepp1+/+ mice. All mice studied were males fed a semipurified diet with defined selenium content. The Sepp1 protein in the plasma of mice with the C-terminal domain deleted was determined by mass spectrometry to terminate after serine 239 and thus was designated Sepp1Δ240–361. Plasma Sepp1 and selenium concentrations as well as glutathione peroxidase activity were determined in the three types of mice. Glutathione peroxidase and Sepp1Δ240–361 accounted for over 90% of the selenium in the plasma of Sepp1Δ240–361 mice. Calculations using results from Sepp1+/+ mice revealed that Sepp1, with a potential for containing 10 selenocysteine residues, contained an average of 5 selenium atoms per molecule, indicating that shortened and/or selenium-depleted forms of the protein were present in these wild-type mice. Sepp1Δ240–361 mice had low brain and testis selenium concentrations that were similar to those in Sepp1–/– mice but they better maintained their whole body selenium. Sepp1Δ240–361 mice had depressed fertility, even when they were fed a high selenium diet, and their spermatozoa were defective and morphologically indistinguishable from those of selenium-deficient mice. Neurological dysfunction and death occurred when Sepp1Δ240–361 mice were fed selenium-deficient diet. These phenotypes were similar to those of Sepp1–/– mice but had later onset or were less severe. The results of this study demonstrate that the C terminus of Sepp1 is critical for the maintenance of selenium in brain and testis but not for the maintenance of whole body selenium.

Extracellular glutathione peroxidase (Gpx3) binds specifically to basement membranes of mouse renal cortex tubule cells

Glutathione peroxidase-3 (Gpx3), also known as plasma or extracellular glutathione peroxidase, is a selenoprotein secreted primarily by kidney proximal convoluted tubule cells. In this study Gpx3(-/-) mice have been produced and immunocytochemical techniques have been developed to investigate Gpx3 metabolism. Gpx3(-/-) mice maintained the same whole-body content and urinary excretion of selenium as did Gpx3(+/+) mice. They tolerated selenium deficiency without observable ill effects. The simultaneous knockout of Gpx3 and selenoprotein P revealed that these two selenoproteins account for >97% of plasma selenium. Immunocytochemistry experiments demonstrated that Gpx3 binds selectively, both in vivo and in vitro, to basement membranes of renal cortical proximal and distal convoluted tubules. Based on calculations using selenium content, the kidney pool of Gpx3 is over twice as large as the plasma pool. These data indicate that Gpx3 does not serve in the regulation of selenium metabolism. The specific binding of a large pool of Gpx3 to basement membranes in the kidney cortex strongly suggests a need for glutathione peroxidase activity in the cortical peritubular space.

Production of Selenoprotein P (Sepp1) by Hepatocytes Is Central to Selenium Homeostasis

Background: Sepp1 transports selenium, but its complete role in selenium homeostasis is not known. Results: Deletion of Sepp1 in hepatocytes increases liver selenium at the expense of other tissues and decreases whole-body selenium by increasing excretion. Conclusion: Sepp1 production by hepatocytes retains selenium in the organism and distributes it from the liver to peripheral tissues. Significance: Sepp1 is central to selenium homeostasis. Sepp1 is a widely expressed extracellular protein that in humans and mice contains 10 selenocysteine residues in its primary structure. Extra-hepatic tissues take up plasma Sepp1 for its selenium via apolipoprotein E receptor-2 (apoER2)-mediated endocytosis. The role of Sepp1 in the transport of selenium from liver, a rich source of the element, to peripheral tissues was studied using mice with selective deletion of Sepp1 in hepatocytes (Sepp1c/c/alb-cre+/− mice). Deletion of Sepp1 in hepatocytes lowered plasma Sepp1 concentration to 10% of that in Sepp1c/c mice (controls) and increased urinary selenium excretion, decreasing whole-body and tissue selenium concentrations. Under selenium-deficient conditions, Sepp1c/c/alb-cre+/− mice accumulated selenium in the liver at the expense of extra-hepatic tissues, severely worsening clinical manifestations of dietary selenium deficiency. These findings are consistent with there being competition for metabolically available hepatocyte selenium between the synthesis of selenoproteins and the synthesis of selenium excretory metabolites. In addition, selenium deficiency down-regulated the mRNA of the most abundant hepatic selenoprotein, glutathione peroxidase-1 (Gpx1), to 15% of the selenium-replete value, while reducing Sepp1 mRNA, the most abundant hepatic selenoprotein mRNA, only to 61%. This strongly suggests that Sepp1 synthesis is favored in the liver over Gpx1 synthesis when selenium supply is limited, directing hepatocyte selenium to peripheral tissues in selenium deficiency. We conclude that production of Sepp1 by hepatocytes is central to selenium homeostasis in the organism because it promotes retention of selenium in the body and effects selenium distribution from the liver to extra-hepatic tissues, especially under selenium-deficient conditions.

Selenium deficiency activates mouse liver Nrf2–ARE but vitamin E deficiency does not

Selenium (Se) and vitamin E are antioxidant micronutrients. Se functions through selenoproteins and vitamin E reacts with oxidizing molecules in membranes. The relationship of these micronutrients with the Nrf2-antioxidant response element (ARE) pathway was investigated using ARE-reporter mice and Nrf2-/- mice. Weanling males were fed Se-deficient (0 Se), vitamin E-deficient (0 E), or control diet for 16 or 22 weeks. The ARE reporter was elevated 450-fold in 0 Se liver but was not elevated in 0 E liver. Antioxidant enzymes induced by Nrf2-ARE (glutathione S-transferase (GST), NAD(P)H quinone oxidoreductase (NQOR), and heme oxygenase-1 (HO-1)) were elevated in 0 Se livers but not in 0 E livers. Deletion of Nrf2 had varying effects on the inductions, with GST induction being abolished by it but induction of NQOR and HO-1 still occurring. Thus, Se deficiency, but not vitamin E deficiency, induces a number of enzymes that protect against oxidative stress and modify xenobiotic metabolism through Nrf2-ARE and other stress-response pathways. We conclude that Se deficiency causes cytosolic oxidative stress but that vitamin E deficiency does not. This suggests that the oxidant defense mechanisms in which these antioxidant nutrients function are independent of one another.

Dietary selenium deficiency exacerbates DSS-induced epithelial injury and AOM/DSS-induced tumorigenesis.

Selenium (Se) is an essential micronutrient that exerts its functions via selenoproteins. Little is known about the role of Se in inflammatory bowel disease (IBD). Epidemiological studies have inversely correlated nutritional Se status with IBD severity and colon cancer risk. Moreover, molecular studies have revealed that Se deficiency activates WNT signaling, a pathway essential to intestinal stem cell programs and pivotal to injury recovery processes in IBD that is also activated in inflammatory neoplastic transformation. In order to better understand the role of Se in epithelial injury and tumorigenesis resulting from inflammatory stimuli, we examined colonic phenotypes in Se-deficient or -sufficient mice in response to dextran sodium sulfate (DSS)-induced colitis, and azoxymethane (AOM) followed by cyclical administration of DSS, respectively. In response to DSS alone, Se-deficient mice demonstrated increased morbidity, weight loss, stool scores, and colonic injury with a concomitant increase in DNA damage and increases in inflammation-related cytokines. As there was an increase in DNA damage as well as expression of several EGF and TGF-β pathway genes in response to inflammatory injury, we sought to determine if tumorigenesis was altered in the setting of inflammatory carcinogenesis. Se-deficient mice subjected to AOM/DSS treatment to model colitis-associated cancer (CAC) had increased tumor number, though not size, as well as increased incidence of high grade dysplasia. This increase in tumor initiation was likely due to a general increase in colonic DNA damage, as increased 8-OHdG staining was seen in Se-deficient tumors and adjacent, non-tumor mucosa. Taken together, our results indicate that Se deficiency worsens experimental colitis and promotes tumor development and progression in inflammatory carcinogenesis.