Glutathione Peroxidase and Phospholipid ...

Biochemical

and Molecular Roles of Nutrients

Glutathione Peroxidase and Phospholipid Hydroperoxide Glutathione Peroxidase Are Differentially Regulated in Rats by Dietary Selenium1'2'3 XIN GEN LEI,4 JACQUELINE ROGER A. SÃœNDE5

K. EVENSON,

KEVIN M. THOMPSON AND

Nutritional Sciences Group, and Departments of Biochemistry, and Food Science and Human Nutrition, University of Missouri, Columbia, MO 65211

'A preliminary

report of this

work was presented

at Ex

perimental Biology 94, April, 1994, Anaheim, CA [Lei, X. G., Evenson, J. K., Thompson, K. M. & Sunde, R. A. (1994| Effect of dietary selenium on phospholipid hydroperoxide glutathione perox idase activity and gene expression in rats. FASEB J. 8: A540 (abs.)]. 2Supported by the University of Missouri Agricultural Ex periment Station and the Food for the 21st Century program, and by NIH grants no. DK 43491 and no. CA 45164. 3The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734

INDEXING KEY WORDS:

solely to indicate this fact. 4Current address: Department

â€¢gene expression â€¢glutathione peroxidase â€¢selenium deficiency â€¢m/?/VA â€¢rats

University, Ithaca, NY 14853. 5To whom correspondence should be addressed. 6Abbreviations used: GPX, glutathione peroxidase;

of Animal

Science,

Cornell

GPX1, in

tracellular glutathione peroxidase (GSH:H2C>2 oxidoreductase, EC 1.11.1.9); PCOOH, phosphatidyl-choline hydroperoxide; PHGPX, phospholipid hydroperoxide glutathione peroxidase. 0022-3166/95 $3.00 Â©1995 American Institute of Nutrition. Manuscript received 1 September 1994. Initial review completed

3 October 1994. Revision accepted 5 January 1995. 1438

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Phospholipid hydroperoxide glutathione peroxidase (PHGPX)6 is the second intracellular selenoenzyme discovered in mammals (Maiorino et al. 1982). Origi nally it was considered a peroxidation-inhibiting protein and was later identified as a Se-dependent glutathione peroxidase (Ursini et al. 1985). Compared with the classical intracellular glutathione peroxidase (GPX1, EC 1.11.1.9), phospholipid hydroperoxide glutathione peroxidase is a monomer instead of a tetramer, and it reduces bulky phospholipid hydroperoxides that are not substrates of GPX1 (Maiorino et al. 1990). Activity of PHGPX in most tissues is approximately one tenth that of glutathione peroxidase (GPX) (Weitzel et al. 1990). Schuckelt et al. (1991) purified PHGPX from pig heart and determined a partial amino acid sequence. Based on the sequence of several proteolytic polypeptides, they synthesized degenerate oligonucleotide primers to amplify a pig heart cDNA library and obtained a partial nucleic

ABSTRACT Phospholipid hydroperoxide glutathione peroxidase (PHGPX) and classical glutathione peroxidase (GPX1) are encoded by separate genes with only about 40% amino acid and nucleic acid sequence identity. To determine the response of tissue PHGPX expression to dietary Se level and to compare these responses with those for GPX1, weanling male rats were fed amino acid diets containing from 2 (-Se) to 130 i +Se) (ig Se/kg diet or a torula diet containing 5 and 190 HQSe/kg diet as Na2SeO3 for 28 d. Tissues were analyzed for PHGPX and GPX1 activity and mRNA. There was no effect of Se on growth. In -Se rats, GPX1 activity was reduced to 1% in liver and 4-9% in heart, kidney and lung compared with +Se rats; PHGPX activity was reduced only to 25-50% in these four tissues. The Se response curves indicated that the dietary Se re quirement to reach plateau liver PHGPX activity was half that required for plateau GPX activity. In -Se rats, liver and heart GPX1 mRNA levels were reduced to 6 and 12%, respectively, whereas PHGPX mRNA was not sig nificantly affected by Se deficiency. Notably, 65 HQSe/ kg diet resulted in plateau liver GPX1 mRNA levels but not plateau GPX activity. Testis had the lowest GPX activity and GPX1 mRNA of all tissues examined, but had 15-fold higher PHGPX activity and 45-fold higher PHGPX mRNA levels when compared with liver. There was no significant effect of dietary Se on testis GPX1 and PHGPX mRNA levels. This study demonstrates that these two selenoperoxidases are differentially regulated by dietary Se. Differences in Se regulation of mRNA levels in liver and heart were even more pronounced than for enzyme activity. The lack of any significant effect of reduced dietary Se on PHGPX mRNA levels suggests that there are detailed underlying molecular mechanisms whereby Se status regulates GPX1 mRNA levels but not PHGPX mRNA levels. J. Nutr. 125: 1438-1446, 1995.

SELENIUM REGULATION

MATERIALS AND METHODS Animals and diets. All named chemicals, unless otherwise indicated, were purchased from Sigma Chemical (St. Louis, MO). Forty Holtzman male weanling rats (Holtzman, Madison, WI), 21-d-old, were randomly divided into eight groups. Six groups of rats were fed a basal crystalline amino acid diet,7 containing 2 Â¿tgSe/kg by analysis, or supplemented with five graded levels of Se up to 130 /Â¿g/kgdiet as Na2SeOa. The other two groups of rats were fed either a basal torula yeast-based diet8 containing 5 /*g Se/kg by analysis, or that diet with 190 /ig Se/kg as Na2SeOa. Rats were reared in hanging wire cages in temperature-controlled (22Â°C)animal quarters with a 12-h light:dark cycle. Animals were given free access to water and feed. Care and treatment of experimental animals was approved by the Institutional Animal Care and Use Committee at the University of Mis souri.

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Sample collection. On d 28, three rats were selected randomly from each treatment group, anesthetized with ether, and killed by exsanguination via heart puncture using a heparinized syringe. Livers were perfused with ice-cold 0.15 mol/L KC1 in situ and divided into three portions. Heart, kidney, lung and testis were divided into two portions, frozen in liquid N2 and stored at -80Â°C. Plasma samples were prepared and assayed for GPX activity on the same day. Tissue GPX and PHGPX activities were assayed within 2 wk after collection. Substrate synthesis. Phosphatidylcholine hydroperoxide (PCOOH), the substrate for the PHGPX ac tivity assay, was synthesized using a method modified from that of Maiorino et al. (1990) and Weitzel et al. (1990). Briefly, 10 mg L-a-phosphatidylcholine (Type III, from soybean, Sigma P-6263), dried in an argon stream in a 50-mL reaction flask, was dispersed with 4 mL of 3% deoxycholate and diluted with 21 mL of 0.20 mol/L sodium borate, pH 9.0. The reaction was started by bubbling with 99% Ã›2 and addition of 100 jtL (500,000 units) of lipoxidase (EC 1.13.11.12, type IV), and continued at 37Â°C with stirring for 1 h. The reaction mixture was then passed through a Sep-Pak Qg cartridge (Waters, Milford, MA) previously washed with methanol and equilibrated with water. The cartridge was next washed with 20 mL water, and the PCOOH was then eluted with 2 mL of methanol. The hydroperoxide content of PCOOH was determined by reduction of NADPH in the coupled assay with partially purified PHGPX and verified iodometrically (Thomas et al. 1990). Enzyme activity assay. Tissues were thawed on ice and homogenized in three volumes (wt/v) of 0.25 mol/L sucrose, containing 20 mmol/L Tris-HCl, pH 7.4, 0.1% peroxide-free Triton X-100, with a Polytron homogenizer (model PT 10/35, Brinkmann, Westbury, NY), and centrifuged at 10,000 x g for 15 min (JA20, J2-21M, Beckman Instruments, Palo Alto, C A). Super natant was saved for enzyme activity assay. Activity of PHGPX was assayed spectrophotometrically (UVIKON 940, Kontron Instrument, San Diego, CA)

7Amino acid basal diet (g/kg diet, as described by Thompson et al. 1995): crystalline amino acid mix [(in g/kg amino acid mix): Lalanine, 44.6; L-arginine free base, 44.3; L-asparagine-J^O, 37.8; Laspartic acid, 44.6; glycine, 44.6; L-histidine, 26.7; L-isoleucine, 44.6; L-leucine, 66.8; L-lysine hydrochloride, 62.4; L-methionine, 53.5; L-monosodium glutamate, 267.3; L-phenylalanine, 44.6; Lproline, 35.6; L-serine, 44.6; L-threonine, 44.6; L-tryptophan, 13.4; Ltyrosine, 26.7; L-valine, 53.5], 179; sucrose, 674; Solka Floe, 50; lard, 35; cod liver oil, 15; AIN-76A vitamin mix, 10; AIN-76 mineral mix, 35; all-rac-a-tocopheryl acetate, 0.15; choline chloride, 2. 8Torula basal diet (g/kg diet, as described by Knight & Sunde 1987): torula yeast, 300; sucrose, 585.9; lard, 50; mineral mix, 50; vitamin mix, 9; DL-methionine, 4; all-rac-a-tocopheryl acetate, 0.1; choline chloride, 1.


acid sequence of PHGPX. Their results demonstrated that PHGPX is a unique gene product. Recently, we isolated and sequenced two full-length cDNA clones for pig (Sunde et al. 1993) and rat (Sunde 1994) PHGPX and found that the amino acid and nucleotide sequence identities between GPX1 and PHGPX were both less than 40%. The porcine structural gene has now been sequenced (Brigelius-FlohÃ© et al. 1994). Previous work in our laboratory showed that Se deficiency in rats resulted in 90% loss of liver GPX1 mRNA as well as 99% loss of GPX activity (Saedi et al. 1988, Sunde et al. 1993). A single injection of Se to deficient rats rapidly restores both GPX activity and mRNA (Sunde et al. 1989), showing that expression of GPX in vivo is specifically regulated by dietary Se. Knowledge about the response of PHGPX activity to altered dietary Se, however, is limited to Se-deficient and -adequate treatments (Roveri et al. 1992, Weitzel et al. 1990, Zhang et al. 1989). Our initial use of cloned pig PHGPX cDNA to determine liver PHGPX mRNA levels in rats fed Se-deficient torula diets also indicated that PHGPX mRNA was little affected com pared with GPX1 mRNA levels (Sunde et al. 1993). Because PHGPX and GPX differ remarkably in structure, substrate specificity and presumed func tions (Maiorino et al. 1990), we hypothesized (Sunde 1994) that PHGPX expression is regulated by dietary Se differently than is GPX1 expression. The objectives of this study were to elucidate the response of tissue PHGPX expression to dietary Se level, and to compare these response patterns with the response curves for GPX1. Weanling male rats were fed graded levels of dietary Se in basal amino acid or torula diets, and the activity and mRNA level of both selenoperoxidases were determined in several tissues.

OF TWO SELENOPEROXIDASES

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at 25Â°Cas described previously

LEI ET AL.

(Maiorino et al. 1990,

Weitzel et al. 1990). Briefly, 200 /iL of buffer con taining 0.5 mol/L Tris-HCl (pH 7.4), 25 mmol/L EDTA, 0.5 mmol/L NADPH, 5 mmol/L NaN3, 15 mmol/L reduced glutathione, 510 iiL H2O, 10 /Â¿L20% Triton X-100, 10 /iL glutathione reductase (1.2 x 10~5

samples, with one sample from each of the eight different dietary Se groups, were subjected to denaturing agarose gel electrophoresis. Three blots with samples from different rats were conducted for each tissue. Additional blots, with samples from Seadequate rats (130 Â¿tgSe/kg diet), compared relative abundance of GPX1 and PHGPX mRNA for liver, heart and testis. Integrity of RNA and equality of loading for each lane was verified by staining with acridine orange. The RNA was transferred to biotrans membrane (Pall Biosupport, East Hills, NY) and probed with a random-primed 32P-labeled 650 bp EcoRI/XhoI fragment of rat PHGPX cDNA (Sunde unpublished, accession number L-24896). The GPX1 mRNA was detected on the same blot after stripping, using the 700 bp EcoRI fragment of murine GPX1 cDNA (Saedi et al. 1988). The blots were then restripped, and the level of 18S rRNA was determined using an 1.4 kb BamHl fragment of genomic human 18S rRNA (Erickson et al. 1981) as described previ ously (Hesketh et al. 1994). Densitometry (Ultrascan XL 2222, Pharmacia LKB Biotechnology, Uppsala, Sweden) of resulting autoradiograms was used to quantify mRNA. Relative levels of mRNA were ex pressed as the percentage of the density for rats fed the 130 /ig Se/kg diet. Sample selenium analysis. Diet and liver Se con centrations were measured by neutron activation analysis (McKown and Morris 1978). For liver,

RESULTS Diet selenium. The measured Se concentrations of the basal amino acid and torula diets were 2 and 5 /ig/ kg, respectively, and the five Se-supplemented amino acid diets contained 6.5, 13, 33, 65, or 130 /ig Se/kg. The Se-supplemented torula diet contained 190/ig Se/ kg. We originally included torula diets to determine the level of PHGPX expression in torula-fed rats so that the PHGPX values we observed in the amino acid-fed rats could be compared directly with our previous experiments. There was no significant inter action between diet type and Se level, thus data from rats fed the two diets were pooled to focus on the effect of dietary Se concentrations and to extend the biological implications of the experiments. Body weight gain, liver selenium, and plasma glutathione peroxidase activity. There was no sig nificant effect of dietary Se on growth (P = 0.85, Table 1). Liver Se increased significantly with increasing dietary Se supplementation, and plasma GPX activity also increased as dietary Se increased; the steepest increase in plasma GPX activity occurred with sup plementation between 13 and 33 /ig Se/kg diet. Selenoperoxidase activity. Activity of GPX in tissues other than testis was greatly affected by di etary Se levels (Fig. l, P < 0.0001). Compared with Seadequate rats (130 /Â¿gSe/kg), rats fed the basal amino acid diet retained only 1, 6, 4 and 9% of the Seadequate GPX activity in liver, heart, kidney and lung, respectively. The sharpest increase in GPX ac tivity occurred with supplementation between 33 and 65 /ig Se/kg diet. Plateau levels (defined as the level of


U/L), and 200 Â¡iLdiluted tissue sample were mixed in a 1-mL cuvette. The reaction mixture was preincubated for 3 min, and the initial slope of NADPH disappearance (AA34onm) was determined. Then 70 /iL of PCOOH (78 nmol) was added and the PCOOH-dependent slope was measured for 3 min. With proper choice of tissue dilution, the slope remained linear for 10 min. The PHGPX activity was calculated from the difference between the two slopes, and one enzyme unit was defined as one micromole glutathione oxi dized per minute under these conditions. The GPX activity was assayed using initial concentrations of 2 mmol/L glutathione and 120 nmol/L H2Ã›2 as described previously (Lawrence et al. 1974). Protein was determined as described by Lowry et al. (1951). mRNA analysis. To determine the effect of dietary Se on selenoperoxidase mRNA, total cellular RNA was isolated from liver, heart, and testis samples by the guanidinium isothiocyanate method and sub jected to Northern blot analysis as described previ ously (Saedi et al. 1988). Total RNA was quantified at A260nm using an extinction coefficient of 25 mL-mg~1-cm~1. For each blot, eight 30-/ig RNA

triplicate 0.2-0.4 g samples were irradiated for 5 s, and after a 15 s delay, samples were counted for 25 s using a germanium-lithium detector to measure the 77mSe gamma emission. Integrated peak areas were compared with areas of known selenium standards to determine the selenium concentration. Diet samples were digested in nitric acid before irradiation. Bovine liver or orchard leaves standards (National Bureau of Standards, Gaithersburg, MD) were used as standards. Statistical analysis. All analysis was conducted using SAS (release 6.04, SAS Institute, Cary, NC) following established methods (Steel and Torrie 1960). Interaction between diet type and Se level was first checked before pooling data from both types of diets. Main effects of dietary Se on measures were analyzed by one-way ANOVA, and Bonferroni t test was used for multiple mean comparisons. Linear (first-degree polynomial: Y = a + bX} and quadratic (second-degree polynomial: Y = a + bX + cX2) regres sions (Gill 1978) were conducted to determine the overall response patterns of various measures to di etary Se. Relationship between measures was deter mined by correlation (Steel and Torrie 1960).

SELENIUM REGULATION

TABLE

1

Effect of dietary Se on body weight gain, liver Se concentration and plasma glutathione peroxidase (GPX) activity^ Dietary Se2

Weight gain

Liver Se3

Plasma GPX4

Se/kg diets were amino acid diets, and the 5 and 190 (Â¿g Se/kg diets were tonila diets. ^Responses of liver Se concentration [Y] to dietary Se (X) fit a first-degree polynomial: y - 0.278 + 0.038X [R1 Â»0.94, P < 0.0001). Data are expressed on wet tissue basis. 4Responses of plasma GPX activity (Y) to dietary Se (X) fit a second degree polynomial: Y - -2.44 + 1.50X - 0.005X2 |R2 - 0.95, P < 0.0001). Enzyme unit (EU) is micromoles of glutathione oxidized per minute at 25'C. Significance

of overall treatment

effects.

enzyme activity or mRNA for each tissue that is not significantly different from the level in rats fed 130 /ig Se/kg diet) of GPX activity in these four tissues were reached with supplementation between 65 and 130 /ig Se/kg diet. Overall responses of the four tissues' GPX activity to dietary Se fit second-degree polynomials (R2 > 0.93, P < 0.0001). The PHGPX activities in tissues of rats fed the Sedeficient basal amino acid diet were 41, 50, 26 and 25% of Se-adequate PHGPX activities in liver, heart, kidney, and lung, respectively (Fig. 1). The sharpest increase in PHGPX activity was seen with sup plementation between 13 and 65 /ig Se/kg diet in liver and kidney and between 13 and 33 /ig Se/kg diet in heart and lung. Heart PHGPX activity reached plateau levels with supplementation at 33 /ig Se/kg diet; liver, kidney and lung PHGPX activities reached plateau levels at 65 /ig Se/kg. Overall responses of PHGPX activity to dietary Se were also described well by second-degree polynomials (R2 > 0.81, P < 0.0001). Testis had the lowest GPX activity but the highest PHGPX activity among all tissues assayed (Fig. 2). Testis GPX activity was reduced significantly to 53% of the plateau level in rats fed the basal amino acid diet. Mean testis GPX activities increased nonsignificantly with supplementation between 5 and 33 /ig Se/

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g diet, and there was a significant overall quadratic response to dietary Se (P < 0.0005). There also was a significant quadratic response of testis PHGPX ac tivity to dietary Se (P < 0.003), but there were no significant differences among any dietary Se levels (Fig. 2), although rats fed the three lowest Se diets had mean PHGPX activities that were 57-70% of plateau levels. Selenoperoxidase mRNA. Relative abundance of GPX1 mRNA in Se-adequate rats fed the 130 /ig Se/kg diet was 100, 54.1, and 3.64 in liver, heart, and testis, respectively (Fig. 3). Corresponding relative PHGPX mRNA levels were 100, 101, and 4520, respectively, indicating that PHGPX mRNA abundance was 45-fold richer in testis than in liver and heart. Liver GPX1 mRNA levels were reduced to 6-8% of Se-adequate (130 /ig Se/kg diet) plateau levels in rats fed the two basal Se-deficient diets (P < 0.0001, Fig. 4 and 5). Liver GPX1 mRNA responded sigmoidally to increasing dietary Se concentration such that liver GPX1 mRNA levels reached the Se-adequate plateau with supplementation at 65 /tg Se/kg diet. In contrast, liver PHGPX mRNA was not significantly affected by dietary Se (P = 0.37) (Fig. 4, 5); the PHGPX mRNA level in rats fed the 2 /ig Se/kg diet was decreased 39% relative to the Se-adequate plateau but the quad ratic response was also not significant (P = 0.09). Responses of heart GPX1 and PHGPX mRNA to di etary Se were basically the same as those for liver. Overall responses of liver and heart GPX1 mRNA to dietary Se fit second-degree polynomials (R2 > 0.88, P < 0.0001) much better than the same type responses of PHGPX mRNA (liver: R2 = 0.20, P < 0.09 and heart: R2 = 0.29, P < 0.03). Testis GPX1 mRNA was little affected by dietary Se level (P = 0.73) (Fig. 5); the apparent 27-39% reduction of testis GPX1 mRNA in rats fed the two lowest Se diets, however, was not significant. Testis PHGPX mRNA was also not af fected by dietary Se (P = 0.99, Fig. 5). Dietary Se had no effect on the level of 18S rRNA in liver, heart and testis (data not shown, P = 0.62, 0.74, 0.10, respectively). When individual GPX1 and PHGPX mRNA levels were normalized based on 18S rRNA, the response curves were not affected. For instance, normalized Se-deficient liver and heart GPX1 mRNA levels were 7.1 and 11.0%, respectively, of normalized Se-adequate plateau values (data not shown) as compared with 6.0 and 11.9% shown in Fig. 5. An ANO VA of normalized PHGPX mRNA levels also indicated that there was no significant difference due to dietary Se in liver, heart or testis (data not shown). Correlations between measures. Liver Se content was highly correlated with liver GPX activity (R2 = 0.98, P < 0.0001) and liver GPX1 mRNA (R2 = 0.88, P < 0.0001). In contrast, liver Se correlated well with liver PHGPX activity (R2 = 0.95, P < 0.0001), but not with PHGPX mRNA (R2 = 0.03, P = 0.40). There was no correlation between PHGPX mRNA and activity in any tissue (R2 = 0.03-0.10, P > 0.45).


tissue0.16 protein2.00 /g 0.01e0.19 Â± 0.58e4.00 Â± 0.107.65 Â± 0.06e0.28 Â± 0.58e6.33 Â± 0.768.01 Â± Â±0.03e Â± 1.86e Â±0.67 0.54 Â±0.03de 9.67 Â± 0.88e 13 8.07 Â±0.36 0.08d4.02 1.35 Â± 44.00 2.00b78.33 Â± 3365130190 8.52 0.289.30 Â± Â±0.26C Â± 6.17a Â±0.31 0.25b6.74 5.55 Â± 95.33 Â± 14.15a94.33 8.64 0.257.58 Â± Â±0.23a Â± 4.41a Â±0.16 P5