Journal of Inorganic Biochemistry 114 (2012) 47–54

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Journal of Inorganic Biochemistry

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Supranutritional selenium induces alterations in molecular targets related to energymetabolism in skeletal muscle and visceral adipose tissue of pigs

Antonio Pinto a,1, Darren T. Juniper b,1, Mert Sanil c, Linda Morgan c, Lynne Clark b, Helmut Sies a,d,e,Margaret P. Rayman c,1, Holger Steinbrenner a,⁎,1

a Institute for Biochemistry and Molecular Biology I, Heinrich-Heine-University, Universitätsstrasse 1, D-40225 Düsseldorf, Germanyb School of Agriculture, Policy and Development, University of Reading, Reading RG6 6AR, UKc Faculty of Health and Medical Sciences, University of Surrey, Guildford GU2 7XH, UKd Leibniz-Institut für umweltmedizinische Forschung (IUF), Heinrich-Heine-University, Auf'm Hennekamp 50, D-40225 Düsseldorf, Germanye College of Science, King Saud University, Riyadh, Saudi Arabia

⁎ Corresponding author at: Heinrich-Heine-University Dütry andMolecular Biology I, Universitätsstrasse 1, Geb. 22.03Tel.: +49 211 8112712; fax: +49 211 8113029.

E-mail address: Holger.Steinbrenner@uni-duesseldo1 These authors contributed equally to this work.

0162-0134/$ – see front matter © 2012 Elsevier Inc. Alldoi:10.1016/j.jinorgbio.2012.04.011

a b s t r a c t

a r t i c l e i n f o

Article history:Received 23 January 2012Received in revised form 17 April 2012Accepted 17 April 2012Available online 30 April 2012

Keywords:Trace elementSelenium supplementationSelenoproteinInsulinDiabetesAkt

While selenium (Se) is an essential micronutrient for humans, epidemiological studies have raised concern thatsupranutritional Se intake may increase the risk of developing Type 2 diabetes mellitus (T2DM). We aimed todetermine the impact of Se at a dose and source frequently ingested by humans on markers of insulin sensitivityand signalling. Male pigs were fed either a Se-adequate (0.17 mg Se/kg) or a Se-supranutritional (0.50 mg Se/kg;high-Se) diet. After 16 weeks of intervention, fasting plasma insulin and cholesterol levels were non-significantlyincreased in the high-Se pigs, whereas fasting glucose concentrations did not differ between the two groups. Inskeletal muscle of high-Se pigs, glutathione peroxidase activity was increased, gene expression of forkhead boxO1 transcription factor and peroxisomal proliferator-activated receptor-γ coactivator 1α were increased andgene expression of the glycolytic enzyme pyruvate kinase was decreased. In visceral adipose tissue of high-Sepigs, mRNA levels of sterol regulatory element-binding transcription factor 1 were increased, and the phosphory-lation of Akt, AMP-activated kinase and mitogen-activated protein kinases was affected. In conclusion, dietary Seoversupply may affect expression and activity of proteins involved in energy metabolism in major insulin targettissues, though this is probably not sufficient to induce diabetes.

© 2012 Elsevier Inc. All rights reserved.

1. Introduction

Trace elements are known to influence the hormonal control ofenergy metabolism in mammals. Compounds of selenium, chromium,zinc, vanadium and lithium are capable of affecting the activationstate of components of the insulin signalling cascade and thus maymimic, potentiate or interfere with actions of insulin on carbohydrateand lipid metabolism [1]. Key proteins involved in insulin signallinginclude the insulin receptor and its substrate IRS-2, protein kinase B(Akt), glycogen synthase kinase 3β, forkhead box class O (FoxO) tran-scription factors, peroxisomal proliferator-activated receptor-γcoactivator 1α (PGC-1α) and phosphatases such as the lipid phosphatasePTENand the protein tyrosine phosphatase PTP-1B. Dysregulated biosyn-thesis, cellular localisation and/or activity of those proteins may impairthe capacity of liver, skeletal muscle and adipose tissue to respond

sseldorf, Institute for Biochemis-, D-40225 Düsseldorf, Germany.

rf.de (H. Steinbrenner).

rights reserved.

adequately to insulin. The resulting pathophysiological state, termedinsulin resistance, represents a major step in the pathogenesis of Type 2diabetes mellitus (T2DM) [2–4].

In this regard, the impact of the essential micronutrient selenium(Se) on metabolic pathways related to the onset and progression ofT2DM is the subject of ongoing debate [5,6]. Se has been creditedwith insulin-mimetic and anti-diabetic properties, following observa-tions that high doses of sodium selenate stimulated glucose uptake incultivated adipocytes and dissected skeletal muscle of rats [7,8] andimproved glucose homeostasis in diabetic rats [9]. By contrast, morerecent evidence has suggested pro-diabetic effects of supranutritionalSe intake in humans [5,6,10]. Se accomplishes its biological functionsthrough selenoproteins, mostly representing antioxidant enzymes[11]. Abundant expression of selenoproteins such as cytosolic gluta-thione peroxidase (GPx1) and selenoprotein P (SeP) can dysregulateinsulin secretion and impair insulin sensitivity [6,12–14]. Sodiumselenite delayed insulin-induced phosphorylation of Akt and FoxO1a/3in myocytes [15], and conversely, Akt phosphorylation was increasedin skeletal muscle of transgenic mice with deficient selenoprotein bio-synthesis [16].

The recommended levels for adequate Se supply range between 30and 85 μg/day for adults [17]; these doses are based on the Se intake

48 A. Pinto et al. / Journal of Inorganic Biochemistry 114 (2012) 47–54

required to maximise the activity of the selenoenzyme extracellularglutathione peroxidase (GPx3) in plasma [18]. Dietary Se intake inEuropean countries averages 40 μg/day compared to 93 to 134 μg/dayfor males and females, respectively, in the U.S. [17,19]. Although overtSe deficiency is rare in humans, the consumption of Se-enriched dietarysupplements is common in Europe and more so in the U.S. due to theirperceived health benefits. Supplements can provide an additional10–200 μg Se/day [19]. Dietary Se supplementation has been invokedfor the prevention of certain forms of cancer, oxidative stress-relatedchronic diseases of the cardiovascular system and brain and for thetherapy of inflammatory disorders [11,19]. Se appears to exert a benefi-cial influence on the course of autoimmune thyroid disease: dailyadministration of 200 μg selenite improved thequality of life and slowedthe progression of orbitopathy in patients with Graves’ disease [20].However, the action of Se on human health is characterised by a narrowtherapeutic window and a U-shaped dose–response curve. Se intakeabove nutritional requirements could trigger adverse side-effects evenbelow the “tolerable upper intake level” that is currently set at300–450 μg/day for adults [17,19]. This issue has become evidentfollowing a secondary analysis of data from the U.S. Nutritional Preven-tion of Cancer (NPC) trial: trial participants who received a dose of200 μg Se/day for a mean of 4.5 years had a higher risk of developingT2DM over a mean follow-up period of 7.7 years than those assignedto placebo [10].

Cross-sectional epidemiological studies show an associationbetween high plasma Se and hyperglycaemia and dyslipidemia,though prospective analyses do not support the causality of this rela-tionship [5,6,21–23]. To assess the risk potential of Se as a diabeto-genic factor, it is important to understand its impact on insulin-regulated energy metabolism. We here present data from a study inhealthy young pigs fed either a Se-adequate or a Se-supranutritionaldiet, where Se-yeast was the Se source. This treatment scheme waschosen with the intention of mimicking dietary Se supplementationin human populations with adequate Se nutrition. Energy metabolismis regulated similarly in humans and in pigs, and moreover, pigs maydevelop symptoms of the humanmetabolic syndrome (e.g. insulin re-sistance, hypertension, dyslipidemia and arteriosclerotic lesions)[24,25]. Thus, pigs are thought to represent a suitable animal modelfor gaining insight into molecular actions of assumed diabetogenicfactors in the human diet.

2. Materials and methods

2.1. Animals, diet and sample collection

Experimental procedures were performed according to the regula-tions approved by the Home Office under the Animals (Scientific Pro-cedures) Act 1986 UK. A total of 12 male finishing pigs (6–8 weeksold; Reading University farm) were selected and divided into twogroups, a Se-adequate (n=6) and a Se-supranutritional (high-Se)group (n=6). For an intervention period of 16 weeks, pigs in theSe-adequate control group received a vegetable-based diet (predomi-nantly wheat and soy) with 0.17 mg Se/kg dry matter, whereas pigs inthe Se-supranutritional group were fed the basal diet supplementedwith Se-yeast (Sel-Plex®; Alltech; Nicholasville, KY) resulting in atotal content of 0.5 mg Se/kg dry matter. All pigs were allowed adlibitum access to water. Food was issued at a fixed rate per animal perday (initially 1.5 kg/d, increasing to 2.5 kg/d).

Body weights of the pigs were recorded at the start and the end ofthe intervention period. Before blood and tissue collection, pigs werefasted overnight. Bloodwas collected from the jugular vein immediatelyafter the animals were euthanised; plasma samples were obtained bycentrifugation of whole blood. Tissue samples were dissected fromliver (left liver lobe), skeletal muscle (Musculus glutaeus superficialis)and visceral fat, and snap-frozen in liquid nitrogen.

2.2. Determination of biochemical parameters in plasma

Se levels in plasma samples were determined at SAS Trace ElementsCentre (University of Surrey) using a Thermo Elemental X Series Induc-tively Coupled Plasma Mass Spectrometer (ICP-MS) in collision cellmode. All analyses were run in triplicate and averaged. Accuracy of theprocedurewas assured bymeasuring the Se content of certified referencematerials, Seronorm™ trace element control serum (Sero AS; Billingstadt,Norway) and serum samples from the Trace Element Quality AssuranceScheme.

Plasma cholesterol, triacylglycerol and glucose levels were deter-mined on an ILab 650 clinical chemistry analyzer (InstrumentationLaboratory; Warrington, UK) by routine colorimetric assays withintra-assay CVs of b4%, b4% and b1% respectively across the assayranges. Plasma insulin levels were measured using a commercialimmunoassay kit (Invitron; Monmouth, UK) with an intra-assay CVof b6.5% at 9.7 pmol/L.

Alanine aminotransferase (ALT) activity was measured in plasmasamples using the Fluitest®ALT assay (Analyticon; Lichtenfels, Germany).NADH consumption after addition of samples to a reaction mixturecontaining 100 mM Tris/HCl pH 7.8, 500 mM L-alanine, 1200 U/L lactatedehydrogenase, 0.18 mM NADH and 15 mM 2-oxoglutarate was mea-sured at 340 nm with a Lambda 25 spectrophotometer (Perkin-Elmer;Waltham, MA). ALT activity was calculated using the extinction coeffi-cient of NADH at 340 nm (ε=6.2 mM−1 cm−1). For quality assessment,ALT activity was determined in Precinorm U control serum (Roche;Mannheim, Germany).

2.3. Western blot analysis

A portion of each tissue was lysed in ProteoJET Mammalian CellLysis Reagent (Fermentas; St Leon-Rot, Germany) containing proteaseinhibitors (Merck; Darmstadt, Germany) and phosphatase inhibitors(Sigma; Taufkirchen, Germany). Protein content of the lysates wasmeasured by DC Protein Assay (Bio-Rad; München, Germany). Foranalysis of protein levels and/or phosphorylation, standard immunoblottechniques were applied as described [26]. The primary antibodies usedwere: anti-Akt (total), anti-phospho-Akt (Ser473), anti-phospho-Akt(Thr308), anti-AMPKα (total), anti-phospho-AMPKα (Thr172), anti-phospho-ERK1/2 (Thr202/Tyr204), anti-phospho-p38 MAPK (Thr180/Tyr182), anti-β-actin (Cell Signaling Technology; Beverly, MA), anti-GPx1 (Epitomics; Burlingame, CA), and anti-TrxR1 (Santa Cruz Biotech-nology; Santa Cruz, CA). The secondary HRP-coupled anti-rabbit IgG andanti-mouse IgG antibodies were from Dianova (Hamburg, Germany)and Thermo Scientific Pierce (Rockford, IL).

2.4. Determination of selenoenzyme activities

GPx activity in the whole tissue lysates was measured as described[27] with slight modifications. Briefly, NADPH consumption was mea-sured spectrophotometrically at 340 nm after addition of the substratetert-butyl hydroperoxide to a final concentration of 50 μM into the reac-tion mixture containing 3 mM glutathione, 600mU/ml glutathionereductase and 0.2 mM NADPH in GPx assay buffer (100 mM Tris/HCl pH7.6, 5 mM EDTA, 1 mM sodium azide, 0.1% Triton X-100).

TrxR activity in homogenates of liver lobe and skeletal muscle wasdetermined by monitoring the reduction of DTNB (5,5′-dithiobis-2-nitrobenzoic acid) to 5-thio-2-nitrobenzoic acid. 25 μl homogenatewere added to a reaction mixture of phosphate buffer (100 mM, pH7.0) containing 2 mM EDTA, 9 mM DTNB and 0.4 mM NADPH withor without 25 μM auranofin. Linear increase in absorbance at412 nm was measured in 96-well plates in a BioTek KC4 plate reader(BioTek; Winooski, VT). TrxR activity in visceral adipose tissue wasdetermined spectrophotometrically at 412 nm by end-point mea-surement of DTNB reduction using insulin as substrate as described[27].

49A. Pinto et al. / Journal of Inorganic Biochemistry 114 (2012) 47–54

2.5. Real-time RT-PCR

Total RNA from liver and skeletal muscle was prepared using theRNeasy Fibrous Tissue Mini Kit (Qiagen; Hilden, Germany). Fromvisceral adipose tissue, total RNA was extracted using the RNeasy LipidTissue Kit (Qiagen). From each sample, 2 μg RNA were transcribed intocDNA with SuperScript II reverse transcriptase (Invitrogen; Karlsruhe,Germany) and p(dT)15 primers (Roche). Expression of mRNA wasanalysed by real-time RT-PCR using the FastStart DNA Master SYBRGreen I Reaction Mix (Roche) in a LightCycler 2.0 qPCR system(Roche) as described [26]. In consideration of a comparative analysisof reference genes that identified beta-actin as the most stablyexpressed housekeeping gene in skeletal muscle and adipose tissue ofpigs [28], beta-actin was employed for internal normalisation. An addi-tional analysiswasperformedwith hypoxanthinephosphoribosyl trans-ferase (HPRT1) as reference gene, yielding similar results (data notshown). Primers were designed using Primer-BLAST of the NationalCenter for Biotechnology Information (NCBI) or taken from a study onselenoprotein gene expression in pigs [29]. Primers were synthesizedby Invitrogen; their specificity was confirmed bymelting curve analysisand agarose gel electrophoresis of PCR products. Primer sequences aregiven as supplemental material, listed in the Supplementary Table S1.

2.6. Statistical analysis

Mean values were calculated from the six animals of each group,and error bars represent standard error of the mean (S.E.M.). Analysisof statistical significance was done by Student's t-test with *Pb0.05considered to be significant.

3. Results

3.1. Metabolic characteristics of the animals

Healthy young male pigs (n=6 per group) were fed either aSe-adequate or a Se-supranutritional diet for 16 weeks. In order to simu-late the intake of Se-containing mineral supplements by humans, thebasal (Se-adequate) dietwith a natural content of 0.17 mg Se/kg drymat-ter was supplemented with Se-yeast to obtain the Se-supranutritional(high-Se) level of 0.50 mg Se/kg. At the beginning of the interventionperiod, body weights of the two groups of animals were similar withmeans±SD of 15.8±1.47 kg and 16.1±1.38 kg, respectively. Finalbody weights were greater in the high-Se pigs (150.5±13.75 kg) thanin the Se-adequate pigs (136.0±15.30 kg), the difference almostreaching significance (P=0.053). The metabolic characteristics of thepigs, as determined at the end of the trial after 16 weeks of Se supple-mentation, are summarised in Table 1. As expected, plasma selenium

Table 1Metabolic characteristics of pigs fed either a Se-adequate or a Se-supranutritional dietfor 16 weeks.

Parameter Adequate-Se(0.17 mg Se/kg)

Supranutritional-Se(0.50 mg Se/kg)

p value

Selenium (μg/L) 159±8.0 199±7.4 0.003**Fasting insulin (pM) 7.9±2.3 11.4±2.3 0.315Fasting glucose (mM) 11.2±1.6 12.0±1.9 0.784HOMA-IR 0.73±0.27 0.97±0.19 0.477Triacylglycerols (mM) 0.32±0.05 0.37±0.02 0.380Cholesterol (mM)

- total 2.02±0.12 2.36±0.08 0.077HDL 0.87±0.06 1.04±0.04 0.058non-HDL 1.15±0.09 1.32±0.09 0.275

ALT (U/L) 39.1±1.4 42.5±3.2 0.380

Data represent means±S.E.M. from plasma samples of 6 animals in each group. Thehomeostatic model assessment insulin resistance score (HOMA-IR) was calculated foreach individual as fasting plasma insulin (μU/mL)×fasting plasma glucose (mM)/22.5[30]. Differences between treatments were determined by ANOVA using a generallinear model with final weight as a covariate term.

concentrations were significantly enhanced (Pb0.01) in pigs fed theSe-supranutritional diet. Fasting plasma glucose and triacylglycerol(TAG) levels were similar in both groups. Cholesterol (total, HDL andnon-HDL cholesterol) concentrations were elevated in the plasma ofhigh-Se pigs, although this difference failed to achieve statistical signif-icance (P=0.077, 0.058 and 0.275, respectively). There was a trend forincreased fasting plasma insulin concentrations in the high-Se pigs(P=0.315). A secondary analysis revealed aberrant insulin plasmalevels in two pigs that exhibited overt signs of stress prior to slaughter.After excluding these outlier animals (one in each group), the differencein insulin plasma levels of Se-adequate (6.06±1.04 pM) and Se-supranutritional (8.79±0.46 pM) pigs reached statistical significance(P=0.041). The HOMA-IR score was calculated from fasting plasma con-centrations of insulin and glucose [30]: the group of Se-supranutritionalpigs showed a trend to an increased HOMA-IR score (P=0.477),suggesting impaired insulin sensitivity in the Se-supplemented animals.However, plasma levels of the liver enzyme alanine aminotransferase(ALT), an independent predictive marker for T2DM [31], were not signif-icantly enhanced in the Se-supranutritional pigs.

3.2. Impact of Se supplementation on biosynthesis of selenoproteins andantioxidant enzymes

As abundant expression of selenoproteins has been reported topromote the development of insulin resistance [12–14],we investigatedthe influence of dietary Se supplementation on protein expression andactivity of the selenoenzymes GPx1 and thioredoxin reductase 1(TrxR1) in the major insulin target tissues of the pigs. Both GPx1 andTrxR1 were highly expressed in liver and poorly expressed in VAT;their protein expressionwas not altered by Se supplementation. In skel-etal muscle, protein levels of the two selenoenzymes differed strongly:TrxR1 exhibited high expression, whereas GPx1 was not detectablewith the standard procedure (Fig. 1A). To provide evidence of GPx1 pro-tein in skeletal muscle, we had to apply our most sensitive method ofdetection: by using SuperSignal West Femto Substrate (Pierce), verylow GPx1 protein expression was demonstrated that was more pro-nounced in the high-Se animals (Supplementary Fig. S1). The differentprotein levels of GPx1 and TrxR1 were reflected in the overall GPx andTrxR enzymatic activities in the three tissues. Liver exhibited by far thehighest activity of both selenoenzymes, whereas the lowest GPx activitywas found in skeletalmuscle and the lowest TrxR activitywasmeasuredin VAT. An increase in GPx activity was observed in the skeletal muscleof high-Se pigs (Pb0.05), which was the only significant differencebetween the Se-adequate and the Se-supranutritional group in termsof expression/activity of selenoproteins in the key insulin-sensitivetissues (Fig. 1B). This result indicates that the biosynthesis ofselenoproteins was already saturated to a large extent in the animalsfed a Se-adequate diet. Consistent with this notion, no significantchanges in steady-state mRNA levels of any of the selenoproteins inves-tigated in liver, skeletal muscle or visceral adipose tissue (VAT) werefound in the pigs fed a Se-supranutritional diet when compared tothose fed a Se-adequate diet (Supplementary Table S2).

In order to test whether Se supplementation had an impact on geneexpression of other proteins involved in cellular redox homeostasis andantioxidant protection, steady-state mRNA levels of superoxidedismutases 1 and 2, heme oxygenase 1, catalase, glutathione reductaseand uncoupling proteins 2 and 3 were assessed in liver, skeletal muscleand VAT. There was no significant difference between the two groups(Supplementary Table S2).

3.3. Impact of Se supplementation on enzymes and transcription factorsrelated to energy metabolism in visceral adipose tissue

Protein kinaseB (Akt) is activated by insulin-inducedphosphorylationof Thr308 and Ser473 residues, and plays a key role in integrating theactions of insulin on energy metabolism [2]. Chronic hyperinsulinaemia

Fig. 1. Expression and activity of the selenoenzymes glutathione peroxidase (GPx) andthioredoxin reductase (TrxR) inmajor insulin target tissues of pigs. (A) Protein expressionof GPx1 and TrxR1 was determined by immunoblotting in whole tissue lysates of liver,skeletal muscle (SM) and visceral adipose tissue (VAT), obtained from pigs fed a Se-adequate (ad) or a supranutritional-Se (high-Se, hi) diet for 16 weeks. A representativeblot with lysates from two animals (one of each group) is shown. (B) In whole tissuelysates (liver, SM and VAT) of adequate-Se (ad) and high-Se (hi) pigs, GPx activitieswere measured with 50 μM tert-butyl hydroperoxide as substrate and TrxR activitieswere determined by monitoring the reduction of DTNB (5,5’-dithiobis-2-nitrobenzoicacid). Data represent means±S.E.M. from measurements in tissue lysates of 6 animalsin each group.

50 A. Pinto et al. / Journal of Inorganic Biochemistry 114 (2012) 47–54

has been demonstrated to result in elevated basal serine phosphorylationof Akt in white adipose tissue of rats [32]. In the VAT of the high-Se pigs,there was a trend for elevated basal phosphorylation of Akt at bothSer473 (P=0.27) and Thr308 (P=0.43), whereas total Akt protein levelsremained unchanged (Fig. 2A).

In response to stimulation with insulin, mitogen-activated proteinkinases (MAPKs) i.e. the extracellular signal-regulated kinases (ERKs),p38 and c-Jun amino-terminal kinases (JNKs) become phosphorylated(activated). MAPKs are involved in the control of lipid homeostasisand adipocyte differentiation [33]. Adipocytes isolated from hyper-insulinaemic patients with Type 2 diabetes exhibit elevated basal phos-phorylation of MAPKs compared to adipocytes from healthy subjects[34]. A trend for increased basal phosphorylation of ERK1/2 at Thr202/Tyr204 (P=0.54) and p38-MAPK at Thr180/Tyr182 (P=0.15) wasobserved in the VAT of the high-Se pigs (Fig. 2B). Even though the

alterations failed to reach significance, the observed increases in basalphosphorylation of Akt, ERK1/2 and p38-MAPK appear to be consistentwith the elevated fasting plasma insulin concentrations of the high-Sepigs (Table 1).

AMP-activated protein kinase (AMPK) becomes activated at a lowcellular energy status by phosphorylation of the AMPKα subunit atThr172, and functions by switching on catabolic pathways, switchingoff anabolic pathways and increasing insulin sensitivity [35]. Recently,selenoprotein P has been shown to decrease AMPKα phosphorylation[14]. Consistent with the elevated plasma Se levels of pigs fed a Se-supranutritional diet (Table 1), a trend for decreased basal AMPKαphosphorylation (P=0.18) was observed in the VAT of the high-Sepigs (Fig. 2C), suggesting impaired AMPK activity.

Next, we determined whether Se supplementation may haveaffected the gene expression of proteins related to insulin signaltransduction and insulin-regulated energy metabolism. Relativechanges in mRNA levels of key components of the canonical insulinsignal cascade, key enzymes of glucose and lipidmetabolism and factorsinvolved in their transcriptional regulation were examined by real-timePCR. As shown in Table 2, mRNA levels of most of the examined genesremained unchanged. Sterol regulatory element-binding transcriptionfactor 1 (SREBP1c) was significantly up-regulated (1.60 fold; Pb0.05)in the VAT of high-Se pigs compared to Se-adequate animals, andthere was a trend to increased mRNA levels of lipoprotein lipase (LPL)(1.90 fold; P=0.17) and decreased mRNA levels of peroxisomalproliferator-activated receptor-γ coactivator 1α (PGC-1α) (0.55 fold;P=0.27). This pattern is in agreement with the altered kinase activityand elevated plasma insulin concentrations, and it indicates an increasedlipid turnover in the VAT of the supranutritional-Se pigs [35–38].

3.4. Impact of Se supplementation on enzymes and transcription factorsrelated to energy metabolism in liver and skeletal muscle

In the liver of pigs fed a Se-supranutritional diet, we found nomolecular alterations associated with impaired insulin sensitivity[2–4,39]. Basal Ser473 phosphorylation and expression of Akt, basalThr172 phosphorylation and expression of AMPKα as well as basalThr202/Tyr204 phosphorylation of ERK1/2 and basal Thr180/Tyr182phosphorylation of p38-MAPK in the liver remained unchanged(Supplementary Fig. S2). Likewise, gene expression was not alteredin the liver of the high-Se pigs. In particular, neither the mRNA levelsof sterol regulatory element-binding transcription factors SREBP1c andSREBP2 controlling hepatic triacylglycerol and cholesterol biosynthesisnor mRNA levels of FoxO1 transcription factor and its co-activatorPGC-1α controlling hepatic gluconeogenesis, nor their respective targetgenes was affected (Table 2).

In the skeletal muscle of high-Se pigs, basal Ser473 phosphorylationand expression of Akt were not altered in comparison to those of Se-adequate animals (Fig. 3A). Basal Thr180/Tyr182 phosphorylation ofp38-MAPK was not affected, whereas basal Thr202/Tyr204 phosphory-lation of ERK1/2 tended to be decreased (P=0.14) in high-Se pigs(Fig. 3B). We detected no basal Thr172 phosphorylation of AMPKα;however, we observed a non-significant increase in total protein levelsof AMPKα (P=0.10) in the skeletal muscle of high-Se pigs (Fig. 3C).

The analysis of gene expression in skeletal muscle revealed aparadoxical pattern of alterations induced by Se supplementation, assteady-state mRNA levels of FoxO1 (1.36 fold; Pb0.01) and PGC-1α(1.89 fold; Pb0.05) were significantly increased in the high-Se pigs.These factors, which have key importance in the control of energymetabolism, are conversely regulated in the skeletal muscle of patientssuffering from type 2 diabetes, FoxO1 exhibiting up-regulation andPGC-1α exhibiting down-regulation [3,4,40]. In addition, the glycolyticenzyme pyruvate kinase was significantly down-regulated (0.61fold; Pb0.05) in high-Se pigs. We also observed trends for in-creased mRNA levels of LPL (1.39 fold; P=0.10) and for decreasedmRNA levels of IRS1 (0.46 fold; P=0.11), SREBP1c (0.75 fold;

Fig. 2. Impact of Se supplementation on the basal phosphorylation state of protein kinases in the visceral adipose tissue of pigs. Lysates from visceral adipose tissue of high-Se (hi)and adequate-Se (ad) pigs were analysed by immunoblotting for phosphorylation of Akt, MAPK and AMPK protein kinases. Representative blots of lysates from six selected animals(three of each group) are shown. For the presentation of densitometric analyses of the blots, phosphorylation levels were normalised against the respective loading controls; thedata represent means±S.E.M. from whole tissue lysates of 6 animals in each group. (A) Phosphorylation of Akt at Ser473 and Thr308; total Akt levels served as loading control.(B) Phosphorylation of the mitogen-activated protein kinases ERK1/2 at Thr202/Tyr204 and p38-MAPK at Thr180/Tyr182; total protein levels of β-actin served as loading control.(C) Phosphorylation of AMPKα at Thr172; total AMPKα levels served as loading control.

51A. Pinto et al. / Journal of Inorganic Biochemistry 114 (2012) 47–54

P=0.27), carbohydrate responsive element-binding protein (0.55fold; P=0.31), glycerinaldehyde-3-phosphate dehydrogenase(GAPDH) (0.80 fold; P=0.10) and pyruvate dehydrogenase kinase4 (PDK4) (0.37 fold; P=0.21) in the skeletal muscle of high-Se pigs(Table 2). Altogether, these results indicate that Se supplementationmay have promoted a switch in the preferred energy source fromcarbohydrates to fatty acids [2–4,39,40].

4. Discussion

In this study, we investigated in healthy male pigs how a sup-ranutritional seleniumdiet affectedmetabolic andmolecular parametersrelated to the hormonal control of energy metabolism. Both the Sesource (Se-yeast) and the Se doses fed to the two groups of animals(0.17 mg Se/kg for a Se-adequate versus 0.50 mg Se/kg for a Se-supranutritional diet) are of physiological relevance for humans, beingselected with respect to dietary and supplemental Se intake. After16 weeks of Se supplementation, some metabolic parameters werenon-significantly increased (fasting plasma insulin concentrations,plasma cholesterol levels and ALT activity, HOMA-IR score) in thesupranutritional-Se group, while fasting plasma glucose concentrationswere not affected (Table 1). Combined, the observed alterations mightbe early indicators of a predisposition to Type 2 diabetes mellitus.

Interestingly, hyperinsulinaemia has emerged as a common attri-bute in mice fed a high-Se diet (0.40 mg Se/kg) for three monthsand in mice overexpressing GPx1 [12,13]. Compared to other tissues,the insulin-producing beta cells in pancreatic islets express very lowlevels of antioxidant enzymes [41]. Stimulation of GPx1 biosynthesisabove physiological levels, either through nutritional or genetic ma-nipulation, appears to result in dysregulation of pancreatic insulin se-cretion. Moreover, selenoproteins may affect insulin-regulatedmetabolic pathways. Combined evidence from studies in rodentsindicates that abundant expression of selenoproteins impairs the insulinsensitivity of liver and skeletal muscle by counteracting insulin-inducedphosphorylation of protein kinases in the insulin signalling cascade,resulting in altered expression/activity of transcription factors andenzymes involved in insulin-regulated energy metabolism [12–14,42].Conversely, genetic knock-downof selenoprotein biosynthesis increasedthe sensitivity of liver and skeletal muscle to insulin [14,16,43]. A ratio-nale for those observations is provided by the antioxidant activity ofmany selenoenzymes thatmay interferewith insulin signal transduction[6], as hydrogen peroxide is required as second messenger in the earlyinsulin signalling cascade [44]. On the other hand, the applicability ofthose results to healthy humans ingesting Se-containing supplementsmight be limited. Se deficiency is rare in humans and individuals ofadequate Se status are likely to exhibit saturated biosynthesis of mostselenoproteins, as did the animals in our study (Fig. 1 and Supplementary

Table 2Influence of Se supplementation on gene expression of enzymes and transcription factors involved in the control of energy metabolism in major insulin target tissues.

Protein Liver p value Skeletal muscle p value VAT p value

insulin receptor 0.79±0,42 0.68 1.20±0.44 0.71 0.53±0.22 0.08insulin receptor substrate 1 1.09±0.16 0.67 0.46±0.27 0.11 0.94±0.30 0.92glucose transporter 4 n.d. 0.92±0.03 0.16 1.19±0.12 0.58protein tyrosine phosphatase type 1 1.06±0.37 0.92 0.53±0.14 0.17 0.97±0.23 0.93pyruvate dehydrogenase kinase 4 1.00±0.01 0.97 0.37±0.42 0.21 0.70±0.41 0.48glyceraldehyde-3-phosphate dehydrogenase 0.98±0.18 0.92 0.80±0.10 0.10 1.34±0.18 0.29pyruvate kinase muscle n.d. 0.61±0.11 0.04* 1.16±0.22 0.66phosphoenolpyruvate carboxykinase 1 1.03±0.17 0.86 n.d. n.d.glucose-6-phosphatase 1.05±0.10 0.68 n.d. n.d.lipoprotein lipase n.d. 1.39±0.13 0.10 1.90±0.30 0.17adipose triglyceride lipase n.d. n.d. 1.16±0.31 0.76hormone-sensitive lipase n.d. n.d. 0.86±0.24 0.74fatty acid synthase 0.78±0.23 0.57 n.d. n.d.carnitine palmitoyl transferase 1 liver 1.63±0.55 0.54 n.d. n.d.diacylglycerol acyltransferase 0.98±0.16 0.91 1.16±0.12 0.35 1.27±0.20 0.47hydroxy-3-methylglutaryl-coenzyme A reductase 1.04±0.27 0.92 1.00±0.20 0.99 0.86±0.30 0.87forkhead box O1 0.87±0.14 0.47 1.36±0.05 0.001** 0.97±0.21 0.91peroxisomal proliferator-activated receptor-γ coactivator 1α 1.05±0.10 0.74 1.89±0.20 0.04* 0.55±0.18 0.27hepatocyte nuclear factor 4α 1.10±0.11 0.52 n.d. 0.71±0.25 0.27carbohydrate responsive element-binding protein 0.91±0.14 0.70 0.55±0.18 0.31 1.21±0.15 0.39sterol regulatory element-binding transcription factor 1 1.26±0.52 0.77 0.75±0.10 0.27 1.60±0.10 0.04*sterol regulatory element-binding transcription factor 2 0.97±0.16 0.87 0.93±0.12 0.63 0.93±0.35 0.87

Steady-state mRNA levels were determined by real-time RT-PCR with normalisation against beta-actin and are expressed as changes in supranutritional-Se pigs relative toadequate-Se pigs. Values are given as means±S.E.M., comprising 6 animals per group. Significant changes are marked in bold. (n.d., not determined).

52 A. Pinto et al. / Journal of Inorganic Biochemistry 114 (2012) 47–54

Table S2). Thus, adverse effects of supranutritional Se intake on insulin-regulatedmetabolic pathwaysmay not derive exclusively from impairedcellular redox homeostasis due to abundant activity of selenoenzymes,degrading reactive oxygen species (ROS).

Fig. 3. Impact of Se supplementation on the basal expression and phosphorylation state of protadequate-Se (ad) pigs were analysed by immunoblotting for protein levels and phosphorylaanalyses, the data represent means±S.E.M. from whole tissue lysates of 6 animals in each(B) Phosphorylation of the mitogen-activated protein kinases ERK1/2 at Thr202/Tyr204 and(C) Total protein levels of AMPKα; total protein levels of β-actin served as loading control.

The results presented here provide evidence for tissue-selectiveeffects of Se supplementation. We found several differences in geneexpression and phosphorylation status of proteins in the VAT (Table 2and Fig. 2) that could result from an increase in plasma insulin

ein kinases in the skeletal muscle of pigs. Lysates from skeletal muscle of high-Se (hi) andtion of Akt, MAPK and AMPK protein kinases as described in Fig. 2. In the densitometricgroup. (A) Phosphorylation of Akt at Ser473; total Akt levels served as loading control.p38-MAPK at Thr180/Tyr182; total protein levels of β-actin served as loading control.

53A. Pinto et al. / Journal of Inorganic Biochemistry 114 (2012) 47–54

concentrations induced by supranutritional Se intake. The molecularpattern observed in skeletal muscle (Table 2 and Fig. 3) might beexplained by a high Se-induced switch in fuel usage from carbohydratesto fatty acids. By contrast, no alteration in gene expression or phosphor-ylation status of any of the selected proteins was detected in the liver ofsupranutritional-Se pigs (Table 2 and Supplementary Fig. S2),most likelyrelated to the high levels of GPx1 and TrxR1 in the liver. A microarrayanalysis of Se-induced alterations in hepatic gene expression in rodentssupports this notion: Se intake up to 20-times the dietary requirementcaused very little change in comparison to adequate Se supply and onlytoxic Se doses (5 mg Se/kg) vastly altered the hepatic transcriptome[45]. In apparent contrast to our observations, dietary Se supplementa-tion has been reported to increase hepatic triacylglycerol synthesis inrats via redox-sensitive activation of protein tyrosine phosphatase 1B(PTP-1B) and transcriptional up-regulation of SREBP1c. However, theauthors compared Se-deficient and Se-adequate animals, exhibitinghuge differences in hepatic activities of selenoenzymes [42]. This groupreported in another study significant Se-induced changes in hepaticexpression of enzymes involved in cholesterol, homocysteine and gluta-thionemetabolism only between Se-deficient and Se-supplemented rats,whereas no significant differences were observed between animals fed aSe-adequate (0.15 mg Se/kg) and a Se-supranutritional (0.45 mg Se/kg)diet [46].

Skeletal muscle was the only insulin target tissue with a significantstimulation of GPx activity in the high-Se pigs (Fig. 1B). Se supplemen-tation significantly stimulated the gene expression of the transcriptionfactor FoxO1 and its coactivator PGC-1α in skeletal muscle (Table 2).FoxO1 blunts glycolysis, inhibits lipogenesis and promotes the hydro-lysis of lipoproteins and the uptake of free fatty acids, thusmediating aswitch in fuel usage. Hyper-activation of FoxO1 in insulin-resistantskeletal muscle is associated with metabolic disturbances [4]. PGC-1α has a similar impact on fuel selection in skeletal muscle as doesFoxO1, mediating repression of glucose oxidation while increasingfatty acid oxidation [3]. PGC-1α in skeletal muscle is decreased underdiabetic conditions together with genes of oxidative metabolism [40],and conversely, overexpression of PGC-1α has been found to protectagainst the development of insulin resistance [3]. The exceptionalconcomitant up-regulation of FoxO1 and PGC-1α argues against a sup-ranutritional Se-induced insulin resistance.

We observed an altered basal phosphorylation of several proteinkinases in the VAT of the high-Se pigs (Fig. 2). It is conceivable thatthe enhanced phosphorylation of Akt, ERK1/2 and p38-MAPK haveresulted from the increase in plasma insulin concentrations inducedby high Se. A tissue-selective effect of hyperinsulinaemia has beendemonstrated in rats by a hyperinsulinaemic-euglycaemic clamp:hyperinsulinaemic animals exhibited increased basal, but normalinsulin-induced Akt phosphorylation in white adipose tissue, whereasunaltered basal, but impaired insulin-stimulated Akt phosphorylationwas found in skeletal muscle and liver [32].

5. Concluding remarks

In conclusion, our study provides evidence that selenium over-supplymay affect expression and activity of proteins involved in energymetabolism in major insulin target tissues, though supranutritional Seintake at the levels employedwas not sufficient to induce overt diabetesin healthy animals. Indeed, humans supplemented with 200 μg Se/dayin the NPC trial did not have a significantly increased risk of developingType 2 diabetes mellitus unless their baseline plasma Se exceeded121.6 μg/L (top tertile) [10]. Similarly, there was no significant increasein T2DM risk in Se-supplemented men of the Se and Vitamin E CancerTrial (SELECT) [47,48]. Nevertheless, we recommend that individualswith high Se status should not ingest Se-containing supplements, asoptimising, rather than maximising, exposure is the key to benefitmost from Se while avoiding potential adverse effects [49].

Abbreviations

Akt protein kinase BALT alanine aminotransferaseAMPK AMP-activated protein kinaseERK extracellular signal-regulated kinaseFoxO forkhead box protein class OGPx glutathione peroxidaseHOMA-IR homeostatic model assessment insulin resistanceIRS insulin receptor substrateLPL lipoprotein lipaseMAPK mitogen-activated protein kinaseNPC Nutritional Prevention of Cancer TrialPGC-1α peroxisomal proliferator-activated receptor-γ coactivator

1αROS reactive oxygen speciesRT-PCR reverse transcription polymerase chain reactionSe seleniumSELECT Selenium and Vitamin E Cancer TrialSeP selenoprotein PSREBP sterol element-binding transcription factorT2DM type 2 diabetes mellitusTAG triacylglycerolTrxR thioredoxin reductaseVAT visceral adipose tissue

Acknowledgements

This work was supported by a grant of DeutscheForschungsgemeinschaft (DFG) (Bonn, Germany) to H. Steinbrenner(STE 1782/2-2). H. Sies is a Fellow of the National Foundation forCancer Research (NFCR), Bethesda, MD. We thank Dr. S. Schinner(Department of Endocrinology, Diabetes and Rheumatology, UniversityHospital Düsseldorf) for the helpful discussions and A. Borchardt for theexcellent technical assistance.

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jinorgbio.2012.04.011.

References

[1] N. Wiernsperger, J. Rapin, Diabetol. Metab. Syndr. 2 (2010) 70.[2] S. Schinner, W.A. Scherbaum, S.R. Bornstein, A. Barthel, Diabet. Med. 22 (2005)

674–682.[3] B.N. Finck, D.P. Kelly, J. Clin. Invest. 116 (2006) 615–622.[4] Z. Cheng, M.F. White, Antioxid. Redox Signal. 14 (2011) 649–661.[5] S. Stranges, A. Navas-Acien, M.P. Rayman, E. Guallar, Nutr. Metab. Cardiovasc. Dis.

20 (2010) 754–760.[6] H. Steinbrenner, B. Speckmann, A. Pinto, H. Sies, J. Clin. Biochem. Nutr. 48 (2011)

40–45.[7] O. Ezaki, J. Biol. Chem. 265 (1990) 1124–1128.[8] C. Fürnsinn, R. Englisch, K. Ebner, P. Nowotny, C. Vogl, W. Waldhäusl, Life Sci. 59

(1996) 1989–2000.[9] D.J. Becker, B. Reul, A.T. Ozcelikay, J.P. Buchet, J.C. Henquin, S.M. Brichard,

Diabetologia 39 (1996) 3–11.[10] S. Stranges, J.R. Marshall, R. Natarajan, R.P. Donahue, M. Trevisan, G.F. Combs, F.P.

Cappuccio, A. Ceriello, M.E. Reid, Ann. Intern. Med. 147 (2007) 217–223.[11] H. Steinbrenner, H. Sies, Biochim. Biophys. Acta 1790 (2009) 1478–1485.[12] J.P. McClung, C.A. Roneker, W. Mu, D.J. Lisk, P. Langlais, F. Liu, X.G. Lei, Proc. Natl.

Acad. Sci. U. S. A. 101 (2004) 8852–8857.[13] V.M. Labunskyy, B.C. Lee, D.E. Handy, J. Loscalzo, D.L. Hatfield, V.N. Gladyshev,

Antioxid. Redox Signal. 14 (2011) 2327–2336.[14] H. Misu, T. Takamura, H. Takayama, H. Hayashi, N. Matsuzawa-Nagata, S. Kurita,

K. Ishikura, H. Ando, Y. Takeshita, T. Ota, M. Sakurai, T. Yamashita, E. Mizukoshi,T. Yamashita, M. Honda, K. Miyamoto, T. Kubota, N. Kubota, T. Kadowaki, H.J.Kim, I.K. Lee, Y. Minokoshi, Y. Saito, K. Takahashi, Y. Yamada, N. Takakura, S.Kaneko, Cell Metab. 12 (2010) 483–495.

[15] A. Pinto, B. Speckmann, M. Heisler, H. Sies, H. Steinbrenner, J. Inorg. Biochem. 105(2011) 812–820.

54 A. Pinto et al. / Journal of Inorganic Biochemistry 114 (2012) 47–54

[16] T.A. Hornberger, T.J. McLoughlin, J.K. Leszczynski, D.D. Armstrong, R.R. Jameson,P.E. Bowen, E.S. Hwang, H. Hou, M.E. Moustafa, B.A. Carlson, D.L. Hatfield, A.M.Diamond, K.A. Esser, J. Nutr. 133 (2003) 3091–3097.

[17] M.P. Rayman, Br. J. Nutr. 100 (2008) 254–268.[18] A.J. Duffield, C.D. Thomson, K.E. Hill, S. Williams, Am. J. Clin. Nutr. 70 (1999)

896–903.[19] S.J. Fairweather-Tait, Y. Bao, M.R. Broadley, R. Collings, D. Ford, J.E. Hesketh, R.

Hurst, Antioxid. Redox Signal. 14 (2011) 1337–1383.[20] C. Marcocci, G.J. Kahaly, G.E. Krassas, L. Bartalena, M. Prummel, M. Stahl, M.A.

Altea, M. Nardi, S. Pitz, K. Boboridis, P. Sivelli, G. von Arx, M.P. Mourits, L.Baldeschi, W. Bencivelli, W. Wiersinga, N. Engl. J. Med. 364 (2011) 1920–1931.

[21] J. Bleys, A. Navas-Acien, E. Guallar, Diabetes Care 30 (2007) 829–834.[22] S. Stranges, F. Galletti, E. Farinaro, L. D'Elia, O. Russo, R. Iacone, C. Capasso, V.

Carginale, V. De Luca, E. Della Valle, F.P. Cappuccio, P. Strazzullo, Atherosclerosis217 (2011) 274–278.

[23] S. Czernichow, A. Couthouis, S. Bertrais, A.C. Vergnaud, L. Dauchet, P. Galan, S.Hercberg, Am. J. Clin. Nutr. 84 (2006) 395–399.

[24] J.K. Patterson, X.G. Lei, D.D. Miller, Exp. Biol. Med. (Maywood) 233 (2008)651–664.

[25] E. Bendixen, M. Danielsen, K. Larsen, C. Bendixen, Brief. Funct. Genomics 9 (2010)208–219.

[26] B. Speckmann, P.L. Walter, L. Alili, R. Reinehr, H. Sies, L.O. Klotz, H. Steinbrenner,Hepatology 48 (2008) 1998–2006.

[27] H. Steinbrenner, E. Bilgic, L. Alili, H. Sies, P. Brenneisen, Free Radic. Res. 40 (2006)936–943.

[28] T. Erkens, M. Van Poucke, J. Vandesompele, K. Goossens, A. Van Zeveren, L.J.Peelman, BMC Biotechnol. 6 (2006) 41.

[29] J.C. Zhou, H. Zhao, J. Li, X.J. Xia, K.N. Wang, Y.J. Zhang, Y. Liu, Y. Zhao, X.G. Lei, J.Nutr. 139 (2009) 1061–1066.

[30] D.R. Matthews, J.P. Hosker, A.S. Rudenski, B.A. Naylor, D.F. Treacher, R.C. Turner,Diabetologia 28 (1985) 412–419.

[31] A.J. Hanley, K. Williams, A. Festa, L.E. Wagenknecht, R.B. D'Agostino, J. Kempf, B.Zinman, S.M. Haffner, Diabetes 53 (2004) 2623–2632.

[32] M. Ueno, J.B. Carvalheira, R.C. Tambascia, R.M. Bezerra, M.E. Amaral, E.M. Carneiro,F. Folli, K.G. Franchini, M.J. Saad, Diabetologia 48 (2005) 506–518.

[33] H. Gehart, S. Kumpf, A. Ittner, R. Ricci, EMBO Rep. 11 (2010) 834–840.

[34] C.J. Carlson, S. Koterski, R.J. Sciotti, G.B. Poccard, C.M. Rondinone, Diabetes 52(2003) 634–641.

[35] D.G. Hardie, Am. J. Clin. Nutr. 93 (2011) 891S–896S.[36] S.J. Kim, C. Nian, C.H. McIntosh, J. Lipid Res. 51 (2010) 3145–3157.[37] H. Daitoku, K. Yamagata, H. Matsuzaki, M. Hatta, A. Fukamizu, Diabetes 52 (2003)

642–649.[38] K.J. Nadeau, J.W. Leitner, I. Gurerich, B. Draznin, J. Biol. Chem. 279 (2004)

34380–34387.[39] H. Shimano, FEBS J. 276 (2009) 616–621.[40] M.E. Patti, A.J. Butte, S. Crunkhorn, K. Cusi, R. Berria, S. Kashyap, Y. Miyazaki, I.

Kohane, M. Costello, R. Saccone, E.J. Landaker, A.B. Goldfine, E. Mun, R.DeFronzo, J. Finlayson, C.R. Kahn, L.J. Mandarino, Proc. Natl. Acad. Sci. U. S. A.100 (2003) 8466–8471.

[41] M. Tiedge, S. Lortz, J. Drinkgern, S. Lenzen, Diabetes 46 (1997) 1733–1742.[42] A.S. Mueller, S.D. Klomann, N.M. Wolf, S. Schneider, R. Schmidt, J. Spielmann, G.

Stangl, K. Eder, J. Pallauf, J. Nutr. 138 (2008) 2328–2336.[43] K. Loh, H. Deng, A. Fukushima, X. Cai, B. Boivin, S. Galic, C. Bruce, B.J. Shields, B.

Skiba, L.M. Ooms, N. Stepto, B. Wu, C.A. Mitchell, N.K. Tonks, M.J. Watt, M.A.Febbraio, P.J. Crack, S. Andrikopoulos, T. Tiganis, Cell Metab. 10 (2009) 260–272.

[44] K. Mahadev, A. Zilbering, L. Zhu, B.J. Goldstein, J. Biol. Chem. 276 (2001)21938–21942.

[45] A.M. Raines, R.A. Sunde, BMC Genomics 12 (2011) 26.[46] N.M. Wolf, K. Mueller, F. Hirche, E. Most, J. Pallauf, A.S. Mueller, Br. J. Nutr. 104

(2010) 520–532.[47] E.A. Klein, I.M. Thompson, C.M. Tangen, J.J. Crowley, M.S. Lucia, P.J. Goodman, L.M.

Minasian, L.G. Ford, H.L. Parnes, J.M. Gaziano, D.D. Karp, M.M. Lieber, P.J. Walther,L. Klotz, J.K. Parsons, J.L. Chin, A.K. Darke, S.M. Lippman, G.E. Goodman, F.L.Meyskens, L.H. Baker, JAMA 306 (2011) 1549–1556.

[48] S.M. Lippman, E.A. Klein, P.J. Goodman, M.S. Lucia, I.M. Thompson, L.G. Ford, H.L.Parnes, L.M. Minasian, J.M. Gaziano, J.A. Hartline, J.K. Parsons, J.D. Bearden 3rd,E.D. Crawford, G.E. Goodman, J. Claudio, E. Winquist, E.D. Cook, D.D. Karp, P.Walther, M.M. Lieber, A.R. Kristal, A.K. Darke, K.B. Arnold, P.A. Ganz, R.M.Santella, D. Albanes, P.R. Taylor, J.L. Probstfield, T.J. Jagpal, J.J. Crowley, F.L.Meyskens Jr, L.H. Baker, C.A. Coltman Jr, JAMA 301 (2009) 39–51.

[49] M.P. Rayman, Lancet 379 (2012) 1256–1268.