Abstract
BACKGROUND:
The amino acid selenocysteine (Sec) is an integral part of selenoproteins, a class of proteins mostly involved in strong redox reactions. The enzyme Sec lyase (SCLY) decomposes Sec into selenide allowing for the recycling of the selenium (Se) atom via the selenoprotein synthesis machinery. We previously demonstrated that disruption of the Scly gene (Scly KO) in mice leads to the development of obesity and metabolic syndrome, with effects on glucose homeostasis, worsened by Se deficiency or a high-fat diet, and exacerbated in male mice. Our objective was to determine whether Se supplementation could ameliorate obesity and restore glucose homeostasis in the Scly KO mice.
METHODS:
Three-weeks old male and female Scly KO mice were fed in separate experiments a diet containing 45% kcal fat and either sodium selenite or a mixture of sodium selenite and selenomethionine (selenite/SeMet) at moderate (0.25 ppm) or high (0.5 – 1 ppm) levels for 9 weeks, and assessed for metabolic parameters, oxidative stress and expression of selenoproteins.
RESULTS:
Se supplementation was unable to prevent obesity and elevated epididymal white adipose tissue weights in male Scly KO mice. Serum glutathione peroxidase activity in Scly KO mice was unchanged regardless of sex or dietary Se intake; however, supplementation with a mixture of selenite/SeMet improved oxidative stress biomarkers in the male Scly KO mice.
CONCLUSION:
These results unveil sex- and selenocompound-specific regulation of energy metabolism after the loss of Scly, pointing to a role of this enzyme in the control of whole-body energy metabolism regardless of Se levels.
Keywords: selenium, selenocysteine lyase, diet-induced obesity
1. Introduction
Selenium (Se) is a trace element essential to health and acquired from dietary sources such as brazil nuts, seafood and plants growing in Se-rich soils. In our diets, Se is available as various chemical forms, with the predominance of inorganic selenite or selenate, and organic selenomethionine (SeMet) or selenocysteine (Sec) [1]. Dietary selenocompounds differ significantly in their metabolic pathways and in their abilities to produce various Se metabolites [2, 3]. To become bioavailable, selenocompounds are metabolized in cells to selenide. Selenide is utilized to produce the amino acid Sec, present in the primary structure of proteins deemed selenoproteins, and allowing for a strong redox potential. Interestingly, Sec needs to be synthesized from selenide in its tRNA[Ser]Sec in order to be co-translationally incorporated into selenoproteins [4, 5]. The mechanism of Sec synthesis and selenoprotein production has been well-characterized by several groups [6–9].
In the liver, inorganic selenite and selenate are reduced via the glutathione/thioredoxin redox cycle. Organic SeMet can be metabolized by the enzymes of the methionine-decomposing transsulfuration pathway, such as cystathionine beta-synthase (CBS) [10] and cystathionine gamma-lyase (CTH) [11], or can be transaminated by kynurenine aminotransferase III (KYAT3, also known as Ccbl2) into α-ketomethylselenobutyrate [12]. The bioavailability of SeMet has been reported to be higher than that of selenite [13]. Sec is decomposed into alanine and selenide by the enzyme Sec lyase (SCLY) [14]. Other Se-conjugates of Sec can be metabolized by the same KYAT3 into β-methylselenopyruvate [12]. Sec may also be decomposed, in a less efficient manner, by cysteine desulfurases such as NFS1 [15].
SCLY utilizes Sec either from dietary sources, selenoprotein degradation, or derived of SeMet metabolism [16]. Free Sec has not been detected in cells, suggesting that after selenoproteins exert their biological function and are degraded, the Sec residue should be promptly decomposed to yield selenide. This Se recycling process may be a significant source of Se when this micronutrient is limiting.
We previously uncovered that male mice with a disruption in the Scly gene (Scly KO) became obese, glucose intolerant and hyperinsulinemic, marked characteristics of metabolic syndrome development, and also had reduced hepatic Se levels [17]. Obesity in the Scly KO mice was sexually dimorphic [18], and exacerbated by either feeding of a Se-adequate, high-fat diet [19] or by Se deficiency [17, 18], conditions in which mild hepatic Se deficiency was observed when amino acid pathways were altered, possibly to cope with the disruption in Se recycling [20]. Nevertheless, it is unknown whether diet-induced obesity in the Scly KO mice could be ameliorated by dietary Se supplementation.
In this study, we investigated whether Se supplementation can rescue the obese phenotype of the Scly KO mouse model fed a high-fat diet. We also determined that Se supplementation delivers a differential metabolic response depending on the selenocompound present in diets. We found that male Scly KO mice gained more weight than WT controls after Se supplementation, a phenotype even more pronounced in female Scly KO mice. Moreover, expression of selenoproteins glutathione peroxidase 1 (GPX1) and selenoprotein P (SelenoP) was differentially regulated in the Scly KO mice by the dietary Se chemical form ingested.
2. Materials and Methods
2.1. Chemicals
All reagents are from Sigma-Aldrich/MilliporeSigma (Burlington, MA, USA) unless otherwise noted.
2.2. Animals, diets and experimental design
Homozygous Scly KO mice and C57BL/6N wild-type (WT) homozygous animals derived from The Jackson Laboratory (Bar Harbor, ME) were born and raised in our vivarium in separate colonies and used in experiments after weaning in accordance with the Institutional Animal Care and Use Committee of the University of Hawaii (Protocol #17–2616). Scly KO mice have been generated in a C57BL/6 background and backcrossed into the C57BL/6N strain for at least 4 generations prior to use in experiments. Starting at 3 weeks of age, animals were group-caged in box cages with corn cob bedding, and fed for 9 weeks with a customized high-fat diet containing 45 kcal% fat as lard, 35 kcal% carbohydrate as a mixture of sucrose and corn starch (Research Diets, Inc., New Brunswick, NJ) and basal 0.155 mg/kg of sodium selenite as part of the mineral mix (Research Diets, New Brunswick, NJ). In the first study (Experiment 1), diets contained basal sodium selenite plus either 0.34 or 1.77 mg/kg of sodium selenite, hence providing 0.25 or 1 ppm of Se. The second study (Experiment 2) utilized a blend diet containing basal sodium selenite plus either 0.22 or 0.87 mg/kg of SeMet, providing 0.25 or 0.5 ppm of Se. At 12 weeks of age (9 weeks on above diets), animals were euthanized via CO2 asphyxiation and tissues were harvested for further analysis. Serum, liver, epididymal white adipose tissue (eWAT), and gonadal WAT (gWAT) tissues were collected, weighed, and frozen in liquid nitrogen. eWAT and gWAT weights were normalized to the body weight of the animal at the time of euthanasia. Frozen tissues from all experiments were thawed together for assays and qPCR analysis.
2.3. Glucose tolerance test
Glucose tolerance test (GTT) was performed at 10 weeks of age (following 7 weeks of dietary intervention) to assess glucose sensitivity. For the GTT, 10 week-old mice were fasted for 4 h and injected intraperitoneally with glucose (1 g/kg of body weight). Tail vein blood was collected at 0, 30, 60, and 120 min post-injection and tested for glucose using glucose strips and a glucometer (LifeScan, Milpitas, CA, USA). The area under the curve (AUC) was calculated for individual mice, averaged for the group and plotted as bar graphs.
2.4. Glutathione peroxidase (GPX) activity assay
Total serum or liver GPX activity was measured using a colorimetric assay kit (Abcam, Cambridge, UK, catalog no. Ab102530) following the manufacturer’s protocol. In the GPX assay protocol, GPX oxidizes glutathione (GSH) to produce diglutathione (GSSG) as part of the reaction in which it reduces cumene hydroperoxide. Glutathione reductase (GR) then reduces the GSSG to produce GSH, and in the same reaction consumes NADPH. The decrease of NADPH (measured at OD=340 nm) is proportional to GPX activity.
2.5. Measurement of oxidative stress
Oxidative stress status was assayed by measuring lipid peroxidation in the liver tissues using the OxiSelect HNE-his Adduct ELISA Kit (Cell Biolabs, Inc., San Diego, CA, USA, catalog number: STA – 838–5), according to the manufacturer’s protocol.
2.6. Quantitative real-time PCR (qPCR)
One microgram of total RNA was reverse-transcribed using High Capacity cDNA Reverse Transcription Kits (Applied Biosciences/Thermo Fisher Scientific, Waltham, MA, USA), with 10 ng of resulting cDNA used for qPCR with PerfeCTa SYBR Green FastMix (Quantabio, Beverly, MA, USA) and 45 amplification cycles in a 384-well plate platform of a LightCycler 480 II (Roche, Basel, Switzerland). Relative quantification used the Δ−CT method, normalized to hypoxanthine-guanine phosphoribosyltransferase (Hprt1) expression levels. All primers were evaluated for their efficiency prior to use in experiments. Primer sequences are found in Table 1.
Table 1.
Primer sequences used for qPCR analysis in this study.
| Gene | Forward | Reverse |
|---|---|---|
| Ccbl2 | 5′-CCGTAGAAAATGGCTTTGAAAT | 5′-AGTAAATTCAACCCACACATTG |
| Cth | 5′-TGCCACCATTACGATTACCCAT | 5′-TTGGTGCCTCCATACACTTCAT |
| Gpx1 | 5′-ACAGTCCACCGTGTATGCCTTC | 5′-CTCTTCATTCTTGCCATTCTCCTG |
| Hprt1 | 5′-TCCTCCTCAGACCGCTTTT | 5′-CCTGGTTCATCATCGCTAATC |
| Nfs1 | 5′-AGTGGAGCTACTGAGTCCAA | 5′-CACATTTGTGTTCTGTCTGG |
| Pparg | 5′-TTGATTTCTCCAGCATTTCT | 5′-TGTTGTAGAGCTGGGTCTTT |
| Selenbp2 | 5′-ATACTGCCTGGTCTCATGTC | 5′-AAGGTGTTGACCATGACTTC |
| Selenop | 5′-CCTTGGTTTGCCTTACTCCTTCC | 5′-TTTGTTGTGGTGTTTGTGGTGG |
| Txnrd1 | 5′-CCTATGTCGCCTTGGAATGTGC | 5′-ATGGTCTCCTCGCTGTTTGTGG |
2.7. Statistical analysis
Data were plotted in GraphPad Prism software (GraphPad Software Inc., San Diego, CA). Applied statistical tests varied depending on the experiment and differences were considered significant if they resulted in p-values under 0.05. Body weight assessment was evaluated after mixed-effects analysis to include the three experimental variables.
3. Results
We previously reported that male Scly KO mice are more susceptible to diet-induced obesity than their WT counterparts, despite receiving adequate Se supply. In the present study, male and female Scly KO mice fed the 45% kcal high-fat diet showed increased weight gain relative to WT mice. Supplementation with high Se, either as selenite or selenite/SeMet, resulted in an ~20% increase in weight in female but not male SclyKO mice (Figures 1A and 1C). High selenite (1 ppm) further exacerbated weight gain in female Scly KO mice by ~10%, compared to moderate (0.25 ppm) selenite-supplemented Scly KO mice. Male Scly KO mice developed obesity with similar weight gains regardless of dietary Se levels or chemical form ingested through their diets (Figures 1B and 1D). We did not observe differences in body weight for WT mice in response to varying Se, regardless of sex.
Figure 1.
Body weight increases upon feeding a high-fat diet in female mice supplemented with selenite (A) or selenite/SeMet (C), and male mice supplemented with selenite (B) or with selenite/SeMet (D). Values are mean ± SEM. Mixed-effects analysis of variance was applied, followed by Bonferroni’s post-hoc test. **, p <0.01; ***, p <0.001, ****, p<0.0001, n is displayed in graphs per group. Circle, female mice; square, male mice; open circle or square, 0.25 ppm of selenocompound; filled circle or square, 0.5 or 1 ppm of selenocompound; black, WT; gray, Scly KO.
White adipose tissue weights were considerably higher in Scly KO mice versus WT mice fed either Se concentration, and more than doubled with high selenite/SeMet supplementation in Experiment 2 (Figure 2A, 2C). Male Scly KO mice showed eWAT weight also increased with either level of supplementation, regardless of the chemical form, with 0.25 ppm selenite/SeMet blend leading to three times heavier eWAT in male Scly KO mice than their WT counterparts.
Figure 2.
White adipose tissue weights after a high-fat diet in female mice supplemented with selenite (A) or selenite/SeMet (C), and male mice supplemented with selenite (B) or with selenite/SeMet (D). Values are mean ± SEM, and were normalized by total body weight per mouse. Two-way analysis of variance was applied, followed by Bonferroni’s post-hoc test. *, p<0.05; **, p <0.01; ***, p <0.001, n=9–11 per genotype. eWAT, epididymal white adipose tissue; gWAT, gonadal white adipose tissue. Filled bars, WT; open bars, Scly KO.
After 7 weeks of selenite supplementation for Experiment 1, Scly KO mice of both sexes were more severely glucose intolerant when compared with their WT counterparts, and the level of Se supplementation had little additional effect on glucose intolerance (Figure 3A, 3B). With the blend selenite/SeMet in Experiment 2, there was little difference in glucose intolerance in female mice regardless of genotype or level of Se supplementation (Figure 3C). In male Scly KO mice, supplementation with 0.25 ppm selenite/SeMet further enhanced glucose intolerance compared to 0.25 ppm as selenite alone, but 0.5 ppm selenite/SeMet reduced glucose intolerance in male SclyKO mice (Fig 3D). Overall, the effect of lacking Scly on glucose tolerance was more dramatic in male Scly KO mice.
Figure 3.
Glucose tolerance test after a glucose overload with area under the curve (AUC) quantification plotted as bar graph. GTT was performed at 10-weeks of age after mice were fed a high-fat diet for 7 weeks. Female (A,C) and male (B,D) mice were supplemented with selenite in Experiment 1 (A, C) or a mixture of selenite/SeMet in Experiment 2 (B,D). Values are mean ± SEM. Two-way analysis of variance (ANOVA) was applied followed by Bonferroni’s post-hoc test. *, p<0.05, **, p <0.01; ***, p <0.001; sample size is displayed inside bars. Circle, female mice; square, male mice; open circle or square, 0.25 ppm of selenite or selenite/SeMet; filled circle or squares, 1 ppm of selenite or 0.5 ppm of selenite/SeMet; black, WT; gray, Scly KO.
Different forms of Se supplementation influenced oxidative stress parameters in distinct ways. Se supplementation elevated serum GPX activity 5–10% in the Scly KO mice regardless of sex or chemical form of Se (Tables 2 and 3). This effect occurred despite unchanged hepatic GPX activity in most groups except female Scly KO mice fed selenite (Table 2). Selenite supplementation in Experiment 1 did improve, however, male WT oxidative stress status as lipid peroxidation measured by HNE decreased in the liver, but this effect was absent in Scly KO mice (Table 2). On the other hand, supplementation with selenite/SeMet in Experiment 2 increased circulating GPX activity of Scly KO mice while also improving by ~20% the hepatic oxidative status of Scly KO mice measured by HNE adduct formation (Table 3).
Table 2.
Serum and hepatic parameters of oxidative stress in the Experiment 1, in which WT and Scly KO mice were fed a high-fat diet supplemented with selenite for 9 weeks. Values are mean ± SEM. Two-way ANOVA was applied, and different letters represent statistical significance after Bonferroni post-hoc test. P-values under 0.05 were deemed significant; n = 4–6. WT, wild type; KO, knockout; GPX, glutathione peroxidase; HNE, 4-Hydroxy-Trans-2-Nonenal.
| MALES | |||||||
|---|---|---|---|---|---|---|---|
| WT | Scly KO | 2-way ANOVA | |||||
| 0.25 ppm | 1 ppm | 0.25 ppm | 1 ppm | Pinteraction | PSe | PGenotype | |
| Serum | |||||||
| GPX activity (nmol/mL-1) | 37.6±0.6a | 37.0±0.7a | 38.7±1.2a | 40.9±1.9b | 0.026 | 0.16 | 0.0004 |
| Liver | |||||||
| GPX activity (nmol/mg-1) | 13.5±0.09 | 12.7±1.1 | 13.3±0.3 | 13.2±0.2 | 0.194 | 0.16 | 0.6859 |
| HNE (mg/mL) | 16.9±4.9a | 9.98±3.3b | 11.5±4.8a,b | 13.6±7.5a,b | 0.09 | 0.35 | 0.734 |
| FEMALES | |||||||
| WT | Scly KO | 2-way ANOVA | |||||
| 0.25 ppm | 1 ppm | 0.25 ppm | 1 ppm | Pinteraction | PSe | PGenotype | |
| Serum | |||||||
| GPX activity (nmol/mL-1) | 39.8±1.6a | 40.75±1.0a | 41.76±1.6a | 44.29±1.0b | 0.24 | 0.02 | 0.0008 |
| Liver | |||||||
| GPX activity (nmol/mg-1) | 12.8±0.2a | 12.9±0.3b | 12.8±0.3a,b | 12.4±0.2c | 0.04 | 0.25 | 0.115 |
| HNE (mg/mL) | 11.7±7.7 | 6.6±1.5 | 10.4±1.4 | 10.73±5.2 | 0.23 | 0.29 | 0.5294 |
Table 3.
Serum and hepatic parameters of oxidative stress in the Experiment 2, in which WT and Scly KO mice were fed a high-fat diet supplemented with selenite + selenomethionine for 9 weeks. Values are mean ± SEM. Two-way ANOVA was applied, and different letters represent statistical significance after Bonferroni post-hoc test. P-values under 0.05 were deemed significant; n = 4–6 per group. WT, wild type; KO, knockout; GPX, glutathione peroxidase; HNE, 4-Hydroxy-Trans-2-Nonenal.
| MALES | |||||||
|---|---|---|---|---|---|---|---|
| WT | Scly KO | 2-way ANOVA | |||||
| 0.25 ppm | 0.5 ppm | 0.25 ppm | 0.5 ppm | Pinteraction | PSe | PGenotype | |
| Serum | |||||||
| GPX activity (nmol/mL-1) | 41.2±3.8a | 43.6±1.8a,b | 46.6±1.2b | 45.8±2.4b | 0.1666 | 0.4905 | 0.0027 |
| Liver | |||||||
| GPX activity (nmol/mg-1) | 13.3±0.2 | 13.3±0.1 | 13.4±0.08 | 13.4±0.3 | 0.908 | 0.7292 | 0.3062 |
| HNE (mg/mL) | 6.8±1.4a | 7.6±3.4a | 12.8±1.8b | 7.9±2.1a | 0.008 | 0.0495 | 0.0043 |
| FEMALES | |||||||
| WT | Scly KO | 2-way ANOVA | |||||
| 0.25 ppm | 0.5 ppm | 0.25 ppm | 0.5 ppm | Pinteraction | PSe | PGenotype | |
| Serum | |||||||
| GPX activity (nmol/mL-1) | 40.4±0.8a | 40.9±1.3a | 42.2±1.1b | 45.2±0.8c | 0.0155 | 0.001 | <0.001 |
| Liver | |||||||
| GPX activity (nmol/mg-1) | 13.3±0.3 | 13.3±0.2 | 13.4±0.08 | 13.5±0.3 | 0.6771 | 0.6771 | 0.4564 |
| HNE (mg/mL) | 10.4±2.2a | 9.4±7.3a | 20.4±4.3b | 16.2±7.2b | 0.293 | 0.524 | 0.0025 |
Scly is known to be involved in selenoprotein synthesis [21]. Lack of Scly disrupted energy metabolism in the liver, upregulating mRNA expression of selenoproteins and metabolic genes [17]. To investigate the effects of distinct chemical forms of Se supplementation in the Scly KO mice we determined mRNA expression of selenoprotein genes (Tables 4 and 5). Overall, there was no effect of Se supplementation on transcript levels of selenoproteins in female or male mice regardless of form of supplemental Se (Se effect p>0.05; Tables 4 and 5). These results demonstrate that a saturation of selenoprotein gene expression possibly occurred at lower Se levels than available on tested diets.
Table 4.
Hepatic gene expression of selenoprotein and metabolic enzymes of WT and Scly KO mice after feeding on a high-fat diet supplemented with selenite (Experiment 1), assessed by qPCR. Values are mean ± SEM, were normalized to Hprt1 mRNA levels, and presented as arbitrary units. Two-way ANOVA was used to compare averages. P-values under 0.05 were deemed significant; n = 4–6 per group.
| SELENITE | |||||||
|---|---|---|---|---|---|---|---|
| 0.25 ppm | 1 ppm | 2-way ANOVA (p-value) | |||||
| MALES | WT | Scly KO | WT | Scly KO | Pinteraction | PSe | PGenotype |
| Selenop | 413.8±42.9 | 382.1±24.6 | 390.3±33.1 | 388.8±61.2 | 0.7291 | 0.8478 | 0.705 |
| Gpx1 | 80.4±3.7 | 64.7±6.4 | 67.7±6.1 | 65.2±3.8 | 0.2289 | 0.2593 | 0.1024 |
| Txnrd1 | 1.8±0.6 | 1.6±0.05 | 2.1±0.7 | 1.6±0.7 | 0.5055 | 0.4433 | 0.1887 |
| Pparg | 0.1±0.02 | 0.4±0.1 | 0.2±0.05 | 0.5±0.1 | 0.7851 | 0.7558 | 0.0063 |
| Selenbp2 | 3.2±0.8 | 1.7±0.4 | 3.9±0.7 | 1.3±0.3 | 0.3871 | 0.8573 | 0.0023 |
| Nfs1 | 0.3±0.05 | 0.3±0.04 | 0.4±0.05 | 0.4±0.05 | 0.4112 | 0.083 | 0.9016 |
| Ccbl2 | 2.5±0.9 | 1.2±0.3 | 2.9±0.5 | 1.5±0.4 | 0.9311 | 0.2877 | 0.0001 |
| 0.25 ppm | 1 ppm | 2-way ANOVA (p-value) | |||||
| FEMALES | WT | Scly KO | WT | Scly KO | Pinteraction | PSe | PGenotype |
| Selenop | 874.0±186.8 | 517.7±104.4 | 757.2±355.9 | 584.2±55.7 | 0.3369 | 0.7895 | 0.0113 |
| Gpx1 | 66.7±4.8 | 55.6±13.1 | 105.0±50.5 | 67.4±13.0 | 0.2976 | 0.0582 | 0.0653 |
| Txnrd1 | 1.6±0.7 | 2.1±0.3 | 2.4±1.6 | 2.8±1.1 | 0.9452 | 0.1451 | 0.2847 |
| Pparg | 0.3±0.1 | 0.5±0,3 | 0.3±0.2 | 0.6±0.2 | 0.3533 | 0.4922 | 0.0083 |
| Selenbp2 | 2.6±1.4 | 3.7±1.0 | 2.5±1.6 | 3.6±1.2 | 0.9897 | 0.8237 | 0.0851 |
| Nfs1 | 0.3±0.06 | 0.5±0.05 | 0.4±0.08 | 0.5±0.05 | 0.2237 | 0.3922 | 0.03 |
| Ccbl2 | 2.2±0.6 | 4.0±1.1 | 4.9±2.5 | 3.6±0.8 | 0.0444 | 0.1007 | 0.7041 |
Table 5.
Hepatic gene expression of selenoprotein and metabolic enzymes of WT and Scly KO after feeding on a high-fat diet supplemented with a mixture of selenite + selenomethionine (Experiment 2) assessed by qPCR. Values are mean ± SEM, were normalized to Hprt1 mRNA levels, and presented as arbitrary units. Two-way ANOVA was used to compare averages. P-values under 0.05 were deemed significant; n = 4–6 per group.
| SELENITE + SEMET | |||||||
|---|---|---|---|---|---|---|---|
| 0.25 ppm | 0.5 ppm | 2-way ANOVA (p-value) | |||||
| MALES | WT | Scly KO | WT | Scly KO | Pinteraction | PSe | PGenotype |
| Selenop | 618.0±47.6 | 406.6±48.1 | 425.1±65.7 | 441.9±56.1 | 0.0552 | 0.1744 | 0.0978 |
| Gpx1 | 73.1±4.7 | 48.0±6.3 | 53.0±8.1 | 51.6±8.3 | 0.1066 | 0.2441 | 0.0749 |
| Txnrd1 | 4.3±1.4 | 4.7±1.5 | 4.5±2.5 | 5.5±2.6 | 0.6958 | 0.5343 | 0.4542 |
| Pparg | 0.2±0.04 | 0.4±0.05 | 0.2±0.09 | 0.4±0.08 | 0.7356 | 0.7312 | 0.0006 |
| Selenbp2 | 10.9±3.6 | 3.1±0.4 | 8.2±1.8 | 3.2±0.5 | 0.4553 | 0.4925 | 0.0028 |
| Nfs1 | 0.5±0.09 | 0.4±0.08 | 0.4±0.03 | 0.5±0.02 | 0.6673 | 0.571 | 0.9033 |
| Ccbl2 | 3.8±1.6 | 3±0.9 | 3.3±1.2 | 3.9±1.3 | 0.2109 | 0.7612 | 0.8552 |
| Cth | 6.0±0.7 | 5.7±0.5 | 5.3±0.3 | 5.0±0.5 | 0.9093 | 0.2389 | 0.5703 |
| 0.25 ppm | 0.5 ppm | 2-way ANOVA (p-value) | |||||
| FEMALES | WT | Scly KO | WT | Scly KO | Pinteraction | PSe | PGenotype |
| Selenop | 658.8±86.1 | 958.4±145.6 | 999.5±122.3 | 713.2±30.4 | 0.0132 | 0.6593 | 0.9508 |
| Gpx1 | 76.3±36.0 | 125.7±41.6 | 109.4±222.2 | 81.6±25.2 | 0.0116 | 0.6916 | 0.441 |
| Txnrd1 | 2.9±0.7 | 4.3±2.2 | 3.9±0.9 | 2.6±1 | 0.0294 | 0.5586 | 0.8339 |
| Pparg | 0.3±0.1 | 0.7±0.2 | 0.3±0.1 | 0.6±0.2 | 0.9564 | 0.5791 | 0.0021 |
| Selenbp2 | 2.7±0.8 | 4.1±0.5 | 2.8±+0.8 | 3.6±0.5 | 0.316 | 0.7191 | 0.0016 |
| Nfs1 | 0.5±0.2 | 0.8±0.08 | 0.6±0.1 | 0.7±0.08 | 0.0939 | 0.7087 | 0.0066 |
| Ccbl2 | 3.7±0.9 | 5.5±2.5 | 5.9±0.9 | 3.4±1.0 | 0.0037 | 0.9297 | 0.5748 |
| Cth | 5.9±1.5 | 11.1±2.3 | 6.5±0.8 | 9.0±2.7 | 0.1138 | 0.3548 | 0.0001 |
Regarding genes involved in Se and energy metabolism, we have previously determined that expression of the master regulator of lipid metabolism peroxisome proliferator-activated receptor gamma (Pparg) and of Se-related selenium-binding protein 2 (Selenbp2) was differentially regulated in the livers of Scly KO mice compared to their WT counterparts when dietary Se was limiting [20]. In this study, we observed that lack of Scly KO typically at least doubled Pparg expression, regardless of Se form or sex (Tables 4 and 5). In terms of Se metabolism, Selenbp2 expression was downregulated by one third in male Scly KO mice compared to WT, regardless of Se supplementation levels, whereas female Scly KO mice upregulated Selenbp2 expression (Tables 4 and 5). In addition, genes that express enzymes of the transsulfuration pathway, shared by the SeMet metabolism, were assessed. Ccbl2 gene expression was lowered by half only in male Scly KO mice supplemented with selenite in Experiment 1. In female Scly KO mice fed 0.25 ppm diets, Nfs1 expression was approximately 70% higher than in WT mice, and also independent of the Se form fed. Increased levels of Cth were only observed with selenite/SeMet supplementation in female Scly KO mice of Experiment 2.
4. Discussion
This is the first study to characterize the effects of Se supplementation in a mouse model lacking the Scly gene and fed a high-fat diet. We previously noted that Scly KO mice had aggravated obesity when fed either a high-fat, Se-adequate diet [19] or a Se-deficient diet, with a milder phenotype when dietary Se levels were adequate [17]. Here we attempted to assess whether Se supplementation could rescue, at least to some extent, the metabolic phenotype of the high-fat diet-fed Scly KO mice.
To our surprise, Se supplementation with selenite in Experiment 1 rescued neither the weight gain nor the glucose tolerance of Scly KO mice, even worsening obesity in females. Diets with a blend of selenite/SeMet in Experiment 2 still led to obesity in male Scly KO mice, regardless of Se concentration, despite improving their glucose tolerance. These findings uncover the possibility that SCLY may have a secondary role beyond providing selenide for selenoprotein synthesis, consisted with our previous observation of effects on lipid metabolism to be independent of selenoprotein levels [19].
The differential effects of dietary Se intake on the development of obesity in the Scly KO mice according to the chemical form also attest to the different pathways that inorganic and organic Se undergo [22]. Particularly in the case of the selenite/SeMet supplementation in Experiment 2, it points to a pivotal role for SCLY in the transselenation pathway that metabolizes SeMet [16, 23] at least in males. Notably, only female Scly KO livers presented elevated gene expression of the transselenation enzyme Cth, indicating not only that SeMet supplementation is activating the transselenation pathway in a sex-specific manner, but also raising the possibility that male Scly KO mice might favor the metabolism of SeMet through the alternative transamination pathway that produces α-ketomethylselenobutyrate [3, 24]. It is also possible that male Scly KO mice have a higher rate of misincorporation of SeMet into Met residues of proteins as has been suggested to occur when the SeMet pool is larger [25], while females are possibly metabolizing SeMet via α, γ elimination by CTH [11], hence favoring excretion of excess Se. Furthermore, this potential Se species-dependent sex difference may contribute to the finding that weight gain in female Scly KO mice was augmented by higher amounts of selenite, but not by the highest Se supplementation in the form of a selenite/SeMet combination. CTH contributes to the elevation of GPX activity in a dose-dependent manner following SeMet supplementation [26]. As we did not observe changes in hepatic GPX activity for the male Scly KO mice after Se supplementation, it is possible that the enhanced oxidative stress of the energy overload already induces and maximizes GPX activity, and the Scly requirement for its synthesis is circumvented by the amount of selenite the supplementation blend contains. Another possibility for the fate of SeMet is that excess of this selenocompound leads to misincorporation of Sec in place of cysteine via mechanisms independent of oxidative stress levels [27, 28]; however, to date this possibility has been reported only in yeast. Regardless of which pathway is favored in mice with disruption of the Scly gene, it is clear that the prevalence of each varies with sex.
Male WT mice enhance their plasma GPX activity when supplemented with 0.75 ppm of SeMet, but not selenite, and hepatic GPX activity follows this pattern [29]. Our current results add a layer of complexity, as mice were fed a high-fat, Se-supplemented diet. Still, we uncovered the same patterns for GPX activity in male WT mice. Moreover, we found that serum and hepatic activity of GPX in female WT mice does not respond to Se supplementation. In the Scly KO mice, neither sex nor supplementation level affects GPX activity in the serum. It is possible that Scly participation in the synthesis of GPX3 in the kidneys is crucial to gauge and maintain levels of circulating GPX, as most serum GPX activity comes from GPX3, produced by the kidneys [30].
Classically, SCLY is postulated to provide selenide for selenoprotein synthesis [21], possibly via delivery to selenophosphate synthetases [22]. Selenoproteins, thus, are considered the main effectors of the biological actions of Se in organisms [8]. Previous studies observed that Se concentration requirements for maximal selenoprotein expression, were lower than those utilized in our study [31, 32]. At optimal Se levels, selenoprotein mRNAs are able to circumvent nonsense-mediated decay, whereas Se limitation to levels that jeopardize insertion of the UGA-coded Sec amino acid lead to degradation of some selenoprotein mRNAs [33]. Hence, at 0.25 ppm of dietary Se form, its availability should not be a limiting factor for appropriate selenoprotein expression. Nevertheless, when selenite was the dietary source of Se as in Experiment 1, we observed that Selenop expression was still diminished in the female Scly KO mouse, but not in the males, as we have determined before [17, 19]. It is plausible that Selenop mRNA expression does not correspond to increases in SelenoP levels, or that sex-dependent regulation of Selenop expression occurs when mice are fed selenite [34]. Interestingly, the expression of the Scly gene in the male mouse liver depends on the presence of SelenoP [35]. Paradoxically, Selenop expression was not regulated by sex in Experiment 2, when a mixture of selenite/SeMet at moderate levels was fed to WT mice, with ~30% increase in expression occurring at supplemented levels in females but ~ 30% downregulation in males. Based on our results, expression of Selenop is differentially regulated according to the chemical form of Se taken, and these differences have been investigated in WT mice at the proteome level [29]. Overall, even though selenide is the common Se form for selenoprotein synthesis, the upstream reactions that form selenide are relevant to the gene regulation of selenoproteins, possibly even to the hierarchy of expression that this group of proteins present [32, 36].
In conclusion, we found that the weight gain and glucose intolerance displayed by the Scly KO mice on a hypercaloric diet are not rescued by Se supplementation. Furthermore, depending on the dietary chemical form of Se, lack of Scly produces a differential metabolic response that is attuned to the role of this enzyme in the transselenation pathway, or potentially to a role in other pathways beyond Se metabolism, possibly cysteine or methionine metabolism. Metabolomic studies may shed light on this alternative role. As an energy-rich, Se-sufficient diet has become common in several countries, including the United States [37–39], our results also steer future studies in human populations to account for the differential, sex-dependent metabolism of Se, particularly when involving Scly, in improving our understanding of energy metabolism and obesity development
Acknowledgments
5. Funding Sources
This work was supported by NIH grants R01DK047320 to MJB; U54MD007601 – Subproject 5544 to LAS; R01DK047320–22S1 to MJB, an Administrative Supplement for Research on Dietary Supplements from the Office of the Director (OD) and co-funded by the Office of Dietary Supplements (ODS); R01DK047320–22S2 to MJB, a Research Supplement to Promote Diversity in Health-Related Research and fellowship 2018/09478–4 from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) to LMW.
Footnotes
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7. Conflict of Interest
The authors declare no conflicts of interest.
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