Serum vitamin C levels modulate the lifespan and endoplasmic reticulum stress response pathways in mice synthesizing a nonfunctional mutant WRN protein

Histologic examination

Tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Thin sections were mounted on glass slides and stained with hematoxylin and eosin (H&E). TUNEL assays for detection of apoptotic cells were performed on 4–5 µm paraffin-embedded sections with an In situ Apoptosis Detection Kit (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s recommendations.

Metabolite measurements

Blood was drawn at 10 am by cardiac puncture and exsanguination under anesthesia at the age of 4 mo and processed (21). Ascorbic acid in serum was measured with the ferric reducing ascorbate assay kit from BioVision Research Products (Mountain View, CA, USA). Metabolite measurements were performed by Biocrates Life Sciences metabolomic services (Biocrates Life Sciences, Innsbruck, Austria). In brief, Biocrates’ commercially available kit plates were used for quantification of amino acids, acylcarnitines, sphingomyelins, phosphatidylcholines, hexoses, and biogenic amines. The fully automated assay was based on phenylisothiocyanate derivatization in the presence of internal standards followed by flow injection analysis–tandem mass spectrometry (MS) (acylcarnitines, lipids, and hexose) and liquid chromatography (LC)/MS (amino acids, biogenic amines) with a 4000 QTrap mass spectrometer (AB Sciex, Darmstadt, Germany) with electrospray ionization. The experimental metabolomics measurement technique has been described in Illig et al. (22). Eicosanoids and other oxidized polyunsaturated fatty acids were extracted from samples with aqueous acetonitrile that contained deuterated internal standards. The metabolites were determined by LC-MS/MS with multiple-reaction monitoring in negative mode on a 4000 QTrap mass spectrometer with electrospray ionization. The LC-MS/MS method used for the analytical determination of eicosanoids has been published (23). Accuracy of the measurements was in the normal range of the method (deviations from target, ≤20%) for all analytes. In total, 203 different metabolites were measured. The metabolomics data set contains 21 aa, 19 biogenic amines, 1 hexose (H1), free carnitine (C0), 40 acylcarnitines (Cx:y), hydroxylacylcarnitines [C(OH)x:y], and dicarboxylacylcarnitines (Cx:y-DC), 15 sphingomyelins (SMx:y), N-hydroxylacyloylsphingosylphosphocholine [SM (OH)x:y], 77 phosphatidylcholines (PC), 14 lyso-phosphatidylcholines, and 17 eicosanoid acids and prostaglandins. Lipid side-chain composition is abbreviated as Cx:y, where x denotes the number of carbons in the side chain and y the number of double bonds. For example, PC acyl-alkyl (PC ea) C30:1 denotes an acyl-alkyl phosphatidylcholine with 30 carbons in the 2 fatty acid side chains and 1 double bond in one of them (22). The precise position of the double bonds and the distribution of the carbon atoms in different fatty acid side chains cannot be determined with this technology. Full biochemical names and raw data are provided in the Excel spreadsheets of Supplemental Data S1.

Cytokine measurements

The following cytokines were assessed in the serum (diluted 1:4) using the multiplex kit from Bio-Rad Laboratories (MD000000EL; Mississauga, ON, Canada): IL-15, IL-18, leukemia inhibitory factor, macrophage–colony-stimulating factor (M-CSF), monokine induced by γ-interferon (MIG), macrophage inflammatory protein (MIP)-2, platelet-derived growth factor-BB, VEGF, and fibroblast growth factor (FGF) basic. The following cytokines were assessed in the serum (diluted 1:4) using the Multiplex Kit from Bio-Rad Laboratories (MD60009RDPD): IL-1α, -1β, -2, -3, -4, -5, -6, -10, -12 (p40), -12 (p70), -13, and -17; katacalcin (KC); monocyte chemoattractant protein-1; MIP-1α and -1β; TNF-α; regulated on activation, normal T-cell–expressed and –secreted (RANTES); Eotaxin; granulocyte–colony-stimulating factor (G-CSF); granulocyte-macrophage–colony-stimulating factor (GM-CSF); and IFN-γ. The metabolic hormones C-peptide 2, gastric inhibitory polypeptide, leptin, and resistin were assessed in serum with the Milliplex Mouse Metabolic Magnetic Bead Panel (MMHMAG-44K) from MilliporeSigma (Billerica, MA, USA). The following cardiovascular risk factors were assessed in the serum (diluted 1:20) using the Milliplex Mouse CVD Panel 1 Kit from MilliporeSigma (MCVD1MAG-77K): soluble E- and P-selectin, intercellular adhesion molecule-1, platelet endothelial cell adhesion molecule-1, pro-matrix metalloproteinase (MMP)-9, thrombomodulin, and total plasminogen activation inhibitor (PAI)-1. Full biologic names and raw data are provided in the Supplemental Table S1. All measurements were performed on a Bio-Plex 200 System (Bio-Plex Manager Software v.6.0; Bio-Rad Laboratories.)

Microcomputerized tomography analysis of trabecular and cortical bone

High-resolution images of femurs were acquired by using a Scanco VivaCT 40 (Bruettisellen, Switzerland). The femurs were scanned with the source voltage of 55 kV, a source current of 142 µA, and an isotropic voxel size of 10.5 µm. After the scanning, 3D microstructural image data were reconstructed and structural indices were calculated with Scanco µCT V6.1 Software. The number of femurs from 4-mo-old mice analyzed by micro-CT analysis were n = 8 WT, n = 10 Wrn^Δhel/Δhel, n = 10 Gulo^−/−, n = 12 Wrn^Δhel/Δhel/Gulo^−/− mice with supplemental 0.01% ascorbate, and n = 8 Wrn^Δhel/Δhel/Gulo^−/− mice treated with supplemental 0.4% ascorbate.

The area for the trabecular analysis started 57.5 µm proximal from the growth plate at the distal end of the femur and extended proximally 1050 µm toward the femoral head. The region of interest (ROI) for trabecular microarchitectural variables was defined by manually outlining the bone within the endocortical margins, allowing a few voxels between the ROI and the endocortical margin (24). An upper threshold of 1000 Hounsfield units and a lower threshold of 220 Hounsfield units were used to delineate each pixel as bone or nonbone. Representative 2-dimensional cross-sectional images for each genotype within the young and aged groups are shown in gray scale. Trabecular bone volume per total volume (%), mean trabecular thickness (mm), mean trabecular number (1/mm), and mean trabecular separation (mm) indices were computed.

Fractionation procedures for tissues

Total liver protein extracts were prepared as described elsewhere (21). Total endoplasmic reticulum (ER) fractions from mouse livers were obtained with an ER Enrichment Assay Kit (Novus Biologicals, Burlington, ON, Canada) according to the manufacturer’s protocol. Enriched ER pellets were resuspended in suspension buffer containing phosphatase inhibitor cocktail (PhosStop Easy Pack) from Roche Applied Sciences (Indianapolis, IN, USA).

Western blot analyses

Western blot analyses were performed as described in Aumailley et al. (21). When indicated, immunoblots were probed with the following antibodies: rabbit polyclonal antibodies raised against the eukaryotic translation initiation factor 2-α kinase 3 [anti-PERK (H300): sc-13073]; eukaryotic translation factor 2-α [anti-eIF2α (FL-315): sc-11386]; phosphorylated eukaryotic translation factor 2-α [anti-p-eIF2α (Ser 52): sc-101670]; heat shock cognate (HSC) 70 kDa protein [anti-HSP70/HSC70 (H300): sc-33575]; cAMP-dependent transcription factor (ATF)-4 [anti-CREB-2 (C-20): sc-200]; ubiquitin [anti-Ub (FL-76): sc-9133]; homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member-1 [anti-HERP (H-59): sc-98669]; and Werner syndrome protein [anti-WRN (H-300): sc-5629] were from Santa Cruz Biotechnology (Dallas, TX, USA). A mouse mAb against ATF-6α [anti-ATF-6α (F-7): sc-166659] and C/EBP-homologous protein (CHOP) [anti-GADD153 (B-3): sc-7351] was from Santa Cruz Biotechnology; rabbit mAb against inositol-requiring kinase-1α [anti-inositol-requiring enzyme (IRE)-1 (14C10) 3294], protein disulfide isomerase [anti-protein disulfide isomerase (PDI) (C81H6) 3501] from Cell Signaling Technology (Beverly, MA, USA); a rabbit pAb against glucose-related protein-78 (anti-GRP78) from Proteintech (Chicago, IL, USA); a rabbit pAb against calreticulin (anti-calreticulin; ER marker ab39818) from Abcam (Cambridge, United Kingdom); and a mouse mAb against actin (A5441) from MilliporeSigma.

Reactive oxygen species measurements in liver tissues and ER fractions from mouse livers

Liver lysates in RIPA buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM PMSF, and complete protease inhibitor cocktail (Roche Applied Science)] or ER enriched fractions from mouse livers were incubated with 10 µg/ml of the dye 2′-7′ dichlorofluorescein diacetate (DCFA) (MilliporeSigma) for 1 h at 37°C. As controls, RIPA buffer or ER suspension buffer was also incubated with DCFA, and 100 µl (500 µg of liver proteins or 100 µg of ER fractions) of the samples was put into 96-well plates. Fluorescence was measured with a SpectraMax i3x Multi-Mode microplate reader (Molecular Devices, Sunnyvale, CA, USA). The excitation and emission wavelengths used were 485 and 527 nm, respectively.

Protein sulfhydryl measurements in total liver tissues

Weighed liver tissues were homogenized on ice in 20 mM sodium phosphate buffer (pH 7.4) containing 140 mM potassium chloride. Samples were then incubated in lysis buffer for 30 min on a rotary device at 4°C. Protein sulfhydryls were measured by monitoring the reaction between free thiol groups on proteins (240 µg of liver proteins) and the colorimetric reagent 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB; MilliporeSigma) (25, 26).

Impact of 0.01 and 0.4% ascorbate supplementation on Wrn^Δhel/Δhel/Gulo^−/− mouse fertility

Mating between Wrn^{∆hel/∆hel}/Gulo^−/− males and females was unsuccessful in producing offspring when drinking water was supplemented with 0.01% ascorbate. However, when drinking water was supplemented with 0.4% ascorbate at weaning, adult Wrn^{∆hel/∆hel}/Gulo^−/− mice produced offspring. Testes from each genotype were weighed at 4 mo of age and Wrn^{∆hel/∆hel}/Gulo^−/− males exhibited a 25% decrease in size compared with WT, Wrn^{∆hel/∆hel}, and 0.01% ascorbate–treated Gulo^−/− mice (Fig. 2A). Note that the testes’ wet weight did not correlate with the total body weight observed for the different genotypes at 4 mo of age (Pearson’s correlation r = 0.6493; P = 0.24). The addition of 0.4% ascorbate in drinking water re-established the normal (WT) size of Wrn^{∆hel/∆hel}/Gulo^−/− male testes. We performed histologic examination of the testes of Wrn^{∆hel/∆hel}/Gulo^−/− males treated with 0.01% ascorbate and all anatomic structures (spermatids and Leydig and Sertoli cells) looked intact (Fig. 2B). Closer histologic examination of testicular sections at 4 mo of age by TUNEL assay revealed a major increase in overall cell death in Wrn^{∆hel/∆hel}/Gulo^−/− mice compared with WT, Wrn^{∆hel/∆hel}, and 0.01% ascorbate–treated Gulo^−/− mice. Cell death was significantly reduced in Wrn^{∆hel/∆hel}/Gulo^−/− males treated with 0.4% ascorbate (Fig. 2C, D).

Open in a separate window

Figure 2

Effect of ascorbate on testicular morphology of Wrn^Δhel/Δhel/Gulo^−/− mice. A) Testicular wet weight of mice (n = 10) at 4 mo of age. *P < 0.019 vs. all other groups of mice (by post-ANOVA Tukey test). B) H&E staining and TUNEL assay of seminiferous tubules of WT, Wrn^Δhel/Δhel, Gulo^−/−, and Wrn^Δhel/Δhel/Gulo^−/− mice at 4 mo of age. Apoptotic cells are circled in red. C) H&E staining and TUNEL assay of seminiferous tubules of Wrn^Δhel/Δhel/Gulo^−/− mice at 4 mo of age treated with 0.01 or 0.4% ascorbate. Apoptotic cells contain a brown precipitate (arrows). D) Average number of apoptotic cells per histologic section of testes from WT (n = 3), Wrn^Δhel/Δhel (n = 4), Gulo^−/− mice treated with 0.01% ascorbate (n = 4), Wrn^Δhel/Δhel/Gulo^−/− mice treated with 0.01% ascorbate (n = 6), and Wrn^Δhel/Δhel/Gulo^−/− mice treated with 0.4% ascorbate (n = 10) at 4 mo of age. *P < 0.001 vs. all other groups of mice (by Tukey test). Bars in graphs represent sem. Groups of mice are labeled as in Fig. 1B.

Finally, Wrn^{∆hel/∆hel}/Gulo^−/− males treated with 0.01% ascorbate were crossed to WT females but did not produce vaginal plugs underlying other unknown physiologic problems during breeding. Adding 0.4% ascorbate in the drinking water reversed this breeding defect. Wrn^{∆hel/∆hel}/Gulo^−/− females treated with 0.01% ascorbate were crossed to WT males. Although we could detect vaginal plugs, these females did not produce offspring. We performed histologic examination of the ovaries from Wrn^{∆hel/∆hel}/Gulo^−/− adult females treated with 0.01% ascorbate and did not find any obvious anatomic abnormality (data not shown).

Wrn^Δhel/Δhel/Gulo^−/− mice exhibit bone structure anomalies

Osteoporosis of the limbs has been described for patients with WS (27). We thus examined the bone structure of hind limbs in our mice. Upon tissue harvesting for histologic analysis, we found the hind limb femurs of Wrn^{∆hel/∆hel}/Gulo^−/− mice to break more easily than those of WT mice. We performed micro-CT analysis of the femurs of 3-mo-old males from each genotype. Figure 3A shows representative images of the trabecular ROI for each of the genotypes. The bone volume decreased significantly in Gulo^−/− and Wrn^{∆hel/∆hel}/Gulo^−/− mice treated with 0.01% ascorbate compared with WT and Wrn^{∆hel/∆hel} mice. Supplementation of 0.4% ascorbate to Wrn^{∆hel/∆hel}/Gulo^−/− mice reversed bone volume to WT levels (Fig. 3B). The decrease in bone volume was accompanied by a significant decrease in trabecular number with a commensurate increase in trabecular separation in Gulo^−/− and Wrn^{∆hel/∆hel}/Gulo^−/− mice treated with 0.01% ascorbate (Fig. 3C, D). Trabecular thickness increased significantly in Wrn^{∆hel/∆hel} and 0.01% ascorbate–treated Wrn^{∆hel/∆hel}/Gulo^−/− mice (Fig. 3E) compared with WT and 0.01% ascorbate–treated Gulo^−/− mice. All these phenotypes in Wrn^{∆hel/∆hel}/Gulo^−/− mice were reversed by the supplementation of the drinking water with 0.4% ascorbate.

Open in a separate window

Figure 3

Impact of ascorbate on the hind limb bone structures of Wrn^Δhel/Δhel/Gulo^−/− mice at 3 mo of age. A) Representative 3-dimensional reconstructions of the trabecular region of interest. B) Trabecular bone volume per total bone volume (%). C) Trabecular separation. D) Trabecular number. E) Trabecular thickness. F) Representative cross-sectional images of distal femurs from the indicated genotypes are shown. G) Cross-sectional images of femurs from Wrn^Δhel/Δhel/Gulo^−/− mice treated with 0.01% ascorbate since weaning are overlaid onto WT, Wrn^Δhel/Δhel, and Gulo^−/− mutant slices to illustrate bony abnormalities. Note the unusual distribution of trabecular bone (sequestration of bone toward the left quadrants), and misshapen cortical shell (arrow). H) Cross-sectional images of femurs from Wrn^Δhel/Δhel/Gulo^−/− mice treated with 0.4% ascorbate overlaid onto femurs from Wrn^Δhel/Δhel/Gulo^−/− mice treated with 0.01% ascorbate since weaning and femurs from untreated WT mice. Note that the aberrant features of the Wrn^Δhel/Δhel/Gulo^−/− mutant are absent with the 0.4% ascorbate treatment. Labels in each panel: WT, wild type; Wrn, Wrn^Δhel/Δhel mice; Gulo, Gulo^−/− mice; Wrn/Gulo, Wrn^Δhel/Δhel/Gulo^−/− mice. Bars in graphs represent sem. **P > 0.01; ***P > 0.005; ****P > 0.0001 vs. WT; ^###P > 0.005; ^####P > 0.0001 Wrn/Gulo 0.01% asc vs. Wrn/Gulo 0.01% asc (by 1-way ANOVA, with Sidak’s multiple comparisons post hoc test).

Cross-sectional images of Wrn^{∆hel/∆hel}/Gulo^−/− femurs were overlaid onto WT, Wrn^{∆hel/∆hel}, and Gulo^−/− mutant slices to illustrate bony abnormalities (Fig. 3F–H). There was an unusual distribution of trabecular bone (sequestration of bone toward the left quadrants) and misshapen cortical shell in Wrn^{∆hel/∆hel}/Gulo^−/− mice treated with 0.01% ascorbate (Fig. 3G). These aberrant features in the Wrn^{∆hel/∆hel}/Gulo^−/− mice were absent with 0.4% ascorbate (Fig. 3H).

Metabolomic profiling of mutant mice

As ascorbate has an impact not only on the lifespan of Wrn^{∆hel/∆hel}/Gulo^−/− mice but also on their total visceral fat depots, we next examined 203 metabolites (including different lipid species) in the serum of the mouse cohorts described in Fig. 1B using a targeted MS approach. The serum metabolite concentrations in 6 males of each group are shown for all the metabolites in Supplemental Data S1. To identify the metabolites significantly altered between mouse cohorts, a nonparametric Kruskal-Wallis test was applied to the Supplemental Data S1. A summary of the metabolites significantly altered (P < 0.01) in at least 1 of the cohorts is given in a form of a heat map in Fig. 4. Changes were indicated as a z-score value for each metabolite in the serum of each animal. The heat map revealed 104 metabolites that significantly differed between groups (Fig. 4). These included 52 phosphatidylcholines, 6 lysophosphatidylcholines, 21 acylcarnitines, 8 biogenic amines, 5 prostaglandins, 7 sphingomyelins, hexoses, and 4 aa. The dendrogram on top of the heat map indicated that the 4-mo-old Wrn^{∆hel/∆hel} and the WT mice were clustered together. Wrn^{∆hel/∆hel}/Gulo^−/− mice given 0.01% ascorbate in their drinking water formed a distinct group. Wrn^{∆hel/∆hel}/Gulo^−/− mice with 0.4% ascorbate in their drinking water formed other clusters with the old WT mice. We further explored the metabolic profile of each group by employing an unsupervised PCA on all the 203 metabolites (PCA in Fig. 5). The WT and the Wrn^{∆hel/∆hel} mice clustered together in the middle of the upper part of the graph. The Wrn^{∆hel/∆hel}/Gulo^−/− mice treated with 0.01% ascorbate clustered in the lower left quadrant of the graph. The Wrn^{∆hel/∆hel}/Gulo^−/− mice treated with 0.4% ascorbate formed a cluster with the old WT (20-mo-old) mice in the middle of the lower part of the graph. Gulo^−/− mice treated with 0.01% ascorbate clustered in the lower right quadrant of the graph. These results indicate that the overall metabolic profile of 4-mo-old Wrn^{∆hel/∆hel}/Gulo^−/− mice treated with 0.01% ascorbate was very different from the other genotypes. The metabolic profile of Wrn^{∆hel/∆hel}/Gulo^−/− mice treated with 0.4% ascorbate resembled the metabolic profile of 20-mo-old WT mice.

Open in a separate window

Figure 4

Heat map depicting the z score of the log base 10 in serum metabolites concentration (rows) between individual (columns) WT and mutant mice treated with different amounts of ascorbate. Columns are reordered by hierarchical clustering using the genotype and ascorbate treatments. Metabolites are grouped according to chemical classification. Young (4 mo) WT mice are labeled WT.i (where i = 1–6). Old (20 mo) WT mice are labeled Old WT.i. Wrn^Δhel/Δhel mice are labeled Wrn.i. Gulo^−/− mice treated with 0.01% ascorbate are labeled Gulo 0.01% ascorbate.i. Wrn^Δhel/Δhel/Gulo^−/− mice treated with 0.01% ascorbate are labeled Wrn/Gulo 0.01% ascorbate.i. Wrn^Δhel/Δhel/Gulo^−/− mice treated with 0.4% ascorbate are labeled Wrn/Gulo 0.4% ascorbate.i. The Euclidian distance and complete agglomerative methods were used for clustering.

Open in a separate window

Figure 5

PCA graph demonstrating the differentiation effect of ascorbate and genotype on the metabolomic profiles of mice. X axis: principal component 1; Y axis: principal component 2.

Inflammatory and cardiometabolic profiles of mutant mice

As metabolite disturbances can lead to an inflammatory response or changes in cardiovascular risk factors (22), we measured the levels of 42 cytokines/chemokines, metabolic hormones, and cardiovascular risk factors in the serum of our different mouse cohorts. Serum cytokine concentrations in 8 males of each group are shown in the Supplemental Table S1. To identify the cytokines significantly altered in either mouse groups, a nonparametric Kruskal-Wallis test was applied to the data. A summary of the cytokines significantly altered in at least one of the groups is given in a form of a heat map in Fig. 6A. Changes were indicated as a z-score value for each cytokine in the serum of each animal. The dendrogram on top of the heat map indicated 2 major leaves. The young WT and the Wrn^{∆hel/∆hel} mice tended to cluster together. All the Wrn^{∆hel/∆hel}/Gulo^−/− mice treated with 0.01% ascorbate clustered together. The old WT, the Gulo^−/− mice treated with 0.01% ascorbate, and the Wrn^{∆hel/∆hel}/Gulo^−/− mice treated with 0.4% ascorbate were distributed unequally within the 2 major leaves of the dendrogram. To get a better picture for each group of mice, we performed an unsupervised PCA using data from all the cytokines (Fig. 6B). The PCA approach showed that all Wrn^{∆hel/∆hel}/Gulo^−/− mice treated with 0.01% ascorbate were clustered in the lower left quadrant of the graph. The young (4 mo) WT mice were mainly clustered in the right quadrants of the graph with the Wrn^{∆hel/∆hel} mice. The old (20 mo) WT mice were distributed in the upper quadrants and showed a large variability between individuals. The Wrn^{∆hel/∆hel}/Gulo^−/− mice treated with 0.4% ascorbate were also clustered in the upper quadrants of the graph and showed less variability between individuals. The Gulo^−/− mice treated with 0.01% ascorbate were distributed in all 4 quadrants of the graph. To summarize the major finding of this PCA data, the serum cytokine profile of Wrn^{∆hel/∆hel}/Gulo^−/− mice treated with 0.01% ascorbate was unique in our cohorts of mice and did not overlap with any other mouse groups.

Open in a separate window

Figure 6

Impact of ascorbate and genotype on the cytokinome of mice. A) Heat map depicting the z score of log base 10 in serum cytokine concentrations (rows) between individual (columns) WT and mutant mice treated with different amounts of ascorbate. Significantly altered cytokines with a value of P < 0.01 are shown. Columns are reordered by hierarchical clustering by using the genotype and ascorbate treatments. The Euclidian distance and complete agglomerative methods were used for clustering. B) PCA graph demonstrating the differentiation effect of ascorbate and genotype on the cytokinome profiles of mice. The labels and symbols on the heat map and the PCA graph are identical to Figs. 4A and ​and5,5, respectively.

The heat map revealed 9 cytokines or factors that significantly differed between groups (Fig. 6A). Graphs representing the levels of secreted factors in each group are shown in Supplemental Fig. S2. The most marked findings were a decreased in IL-12 (p70), G-CSF, MIP-1α, soluble E-selectin, and leptin in Wrn^{∆hel/∆hel}/Gulo^−/− mice treated with 0.01% ascorbate than in WT mice. PAI-1 increased in Wrn^{∆hel/∆hel}/Gulo^−/− mice treated with 0.01% ascorbate compared with WT mice. Treatment of Wrn^{∆hel/∆hel}/Gulo^−/− mice with 0.4% ascorbate reversed the IL-12 (p70), G-CSF, MIP-1α, leptin, and PAI-1 phenotypes (Supplemental Fig. S2).

Ratios of specific metabolites indicate various pathway anomalies in Wrn^Δhel/Δhel/Gulo^−/− mice

Figure 7 provides metabolite ratios that showed a significant difference between groups of mice. Methionine-sulfoxide (Met-SO) can be generated via a 2-electron–dependent mechanism and the Met-SO:Met ratio in the serum can be regarded as a marker of oxidative stress (28). As indicated in Fig. 7A, the Met-SO:Met ratio was significantly higher in Wrn^{∆hel/∆hel}/Gulo^−/− mice treated with 0.01% ascorbate than in WT mice, indicating an increase oxidative stress in these mice. Wrn^{∆hel/∆hel}/Gulo^−/− mice given supplemental 0.4% ascorbate only slightly, but not significantly, decreased this ratio.

Open in a separate window

Figure 7

Ratios of metabolite significantly altered in Wrn^Δhel/Δhel/Gulo^−/− mice. A) Met-SO:Met ratio. *P = 0.046 vs. WT mice (by post-ANOVA Tukey test). B) Free carnitine (C0):acylcarnitine ratio. *P < 0.012 vs. all other groups of mice and ^†P = 0.001 vs. Wrn^Δhel/Δhel/Gulo^−/− mice given 0.01% supplemental ascorbate (by Tukey test). C) Saturated:unsaturated phosphatidylcholines ratio. Groups of mice are labeled as in Fig. 1B. *P = 0.021 vs. WT mice and ^†P = 0.027 vs. Wrn^Δhel/Δhel/Gulo^−/− mice given 0.01% supplemental ascorbate (by unpaired Student t tests). Bars represent sem.

Acylcarnitines are important molecules for mitochondrial function and lipid metabolism. As indicated in Fig. 4, 21 acylcarnitine species were significantly different among our different cohorts of mice. We examined the ratio of free carnitine over the sum of these 21 acylcarnitine species. As indicated in Fig. 7B, this ratio increased significantly in Wrn^{∆hel/∆hel}/Gulo^−/− mice treated with 0.01% ascorbate compared with all the other groups of mice. Wrn^{∆hel/∆hel}/Gulo^−/− mice given supplemental 0.4% ascorbate significantly reversed this ratio to WT. We also examined the ratio of saturated to unsaturated phosphatidylcholine species. We pooled the serum concentrations of saturated phosphatidylcholines and of unsaturated phosphatidylcholines to calculate a ratio. [For example, the concentrations of PC diacyl (PC aa) C26:0, PC aa C36:0, PC ae C36:0, PC ae C38:0, PC ae C40:0, and PC ae C42:0 that are significantly changed in our mouse cohorts based on Fig. 4 were summed to obtain the numerator of the ratio]. As indicated in Fig. 7C, the ratio of saturated:unsaturated phosphatidylcholines was not significantly different between groups, according to results of ANOVA. However, if we compared only the 0.01% ascorbate–treated Wrn^{∆hel/∆hel}/Gulo^−/− mice with the WT mice, an unpaired Student t test showed a significant difference. Wrn^{∆hel/∆hel}/Gulo^−/− mice given supplemental 0.4% ascorbate significantly reversed this ratio to WT levels.

Oxidative stress in the liver tissue of Wrn^Δhel/Δhel/Gulo^−/− mice

We had found that the liver of Wrn^{∆hel/∆hel} and Gulo^−/− mice treated with low amounts of ascorbate (<0.01% in drinking water) exhibit an oxidative stress in the ER (11, 21, 29). The liver plays a pivotal role in nutrient, hormone, lipid, and metabolic waste product processing, thereby maintaining body homeostasis (30). The liver ER is the normal location of ascorbate synthesis in mice (31). Furthermore, the active catalytic site of the membrane-associated Gulo enzyme is located in the ER lumen (32). We did not detect hepatic steatosis or significant difference in fat content between Wrn^{∆hel/∆hel}/Gulo^−/− mice treated with 0.01% ascorbate compared with age-matched (4-mo-old) mice of the other groups (data not shown). However, we observed a significant increase in the mitochondrial DNA mutation rate in Wrn^{∆hel/∆hel}/Gulo^−/− mice treated with 0.01% ascorbate compared with age-matched (4-mo-old) WT mice (Table 1). It was comparable to older (20-mo-old) WT mice. Treatment of Wrn^{∆hel/∆hel}/Gulo^−/− mice with 0.4% ascorbate in drinking water decreased the mutation rate to young WT level (Table 1). The mutation rate in mitochondrial DNA of Wrn^{∆hel/∆hel} and Gulo^−/− mice treated with 0.01% ascorbate was not significantly different from that of age-matched WT mice. We next focused on reactive oxygen species (ROS) levels in the liver of Wrn^{∆hel/∆hel}/Gulo^−/− mice and age-matched WT mice. We quantified ROS levels in total liver tissues of WT and Wrn^{∆hel/∆hel}/Gulo^−/− mice treated with 0.01 or 0.4% ascorbate. ROS levels in total liver lysate from Wrn^{∆hel/∆hel}/Gulo^−/− mice treated with 0.01% ascorbate increased significantly (∼10%) compared with age-matched WT mice (Fig. 8A). Treatment of Wrn^{∆hel/∆hel}/Gulo^−/− mice with 0.4% ascorbate significantly decreased total ROS in the liver by ∼16%.

Open in a separate window

Figure 8

ROS levels in total liver extract and the ER enriched fraction of 4-mo-old Wrn^Δhel/Δhel/Gulo^−/− mice. A) ROS levels in total liver extract. *P < 0.05 vs. WT mice and ^†P = 0.01 vs. Wrn^Δhel/Δhel/Gulo^−/− mice given 0.01% supplemental ascorbate, by Tukey test. B) ROS levels in the ER enriched fraction of WT and Wrn^Δhel/Δhel/Gulo^−/− mice given drinking water supplemented with 0.01 or 0.4% ascorbate. ^†P < 0.01 vs. WT mice; ^‡P = 0.01 vs. Wrn^Δhel/Δhel/Gulo^−/− mice given 0.01% supplemental ascorbate (by Tukey test). ROS was detected with DCFA and is shown as relative fluorescence units (RFU). Bars in each graph represent sem.

We previously found that Wrn^{∆hel/∆hel} mice exhibited a significant increase (∼15%) in ROS levels in the ER fraction of their liver compared with WT mice (29). We next examined ROS levels in the ER fraction of liver from our Wrn^{∆hel/∆hel}/Gulo^−/− mice. ROS level in the ER fraction of Wrn^{∆hel/∆hel}/Gulo^−/− mice treated with 0.01% ascorbate increased significantly (2.2-fold) compared with that in age-matched WT mice (Fig. 8B). Treatment of Wrn^{∆hel/∆hel}/Gulo^−/− mice with 0.4% ascorbate significantly decreased ER ROS to WT levels. These results indicate that 4-mo-old Wrn^{∆hel/∆hel}/Gulo^−/− mice treated with 0.01% ascorbate exhibit greater oxidative ER stress levels than Wrn^{∆hel/∆hel} mice (29). We thus examined different ER stress markers in our WT and Wrn^{∆hel/∆hel}/Gulo^−/− mice. PERK and IRE1α were first analyzed by Western blot analysis in ER-enriched fractions (33). Calreticulin, a protein associated with the ER, was used as a loading control. The PERK protein level increased significantly in the liver ER of Wrn^{∆hel/∆hel}/Gulo^−/− mice treated with 0.01% ascorbate compared with WT mice (Fig. 9A, B). Treatment of Wrn^{∆hel/∆hel}/Gulo^−/− mice with 0.4% ascorbate significantly decreased PERK to the WT level (Fig. 9B). IRE1α protein increased significantly in the liver ER enriched fraction of Wrn^{∆hel/∆hel}/Gulo^−/− mice treated with 0.01 and 0.4% ascorbate compared with age-matched WT mice (Fig. 9A, C). There was no significant difference between the effects of the 0.01 and 0.4% ascorbate treatments in Wrn^Δhel/Δhel/Gulo^−/− mice.

Open in a separate window

Figure 9

Activation of ER stress protein markers in the liver of 4-mo-old Wrn^Δhel/Δhel/Gulo^−/− mice. A) Western blots for the indicated proteins in 4-mo-old WT and Wrn^Δhel/Δhel/Gulo^−/− mice given supplemental 0.01 or 0.4% ascorbate. B) Ratio of total PERK signal over calreticulin signal in ER enriched fractions from the Western blots. **P < 0.05 compared with WT and Wrn^Δhel/Δhel/Gulo^−/− mice treated with 0.4% ascorbate (by Tukey post-ANOVA test). C) Ratio of total IRE1α signal over calreticulin signal in ER enriched fractions from the Western blots. *P < 0.05 compared with WT mice (by Tukey post-ANOVA test). D) Ratio of CHOP signal over β-actin signal in total liver lysates from the Western blots *P < 0.05 compared with WT mice (by Tukey post-ANOVA test). E) Sum of the cleaved and full-length ATF6 signals in total liver extracts. F) Ratio of the cleaved over total (cleaved and full length) ATF6 signals in total liver extracts. **P < 0.01 compared with WT mice; ^††P < 0.01 compared with WT and Wrn^Δhel/Δhel/Gulo^−/− mice treated with 0.01% ascorbate (by Tukey post-ANOVA test). G) Signal of PDI signal over calreticulin signal in ER enriched fractions from the Western blots. *P < 0.05 compared with WT mice (by Tukey post-ANOVA test). H) Ratio of mutant Wrn^Δhel signal over calreticulin signal in ER enriched fractions from the Western blots. I) Ratio of HERPUD1 signal over β-actin signal in total liver lysates from the Western blots. ^††P < 0.01 compared with WT and Wrn^Δhel/Δhel/Gulo^−/− mice treated with 0.4% ascorbate (by Tukey post-ANOVA test). Bars in all histograms represent SEM of 4 mice.

The PERK and IRE1α pathways are known to activate the transcription of CHOP (34). Since CHOP is not found in the ER, proteins from total liver lysates were analyzed by Western blot. β-actin was used as a loading control. There was only a tendency for CHOP to be increased in Wrn^{∆hel/∆hel}/Gulo^−/− mice treated with 0.01% ascorbate compared with age-matched WT mice. By contrast, CHOP protein level increased significantly (∼2-fold) in Wrn^{∆hel/∆hel}/Gulo^−/− mice treated with 0.4% ascorbate compared with age-matched WT mice (Fig. 9A, D).

Upon ER stress, the ER membrane-associated ATF6 protein is cleaved into a subunit that translocates into the nucleus to activate the transcription of target genes (33). We thus examined the levels of full length and cleaved ATF6 in total liver extracts (Fig. 9A, E, F). There was only a tendency for the sum of full-length and cleaved ATF6 signals to be higher in the liver of Wrn^Δhel/Δhel/Gulo^−/− mice treated with 0.01% ascorbate than in the liver of WT and Wrn^Δhel/Δhel/Gulo^−/− mice treated with 0.4% ascorbate (Fig. 9E). However, when we calculated the ratio of cleaved ATF6 over the sum of full-length and cleaved signals, the levels of cleaved ATF6 was higher in the Wrn^Δhel/Δhel/Gulo^−/− mice treated with 0.01% ascorbate than in WT mice. The levels of cleaved ATF6 were even higher in the liver of Wrn^Δhel/Δhel/Gulo^−/− mice treated with 0.4% ascorbate (Fig. 9F).

We have reported that the mutant Wrn protein (Wrn^Δhel) is mislocalized in the cytoplasm of Wrn^{∆hel/∆hel} mice (11). Further microscopy analyses indicated a colocalization of Wrn^Δhel proteins with the PDI. We examined the levels of PDI in our mouse cohort. As indicated in Fig. 9A, G, the levels of PDI increased significantly in Wrn^{∆hel/∆hel}/Gulo^−/− mice treated with 0.01 and 0.4% compared with untreated WT mice. This PDI increase in the liver ER fraction of Wrn^{∆hel/∆hel}/Gulo^−/− mice corresponded to the presence of Wrn^Δhel protein in this ER fraction (Fig. 9A, H). No normal Wrn protein was detected in the ER fraction of WT mice. As previously found with Wrn^{∆hel/∆hel} mice (29), the treatment of Wrn^{∆hel/∆hel}/Gulo^−/− mice with 0.4% ascorbate in drinking water did not decrease the expression level of the mutant Wrn^Δhel protein (Fig. 9H).

Total eIF2α, ATF4, glucose-regulated protein–78, and HSC70 protein levels were not different between WT and Wrn^Δhel/Δhel/Gulo^−/− mice treated with different concentrations of ascorbate in drinking water (Supplemental Fig. S3). Phosphorylation of eIF2α was not significantly different between WT and ascorbate treated Wrn^Δhel/Δhel/Gulo^−/− mice (Supplemental Fig. S3).

We also analyzed ATF4, CHOP, HSC70, and full-length and cleaved ATF6 protein levels in age-matched WT, Wrn^{∆hel/∆hel}, and 0.01% ascorbate–treated Gulo^−/− mice, because it had not been examined in our other studies (11, 21, 29). There was no significant difference in CHOP, HSC70, or full-length and cleaved ATF6 protein levels between these groups of mice, according to ANOVA results (Supplemental Fig. S4). ATF4 was lower in Wrn^{∆hel/∆hel}, and 0.01% ascorbate–treated Gulo^−/− mice compared with WT mice, but it was not significant according to ANOVA.

Body weight and metabolic profile of Wrn^Δhel/Δhel/Gulo^−/− mice

A common feature among patients with WS is their short stature (27). Wrn^{∆hel/∆hel}/Gulo^−/− mice treated with 0.01% ascorbate were smaller than all the other groups of mice, including untreated Wrn^Δhel/Δhel and 0.01% ascorbate–treated Gulo^−/− mice (Fig. 1D). However, contrary to what has been described for patients with WS (41), visceral fat weight was significantly lower in Wrn^{∆hel/∆hel}/Gulo^−/− mice treated with 0.01% ascorbate (Supplemental Fig. S1). Concomitantly, our targeted MS analyses of serum metabolites revealed a decrease in the levels of several lysophosphatidylcholine and phosphatidylcholine lipid types and hexose molecules in Wrn^Δhel/Δhel/Gulo^−/− mice treated with 0.01% ascorbate compared with levels in the other groups of mice. The serum leptin level in Wrn^{∆hel/∆hel}/Gulo^−/− mice treated with 0.01% ascorbate was also severely lower than in the other groups of mice (Supplemental Fig. S2). Leptin is an important hormone that regulates food intake according to body fat mass, and its secretion correlates with the size of adipose mass in mice (42–44). Wrn^{∆hel/∆hel}/Gulo^−/− mice treated with 0.01% ascorbate ate less than the other groups of mice. Upon 0.4% ascorbate treatment, serum leptin level significantly increased and reached serum concentrations above 7000 pg/ml in Wrn^{∆hel/∆hel}/Gulo^−/− mice. A study on serum leptin levels in Gulo^−/− mice indicated no significant difference between the 0.01 and 0.4% treatments with concentrations reaching 4000–6000 pg/ml (21). Leptin serum levels are ∼3000–4000 pg/ml in age-matched WT mice (this study and reference 21). In addition, Gulo^−/− mice treated with 0.4% ascorbate do not drink or eat more than Gulo^−/− mice treated with 0.01% ascorbate (21). These results indicate that Wrn^{∆hel/∆hel}/Gulo^−/− mice treated with 0.01 or 0.4% ascorbate behave very differently from Gulo^−/− mice.

In addition to lower levels of several lipid types in the serum, Wrn^{∆hel/∆hel}/Gulo^−/− mice treated with 0.01% ascorbate exhibited a significant increase in the ratio of serum saturated and unsaturated lipids and the ratio of free carnitine:acylcarnitine compared with WT mice (Fig. 7B, C). Of note, dyslipidemia has been associated with 85% of patients with genetically confirmed WS (40). A decrease in unsaturated lipid species may lead to perturbation of cellular membrane fluidity with age (45). An abnormal increase in saturated lipids, in return, may contribute to the development of inflammatory and oxidative stress (46). Carnitine is necessary for the transport of fatty acids across the mitochondrial membrane for their catabolism during cycles of β-oxidation (47). It is possible that the lower levels of several acylcarnitine molecules (Fig. 4) are imputable to the overall decrease of several lipid types in the serum of Wrn^{∆hel/∆hel}/Gulo^−/− mice treated with 0.01% ascorbate. This phenotype was not observed in all the other groups of mice.

As published for the Gulo^−/− and Wrn^Δhel/Δhel mice (21, 29), addition of 0.4% ascorbate to the drinking water also significantly improved the altered metabolic phenotypes observed in Wrn^Δhel/Δhel/Gulo^−/− mice. The PCA clearly indicated that the metabolic profile of 4-mo-old Wrn^{∆hel/∆hel}/Gulo^−/− mice treated with 0.01% ascorbate clustered together and did not overlap any other mouse group (Fig. 5). The metabolic profile of 4-mo-old Wrn^{∆hel/∆hel}/Gulo^−/− mice treated with 0.4% ascorbate did not overlap the profile of 4-mo-old WT mice, but it did overlap the profile of 20-mo-old WT mice (Fig. 5). The reason for this overlap in metabolic profiles is unknown. We observed that Wrn^{∆hel/∆hel}/Gulo^−/− mice treated with 0.4% ascorbate gained weight more rapidly than age-matched WT mice during the first 10–16 wk of life (Fig. 1E), which certainly affected their overall metabolic profile. In addition, the ratio of visceral fat weight to total body weight was similar between Wrn^{∆hel/∆hel}/Gulo^−/− mice treated with 0.4% ascorbate and untreated 20-mo-old WT mice (Supplemental Fig. S1). Finally, the PCA indicated very little overlap between Gulo^−/− and Wrn^{∆hel/∆hel}/Gulo^−/− mice treated with 0.01% ascorbate. A recently published PCA showed that the metabolic profile of Gulo^−/− mice treated with 0.4% ascorbate was similar to that of 4-mo-old Gulo^−/− mice treated with 0.01% ascorbate and did not overlap the profile of age-matched untreated WT mice (21) and thus would not overlap that of 20-mo-old WT mice. Future longitudinal studies on the metabolic profiles of these mice will provide a more complete picture of what is happening to these different mouse cohorts with age.

Cytokinome profile of Wrn^Δhel/Δhel/Gulo^−/− mice

Metabolite disturbances can lead to an inflammatory response or changes in cardiovascular risk factors (22), and we therefore measured the levels of 42 cytokines/chemokines, hormones, and cardiovascular risk factors in the serum of our different mouse cohorts. The PCA clearly indicated that the profile of 4-mo-old Wrn^{∆hel/∆hel}/Gulo^−/− mice treated with 0.01% ascorbate was clustered and did not significantly overlap that of the other mouse groups (Fig. 6B). In contrast to the metabolic profile, the cytokinome profile of 4-mo-old Wrn^{∆hel/∆hel}/Gulo^−/− mice treated with 0.4% ascorbate was much closer to the profile of 4-mo-old WT mice than that of 20-mo-old WT mice.

When we examined each analyte, the most marked findings were a significant decrease in IL-12 (p70), G-CSF, MIP-1α, and E-selectin, as well as a significant increase in PAI-1 in Wrn^{∆hel/∆hel}/Gulo^−/− mice treated with 0.01% ascorbate compared with WT mice. IL-12(p70) promotes the differentiation of type-1 helper T (Th1) cells, thereby supporting cellular immunity (48). It is naturally produced by dendritic cells, macrophages, and neutrophils. G-CSFs and M-CSFs are hematopoietic cytokines that stimulate the bone marrow to produce granulocytes and macrophages for release into the bloodstream (49–51). G-CSF is produced by the endothelium, macrophages, and several other immune cells. M-CSF is produced in the bone marrow and by endothelial cells, fibroblasts, osteoblasts, and smooth muscle cells (52). MIP-1α is produced by macrophages and acts as a chemoattractant to a variety of cells, including monocytes, T cells, B cells, and eosinophils (53). E-selectin plays an important role in recruiting leukocytes to sites of vascular injury and allows leukocytes to tightly bind to the endothelial surface (54). The observed decrease of these cytokines in Wrn^{∆hel/∆hel}/Gulo^−/− mice treated with 0.01% ascorbate may underscore a dysfunction of part of the immune system which may be less efficient to respond to tissue damage. Notably, intracellular ascorbate concentration is important for the activity of macrophages (55). A thorough analysis of the immune cells from these mice is necessary to determine which biologic processes are affected by the accumulation of an abnormal Wrn mutant protein in such cell types.

PAI-1 is a serine protease inhibitor that functions as the principal inhibitor of tissue plasminogen and hence the physiologic breakdown of blood clots (56). It has been reported that a depletion of human WRN protein with small interference RNA molecules leads to an overexpression of PAI-1 in human fibroblasts (57). Similarly, we have found a significant increase in serum PAI-1 level in Wrn^Δhel/Δhel mice (29). Moreover, the serum concentration of ascorbate seems to regulate the expression of PAI-1. Indeed, we have reported that Gulo^−/− mice without ascorbate treatment exhibit high serum levels of PAI-1 that is decreased upon ascorbate treatment (21). In the present study, we also observed an increase in serum PAI-1 in Gulo^−/− mice treated with 0.01% ascorbate compared with WT mice. More important, we observed an additive effect of the Wrn and Gulo mutations as PAI-1 levels were even higher in Wrn^Δhel/Δhel/Gulo^−/− mice treated with 0.01% ascorbate than in WT mice. This phenotype was completely reversed by 0.4% ascorbate treatment. PAI-1 is present in increased levels in various disease states such as cardiovascular diseases (during the development of vessel wall damage) (58) and metabolic syndrome (59). In inflammatory conditions, PAI-1 is secreted by endothelial cells and appears to play a significant role in the progression to fibrosis (60, 61). We found in other work that we can detect an increase in this cardiovascular risk factor in young Wrn^Δhel/Δhel mice before they exhibit cardiac fibrosis and valvular aortic stenosis (13, 37).

This work was supported by funding from the Canadian Institutes of Health Research (to M.L. and A.M.), and by U.S. National Institutes of Health (NIH) National Institute on Aging Grant R01AG028873 (to R.J.P.). L.A. is a scholar from the Fondation du Centre Hospitalier Universitaire de Québec. The authors declare no conflicts of interest.