Vitamin C modulates the metabolic and cytokine profiles, alleviates hepatic endoplasmic reticulum stress, and increases the life span of Gulo^−/− mice

Impact of vitamin C (ascorbate) on life span and body weight of Gulo^−/− mice

We first investigated the effect of ascorbate supplementation on the median life span of Gulo^−/− mice. Generally, four to six weeks after removal of ascorbate in drinking water, Gulo^−/− mice lost more than 20% of their total body weight, looked moribund, and had to be euthanized. In contrast, increasing ascorbate concentration in drinking water improved the survival of these mutant mice in a dose dependent manner (Figure 1A). Gulo^−/− mice supplemented with 0.005% of ascorbate (w/v) at weaning rapidly became sick and moribund within four weeks of treatment (data not shown). Therefore this concentration of ascorbate was not used for further analysis. The median life span of Gulo^−/− mice treated with 0.01% ascorbate was 8 ½ months and the maximum life span was 16 months. All of these animals lost more than 20% of their total body weight, became moribund at some point during this 16 months period and had to be euthanized. Supplementation of ascorbate up to 0.4% (w/v) significantly increased the median life span to 23 months, which was close to the median life span of wild type mice (23.8 months). The maximum life span Gulo^−/− mice treated with 0.4% ascorbate was ∼32 months compared to ∼30 months for untreated wild type animals. The illnesses associated with old Gulo^−/− mice treated with 0.4% vitamin C included either hepatocarcinomas or myeloid leukemias. Several mice were also found dead in the cages in the morning with no sign of distress in the previous days (undetermined cause of death due to tissue autolysis). Overall, these results indicated that the life span of Gulo^−/− mice increased with ascorbate concentration in drinking water. The amount of ascorbate required to achieve a wild type normal life span (log rank test P > 0.05) was reached with 0.4% ascorbate in drinking water (Figure 1A). As we have previously found that 0.4% ascorbate did not increase the life span of wild type animals [18], we did not pursue analyses with higher concentrations of ascorbate in the drinking water of Gulo^−/− mice.

Figure 1. Impact of vitamin C (ascorbate) on the life span and the body weight of Gulo^−/− mice. (A) Percentage of disease-free animals with age. The number of animals in each group is indicated. Gulo^−/− mice were treated with the indicated concentration of ascorbate in drinking water from weaning until they had to be euthanized due to illness. Wild type mice were not treated with ascorbate. (B) Serum ascorbate levels in each indicated cohort (n=5 males at four months of age). (Tukey post ANOVA test **P < 0.01 compared to wild type and 0.4% treated Gulo^−/− mice). (C) Histogram showing the mean total body weight of each mouse cohort (n=6 males) at four month of age. (Tukey post ANOVA test **P < 0.01 compared to all other groups). (D) Histogram showing a significant visceral fat weight loss in ascorbate deficient Gulo^−/− mice (n=6 males) at four month of age. (Tukey post ANOVA test *P < 0.05 compared to all other groups). (B-D) Bars in all histograms represent SEM. One cohort of Gulo^−/− mice was treated with 0.01% of ascorbate (w/v) in drinking water from weaning to four months of age. A second cohort of Gulo^−/− mice was treated with 0.4% of ascorbate from weaning to four months of age. In a third cohort of Gulo^−/− mice, ascorbate was omitted from the drinking water at the age of three months for four weeks. Wild type mice were not treated with ascorbate.

Next we measured the amount of ascorbate in the serum of mice from each treatment group (n=5). As indicated in Figure 1B, minimal ascorbate could be detected in the serum of ascorbate-depleted Gulo^−/− mice (0% of ascorbate in drinking water for one month). There was a 100-fold significant increase in serum ascorbate in Gulo^−/− mice treated with 0.01% ascorbate compared to Gulo^−/− mice that had been depleted of ascorbate for one month (P < 0.01). Furthermore, there was a 5.4-fold increase when Gulo^−/− mice were supplemented with 0.4% ascorbate compared to 0.01% ascorbate treated mice (P < 0.01). Gulo^−/− mice treated with 0.4% ascorbate had similar levels of serum ascorbate compared to wild type untreated animals (Figure 1B).

There was a significant effect of ascorbate supplementation on body weight in the Gulo^−/− animals. As indicated in Figure 1C, Gulo^−/− mice without ascorbate treatment were significantly leaner than Gulo^−/− mice treated with 0.01% or 0.4% ascorbate. The mean total body weight of Gulo^−/− mice treated with 0.01% or 0.4% ascorbate at four months of age were not significantly different from age-matched wild type animals. Analysis of the four-month old animals revealed that Gulo^−/− mice depleted of ascorbate for one month had significantly less visceral fat in proportion to total body weight compared to all Gulo^−/− mice treated with ascorbate (Figure 1D). The weight of visceral fat in ascorbate treated Gulo^−/− mice (0.01% or 0.4% in drinking water) was not significantly different from untreated wild type animals (Figure 1D).

The spleen, kidneys, heart, and liver were also weighed (Supplementary Figure S1). The spleen tended to be larger in all the Gulo^−/− mice compared to age-matched four-month old wild type animals. However, only the Gulo^−/− mice treated with 0.01% ascorbate showed a significant difference compared to wild type animals (Supplementary Figure S1A). The weight of the kidneys in Gulo^−/− mice depleted of ascorbate was increased (∼1.2-fold) compared to age-matched wild type but was significantly decreased in Gulo^−/− mutant mice treated with 0.01% or 0.4% ascorbate (Supplementary Figure S1B). The weights of the cardiac and hepatic tissues were not significantly different between cohorts (Supplementary Figure S1C and D).

Finally, Gulo^−/− mice depleted of ascorbate for one month ate and drank less than wild type (based on student t-test: P < 0.05; supplementary Figure S1E and F).

Effect of ascorbate on the metabolic profile of Gulo^−/− mice

We next measured 203 metabolites employing a targeted mass spectrometry approach in the serum of Gulo^−/− mice treated with different amounts of ascorbate at four months of age. (Full biochemical names are provided in Supplementary Table S1). Untreated age-matched wild type animals were used as the reference control. Serum metabolite concentrations in six animals of each group are shown in the supplementary Table S2. To identify the metabolites significantly altered in either wild type, Gulo^−/− mice treated with 0%, 0.01%, or 0.4% ascorbate, a nonparametric Kruskal-Wallis test was applied to the data. A summary of the metabolites significantly altered (P < 0.01) in at least one of the groups is given in a form of a heatmap in Figure 2. Changes were indicated as a Z-score value for each metabolite in the serum of each animal. The heatmap revealed 61 metabolites that significantly differed between groups (Figure 2). These included 39 phosphatidylcholines, seven lysophosphatidylcholines, two acylcarnitines, five biogenic amines, three prosta-glandins, three sphingomyelins, hexoses, and the amino acid glycine. The dendrogram on top of the heatmap indicated that the Gulo^−/− mice depleted of ascorbate clustered together except for one mouse (mouse identified as GNV.3). The wild type animals also formed a cluster. Gulo^−/− mice supplemented with either 0.01% or 0.4% ascorbate in their drinking water formed another cluster. We further explored the metabolic profile of each group by employing principal component analysis (PCA in Figure 3). The PCA approach showed a good clustering of Gulo^−/− mice supplemented with either 0.01% or 0.4% ascorbate in the lower right quadrant of the graph. Five Gulo^−/− mice depleted of ascorbate were found in the lower left quadrant (and one animal in the lower right quadrant). All wild type animals were found in the upper quadrants of the graph and did not overlap with any mouse of the Gulo^−/− genotype. The PCA analysis indicated that although there was a larger variability within the ascorbate depleted Gulo^−/− mouse group, they were localized at great distance from the wild type mice in the graph (Figure 3). The clustering of Gulo^−/− mice supplemented with either 0.01% or 0.4% ascorbate suggested similar metabolic profiles at four months of age. Such profiles were different from both wild type and ascorbate depleted Gulo^−/− mice. Finally, even though Gulo^−/− mice supplemented with 0.4% ascorbate had a life span and serum ascorbate levels similar to wild type animals (Figure 1A and B), their metabolic profile did not significantly overlap with any wild type individual (Figure 3).

Figure 2. Heatmap depicting the Z-score of the log base ten in serum metabolites concentration (rows) between individual (columns) wild type and Gulo^−/− mice treated with different amounts of ascorbate. Columns are reordered by hierarchical clustering using the genotype and ascorbate treatments to label the three leaves. Metabolites are grouped according to chemical classification. Wild type animals are labeled WT.1 to WT.6. Gulo^−/− mice treated with 0.4% ascorbate in drinking water are labeled GV.1 to GV.6. Gulo^−/− mice treated with 0.01% ascorbate are labeled G.1 to G.6. Gulo^−/− mice treated with 0% ascorbate in drinking water from the age of three to four months are labeled GNV.1 to GNV.6. The Euclidean distance and complete agglomerative methods were used for clustering.

Figure 3. Principal component analysis (PCA) graph demonstrating the differentiation effect of ascorbate on the metabolomic profiles of wild type and Gulo^−/− mice treated with different amounts of ascorbate. X axis: Principal component 1, Y axis: Principal component 2. Wild type animals are labeled WT.1 to WT.6. Gulo^−/− mice treated with 0.4% ascorbate in drinking water are labeled GV.1 to GV.6. Gulo^−/− mice treated with 0.01% ascorbate are labeled G.1 to G.6. Gulo^−/− mice treated with 0% ascorbate in drinking water from the age of three to four months are labeled GNV.1 to GNV.6.

Effect of ascorbate on specific metabolites in Gulo^−/− mice

The levels of phosphatidylcholines, lysophosphatidyl-cholines, sphingomyelins, and hexoses were generally lower in the ascorbate deficient Gulo^−/− mice compared to wild type mice. Supplementation with ascorbate increased these levels equal to or above the wild type levels (Figure 2 and supplementary Table S2). The 15S-HETE (15(S)-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid) and 6-keto-PGF1a (6-keto prostaglandin F1α) were significantly up regulated in the serum of ascorbate deficient Gulo^−/− mice compared to wild type animals (Figure 2 and supplementary Table S2). Supplementation of drinking water with 0.4% ascorbate decreased both serum 15S-HETE and 6-keto-PGF1a to wild type levels in Gulo^−/− mice (Figure 2 and supplementary Table S2). LTB4 (leukotriene B4) was lower in ascorbate deficient Gulo^−/− mice compared to wild type animals. Supplementation of ascorbate in drinking water decreased serum LTB4 even further in Gulo^−/− mice (Figure 2 and supplementary Table S2). Four biogenic amines (spermine, spermidine, histamine, and alpha-aminoadipic acid) were significantly increased in ascorbate deficient Gulo^−/− mice compared to wild type animals (Figure 2 and supplementary Table S2). Histamine, spermine and its precursor spermidine were decreased to normal wild type levels when Gulo^−/− mice were treated with 0.4% ascorbate (Figure 2 and supplementary Table S2). In contrast, the α-aminoadipic acid (alpha-AAA) remained elevated in Gulo^−/− mice compared to wild type animals in both the absence and presence of ascorbate in drinking water (Figure 2 and supplementary Table S2). Finally, the amino acid glycine was significantly increased in ascorbate deficient Gulo^−/− mice compared to wild type animals (Figure 2 and supplementary Table S2). Supplementation of drinking water with 0.4% ascorbate in Gulo^−/− mice decreased serum glycine to wild type levels.

Effect of ascorbate on inflammatory cytokines and metabolic hormones in Gulo^−/− mice

As metabolite disturbances can lead to an inflammatory response or changes in cardiovascular risk factors, we measured the levels of 42 cytokines (including hormones) in the serum of our different mouse cohorts. Serum cytokine concentrations in eight males of each group are shown in the supplementary Table S3. To identify the cytokines significantly altered in either wild type, Gulo^−/− mice treated with 0%, 0.01%, or 0.4% ascorbate, the nonparametric Kruskal-Wallis test was applied to the data. A summary of the metabolites significantly altered (P < 0.01) in at least one of the groups is given in a form of a heatmap in Figure 4A. Changes were indicated as a Z-score value for each metabolite in the serum of each animal. The dendrogram on top of the heatmap indicated that the Gulo^−/− mice depleted of ascorbate tended to cluster together. The wild type animals also tended to form a cluster. We further explored the cytokine profile of each group by employing principal component analysis (PCA in Figure 4B). The PCA approach showed that wild type mice were mainly clustered in the upper left quadrant of the graph. Gulo^−/− mice supplemented with either 0.01% or 0.4% ascorbate were distributed in all four quadrants but their localization overlapped with the wild type animals. Ascorbate deficient Gulo^−/− mice were localized on the right side of the graph (except for mouse GNV.4). To summarize the PCA data, the serum cytokine profile of the ascorbate deficient Gulo^−/− mice was quite different from ascorbate treated Gulo^−/− and wild type mice.

Figure 4. Impact of ascorbate on the cytokinome of Gulo^−/− mice. (A) Heatmap depicting the Z-score of log base ten in serum cytokine concentrations (rows) between individual (columns) wild type and Gulo^−/− mice treated with different amounts of ascorbate. Columns are reordered by hierarchical clustering using the genotype and ascorbate treatments to label the three leaves. (B) Principal component analysis (PCA) graph demonstrating the differentiation effect of ascorbate on the cytokinome profiles of wild type and Gulo^−/− mice treated with different amounts of ascorbate. Wild type animals are labeled WT.1 to WT.6. Gulo^−/− mice treated with 0.4% ascorbate in drinking water are labeled GV.1 to GV.6. Gulo^−/− mice treated with 0.01% ascorbate are labeled G.1 to G.6. Gulo^−/− mice treated with 0% ascorbate in drinking water from the age of three to four months are labeled GNV.1 to GNV.6. The Euclidean distance and complete agglomerative methods were used for clustering.

The heatmap revealed seven cytokines that significantly differed between groups (Figure 4A). These included three pro-inflammatory cytokines (interleukin-18, granulocyte-colony stimulating factor, and the macrophage inflammatory protein 1 beta), two metabolic hormones (leptin and C-peptide 2), and two cardiovascular risk factors (secreted E-selectin and the plasminogen activator inhibitor-1). The most marked findings were a general decrease in the inflammatory factors and sE-selectin in ascorbate deficient Gulo^−/− mice compared to wild type animals (summarized as graphs in the supplementary Figure S2). Ascorbate treatment increased the levels of these cytokines but to a level still below wild type animals. The hormones leptin and C-peptide 2 were significantly decreased in ascorbate depleted Gulo^−/− mice. Supplementation of ascorbate reversed the phenotype. Finally, the plasminogen activator inhibitor-1 (PAI-1) was significantly increased in ascorbate depleted Gulo^−/− mice compared to wild type animals (supplementary Figure S2). Supplementation of ascorbate in drinking water also reversed this phenotype in Gulo^−/− mice.

Correlation between cytokines and metabolites in the different Gulo^−/− cohorts

We determined whether there were any strong correlations (r > 0.99 and P < 0.01) between the cytokines and any of the metabolites that were altered in the Gulo^−/− cohorts. Only two cytokines passed such stringent criteria for these correlations. The serum levels of alpha-aminoadipic acid (alpha-AAA) showed an inverse correlation with serum sE-Selectin (Figure 5A), while the levels of serum 6-keto-PGF1a was positively correlated with serum plasminogen activator inhibitor-1 (PAI-1) (Figure 5B).

Figure 5. Correlation of median life span and serum ascorbate levels in Gulo^−/− mice with different metabolites and cytokines. (A) Correlation between alpha-AAA and sE-selectin with a r > 0.99 and a P-value < 0.01 in our mouse cohorts. (B) Correlation between 6-keto-PGF1a and PAI-1 with a r > 0.99 and a P-value < 0.01 in our mouse cohorts. (C) Graphs showing the correlation between the median life span of mice and serum ascorbate level. (D) Correlation between serum ascorbate and resistin. (E) Correlation between serum acylcarnitine C18:2 and median life span. (F) Correlation between resistin and median life span. The Pearson's correlation r values and P-values are indicated on each graph.

Correlation between ascorbate and the median life span of Gulo^−/− mice with metabolites or cytokines

We then determined which metabolites and cytokines were correlated with the median life span of our Gulo^−/− cohorts. Gulo^−/− mice without ascorbate in the drinking water lived approximately one month as indicated earlier. The median life span of wild type and Gulo^−/− mice treated with either 0.01% or 0.4% ascorbate was obtained from the graph in Figure 1A. As such, the median life span of Gulo^−/− mice correlated positively with serum ascorbate levels (r = 0.9918; P < 0.01 in Figure 5C). The molecule that showed strongest correlation with serum ascorbate level was serum resistin (Figure 5D). Serum resistin levels were inversely correlated with serum ascorbate levels.

The molecules that were correlated to median life span included serum levels of octadecadienoylcarnitine (C18:2) and resistin (Figure 5E and F). C18:2 and resistin exhibited an inverse correlation with the median life span in our mouse cohorts (with a r of at least −0.99 and a P < 0.01). Other metabolites and cytokines measured in the serum of our mouse cohorts did not correlate to ascorbate levels with a Pearson's correlation r value > 0.99. Although we obtained high Pearson's correlation r values between the median life span and the mean levels of serum resistin and C18:2, these molecules exhibited large standard deviations from the means within each group of mice such that differences between groups were not statistically significant (Kruskal-Wallis P-values > 0.01). In fact, no individual metabolite (with a Kruskal-Wallis P-values > 0.01) correlated with life span and/or ascorbate (even with a r value > 0.95 and P-value < 0.05). Next we examined specific ratios of metabolites in our cohorts of mice that could correlate with ascorbate levels or median life span with a r value > 0.95 and P-value < 0.05. Specific metabolite ratios in the serum of mice can provide insight into biological processes relevant to the activity and metabolism of ascorbate.

Figure 6 provides metabolite ratios that showed significant difference between groups of mice (with ANOVA P-values < 0.05). Methionine-sulfoxide (Met-SO) can be generated via a two-electron-dependent mechanism and the ratio of Met-SO/Met in the serum can be regarded as a marker of oxidative stress [19]. As indicated in Figure 6A, Gulo^−/− mice treated with 0.01% ascorbate showed a significant increase in this ratio compared to wild type animals. Ascorbate is also known to affect the hydroxylation of phenylalanine into tyrosine [20]. Figure 6B indicates that the ratio of Tyr/Phe is significantly reduced in ascorbate depleted Gulo^−/− mice compared to wild type animals. Arginine is the only source for nitric oxide production, a molecule with important vasoprotective and anti-atherosclerotic properties [21]. Because lysine uses the same transport system as arginine for intracellular transport, intracellular arginine availability can potentially be affected by lysine [21]. As indicated in Figure 6C, the Arg/Lys ratio is significantly diminished in vitamin C depleted and in Gulo^−/− mice treated with 0.01% ascorbate. Hepatic arginase will also affect the levels of arginine by catabolizing it into ornithine [21]. As indicated in Figure 6D, the Arg/Orn ratio is significantly decreased in ascorbate depleted Gulo^−/− mice compared to wild type animals. Acylcarnitines are important molecules for mitochondrial function and lipid metabolism. Two acylcarnitine species were significantly altered in our mouse cohorts, namely C4:1 and C16-OH (based on Figure 2). Although levels of C4:1 and C16-OH did not correlate significantly with ascorbate levels or median life span, we examined the ratio of carnitine over these two acylcarnitine species. As indicated in Figure 6E and F, the C0/C4:1 and C0/C16-OH ratios were reduced in all Gulo^−/− mice. Finally, we examined the ratio of saturated/unsaturated phosphatidylcholine species. We pooled the serum concentrations of unsaturated phosphatidylcholines together and saturated phosphatidylcholines together to calculate the ratio. (For example, the concentrations of PC aa C32:0, PC aa C36:0, PC ae C36:0, PC ae C40:0, and PC ae C42:0 that are significantly changed in our mouse cohorts based on Figure 2 were added together to obtain the numerator of the ratio). As indicated in the Figure 6G, the ratio of saturated/unsaturated phosphati-dylcholines was significantly increased in Gulo^−/− mice treated with 0.4% ascorbate compared to ascorbate depleted Gulo^−/− mice. Interestingly, among the different species of lipids altered in our mouse cohorts (Figure 2), the specific ratio PC aa C36:0/PC aa C36:2 showed a significant decrease in ascorbate depleted Gulo^−/− mice that was completely reversed by 0.4% ascorbate (Figure 6H). We calculated the Pearson's correlation r values to identify the metabolite ratios that correlated significantly with serum ascorbate levels and/or the median life span of our mice. As indicated in Table 1, the ratios of Arg/Lys, Tyr/Phe, saturated/unsaturated lipids, and PC aa C36:0/PC aa C36:2 correlated significantly with median life span. Serum ascorbate levels correlated significantly with the Arg/Lys, Tyr/Phe, and PC aa C36:0/PC aa C36:2 ratios.

Figure 6. Ratios of metabolites demonstrating significant differences between Gulo^−/− mice treated with different concentrations of ascorbate. (A) Graph showing the Met-SO/Met ratio in the different groups of mice. Tukey post ANOVA test *P-value < 0.05. (B) Graph showing the Tyr/Phe ratio in the different groups of mice. Tukey post ANOVA test *P-value < 0.05. (C) Graph showing the Arg/Lys ratio in the different groups of mice. Tukey post ANOVA test *P-value < 0.05. (D) Graph showing the Arg/Orn ratio in the different groups of mice. Tukey post ANOVA test *P-value < 0.05. (E) Graph showing the C0/C4:1 ratio in the different groups of mice. Tukey post ANOVA test **P-value < 0.01. (F) Graph showing the C0/C16-OH ratio in the different groups of mice. Tukey post ANOVA test P-values are shown (*P < 0.05 and **P < 0.01). (G) Graph showing the ratio of saturated/unsaturated phosphatidylcholine species from the Figure 2. Tukey post ANOVA test *P-value < 0.05. (H) Graph showing the PC aa C36:0/PC aa C36:2 ratio in the different groups of mice. Tukey post ANOVA test **P-value < 0.01.

No correlation between the median life span of Gulo^−/− mice and body weight

Since body mass and visceral fat may have an effect on life span, we determined whether they were significantly correlated in our mouse cohorts. As expected, visceral fat wet weight correlated significantly with total body weight (r = 0.9972; P < 0.01) in these mice. The median life span of Gulo^−/− mice, however, did not correlate with the mean total body weight of mice or the visceral wet weight/body weight ratio (Table 2). The median life span did not correlate with the weight of the other organs. Similarly, serum ascorbate levels did not significantly correlate with total body weight, visceral fat weight or the weight of any organ analyzed (Table 2).

Visceral fat wet weight (and thus total body weight) significantly correlated with the levels of several serum phosphatidylcholine lipid species and serum hexoses (supplementary Table S4). Such phosphatidylcholine lipid species and hexoses, however, did not correlate significantly with the median life span of our mice (P-values > 0.05).

Correlation between hepatic mitochondrial morphology and metabolites or cytokines in Gulo^−/− mice

The liver plays a pivotal role in nutrient, hormone, lipid, and metabolic waste product processing, thereby maintaining body homeostasis [22]. It is also the normal site of ascorbate synthesis in mice [2]. Since ascorbate treated Gulo^−/− mice exhibited alterations in several lipid species, we next measured several cellular morpholo-gical parameters in the hepatic tissue. We first examined sinusoidal endothelial fenestration in liver of mice. Liver endothelial defenestration is a recognized age-related change [23]. Scanning electron microscopy revealed no significant difference in the number of sinusoidal endothelial fenestration between our cohorts of mice (supplementary Figure S3A). In addition, fenestration diameter was not significantly different between cohorts (supplementary Figure S3B and C).

Since the mitochondrion is important in metabolism, we next examined the morphology of mitochondria in the liver of our mouse cohorts. Enlarged mitochondria in liver tissues have been reported in a mouse model of premature aging [24] and in human patients with liver disease [25]. Transmission electron microscopy revealed a significant increase in the dimension of hepatic mitochondria in ascorbate depleted Gulo^−/− mice compared to wild type animals (Figure 7A). Supplementation of ascorbate in drinking water (0.01% or 0.4%) significantly decreased the size of the mitochondria to wild type dimensions. In contrast, the surface density of the mitochondrial envelop was significantly lower in all Gulo^−/− mice (with 0%, 0.01% or 0.4% ascorbate) compared to wild type animals (Figure 7B; Tukey post-ANOVA test; P < 0.01). Finally, the number of mitochondria/hepatic surface area was not significantly different between cohorts of mice (supplementary Figure S4A and B; Tukey post-ANOVA test; P > 0.05). The levels of reactive oxygen species (ROS) were measured in the liver of mice. As indicated in Figure 7C, ROS level was increased in ascorbate depleted and 0.01% ascorbate treated Gulo^−/− mice compared to wild type animals (Tukey post-ANOVA test; P < 0.05). ROS level was decreased in Gulo^−/− mice treated with 0.4% ascorbate to wild type levels. Liver ROS levels significantly correlated inversely with serum ascorbate levels (Figure 7D).

Figure 7. Correlation of mitochondrial morphology in the liver of Gulo^−/− mice with different metabolites and cytokines with a r > 0.99 and a P-value < 0.01. (A) Graph showing the average mitochondrial dimension in different mouse cohorts. (Tukey post ANOVA test: **P < 0.05 compared to all other groups of mice). (B) Graph showing the average surface density of mitochondrial envelop area in different mouse cohorts. (Tukey post ANOVA test: *P < 0.01 compared to all other groups of mice). (C) Graph showing the ROS levels in the liver of our different mouse cohorts. (Tukey post ANOVA test: *P < 0.05 compared to wild type and 0.4% treated Gulo^−/− mice). (D) Correlation between liver ROS levels and serum ascorbate levels. (E) Correlation between serum 15S-HETE and the average mitochondrial dimension in mice. (F) Correlation between serum PAI-1 and the average mitochondrial dimension in mice. (G) Correlation between serum sE-selectin and the average surface density of mitochondrial envelop area in the liver of the different mouse cohorts. (H) Correlation between serum alpha-AAA and the average surface density of mitochondrial envelop area in the liver of the different mouse cohorts. The Pearson's correlation r values and P-values are indicated on each graph.

We determined whether there were significant correlations between cytokines or metabolites and mitochondrial morphology in our mouse cohorts. Figure 7 (panels E-H) gives the molecules with the highest correlations (r > 0.99 and P < 0.01). The prostaglandin derivative 15S-HETE and the secreted protease PAI-1 were positively correlated with hepatic mitochondrial size (Figure 7E and F). Serum sE-selectin levels in return, correlated positively with the surface density mitochondrial envelop area (Figure 7G). In contrast, serum alpha-AAA levels correlated negatively with mitochondrial surface density envelop area (Figure 7H). Finally, hepatic mitochondrial size also significantly (r > 0.95; P < 0.05) correlated positively with histamine, spermidine, spermine, and 6-keto-PGF1a, and inversely with body weight, wet visceral fat weight, PC aa C42:4, and PC aa C42:6 (Table 3). The surface density mitochondrial envelop area significantly correlated with the ratio of C0/C4:1 and inversely with serum C16-OH levels and the Met-SO/Met ratio (Table 3).

Effect of ascorbate on stress markers in the liver of Gulo^−/− mice

Since we could detect changes in serum inflammatory cytokines, lipids, and hepatic ROS in our different cohorts of mice, we analyzed the levels of several stress markers in the liver of these mice. Phosphorylation of IRE1α, PERK, and eIF2α were first analyzed by western blotting (Figure 8A). IRE1α acts as an endoplasmic reticulum (ER) stress sensor and is part of the unfolded protein stress response [26]. As indicated in Figure 8A and B, the levels of phosphorylated IRE1α protein were increased in the liver of Gulo^−/− mice treated with 0.01% or depleted of ascorbate compared to wild type animals. Supplementation of 0.4% ascorbate in drinking water significantly reduced the levels of phosphorylated IRE1α proteins compared to ascorbate depleted Gulo^−/− mice (Figure 8B; shown only by student t-test: P < 0.05). Total IRE1α, in return, was significantly increased in 0.4% ascorbate treated Gulo^−/− mice compared to wild type, ascorbate depleted and 0.01% ascorbate treated Gulo^−/− mice (Figure 8C; Tukey post-ANOVA test: P > 0.01). PERK is a transmembrane protein located in the ER and contains a kinase domain activated upon ER stress that phosphorylates eIF2α, thus inhibiting the translation of mRNAs [27]. ER stress also leads to PERK autophosphorylation. As indicated in Figure 8A and D, the levels of phosphorylated PERK was significantly increased in ascorbate depleted Gulo^−/− mice compared to wild type animals (Tukey post-ANOVA or student t-test: P < 0.05). Total PERK levels were significantly increased in all Gulo^−/− mice compared to wild type animals (Figure 8E; Tukey post-ANOVA or student t-test: P < 0.05). Phosphorylated eIF2α was significantly decreased in 0.4% ascorbate treated Gulo^−/− mice compared to ascorbate depleted Gulo^−/− mice (Figure 8A and F). Total eIF2α level was significantly decreased in 0.01% ascorbate treated Gulo^−/− mice compared to wild type mice (based on student t- test: P < 0.05; Figure 8G).

Figure 8. Impact of ascorbate on the levels of different ER stress markers in the liver of Gulo^−/− mice. (A) Example of western blots showing protein levels of phosphorylated and total IRE1α, GRP78, phosphorylated and total PERK, phosphorylated and total eIF2α. β-actin was used as loading controls. (B) Ratio of phosphorylated IRE1α signal over β-actin signal from the western blots. (ANOVA: P > 0.05; but student t-test: *P < 0.05 for 0.4% ascorbate treated Gulo^−/− mice vs. ascorbate depleted Gulo^−/− mice). (C) Ratio of total IRE1α signal over β-actin signal from the western blots. (Tukey post ANOVA test: **P < 0.01 compared to all other mouse groups). (D) Ratio of phosphorylated PERK signal over β-actin signal from the western blots. (Tukey post ANOVA test: *P < 0.05 compared to ascorbate depleted Gulo^−/− mice). (E) Ratio of total PERK signal over β-actin signal from the western blots. (Tukey post ANOVA test: *P < 0.05 and **P < 0.01 compared to type mice). (F) Ratio of phosphorylated eIF2α signal over β-actin signal from the western blots. (ANOVA: P > 0.05; but student t-test: *P < 0.05 for 0.4% ascorbate treated Gulo^−/− mice vs. ascorbate depleted Gulo^−/− mice). (G) Ratio of total IRE1α signal over β-actin signal from the western blots. (ANOVA: P > 0.05 but student t-test: *P < 0.05 for 0.01% ascorbate treated Gulo^−/− mice vs. wild type mice). (H) Ratio of total GRP78 signal over β-actin signal from the western blots. No significant difference was found between groups (ANOVA: P > 0.05; student t-test P > 0.05). Bars in all histograms represent SEM of three mice.

GRP78, also referred as the immunoglobulin heavy chain-binding protein (BiP), is a member of the heat-shock protein-70 family and is involved in the folding and assembly of proteins in the ER [26]. There was a decrease in GRP78 in Gulo^−/− mice treated with 0.4% ascorbate (Figure 8A), but overall there was no significant difference in the levels of this protein between groups of mice (Figure 8H; Tukey post-ANOVA or student t-test: P > 0.05).

The NFκB transcription factor determines cell response to a wide variety of stresses, including inflammation, and was recently shown to be one of the most strongly age-associated markers [28]. As indicated in Figure 9A and B, phosphorylated NFκB levels were significantly different between groups of mice (Tukey post-ANOVA test: P > 0.01). Phosphorylation (and thus activation of NFκB) was increased in Gulo^−/− mice treated with 0.4% ascorbate compared to wild type and Gulo^−/− mice treated with 0.01% ascorbate. Total NFκB levels were similar in all groups of mice (Figure 9A and C).

Figure 9. Impact of ascorbate on the levels of different stress markers in the liver of Gulo^−/− mice. (A) Example of western blots showing protein levels of phosphorylated and total NFκB, phosphorylated and total p38 MAP kinase. β-actin was used as loading controls. (B) Ratio of phosphorylated NFκB signal over β-actin signal from the western blots. (Tukey post ANOVA test: *P < 0.05 and **P < 0.01 compared to 0.4% ascorbate treated Gulo^−/− mice). (C) Ratio of total NFκB signal over β-actin signal from the western blots. (D) Ratio of phosphorylated p38 MAP kinase signal over β-actin signal from the western blots. (Tukey post ANOVA test: *P < 0.05 compared to wild type mice). (E) Ratio of total p38 MAP kinase signal over β-actin signal from the western blots. (Tukey post ANOVA test: *P < 0.05 compared to wild type mice). Bars in all histograms represent SEM of three mice.

We also examined the levels of total and phosphorylated p38 MAP kinase in the hepatic tissues of our mouse cohorts. As indicated in Figure 9D and E, the addition of ascorbate in drinking water decreased total and phosphorylated levels of p38 in the liver of Gulo^−/− mice compared to wild type animals (Tukey post-post-ANOVA; P < 0.05). However, total and phosphorylated levels of p38 were not higher in the liver of ascorbate depleted Gulo^−/− mice compared to wild type animals.

Levels of phosphorylated IRE1α and eIF2α correlated with levels of ascorbate and median life span

We determined whether there were significant correlations between the stress markers analyzed in our mouse cohorts and the median life span or the serum ascorbate levels. Table 4 provides a summary of the correlations. Phosphorylated IRE1α and phosphorylated eIF2α were the only markers that correlated significantly (and inversely) with the median life span of mice in our different cohorts with a P-value of at least < 0.05. They also significantly correlated inversely with serum ascorbate levels (Table 4). Phosphorylated or total PERK, GRP78, NFκB, and p38 MAP kinase did not correlate significantly with median life span or ascorbate levels (Table 4). Thus, different phosphorylated markers of ER stress response correlated significantly with the median life span of Gulo^−/− mice treated with different concentrations of ascorbate in drinking water.

Correlation between stress markers and metabolites or cytokines

We next looked at the significantly altered metabolites or cytokines (Figures 2 and 4A) that correlated (with a r > 0.95 and a P < 0.05) with the different stress markers analyzed in the liver of our mouse cohorts. Significant correlations with the ER stress markers are shown in Table 5. Importantly, phosphorylated IRE1α and eIF2α correlated positively with the ratio of saturated to unsaturated phosphatidylcholines. Although the phosphorylation of PERK did not correlate significantly with median life span (only a tendency with a r = −0.9196), it positively correlated with the phosphorylation of eIF2α proteins. Total PERK inversely correlated with the surface density of mitochondrial envelop area (Table 5) and with several of the molecules that correlated with this mitochondrial parameter (like sE-selectin, C16-OH, alpha-AAA, and the Met-SO/Met ratio; compare Tables 3 and 5). The amount of the ER stress marker GRP78 correlated inversely with total IRE1α levels in the liver of our mouse cohorts. Finally, the levels of phosphorylated and total eIF2α correlated inversely with several lysophosphatidylcholine and phosphatidylcholine species in the serum of our mice (Table 5).

Total and phosphorylated p38 correlated inversely with several phosphatidylcholine species including lipids with very long carbon chains in our different cohorts of mice (Table 6). Phosphorylated p38 also significantly correlated positively with total p38 in the liver of mice. Finally, phosphorylated NFκB did not significantly correlate with metabolites or cytokines.

Animals and maintenance

Gulo^−/− mice were obtained from the Mutant Mouse Regional Resource Centers (University of California Davis, CA) and were housed at the Centre de Recherche de l'Hôtel-Dieu de Québec animal facility. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Canadian Council on Animal Care in science. The protocol was approved by the Committee on the Ethics and Protection of Animal of Laval University (Permit Number: 2014029). Mice were housed in cages (containing a top filter) at 22 ± 2°C with 40%–50% humidity and a 12-h light–dark cycle (light cycle: 06:00–18:00 hours). All mice were fed ad libitum with Teklad Global (Madison, WI) 18% protein rodent diet, 5% fat, and 0% vitamin C (135 IU/kg of vitamin E and 30 IU/g of vitamin A). Euthanasia was performed by treating mice with 3% isoflurane (general anesthesia) followed by cervical dislocation. Animals were checked every day for any external mass, infection, bleeding, gasping, and overall decrease or change in activity or behavior. Mice that lost 20% of total body weight, became immobile, or moribund were sacrificed for histological examination of their organs as described previously [57]. One cohort of Gulo^−/− mice was maintained on standard diet and supplemented with 0.4% of L-ascorbate (Sigma-Aldrich, Oakville, ON) (w/v) in drinking water from weaning until the age of four months. A second cohort of Gulo^−/− mice was maintained on standard diet and supplemented with 0.01% of L-ascorbate (w/v) in drinking water from weaning until the age of four months. A third cohort of Gulo^−/− mice was maintained on standard diet and supplemented with 0.01% of L-ascorbate (w/v) in drinking water from weaning until the age of three months. Ascorbate was then removed from drinking water for four weeks. Wild type control C57BL/6 mice were maintained in the same room with no ascorbate supplementation in drinking water and were used as our normal reference. The analyses were performed on four month-old animals as more than 85% of Gulo^−/− mice treated with 0.01% ascorbate were healthy at that age.

Serum collection for analysis

Blood was harvested at 10:00 am by cardiac puncture and exsanguination under anesthesia at the age of four months. Blood was allowed to clot for one hour at 4°C and spun on a bench top centrifuge at maximum speed for 15 min. Serum was collected and frozen at −80°C until execution of analyses. Ascorbic acid in serum was measured with the ferric reducing ascorbate assay kit from BioVision Research Products (Mountain View, CA, USA).

Metabolite measurements

Metabolite measurements were performed by the BIOCRATES Life Sciences metabolomic services (BIOCRATES Life Sciences AG, Innsbruck, Austria). Briefly, Biocrates’ commercially available kit plates were used for the quantification of amino acids, acylcarnitines, sphingomyelins, phosphatidylcholines, hexoses, and biogenic amines. The fully automated assay was based on PITC (phenylisothiocyanate)-derivatization in the presence of internal standards followed by FIA-MS/MS (acylcarnitines, lipids, and hexose) and LC/MS (amino acids, biogenic amines) using an AB SCIEX 4000 QTrap® mass spectrometer (AB SCIEX, Darmstadt, Germany) with electrospray ionization. The experimental metabolomics measurement technique has been previously described [53]. Eicosanoids and other oxidized polyunsaturated fatty acids were extracted from samples with aqueous acetonitrile that contained deuterated internal standards. The metabolites were determined by HPLC-tandem mass spectrometry (LC-MS/MS) with Multiple Reaction Monitoring (MRM) in negative mode using a SCIEX API 4000 QTrap mass spectrometer with electrospray ionization. The LC-MS/MS method used for the analytical determination of eicosanoids has been published [58]. Accuracy of the measurements (determined with the accuracy of the calibrators) 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 amino acids, 19 biogenic amines, one hexose (H1), free carnitine (C0), 40 acylcarnitines (Cx:y), hydroxylacylcarnitines (C(OH)x:y), and dicarboxylacylcarnitines (Cx:y-DC), 15 sphingomyelins (SMx:y) and N-hydroxylacyloyl-sphingosylphosphocholine (SM (OH)x:y), 77 phosphatidylcholines (PC, aa = diacyl, ae = acyl-alkyl), 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 ae C30:1” denotes an acyl-alkyl phosphatidylcholine with 30 carbons in the two fatty acid side chains and a single double bond in one of them [53]. Full biochemical names are provided in Supplementary Table S1 and the raw data in the Supplementary Tables S2. Note that 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.

Cytokine measurements

The following cytokines were assessed in the serum (diluted 1:4) using the multiplex kit from Bio-Rad Laboratories Canada Ltd. (catalogue number MD000000EL; Mississauga, ON, Canada): IL-15, IL18, LIF, M-CSF, MIG, MIP-2, PDGF-BB, VEGF, and FGF basic. The following cytokines were assessed in the serum (diluted 1:4) using the multiplex kit from Bio-Rad (catalogue number MD60009RDPD): IL-1α, IL-1β, IL-2, IL3, IL-4, IL-5, IL-6, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-17, KC, MCP-1, MIP-1α, MIP-1β, TNF-α, Rantes, Eotaxin, G-CSF, GM-CSF, and IFN-γ. The metabolic hormones C-peptide 2, GIP, Leptin, and Resistin were assessed in the serum using the Milliplex Mouse Metabolic Magnetic Bead Panel (catalogue number MMHMAG-44K) from EMD Millipore Corp. (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 EMD Millipore Corp. (catalogue number MCVD1MAG-77K): soluble E-selectin, P-selectin, ICAM-1, Pecam-1, proMMP-9, thrombomodulin, and total PAI-1. Full biological names are provided in Supplementary Table S3. All measurements were performed on a Bio-Plex® 200 system (with Bio-Plex Manager™ software version 6.0) from Bio-Rad Laboratories Canada Ltd. (Mississauga, ON, Canada).

Morphological and ultrastructural studies of liver tissues

For liver morphology studies, liver perfusion was performed as described previously [59]. Following fixation, liver samples for transmission electron microscopy were embedded in Spurrs resin, sectioned, and examined using a Philips CM10 transmission electron microscope. Liver morphology was assessed for mitochondrial density, mitochondrial size, surface density of mitochondrial envelop using ImageJ software. Three to five mice per genotype were analyzed by electron microscopy techniques. Fifteen micrographs per animal of hepatocytes cytoplasm were taken at 9,900 × magnification. Mitochondria were manually counted and the number of mitochondria per cytoplasmic unit area was calculated. Mitochondria area was measured by tracing around five random mitochondria in each micrograph. The surface density of mitochondrial envelope (Sv) was estimated using the formula Sv=2*I/Lt where I represents the number of intersections of mitochondria envelops with parallel lines (1μm apart) and Lt the total length of lines as described [60]. Scanning electron microscopy was performed as previously described [61]. Ten images (magnification x 20,000) were taken for each animal for analysis of fenestration diameter and endothelial porosity.

Reactive oxygen species (ROS) measurements in liver tissue

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, 30 mM NaF, 60 mM glycerophosphate, 20 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1mM phenylmethyl-sulfonylfluoride and complete protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN)) were incubated with 10 μg/mL of the dye 2′-7′ dichlorofluorescein diacetate (Sigma-Aldrich Canada Ltd, Oakville, ON) for 1h at 37°C. This dye is highly fluorescent upon oxidation. As control, RIPA buffer was also incubated with dichlorofluorescein diacetate and 100 μL (500 μg of liver proteins) of the samples were put into 96-well plates. Fluorescence was measured with a Fluoroskan Ascent fluorescence spectrophotometer (Thermo Electron Inc., Milford, MA). The excitation and emission wavelengths used were 485 and 527 nm, respectively. Background fluorescence was extracted from the dichlorofluorescein value for each sample and the final result was expressed as units of fluorescence per gram of proteins.

Western blotting

All steps were performed on ice or at 4°C. Tissue protein extraction was carried out in lysis buffer containing 20 mM MOPS (pH 7.0), 2 mM EGTA (pH 8.0), 5 M EDTA (pH 8.0), 30 mM NaF, 60 mM glycerophosphate (pH 7.2), 20 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 3 mM benzamidine, 5 μM pepstatin A, 10 μM leupeptin, 0.5% Triton X-100, 1 mM DTT, 1mM phenylmethyl-sulfonylfluoride and complete protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN). Proteins were then resolved on a 4-12% Criterion XT Bis-Tris gradient gel (Bio-Rad, Mississauga, ON, Canada) and then transferred onto 0.2 μm PVDF membrane (EDM Millipore Corp., Temecula, CA). After incubating 1 h with blocking solution (PBS-T containing 5% nonfat milk), the membrane was probed overnight at 4°C with a primary antibody. After washing with PBS-T, species-specific horseradish peroxidase-conjugated secondary antibody was added for 2 h at room temperature (GE Healthcare Limited, Piscataway, NJ). Signals were generated with Western Lightning Chemiluminescence reagent plus kit (GE Healthcare Limited, Piscataway, NJ). 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-eIF2 α (FL-315): sc-11386), phosphorylated eIF2 α (anti-p-eIF2 α (Ser 52): sc101670) from Santa Cruz Biotechnology (Santa Cruz, CA); a rabbit polyclonal antibody against phospho-inositol-requiring kinase 1α (anti-phospho-IRE1α, NB100-2323) from Novus Biologicals (Burlington, ON, Canada); rabbit monoclonal antibodies against inositol-requiring kinase 1α (anti-IRE1 #3294), phospho-NFκB (anti-phospho-NFκB p65 (Ser536) (93H1)), NFκB (anti-NFκB p65 (D14E12)), and phosphorylated PERK (anti-phospho-PERK (Thr980) (16F8)) from Cell Signaling Technology (Beverly, MA); rabbit polyclonal antibodies against phospho-p38 (anti-phospho-p38 MAP Kinase (Thr180/Tyr182)) and total p38 (anti-p38 MAP Kinase) from Cell Signaling Technology (Beverly, MA); a rabbit polyclonal antibody against glucose-related protein 78 (anti-GRP78) from Proteintech^TM (Chicago, IL); a mouse monoclonal antibody against actin (A5441) from Sigma-Aldrich (Oakville, ON).

Statistical analyses

Identification of significantly different metabolites was achieved by using the Kruskal-Wallis nonparametric test. Principal component analysis (PCA) and presentation of the results using heatmaps were performed using R versions 2.14. To take into account multiple testing, we considered significant events using a P < 0.01. Heatmaps were generated using Euclidean distance and complete agglomerative methods. One-way ANOVA followed by Tukey's HSD (honest significant difference) Test for post-ANOVA pair-wise comparisons were performed for the morphological studies and for the western blot analyses. Differences were considered significant at a P-value < 0.05. These tests were calculated using the http://facultysites.vassar.edu/lowry/ank3.html website. Pearson's correlation r values were calculated with Excel. For our colony of four different groups of mice, the r value must be greater than 0.95 to reach significance with a P-value < 0.05.