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Keywords:

  • aging;
  • fuel oxidation;
  • glutathione;
  • insulin resistance;
  • mitochondria

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. Author contributions
  9. References

Aging is associated with impaired fasted oxidation of nonesterified fatty acids (NEFA) suggesting a mitochondrial defect. Aging is also associated with deficiency of glutathione (GSH), an important mitochondrial antioxidant, and with insulin resistance. This study tested whether GSH deficiency in aging contributes to impaired mitochondrial NEFA oxidation and insulin resistance, and whether GSH restoration reverses these defects. Three studies were conducted: (i) in 82-week-old C57BL/6 mice, the effect of naturally occurring GSH deficiency and its restoration on mitochondrial 13C1-palmitate oxidation and glucose metabolism was compared with 22-week-old C57BL/6 mice; (ii) in 20-week C57BL/6 mice, the effect of GSH depletion on mitochondrial oxidation of 13C1-palmitate and glucose metabolism was studied; (iii) the effect of GSH deficiency and its restoration on fasted NEFA oxidation and insulin resistance was studied in GSH-deficient elderly humans, and compared with GSH-replete young humans. Chronic GSH deficiency in old mice and elderly humans was associated with decreased fasted mitochondrial NEFA oxidation and insulin resistance, and these defects were reversed with GSH restoration. Acute depletion of GSH in young mice resulted in lower mitochondrial NEFA oxidation, but did not alter glucose metabolism. These data suggest that GSH is a novel regulator of mitochondrial NEFA oxidation and insulin resistance in aging. Chronic GSH deficiency promotes impaired NEFA oxidation and insulin resistance, and GSH restoration reverses these defects. Supplementing diets of elderly humans with cysteine and glycine to correct GSH deficiency could provide significant metabolic benefits.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. Author contributions
  9. References

Elderly humans have the highest prevalence of being overweight and obese (Flegal et al., 2010) together with increased accumulation of fat in ectopic tissues such as the liver (Cree et al., 2004; Bianchi et al., 2010) and skeletal muscle (Cree et al., 2004). Ectopic accumulation of fat is linked to insulin resistance. These defects predispose elderly humans to increased risks of developing diabetes, heart disease, metabolic syndrome, and mortality (Harris et al., 1998; Noale et al., 2006, 2012). While underlying mechanisms for these defects are not fully understood, abnormalities in metabolism have been suspected (Johannsen & Ravussin, 2010), and ectopic fat accumulation and insulin resistance have been associated with abnormal mitochondrial function and elevated oxidative stress in aging (Johansson et al., 2005).

Mitochondrial defense against oxidative stress comes from endogenous antioxidants, among which glutathione (GSH, γ-glutamyl-cysteinylglycine) is the most abundant. Induction of acute GSH deficiency in mitochondria results in cell death (Ghosh et al., 2005) or mitochondrial damage (Mastrocola et al., 2005), suggesting that GSH is important for mitochondrial protection. GSH is synthesized in cells from its precursor amino acids cysteine, glutamate, and glycine, but aging humans and rodents are deficient in GSH (Lang et al., 1992; Samiec et al., 1998; Sekhar et al., 2011), and we have shown that this occurs due to decreased synthesis caused by deficiency of cysteine and glycine (Sekhar et al., 2011).

Under physiological conditions, the fuel of choice in the fasted state is nonesterified (‘free’) fatty acids (NEFA) (Kelley & Simoneau, 1994). Because elderly humans have been reported to have GSH deficiency, impaired mitochondrial NEFA oxidation (Calles Escandon et al., 1995; Toth et al., 1996; Levadoux et al., 2001; Solomon et al., 2008), and a higher risk of developing insulin resistance, we hypothesized that GSH deficiency could contribute to these defects, and GSH restoration could potentially reverse these defects. We used a translational approach combining studies in mice and humans to test the following: (i) whether naturally occurring GSH deficiency in 82-week-old C57BL/6 mice fed a regular diet was associated with impaired mitochondrial NEFA oxidation and glucose metabolism compared with young 22-week-old mice, and whether correcting GSH deficiency with dietary cysteine and glycine supplementation improves these defects; (ii) whether acute depletion of GSH in young 20-week-old C57BL/6 mice led to impaired mitochondrial NEFA oxidation and glucose metabolism; and (iii) whether in elderly humans with chronic GSH deficiency and blunted mitochondrial NEFA oxidation and insulin resistance, correcting GSH deficiency improves these defects.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. Author contributions
  9. References

Old C57BL/6 mice have GSH deficiency in liver and skeletal muscle which can be reversed by oral dietary cysteine and glycine supplementation

Compared with young mice, GSH concentrations in unsupplemented old mice (O-CON) mice were 40% lower in liver and 25% lower in soleus muscle (Fig. 1A,B). Pair-feeding a parallel group of old mice with a diet supplemented with GSH precursor amino acids N-acetylcysteine and glycine (O-RX) restored their liver and muscle GSH concentrations to levels seen in young mice (58% increase in liver and 25% increase in soleus muscle compared with O-CON mice) (Fig 1A,B), but concentrations of GSSG (Fig. 1A,B) or GSSG:GSH ratios were not different among the three groups. Supplementation also led to significantly higher RNA levels of the enzymes of glutathione synthesis in O-RX mice—the catalytic subunit of glutamate–cysteine ligase (Gclc, P < 0.05) and glutathione synthetase (Gss, P < 0.01). Because these genes are transcriptionally regulated by Nuclear factor erythroid 2-related factor 2 (NRF2), we measured and found that compared with young mice, O-CON (P < 0.05) had lower transcript levels of Nrf2, and this did not change with supplementation in O-RX mice (Fig. 1C). Reciprocal to GSH levels, hepatic concentrations of TBARS were 83% higher in O-CON mice compared with young mice and decreased by 23% in the O-RX group with GSH restoration compared with O-CON mice (Table 1).

Table 1. Mitochondrial NEFA oxidation and plasma biochemistry in supplemented and unsupplemented old mice
ParametersYoung miceUnsupplemented old mice O-CONSupplemented old mice O-RX P
  1. BUN, blood urea nitrogen; HOMA, homeostatic model assessment; IR, insulin resistance; NEFA, nonesterified fatty acids; TBARS, thiobarbituric acid reducing substances.

  2. All values are means ± SEMs; n = 8 young control mice (Y), 8 old control mice (O-CON), and 8 old mice receiving GSH precursor supplementation (O-RX). Means are significantly different at P < 0.05.

  3. a

    Young mice vs. O-CON mice.

  4. b

    O-CON vs. O-RX mice.

Mitochondrial NEFA oxidation (% dose of 13C1-Palmitate oxidized)32.1 ± 1.021.0 ± 4.5a42.8 ± 4.2b

<0.05a

<0.01b

Liver triglyceride concentration (mg g−1 liver)80 ± 6174 ± 29a65 ± 18b

<0.05a

<0.05b

Muscle triglyceride concentration (mg g−1 muscle)3.4 ± 0.43.5 ± 0.5a3.0 ± 0.5b

0.9a

0.5b

Hepatic TBARS (μm)4.8 ± 0.38.8 ± 0.5a6.8 ± 0.7b

<0.01a

<0.05b

Total bilirubin (μm)2.8 ± 0.23.2 ± 0.3a3.3 ± 0.4b

0.2a

0.7b

Insulin resistance (HOMA-IR)2.6 ± 0.38.9 ± 0.2a4.2 ± 0.7b

<0.05a

<0.05b

Alanine transaminase (U L−1)18.3 ± 2.332.0 ± 6.7a23.8 ± 6.1b

0.3a

0.3b

Aspartate transaminase (U L−1)120.2 ± 21.8183.0 ± 21.3a99.0 ± 19.1b

<0.05a

<0.01b

Alkaline phosphatase (g L−1)40.8 ± 3.649.7 ± 5.3a40.8 ± 2.8b

0.3a

0.3b

Plasma total cholesterol (mm)3.3 ± 0.14.3 ± 0.1a2.1 ± 0.1b

<0.01a

<0.01b

Plasma triacylglycerols (mm)0.7 ± 0.10.9 ± 0.1a0.6 ± 0.1b

0.1a

<0.05b

BUN (mm)6.8 ± 0.37.5 ± 0.2a7.6 ± 0.3b

0.2a

0.9b

Creatinine (μm)1.7 ± 0.02.7 ± 0.0a1.7 ± 0.0b

0.4a

0.4b

image

Figure 1. Cysteine and glycine supplementation in old mice improves glutathione concentrations in liver and skeletal muscle. (A) Liver; (B) Skeletal muscle; (C) Hepatic RNA levels of enzymes of glutathione synthesis and Nrf2 (GSSG = oxidized glutathione; Gclm = modifier subunit of glutamate–cysteine ligase; Gclc = catalytic subunit of glutamate–cysteine ligase; Gss = glutathione synthetase; Nrf2 = Nuclear erythroid factor 2). Means are considered significant at P < 0.05 (* = P < 0.05, θ = P < 0.01).

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GSH-deficient old C57BL/6 mice have impaired mitochondrial NEFA oxidation which can be improved by GSH restoration

GSH-deficient O-CON mice had 14% lower whole-body NEFA oxidation compared with young mice, and this increased by 14% in GSH-replete O-RX mice compared O-CON mice (Fig. 2A). Measurement of 13CO2 recovery from 13C1-palmitate (an index of mitochondrial NEFA oxidation) showed that GSH-deficient O-CON mice had 35% lower mitochondrial oxidation of 13C1-palmitate, and this increased by 104% in GSH-replete O-RX mice (Table 1).

image

Figure 2. GSH restoration in old mice improves fasted NEFA oxidation, lowers total body weight, and decreases hepatic fat content and insulin resistance. (A) NEFA oxidation; (B) body weight expressed as a percentage of basal weight; (C) staining of liver fat by oil red O. The red bar represents 20 μm in length; (D) total hepatic triacylglycerol content; (E) RNA levels of genes regulating hepatic NEFA metabolism; (F) RNA levels of genes regulating muscle NEFA and glucose metabolism; (G) HOMA-IR. Y = young control mice, O-CON = old control mice; O-RX = old treated mice: Pre = before dietary intervention; Post = after dietary intervention; TG = triacylglycerol. Means are considered significant at P < 0.05 (* = P < 0.05, θ = P < 0.01).

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GSH restoration in old C57BL/6 mice results in loss of body weight and body fat

Compared with young mice, GSH-deficient O-CON mice had significantly higher body weight and fat mass. O-CON and O-RX mice were then matched for body weight at baseline (42.4 ± 1.1 vs. 42.0 ± 1.3 g, P = 0.8). After completing the dietary protocol, O-RX mice weighed 17% less than O-CON mice (42.0 ± 1.5 vs. 35.0 ± 1.1 g, P < 0.01) (Fig. 2B) and had significantly lower fat mass (13.8 ± 1.5 vs. 9.6 ± 0.9 g, P < 0.001), but not lean mass (25.7 ± 0.6 vs. 24.8 ± 0.5 g, P = 0.30).

GSH restoration in old C57BL/6 mice lowers hepatic and not skeletal muscle fat content

Compared with young control mice, GSH-deficient O-CON mice had significantly higher liver fat content as seen by liver histology where fat was stained by oil red O (Fig. 2C) and confirmed by measuring hepatic triacylglycerol content (Table 1; Fig. 2D). GSH restoration in O-RX mice was associated with a significant decrease in hepatic fat and triacylglycerol content. We did not find differences between young mice, GSH-deficient O-CON mice, or GSH-replete O-RX mice for skeletal muscle triacylglycerol concentrations (Table 1).

Alterations in expression of enzymes of fat oxidation in liver and skeletal muscle of supplemented and unsupplemented old mice

Liver

Compared with young mice, O-CON mice had significantly lower RNA expression of the fatty acid translocase gene Cd36, carnitine palmitoyl transferase 1 α (Cpt1a), and peroxisome proliferator-activated receptor alpha (Ppara). Compared with young mice, O-RX mice had significantly lower RNA expression of fatty acid synthase (Fasn), Ppara, and peroxisome proliferator-activated receptor gamma coactivator 1 α (Ppargc1a often referred to as Pgc1α). Compared with O-CON mice, O-RX mice had higher transcripts of Cd36, without any change in expression of other genes (Fig. 2E).

Skeletal muscle

We did not find differences in Pgc1a transcripts between O-CON and O-RX, but Ppara transcript levels were significantly lower in O-CON mice compared with young controls, and transcript levels were restored in O-RX. Glucose transporter Glut4 (Slc2a4 gene) transcript levels were significantly higher in the O-RX mice compared with O-CON mice (Fig. 2F).

GSH restoration in old C57BL/6 mice improves insulin sensitivity and lipid profiles

Measurement of insulin resistance (as Homeostasis Model Assessment Insulin Resistance or HOMA-IR) showed that GSH-deficient old mice had 242% higher HOMA-IR compared with young mice, and this fell by 53% on GSH restoration (Fig. 2G). Additional evidence to support an improvement in insulin sensitivity came from glucose and insulin tolerance tests: compared with young mice, O-CON mice had significantly increased glucose and insulin responses to a glucose tolerance test (GTT), and blunted insulin-stimulated glucose disposal during an insulin tolerance test (ITT) suggestive of increased insulin resistance. On GSH restoration, O-RX mice displayed significantly lowered glucose and insulin responses to GTT and significantly improved response to ITT, consistent with improved insulin sensitivity (Fig. 3). GSH restoration in old O-RX mice also significantly lowered fasted plasma concentrations of total cholesterol and triacylglycerols (Table 1).

image

Figure 3. Glutathione restoration improves glucose metabolism in old mice. (A) Glycemic response to glucose tolerance test; (B) Insulin response to glucose tolerance test; (C) glycemic response to insulin tolerance test in young and old mice before and after supplementation with cysteine and glycine. Values are means ± SEMs; Closed triangle = Young; Closed box = O-CON = old control mice; Open box = O-RX = old treated mice. Presupplementation t-tests are Young vs. O-CON mice; postsupplementation t-tests are O-CON vs. O-RX mice. Means are considered significant at P < 0.05.

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Cysteine and glycine supplementation in old C57BL/6 mice is not associated with liver or renal toxicity

Relevant plasma measurements are shown in Table 1. Compared with young mice, GSH-deficient O-CON mice had significantly higher fasted plasma concentrations of aspartate transaminase and total cholesterol. In GSH-replete mice, fasted plasma concentrations of aspartate transaminase decreased significantly compared with O-CON mice. There were no differences between young, O-CON, and O-RX mice for plasma bilirubin, alanine transaminase, alkaline phosphatase, BUN, or creatinine concentrations suggesting lack of toxicity with GSH precursor supplementation. O-CON mice had significantly higher fasted plasma total cholesterol concentrations compared with young mice, and there was a trend for higher plasma triacylglycerol concentrations. Compared with O-CON mice, the O-RX mice had significantly lower concentrations of both total plasma cholesterol and triacylglycerols.

GSH depletion in young C57BL/6 mice decreases mitochondrial fat oxidation and increases fasted plasma LDLC, VLDL, and triacylglycerol concentrations, without any changes in body weight, composition, hepatic fat, or glucose tolerance

Compared with GSH-replete controls, administration of BSO to young, healthy mice for 4 weeks depleted hepatic GSH by 31%, and soleus muscle GSH by 61%, without any significant differences in oxidized glutathione (GSSG) or GSH/GSSG ratios (Table 2). Compared with GSH-replete controls, GSH depletion led to a decrease in both whole-body NEFA oxidation (Fig. 4A) and mitochondrial 13C1-palmitate oxidation (Table 2), without any changes in body weight (31.9 ± 0.8 vs. 31.5 ± 0.5 g), body fat (6.4 ± 0.7 vs. 6.4 ± 0.6 g, P = 1.0), lean mass (22.6 ± 0.5 vs. 22.8 ± 0.6 g, P = 0.84), higher hepatic triacylglycerol content (0.24 ± 0.06 vs. 0.24 ± 0.05 g triacylglycerols/g liver, P = 0.9), or glucose tolerance (Fig. 4B). Depletion of GSH was not associated with any changes in liver profiles (total protein, albumin, globulin, albumin-to-globulin ratio, total bilirubin, alanine transaminase, aspartate transaminase, alkaline phosphatase), blood urea nitrogen, creatinine, total and HDL-cholesterol, but fasted plasma triacylglycerol concentrations increased by 21% (Table 2). However, there was a significant increase in fasted plasma triacylglycerol, VLDL-cholesterol, and LDL-cholesterol concentrations (Table 2).

Table 2. GSH, mitochondrial NEFA oxidation, lipid profile, and plasma biochemistry in GSH-replete and GSH-depleted young mice
ParametersControl young mice (GSH-replete)Young mice on BSO (GSH-deficient) P
  1. All values are means ± SEMs; n = 8 control mice, 8 mice receiving buthionine sulfoximine (BSO). Means are significantly different at P < 0.05.

Hepatic reduced glutathione (GSH, μmol g−1 liver)4.0 ± 0.22.8 ± 0.1<0.001
Hepatic oxidized glutathione (GSSG, μmol g−1 liver)0.3 ± 0.00.3 ± 0.00.9
GSH/GSSG ratio (liver)17.8 ± 3.316.2 ± 5.10.8
Muscle reduced glutathione (GSH, μmol g−1 soleus muscle)1.3 ± 0.10.5 ± 0.1<0.001
Muscle oxidized glutathione (GSSG, μmol g−1 soleus muscle)0.2 ± 0.00.2 ± 0.00.9
GSH/GSSG ratio (muscle)8.0 ± 2.56.0 ± 1.90.4
Mitochondrial NEFA oxidation (% dose of 13C1-Palmitate oxidized)51.5 ± 3.236.8 ± 2.0<0.001
Plasma total cholesterol (mm)2.4 ± 0.12.4 ± 0.10.9
Plasma triacylglycerols (mm)0.7 ± 0.00.9 ± 0.1<0.05
LDL-cholesterol (mm)0.2 ± 0.00.4 ± 0.1<0.05
VLDL-cholesterol (mm)0.3 ± 0.00.4 ± 0.0<0.05
Total protein (g L−1)53 ± 350 ± 10.3
Albumin (g L−1)32 ± 132 ± 10.8
Globulin (g L−1)22 ± 318 ± 10.3
Alanine transaminase (U L−1)26.0 ± 2.323.5 ± 0.90.3
Aspartate transaminase (U L−1)83.6 ± 5.487.0 ± 9.40.8
Alkaline phosphatase (g L−1)43.3 ± 5.434.8 ± 3.60.3
Total bilirubin (μm)1.8 ± 0.31.7 ± 0.10.9
BUN (mm)6.8 ± 1.77.5 ± 1.80.4
Creatinine (μm)8.8 ± 0.08.8 ± 0.01.0
image

Figure 4. Short-term GSH depletion in young mice lowers NEFA oxidation, but does not alter glucose tolerance. (A) Effect of GSH depletion on NEFA oxidation; (B) Glycemic response to glucose tolerance test in GSH-replete and GSH-depleted mice. BSO = buthionine sulfoximine (inhibitor of GSH synthesis). Pre = basal data; Post = after receiving BSO for 4 weeks. Means are considered significant at P < 0.05.

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Correction of GSH deficiency in elderly humans restores fasted mitochondrial fuel oxidation and lowers insulin resistance and NEFA concentrations

After a 16-h fast, compared with young humans, GSH-deficient elderly humans had 41% lower whole-body NEFA oxidation and 116% higher carbohydrate oxidation (Fig. 5A), 110% higher plasma insulin concentrations (44.4 ± 4.8 vs. 93.0 ± 7.2 pm, P < 0.05), 92% higher plasma NEFA concentrations (Fig. 5B), and 160% higher insulin resistance as measured by HOMA-IR (Fig. 5C). GSH restoration in these subjects completely restored fasted whole-body NEFA and carbohydrate oxidation to values seen in younger controls (Fig. 5A). This was associated with 32% lower fasted plasma insulin concentrations (93.0 ± 7.2 vs. 63.6 ± 7.2 pm, P < 0.05), 36% lower fasted plasma NEFA concentrations (Fig. 5B), and 40% lower insulin resistance by HOMA-IR (Fig. 5C), without a significant decrease in their body weight. Cysteine and glycine supplementation in the elderly humans did not alter fasted liver profiles (total or direct bilirubin, alanine transaminase, aspartate transaminase, alkaline phosphatase) or concentrations of BUN, creatinine, LDL-cholesterol, or VLDL-cholesterol.

image

Figure 5. Glutathione repletion restores fasted NEFA and carbohydrate oxidation, and lowers NEFA concentrations and HOMA-IR in fasted elderly humans. (A) Fuel oxidation in fasted elderly humans improved with GSH repletion; (B) Elevated fasted plasma NEFA concentrations in elderly humans improve with GSH repletion; (C) Increased insulin resistance measured by HOMA in elderly humans improve with GSH repletion. NEFA = nonesterified fatty acids; CHO = carbohydrate. Means are considered significant at P < 0.05 (* = P < 0.05, θ = P < 0.01).

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. Author contributions
  9. References

Aging is associated with an increased risk of impaired mitochondrial NEFA oxidation and insulin resistance, but the underlying factors remain poorly understood. We provide novel evidence to support a beneficial role for the endogenous antioxidant GSH on these defects. Decreased GSH was associated with impaired mitochondrial NEFA oxidation and insulin resistance in both old mice and elderly humans, and these defects could be corrected with GSH restoration. Acute pharmacologic induction of GSH deficiency in young mice lowered NEFA oxidation, but did not blunt glucose tolerance, suggesting that chronic GSH deficiency may be required for the development of insulin resistance. These data suggest that chronic GSH deficiency contributes to impaired mitochondrial NEFA oxidation and insulin resistance in aging, and that these defects can be reversed with cysteine and glycine supplementation to restore GSH.

Where is the defect in NEFA oxidation in aging? Data from our studies point to a mitochondrial defect. In mitochondria, metabolism of NEFA and glucose results in the formation of acetyl-CoA, which is further oxidized to CO2 in the Krebs' cycle. A previous rodent study did not find any differences in the oxidation of acetyl-CoA (measured using a 13C-acetate tracer) between young and old mice (Mezzarobba et al., 2000), suggesting that Krebs' cycle function is intact in aging. Consistent with others (Weyer et al., 1999; Karnieli & Armoni, 2008), we also found that fasted carbohydrate oxidation is increased in elderly humans again suggesting that Krebs' cycle function is not impaired in aging. Therefore, the block in NEFA oxidation likely occurs prior to entry of acetyl-CoA into Krebs' cycle, which suggests a defect either in β-oxidation, impaired entry into mitochondria (via CPT1), or both. We found no differences in the transcript levels of liver or muscle CPT1 between GSH-deficient (O-CON) and GSH-replete (O-RX) old mice, even though mitochondrial NEFA oxidation was significantly higher in O-RX mice. Thus, the most parsimonious explanation for impaired NEFA oxidation in aging is a block in mitochondrial β-oxidation. Additional factors affecting NEFA oxidation include fatty acid translocase CD36 regulated cellular entry of NEFA (Abumrad et al., 1998; Febbraio et al., 1999) and regulation by PPARα and PGC1α. Hepatic Cd36 transcripts were lower in O-CON mice (with higher hepatic fat) and higher in O-RX mice (with lower hepatic fat). Could decreased NEFA oxidation occur due to decreased NEFA entry caused by lowered CD36, and not a mitochondrial defect? Evidence against this is indirect and comes from understanding factors governing net cellular regulation of fat, which depends on a balance between factors increasing cellular fat (cellular entry and de novo synthesis) and factors decreasing cellular fat (export and oxidation). Livers of O-CON mice had decreased cellular NEFA entry (lower CD36 expression) and lower NEFA synthesis (lower Fasn transcripts), but had higher cellular fat. This can only be explained by either decreased export of fat in VLDL-triacylglycerol (VLDL-TG), impaired mitochondrial NEFA oxidation, or both. Because fasted plasma triacylglycerols reflect VLDL-TG and were elevated in O-CON mice, it is unlikely that hepatic VLDL-TG export was impaired. Thus, the only remaining explanation is that mitochondrial NEFA oxidation was impaired. That the hepatic levels of Ppara and Pgc1a transcripts did not change despite an increase in NEFA oxidation in O-RX mice argues against a defect in the transcriptional regulation of NEFA oxidation and suggests post-transcriptional defect(s) in enzymes of NEFA oxidation caused by GSH deficiency, unopposed oxidative stress, or both and could involve defects in glutathionylation. In skeletal muscle, however, Cd36 and Pparα transcripts were lower in GSH-deficient older mice with decreased NEFA oxidation and increased in GSH restored old mice with higher NEFA oxidation. Although additional steps by which GSH or oxidative stress derails mitochondrial NEFA oxidation needs to be elucidated in more detail in future studies, the consequences of impaired NEFA oxidation and its correction in aging have important implications for metabolic flexibility, insulin resistance, total body fat, and hepatic fat accumulation in aging, and are discussed next.

Under physiological conditions, the fuel of choice in the fasted state is NEFA and not carbohydrate. In pioneering studies, Kelley & Simoneau (1994) identified that in diabetes, this fasted pattern of fuel oxidation is reversed, with impaired NEFA oxidation and increased glucose oxidation. This abnormal reversal of fasted fuel oxidation is also seen in aging (Calles Escandon et al., 1995; Rising et al., 1996; Toth et al., 1996; Weyer et al., 1999; Levadoux et al., 2001; Solomon et al., 2008). As muscle oxidizes NEFA, muscle loss in older humans has been thought to underlie impaired NEFA oxidation, as a quantitative defect. However, our finding that correcting GSH deficiency in elderly humans is able to fully restore a physiological pattern of fasted fuel oxidation within 14 days, suggests that this is a qualitative defect due to GSH deficiency. Because GSH improves fasted NEFA oxidation (which is a key determinant of metabolic flexibility), our findings suggest a role for GSH in regulating metabolic flexibility.

Insulin resistance in chronically GSH-deficient elderly humans and old mice improved significantly with GSH restoration. Interestingly, acute GSH depletion in young mice did not result in insulin resistance. These data suggest that a longer duration of GSH deficiency is required for insulin resistance to develop. The observation of significant improvement in insulin resistance in elderly humans without a decrease in their body weight suggests that GSH restoration improves insulin resistance independent of changes in body weight. The interplay between ROS and insulin sensitivity is complex. On one end, an obligate amount of ROS appears to be necessary for regulating insulin sensitivity and secretion (Goldstein et al., 2005; Pi et al., 2007; Iwakami et al., 2011), and overexpression of glutathione peroxidase-1 to severely deplete ROS (McClung et al., 2004) results in glucose intolerance. On the other end, elevated levels of reactive oxygen species (ROS) induce insulin resistance by impairing insulin signaling (Dokken et al., 2008; Mouzannar et al., 2011) and decreasing Glut4 transcription (Bloch-Damti & Bashan, 2005; Karnieli & Armoni, 2008). Therefore, a biological system has to maintain a delicate balance between GSH and ROS, and disturbances in either or both can result in insulin resistance. In the O-RX mice in our study, decreasing oxidative stress by restoring GSH improved Glut4 expression, suggesting that lowering excess levels of ROS was beneficial and improved insulin sensitivity. Other factors which could also have contributed to improving insulin sensitivity in O-RX mice include improvements in plasma NEFA and hepatic triacylglycerol concentrations (Bevilacqua et al., 1987; Bruce et al., 1994; Boden, 1997; Kelley & Mandarino, 2000; Corpeleijn et al., 2009). Our results are opposite those of a recent report that GSH depletion could improve insulin sensitivity (Findeisen et al., 2011), but that study did not measure or report GSH levels to support their conclusions (Nguyen & Sekhar, 2012). Supplementing diets with cysteine and glycine is an ideal approach to correct GSH deficiency and improve insulin sensitivity in aging, because this does not disturb cellular autoregulation of a critical balance between GSH and ROS in cells.

Aging is also associated with increased accumulation of total body fat and hepatic fat. Genetic manipulations to induce severe hepatic depletion of GSH have been shown to result in excessive accumulation of fat in the liver of mice (Chen et al., 2007), suggesting that GSH deficiency can promote fat accumulation. We found that old mice with GSH deficiency had significantly higher total body fat and hepatic fat compared with young mice, and that restoring GSH led to significant reductions in both total body fat and hepatic fat, suggesting a novel role for GSH in maintaining body and hepatic fat homeostasis. While GSH restoration in GSH-deficient old mice led to lower liver fat content, acute GSH depletion in young healthy mice did not increase liver fat, suggesting that a longer duration of GSH deficiency may be required for accumulation of hepatic fat, but GSH restoration lowers fat content more rapidly. Our elderly humans had a decreased ratio of fat-to-carbohydrate oxidation which predicts weight gain (Zurlo et al., 1990). GSH restoration in these subjects did not result in weight loss, which we attribute to the short 2-week duration of the study. Longer-term studies are needed to understand the effects of GSH restoration on body and hepatic fat in elderly humans.

Aging is associated with a true deficiency of glutathione, rather than entrapment of GSH in its oxidized form. Glutathione synthesis is catalyzed by glutamate–cysteine ligase (GCL) subunits GCLC and GCLM, and GSS, and their transcription is regulated by the nuclear antioxidant response element (Moinova & Mulcahy, 1999; Wild et al., 1999). Hepatic levels of Nrf2 (a cofactor regulating the antioxidant response to increased oxidative stress), Gclc, Gclm, and Gss transcripts were decreased in O-CON mice, suggesting a blunted antioxidant response to increased oxidative stress in aging. Cysteine and glycine supplementation in O-RX mice was associated with increased transcripts of Gclc and Gss, suggesting that these amino acids may also play a role in the regulation of glutathione synthesis. Concomitant with an increase in GSH concentrations, a reciprocal decline in TBARS could plausibly suggest an improvement in oxidative stress in our old mice.

Collectively, these data suggest a novel role for GSH in regulating NEFA oxidation and insulin resistance in aging. Acute GSH depletion results in decreased mitochondrial NEFA oxidation, but chronic GSH deficiency appears to be required for the additional development of insulin resistance, and these defects are reversed upon GSH restoration. Identifying GSH deficiency and correcting it with dietary precursor supplementation with cysteine and glycine holds the promise of a novel, practical, and innovative nutritional strategy to combat insulin resistance, obesity, and hepatic fat accumulation in elderly humans and warrants further study.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. Author contributions
  9. References

Mouse and human studies

Human studies were approved by the Institutional Review Board at Baylor College of Medicine and were conducted in accordance with the principles of the Declaration of Helsinki as revised in 2000. Mouse protocols were approved by the Institutional Animal Care and Use Committee, and adhered to the criteria outlined in the ‘Guide of the Care and Use of Laboratory Animals’.

GSH restoration study in old mice

Male 16-week (n = 8) and 76-week C57BL/6 mice (Taconic Labs, Hudson, NY) were studied for 6 weeks with final studies carried out at age 22 weeks and 82 weeks, respectively. Old mice were divided into control (O-CON) and treated (O-RX) groups (n = 8 in each group) matched for age, body weight, fasted NEFA oxidation, glucose tolerance, and insulin tolerance. Young and O-CON mice consumed a chow diet ad libitum (protein 25.6%, 3.0 kcal g−1 feed; Harlan Teklad, Indianapolis, IN) for 6 weeks. The O-RX mice were pair fed to O-CON mice using a diet supplemented with cysteine (as N-acetylcysteine) and glycine (protein 23.5%, 3.0 kcal g−1 feed, N-acetylcysteine 1.6 mg g−1 feed and glycine 1.6 mg g−1 feed; Harlan Teklad, Indianapolis, IN, USA) such that this diet was isocaloric and isonitrogenous to the diet of O-CON mice. Thus, O-CON and O-RX mice consumed identical calories and protein nitrogen per day. Weight-based doses of supplemental cysteine and glycine were chosen to approximate doses used in elderly humans (Sekhar et al., 2011). After the 6-week dietary protocol, whole-body NEFA oxidation, mitochondrial 13C1-palmitate oxidation, glucose and insulin tolerances, body weights and body composition were measured. Mice were then sacrificed and glutathione (oxidized and reduced) in liver and skeletal muscle, oxidative stress, liver and muscle fat content, plasma liver and renal profiles, and levels of key hepatic and muscle transcripts were measured.

GSH depletion study in mice

Male 16-week-old C57BL/6 mice (Taconic Labs, Hudson, NY, USA) were studied in two groups (n = 8 in each group) for 4 weeks. Both groups were matched for age, weight, glucose tolerance, and NEFA oxidation and were pair fed with regular chow diet (protein 25.6%, 3.0 kcal g−1 feed, Harlan Teklad), and drank equal volumes of water daily. One group also received L-buthionine-(S,R)-sulfoximine (BSO; Sigma-Aldrich, St. Louis, MO, USA) added to their drinking water (concentration 20 mm) to deplete GSH in liver and skeletal muscle. At 20 weeks, whole-body NEFA oxidation, 13C1-palmitate oxidation, lipid profiles, glucose tolerance, body composition, and body weights were measured. Mice were sacrificed, and liver and skeletal muscle glutathione and hepatic fat content were measured.

GSH restoration study in elderly humans

We previously reported GSH kinetics and oxidative stress in 8 young humans, and before and after 8 GSH-deficient elderly humans received dietary supplementation with cysteine (0.81 mmol kg day−1) and glycine (1.33 mmol kg day−1) for 2-weeks (Sekhar et al., 2011). These doses were well tolerated without alterations in liver or renal functions. However, changes in fuel oxidation, NEFA concentrations or insulin resistance were not previously reported and are presented here. All participants underwent indirect calorimetry after a 16 h fast to calculate NEFA and carbohydrate oxidation, and blood samples were collected for measuring fasting plasma concentrations of fatty acids, glucose, and insulin. Urine was collected over 8 h.

Glutathione

Concentrations of reduced and total GSH in mouse liver and skeletal muscle were measured by HPLC as described in detail by us previously (Sekhar et al., 2011). Briefly, after sacrifice, approximately 10–20 mg piece of liver was immediately weighed and homogenized in a monobromobimane buffer (containing 5 mm monobromobimane) and processed as described previously for red blood cells (Sekhar et al., 2011). Results are expressed per gram of tissue.

Whole-body NEFA oxidation

After acclimatization, mice fasted for 4 h underwent 8-h calorimetry (Oxymax; Columbus Instruments, Columbus, OH, USA) with measurement of oxygen consumption and carbon dioxide production per minute (gas exchange) for a period of 8 h. Under conditions to match the duration and timing of calorimetry on the following day, urine was collected in a mouse metabolic chamber (Harvard Apparatus, Holliston, MA, USA) for measurement of urine urea nitrogen which is used together with gas exchange data to calculate whole-body NEFA oxidation by the method of Frayn (Frayn, 1983).

13C1-palmitate oxidation

After entry into mitochondria, palmitic acid first undergoes β-oxidation with cleavage of 2-carbon fragments of acetyl-CoA, which enters the Krebs' cycle, and the carbon atom from the acetyl-CoA is released as CO2 in the second turn of the Krebs' cycle. We used this principle to develop a simple method where we used recovery of 13CO2 from injected 13C1-palmitate (Cambridge Isotope Laboratories, Andover, MA, USA) as an index of mitochondrial NEFA oxidation. Mice fasted for 4 h were injected with 13C1-palmitate (75 μmol kg−1) intraperitoneally and placed in a closed chamber for 4 h. CO2-free air was pumped in, and breath CO2 in the exiting air was trapped in alkali, released by acidification and collected in vacutainer tubes for measurement of 13CO2 enrichment by gas isotope ratio mass spectrometry (Europa Scientific, Crewe, UK).

13C1-Palmitate oxidation (%) = {[(inline image) × IECO2]/D} × 100, where inline image is the excretion of breath CO2, IECO2 is the isotopic enrichment of 13CO2 (atom% excess), and D is the dose of 99% enriched 13C1-palmitate tracer injected.

Glucose and insulin tolerance tests

Mice fasted for 4 h underwent glucose (GTT) or insulin (ITT) tolerance testing on separate days with intraperitoneal injections of glucose (1.5 g kg−1 or 8.3 μmol kg−1) or insulin (1.5 units kg−1 Humulin R; Eli Lilly and Company, Indianapolis, IN, USA), respectively (Samson et al., 2011). Tail vein blood was collected at 0, 15, 30, 60, 90, and 120 min from the injection measurement of glucose (GTT and ITT) and insulin concentrations (for GTT).

Plasma and urine biochemistry

Mouse plasma glucose, total protein, albumin, total bilirubin, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, blood urea nitrogen, creatinine, total cholesterol and triacylglycerol concentrations, and urine urea nitrogen were measured using the Roche Cobas Integra 400 automated system (Roche Diagnostics, Indianapolis, IN, USA). Plasma insulin concentrations were measured by ELISA (Mercodia Inc., Winston Salem, NC, USA). Insulin resistance was calculated using the homeostatic model assessment (HOMA) index as: fasting plasma insulin (μ IU mL−1) × fasting plasma glucose (mm)/22.4 plasma NEFA concentrations were measured by a spectrophotometric assay (Wako Chemicals, Neusse, Germany).

Body composition

Magnetic resonance imaging (Echo Medical System, Houston, TX, USA) was used at the beginning and end of the protocol, and body weights were measured weekly on a standard calibrated digital scale.

Reverse transcription polymerase chain reaction

Total RNA was isolated using the Aurum Mini RNA extraction kit (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer's instructions. RNA was subject to DNaseI digestion (Invitrogen, Grand Island, NY, USA) and reverse transcription with the Superscript III First Strand Synthesis System (Invitrogen) primed with oligo dT. Quantitative RT-PCR (qRT-PCR) was performed with iQ SYBR Green Supermix (Bio-Rad Laboratories) and Rox reference dye using a Mx3000P quantitative PCR machine (Stratagene). The cDNAs were subject to PCR as follows: one cycle at 95 °C for 5 min, 40 cycles of 95 °C for 1 min, 55 °C for 1 min and 72 °C for 1 min, and one cycle of 72 °C for 5 min. Primers were designed using the Primer 3 program (http://frodo.wi.mit.edu/primer3/input.htm) to cross an intron, to have a Tm of 58–62 °C, and produce an approximately 200 bp product. Primers were synthesized by Sigma Genosys and sequences are available on request. RNA levels are expressed relative to housekeeping genes cyclophilin (liver) and elongation factor 1 gamma and actin (muscle) (Samson et al., 2011).

Tissue triacylglycerol content

Total lipid was extracted from liver and gastrocnemius by the Bligh-Dyer method (Bligh & Dyer, 1959; Samson et al., 2011). The extracted triacylglycerols were quantified by colorimetric assay (Thermo Scientific Infinity, Middletown, VA, USA) and normalized to tissue weight.

Oil red O staining

Liver tissue was frozen at time of sacrifice in O.T.C. Compound (Sakura Finetek, Torrance, CA, USA). Sections (5–10 μm thick) were fixed with 10% formaldehyde, stained with oil red O (Sigma-Aldrich) (0.5% in propylene glycol), and counterstained with Mayer hematoxylin (Sigma-Aldrich). Images were captured using Axiovision 4.8.2 (Carl Zeiss Micro-Imaging, Berlin, Germany) at 20 × magnification.

Statistical analyses

Data are expressed as means ± SEM. An unpaired t-test was used to compute differences in means between the (i) control group and the BSO-treated group of mice (study 1); (ii) control group and old mice (study 2); (iii) GSH-deficient O-CON mice and GSH-replete O-RX mice (study 2); (iv) young and GSH-deficient elderly humans (study 3). A paired t-test was used to compare differences in outcome measures in elderly humans before and after supplementation (study 3). Data analysis was performed with the Statmate statistical software (GraphPad software, La Jolla, CA, USA). Results were considered to be statistically significant at P < 0.05.

Acknowledgments

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. Author contributions
  9. References

This study was supported by the Baylor College of Medicine Faculty Seed Award and the Baylor College of Medicine Alkek Bridge fund (to RVS), the Baylor College of Medicine General Clinical Research Center (NIH RR-0188), and the NIH-Diabetes and Endocrinology Research Center (NIH-P30DK079638). SS is supported by an American Diabetes Association Junior Faculty award and receives funds from the Baylor College of Medicine Alkek Bridge fund. We thank the Baylor adult GCRC nursing staff for adherence to the protocols in the human study. None of the authors have any conflicts of interest to report.

Author contributions

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. Author contributions
  9. References

DN contributed to animal studies, laboratory analyses, and to manuscript preparation. VTR contributed to animal studies and laboratory analyses. EG measured the liver triacylglycerol concentrations in mouse liver. SS contributed to measurement of liver and muscle triacylglycerols, gene expression analyses and data analyses in animal studies, and manuscript review and editing. RVS conceived the project, developed hypotheses, designed studies, and supervised protocols and analyses in mouse and human studies, analyzed and interpreted data analysis, and drafted the manuscript.

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  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. Author contributions
  9. References
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