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. 2015 Mar 19;10(3):e0118436.
doi: 10.1371/journal.pone.0118436. eCollection 2015.

Investigating the Causes for Decreased Levels of Glutathione in Individuals With Type II Diabetes

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Free PMC article

Investigating the Causes for Decreased Levels of Glutathione in Individuals With Type II Diabetes

Minette Lagman et al. PLoS One. .
Free PMC article

Abstract

Tuberculosis (TB) remains an eminent global burden with one third of the world's population latently infected with Mycobacterium tuberculosis (M. tb). Individuals with compromised immune systems are especially vulnerable to M. tb infection. In fact, individuals with Type 2 Diabetes Mellitus (T2DM) are two to three times more susceptible to TB than those without T2DM. In this study, we report that individuals with T2DM have lower levels of glutathione (GSH) due to compromised levels of GSH synthesis and metabolism enzymes. Transforming growth factor beta (TGF-β), a cytokine that is known to decrease the expression of the catalytic subunit of glutamine-cysteine ligase (GCLC) was found in increased levels in the plasma samples from individuals with T2DM, explaining the possible underlying mechanism that is responsible for decreased levels of GSH in individuals with T2DM. Moreover, increased levels of pro-inflammatory cytokines such as interleukin-6 (IL-6) and interleukin-17 (IL-17) were observed in plasma samples isolated from individuals with T2DM. Increased levels of IL-6 and IL-17 was accompanied by enhanced production of free radicals further indicating an alternative mechanism for the decreased levels of GSH in individuals with T2DM. Augmenting the levels of GSH in macrophages isolated from individuals with T2DM resulted in improved control of M. tb infection. Furthermore, cytokines that are responsible for controlling M. tb infection at the cellular and granuloma level such as tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), interleukin-2 (IL-2), interferon-gamma (IFN-γ), and interleukin-12 (IL-12), were found to be compromised in plasma samples isolated from individuals with T2DM. On the other hand, interleukin-10 (IL-10), an immunosuppressive cytokine was increased in plasma samples isolated from individuals with T2DM. Overall, these findings suggest that lower levels of GSH in individuals with T2DM lead to their increased susceptibility to M. tb infection.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist. This research was partially funded by Your Energy Systems. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials. None of the authors are affiliated to this company.

Figures

Fig 1
Fig 1. Assay of total and rGSH in RBCs, plasma, and monocytes from healthy subjects and individuals with T2DM.
RBCs, plasma, and monocytes were isolated by density centrifugation from peripheral blood drawn from healthy volunteers and individuals with T2DM. GSH assay was performed using a colorimetric assay kit from Arbor Assays. Total GSH in RBCs isolated from individuals with T2DM was significantly lower compared to the healthy individuals (Fig. 1A). There was an observable decrease in rGSH in RBCs isolated from individuals with T2DM compared to healthy volunteers (Fig. 1B). Total GSH in plasma samples from individuals with T2DM was significantly lower compared to the healthy individuals (Fig. 1C). There was a significant decrease in rGSH in plasma samples from individuals with T2DM compared to healthy volunteers (Fig. 1D). Total GSH in monocyte samples isolated from individuals with T2DM was significantly lower compared to total GSH measured in monocytes from healthy individuals (Fig. 1E). There was a significant decrease in rGSH in monocyte samples isolated from individuals with T2DM compared to healthy volunteers (Fig. 1F). Data represent means ±SE from 10 healthy individuals and 10 individuals with T2DM.
Fig 2
Fig 2. Assay of TGF-β levels in plasma and western blot analysis of a GSH de novo synthesis enzyme, GCLC, in RBCs from healthy subjects and individuals with T2DM.
Western blot results were corrected for GAPDH and reported as relative intensity compared to GAPDH (Fig. 2A). Results show significantly lower levels of GCLC measured in RBCs isolated from individuals with T2DM compared to healthy subejcts (A; n = 9 healthy, n = 9 T2DM) and B; n = 9 healthy, n = 9 T2DM). Fig. 2B illustrates western blot images of GCLC and GAPDH in RBCs isolated from healthy subjects and individuals with T2DM. Assay of TGF-β was performed using an ELISA Ready-Set-Go kit from eBioscience (Fig. 2C). There was a significant increase in TGF-β production in plasma samples isolated from individuals with T2DM with HbA1c>8 compared to healthy volunteers (n = 5).
Fig 3
Fig 3. Western blot analysis of a GSH de novo synthesis enzyme, GSS, in RBCs of individuals with T2DM compared to healthy subjects.
Western blot results were corrected for GAPDH and reported as relative intensity compared to GAPDH (Fig. 3A). Significant decrease in the levels of GSS was observed in RBCs isolated from individuals with T2DM compared to healthy volunteers. (A; n = 9 healthy, n = 9 T2DM and B; n = 9 healthy, n = 9 T2DM). Fig. 3B illustrates western blot images of GSS and GAPDH in RBCs isolated from healthy subjects and individuals with T2DM.
Fig 4
Fig 4. Western blot analysis of a GSH enzyme, GGT, in RBCs from healthy subjects and individuals with T2DM.
Western blot results were corrected for GAPDH and reported as relative intensity compared to GAPDH (Fig. 4A). In comparison to healthy subjects, we observed significantly lower levels of GGT in RBCs isolated from individuals with T2DM (A; n = 9 healthy, n = 7 T2DM and B; n = 9 healthy, n = 7 T2DM). Fig. 4B illustrates western blot images of GGT and GAPDH in RBCs isolated from healthy subjects and individuals with T2DM.
Fig 5
Fig 5. Western blot analysis of a GSH recycling enzyme, GSR, in RBCs from healthy subjects and individuals with T2DM.
Western blot results were corrected for GAPDH and are reported as relative intensity compared to GAPDH (Fig. 5A). In comparison to healthy subjects, we observed a significant increase in the levels of GSR in RBCs from individuals with T2DM (A; n = 9 healthy, n = 9 T2DM and B; n = 9 healthy, n = 9 T2DM). Fig. 5B illustrates western blot images of GGT and GAPDH in RBCs isolated from healthy subjects and individuals with T2DM.
Fig 6
Fig 6. Assay of IL-6 and IL-17 in healthy subjects and individuals with T2DM (Fig. 6A and B) and Assay of MDA in healthy subjects and individuals with T2DM (Fig. 6C, D, and E).
Assay of IL-6 (Fig. 6A) and IL-17 (Fig. 6B) were performed using an ELISA Ready-Set-Go kit from eBioscience. IL-6 and IL-17 levels were significantly higher in plasma samples from individuals with T2DM. Results on IL-6 and IL-17 represent means ±SE from 10 healthy individuals and 10 individuals with T2DM. MDA assay was performed using a TBARS kit from Cayman Chemical. There was a statistically significant and two-fold increase in the levels of MDA in plasma samples from individuals with T2DM compared to healthy volunteers (Fig. 6C). There was an observable increase in the levels of MDA in monocytes isolated from individuals with T2DM compared to healthy volunteers (Fig. 6D). MDA levels were also assayed in RBCs isolated from individuals with T2DM and healthy subjects. Individuals with T2DM with HbA1c>8 had significantly higher levels of MDA compared to healthy individuals (Fig. 6E).
Fig 7
Fig 7. Measurement of ROS in CD14+ cells, CD4+ T-cells, and CD8+ T-cells by cellROX stain mean intensity in T2DM patients compared to healthy.
CD14+cells were stained with cellROX green reagent, a marker of ROS, and a CD14 cell marker, CD14-PE. CD14+-ROX+ cells’ mean intensity was analyzed by FLOW cytometry. There was an observable increase in ROX mean intensity in CD14+ cells isolated from individuals with T2DM compared to healthy volunteers (Fig. 7A). CD4+cells were also stained with cellROX green reagent and a CD4 cell marker, CD4-Cy5. CD4+-ROX+ cells’ mean intensity was analyzed by FLOW cytometry. There was an observable increase in ROX mean intensity in CD4+ T-cells isolated from individuals with T2DM compared to healthy volunteers (Fig. 7B). CD8+cells were stained with cellROX green reagent and a CD8 cell marker, CD8-Cy5. CD8+-ROX+ cells’ mean intensity was analyzed by FLOW cytometry. There was an observable increase in ROX mean intensity in CD8+ T-cells isolated from individuals with T2DM compared to healthy volunteers (Fig. 7C). Data represents mean ±SE from 5 healthy individuals and 5 individuals with T2DM.
Fig 8
Fig 8. Assay of GSSG in RBCs, plasma, and monocytes isolated from healthy subjects and individuals with T2DM.
GSSG was measured using a GSH colorimetric assay from Arbor Assays. We observed two-fold increase in levels of GSSG in RBCs isolated from individuals with T2DM with HbA1c>8 compared to healthy volunteers (Fig. 8A). There was an observable increase in levels of GSSG in plasma samples isolated from individuals with T2DM with HbA1c>8 compared to healthy volunteers (Fig. 8B). We also observed a significant eight-fold increase in the levels of GSSG in monocytes isolated from individuals with T2DM compared to healthy volunteers (Fig. 8C). Data represent means ±SE from 10 healthy individuals and 10 individuals with T2DM.
Fig 9
Fig 9. Intracellular survival of H37Rv in macrophages from healthy volunteers and individuals with T2DM.
Macrophages were infected with H37Rv, a laboratory strain of M.tb, with multiplicity of infection 10:1. One hour post-infection, infected macrophageswere treated with 10 mM and 20 mM NAC and 10 μM and 20 μM lGSH. Infected macrophages were terminated at 1h and 5 days post-infection. Lysates were plated on 7H11 medium with ADC to determine M.tb growth. Data represent means ±SE from 5 healthy individuals (Fig. 9A) and 5 individuals with T2DM (Fig. 9B). Experiments were performed in triplicates. *p<0.05 when comparing H37Rv-infected macrophages 5 day time point results to any other 5 day time point categories while #p<0.05 when comparing 1 hour and 5 day time points of the same treatment category.
Fig 10
Fig 10. Assay of IL-10 and TNF-α in the plasma samples from healthy subjects and individuals with T2DM.
Assay of IL-10 and TNF-α, in plasma was performed using an ELISA Ready-Set-Go kit from eBioscience. Data represent means ±SE, *p<0.05. There was two-fold increase in IL-10 (Fig. 10A) in plasma samples isolated from individuals with T2DM compared to healthy volunteers (n = 10 healthy, n = 10 T2DM). TNF-α was significantly lower in plasma samples isolated from individuals with T2DM and HbA1c>8 compared to healthy volunteers (Fig. 10B n = 10 healthy, n = 5 T2DM).
Fig 11
Fig 11. Assay of IL-1β, IL-2, IFN-γ, and IL-12 levels, in plasma samples from healthy subjects and individuals with T2DM.
Plasma was isolated from peripheral blood of healthy volunteers and individuals with T2DM. Assay of IL-1β (Fig. 11A), IL-2 (Fig. 11B), IFN-γ (Fig. 11C), and IL-12 (Fig. 11D) levels, in plasma was performed using an ELISA Ready-Set-Go kit from eBioscience. Data represent means ±SE from 10 healthy individuals and 10 individuals with T2DM except panel B in which the sample size for individuals with T2DM and HbA1c>8 is n = 5.

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References

    1. Restrepo BI, Camerlin AJ, Rahbar MH, Wang W, Restrepo MA, et al. (2011) Cross-sectional assessment reveals high diabetes prevalence among newly-diagnosed tuberculosis cases. Bull World Health Organ 89: 352–359. 10.2471/BLT.10.085738 - DOI - PMC - PubMed
    1. World Health Organization (2014) Tuberculosis [Fact sheet].
    1. World Health Organization (2011) Tuberculosis & Diabetes [Fact sheet].
    1. American Diabetes Association (2013) Data and Statistics about Diabetes [Fact sheet].
    1. Center for Disease Control (2014) National Diabetes Statistics Report [Brochure].

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This work was supported in part by Western University of Health Sciences and Your Energy Systems to VV. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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