Glucose metabolism and pyruvate carboxylase enhance glutathione synthesis and restrict oxidative stress in pancreatic islets

Abstract

Glucose metabolism modulates the islet β cell responses to diabetogenic stress, including inflammation. Here, we probed the metabolic mechanisms that underlie the protective effect of glucose in inflammation by interrogating the metabolite profiles of primary islets from human donors and identified de novo glutathione synthesis as a prominent glucose-driven pro-survival pathway. We find that pyruvate carboxylase is required for glutathione synthesis in islets and promotes their antioxidant capacity to counter inflammation and nitrosative stress. Loss- and gain-of-function studies indicate that pyruvate carboxylase is necessary and sufficient to mediate the metabolic input from glucose into glutathione synthesis and the oxidative stress response. Altered redox metabolism and cellular capacity to replenish glutathione pools are relevant in multiple pathologies beyond obesity and diabetes. Our findings reveal a direct interplay between glucose metabolism and glutathione biosynthesis via pyruvate carboxylase. This metabolic axis may also have implications in other settings where sustaining glutathione is essential.

Keywords: ROS; glucose; glutathione; inflammation; nitrosative stress; oxidative stress; pancreatic islets; pyruvate carboxylase.

Figure 1.. Metabolic profiling predicts increased antioxidant potential by phospho-BAD mimicry in human islets undergoing inflammation stress

Quantification of metabolites related to glutathione (GSH) metabolism, including synthesis (methionine, cysteine, serine, glycine, and glutamate) and recycling (oxoproline and cysteinylglycine) pathways in human islets cultured in Prodo Islet Media (Standard) (PIM(S)) media containing 5.8 mM glucose and treated for 24 h with inflammatory cytokines (10 ng/mL TNF-α, 10 ng/mL IL-1β, and 100 ng/mL IFNγ). Values are shown relative to vehicle control (veh) PBS set to 1. All metabolites except GSH, cystathionine, cysteine, γ-glutamylcysteine, and S-adenosylmethionine (SAM) were measured by untargeted metabolomics using human islets from n = 5 donors pooled and split into 8 replicates. For targeted measurement of GSH, cystathionine, cysteine, γ-glutamylcysteine, and SAM, islets from n = 5 donors were used. Single arrows denote direct reactions; two arrows indicate multi-step reactions. Abbreviations are as follows: AHCY, adenosylhomocysteinase; BHMT, betaine-homocysteine S-methyltransferase; CBS, cystathionine beta-synthase; CDO, cysteine dioxygenase type 1; CSAD, cysteine sulfinic acid decarboxylase; CTH, cystathionine gamma-lyase; GCLC/M, glutamate-cysteine ligase; GGCT, gamma-glutamylcyclotransferase; GGT, gamma-glutamyltransferase; GNMT, glycine N-methyltransferase; GPX, GSH peroxidase; GSS, GSH synthase; GSR, GSH-disulfide reductase; KYAT, kynurenine aminotransferase; MAT, methionine adenosyltransferase; OPLAH, 5-oxoprolinase ATP-hydrolyzing; PSPH, phosphoserine phosphatase; SAH, S-adenosylhomocysteine. Data are represented as means ± SEM. Statistical analyses are one-way ANOVAs with comparisons of cytokine-treated cells to PBS control. See also Figure S1 and Tables S1 and S2.

Figure 2.. Glucose metabolism supports GSH synthesis in human islets

(A) Glucose-tracing studies in human islets measuring total ¹³C incorporation from [¹³C₆]-glucose into GSH [¹³C]-GSH and unlabeled GSH, [¹²C]-GSH. Human islets were treated with veh, BAD SAHB_A SD, or BAD SAHB_A AAA in the presence of PBS or a pro-inflammatory cytokine cocktail (TNF-α, IL-1β, and IFNγ) and labeled in RPMI containing 5.8 mM [¹³C₆]-glucose for 24 h (n = 3). (B) Relative labeling of [¹³C]-GSH out of the total sum of GSH pool [¹³C+¹²C] from experiment in (A) normalized to veh PBS. (C) Potential labeling routes and resultant GSH isotopologs from [¹³C₆]-glucose. Only isotopologs from direct incorporation routes are shown without considering multiple rounds of synthesis, recycling steps of GSH, and/or label dilution. (D) Non-normalized total fractional labeling for each mass isotopolog of GSH (M+n), including unlabeled GSH (M+0), quantified in experiment (A). Data are represented as means ± SEM. *p < 0.05; **p < 0.005; n.s., non-significant via two-way ANOVA with Tukey’s adjustment for multiple comparisons.

Figure 3.. The effect of phospho-BAD mimicry on redox coupling in human islets undergoing inflammation stress

(A) Ratios of GSH/GSSG, cysteine/cystine, and pyruvate/malate measured as in Figure 1. (B) Quantification (left) and representative images (right) of ratiometric fluorescence intensity in re-aggregated human islets expressing cytosolic Grx1-roGFP2 that were treated with veh or BAD SAHB_A SD in the presence of PBS or cytokines for 24 h (n = 4). Scale bars, 100 μm at 20× magnification to capture the entire re-aggregated islet without subcellular resolution. Ratiometric images were generated by dividing images captured at 405 nm (oxidized) by those taken at 488 nm (reduced), thus keeping spatial information of the ratio intensities and applying an arbitrary color intensity scale onto the calculated images. (C) Quantification (left) and representative images (right) of ratiometric fluorescence intensity in re-aggregated human islets expressing cytosolic Orp1-roGFP2 that were treated with veh or BAD SAHB_A SD in the presence of PBS or cytokines for 24 h (n = 4). Image outputs calculations were performed as in (B). Data are represented as means ± SEM. *p < 0.05; **p < 0.005; ***p < 0.0005; n.s., non-significant via two-way ANOVA with Tukey’s adjustment for multiple comparisons. See also Figure S2.

Figure 4.. The BAD-GK axis restrains inflammation-induced ROS accumulation by supporting GSH synthesis

(A and B) ROS levels measured as %2’,7’-dichlorodihydrofluorescein diacetate (DCFDA)-positive cells normalized to the maximal signal determined with H₂O₂ in veh-, BAD SAHB_A SD-, or BAD SAHB_A AAA-treated human (A) and mouse (B) islets cultured in the presence of PBS or cytokines for 24 h (n = 4). (C and D) ROS levels in human (C) and mouse (D) islets expressing vector control (VC) or BAD SD that were treated for 24 h with PBS or cytokines in the presence or absence of BSO (n = 5, and n = 3, respectively). (E) ROS levels in mouse islets subjected to GCLC knockdown using two independent shRNAs and treated with veh or BAD SAHB_A SD in the presence of PBS or cytokines for 24 h (n = 4). (F) Viability of mouse islets subjected to phospho-BAD mimicry and GCLC knockdown as in (E) and treated with cytokines for 48 h. Viability was assessed by flow cytometric measurement of Annexin V/7-Aminoactinomycin D (7AAD) incorporation (n = 4). Data are represented as means ± SEM. *p < 0.05; **p < 0.005; ***p < 0.0005; n.s., non-significant via two-way ANOVA with Tukey’s adjustment for multiple comparisons. Glucose concentration was 5.8 mM in PIM(S) media for all human islet cultures and 11 mM in RPMI for all mouse islet cultures. See also Figure S3.

Figure 5.. PC protects against oxidative stress triggered by inflammation

(A and B) The effect of PC knockdown on ROS levels in human (A) and mouse (B) islets treated with veh or BAD SAHB_A SD and cultured in the presence of cytokines for 24 h. Rescue with shRNA-resistant PC cDNA (PC) compared with VC validates the on-target effects of PC shRNA (n = 5). (C) Quantification of ROS levels in mouse islets treated as in (B) in the presence of N-acetylcysteine (NAC) or veh for NAC (H₂O). Values are normalized to PBS-treated shRNA control samples (shCtrl) (n = 3). (D) Quantification of [¹³C₆]-glucose-derived GSH as non-normalized total fractional labeling for each mass isotopolog of GSH (M+n), including unlabeled GSH (M+0). Human islets were treated with veh or BAD SAHB_A SD and transduced with control (Ctrl) or PC shRNA lentiviruses in the presence of cytokines and labeled in media containing 5.8 mM [¹³C₆]-glucose for 24 h (n = 3). Inset magnifies the quantification of M+2 GSH. C and P on the y axis denote Ctrl and PC knockdown, respectively. (E) Relative labeling of [¹³C]-GSH out of the total sum of GSH pool [¹³C+¹²C] from experiment in (D) normalized to veh PBS. (F) ROS levels in human islets transduced with lentiviruses carrying Ctrl or PC shRNA and treated with PBS or cytokines in the absence (H₂O) or presence of NAC (n = 4). (G) ROS levels in human islets transduced with VC- or human PC (PC)-expressing lentiviruses in the presence of PBS or cytokines for 24 h (n = 5). (H) The effect of PC overexpression on the viability of human islets treated with cytokines for 48 h and its reversal upon inhibition of GSH synthesis with BSO. Values are shown relative to VC PBS samples (n = 3). Data are represented as means ± SEM. *p < 0.05; **p < 0.005; ***p < 0.0005; n.s., non-significant via one-way (A and B) or two-way (C–H) ANOVA with Tukey’s adjustment for multiple comparisons. See also Figure S4.

Figure 6.. PC is required for maintenance of GSH pools and protection from nitrosative stress

(A) GSH levels and GSH/GSSG ratios in vehicle- and BAD SAHB_A SD-treated mouse islets exposed to the NO donor (GEA3162 dissolved in DMSO) for 24 h as measured by quantitative colorimetric assays. Data are normalized to protein levels with vehicle DMSO set to 100% (n = 6). (B) ROS levels in mouse islets treated with veh, BAD SAHB_A SD, or BAD SAHB_A AAA and cultured in the presence of the NO donor for 24 h (n = 4). (C) Viability of mouse islets treated with the indicated compounds followed by 48 h of cytokine treatment in the absence or presence of BSO (n = 3). (D) ROS levels in islets expressing VC or BAD SD treated as in (C) (n = 3). (E and F) The effect of PC knockdown on ROS (E) and viability (F) of human islets treated with the NO donor for 24 h (E) or 48 h (F). Rescue with shRNA-resistant PC cDNA validates the on-target effects of PC shRNA (n = 4 and n = 5 for ROS and viability measurements, respectively). (G) The effect of PC knockdown on GSH levels and GSH/GSSG ratios in human islets exposed to the NO donor for 24 h as measured by mass spectrometry. Values are relative to vehicle DMSO-treated islets set to 1 (n = 5). (H) Protective effects of BAD:GK:PC axis in countering inflammation and oxidative/nitrosative stress. Data are represented as means ± SEM. *p < 0.05; **p < 0.005; ***p < 0.0005; n.s., non-significant via one-way ANOVA (A, B, and E–G) or two-way ANOVA (C and D) with Tukey’s adjustment for multiple comparisons.