Pyruvate and related energetic metabolites modulate resilience against high genetic risk for glaucoma

Abstract

A glaucoma polygenic risk score (PRS) can effectively identify disease risk, but some individuals with high PRS do not develop glaucoma. Factors contributing to this resilience remain unclear. Using 4,658 glaucoma cases and 113,040 controls in a cross-sectional study of the UK Biobank, we investigated whether plasma metabolites enhanced glaucoma prediction and if a metabolomic signature of resilience in high-genetic-risk individuals existed. Logistic regression models incorporating 168 NMR-based metabolites into PRS-based glaucoma assessments were developed, with multiple comparison corrections applied. While metabolites weakly predicted glaucoma (Area Under the Curve = 0.579), they offered marginal prediction improvement in PRS-only-based models (p=0.004). We identified a metabolomic signature associated with resilience in the top glaucoma PRS decile, with elevated glycolysis-related metabolites-lactate (p=8.8E-12), pyruvate (p=1.9E-10), and citrate (p=0.02)-linked to reduced glaucoma prevalence. These metabolites combined significantly modified the PRS-glaucoma relationship (P_interaction = 0.011). Higher total resilience metabolite levels within the highest PRS quartile corresponded to lower glaucoma prevalence (Odds Ratio_{highest vs. lowest total resilience metabolite quartile}=0.71, 95% Confidence Interval = 0.64-0.80). As pyruvate is a foundational metabolite linking glycolysis to tricarboxylic acid cycle metabolism and ATP generation, we pursued experimental validation for this putative resilience biomarker in a human-relevant Mus musculus glaucoma model. Dietary pyruvate mitigated elevated intraocular pressure (p=0.002) and optic nerve damage (p<0.0003) in Lmx1b^V265D mice. These findings highlight the protective role of pyruvate-related metabolism against glaucoma and suggest potential avenues for therapeutic intervention.

Keywords: cell biology; genetics; genomics; glaucoma; glaucoma resilience; metabolomics; mouse; pyruvate; tricarboxylic acid cycle.

Figure 1.. Participant flow chart describing inclusion and exclusion criteria from the UK Biobank.

Figure 2.. Study design from the UK Biobank.

(a) 117,698 individuals had metabolomics data available from the UK Biobank, which was divided into a training and test set to formulate a metabolic risk score (MRS) model. (b) The inclusion of metabolites (either 168 metabolites on the nuclear magnetic resonance (NMR) platform or a subset of 27 metabolites with European Union (EU) certification) in relation to prevalent glaucoma risk prediction was studied. (c) A histogram of the polygenic risk score (PRS) distribution is shown. Overall, 4,658 cases and 113,040 individuals without glaucoma are available for analysis. The metabolomic signature of resilience to the top 10% of glaucoma PRS was assessed among 1,693 cases (14.4%) and 10,077 individuals without glaucoma (85.6%). (d) Interactions of prevalent glaucoma with MRS and PRS quartiles were examined.

Figure 3.. Inclusion of metabolite data into glaucoma prediction algorithms.

Model 1 includes metabolites only; Model 2 incorporates additional covariates including age (years), sex, genetic ancestry, season, time of day of specimen collection, and fasting time; Model 3 incorporates covariates in Model 2 and smoking status (never, past, and current smoker), alcohol intake (g/week), caffeine intake (mg/day), physical activity (metabolic equivalent of task [MET], hours/week), body mass index (kg/m²), average systolic blood pressure (mm Hg), history of diabetes, HbA1c (mmol/mol), history of coronary artery disease, systemic beta-blocker use, oral steroid use, and spherical equivalent refractive error (diopters); Model 4 incorporates covariates in model 3 and a glaucoma polygenic risk score (PRS). Each color represents a different panel of metabolites (gray = no metabolites; light blue = 27 metabolites; and dark blue = 168 metabolites). The white text represents the AUC ± 95% confidence interval. Abbreviations: ROC, receiver operator curve; AUC, area under the curve; EU, European Union.

Figure 4.. The distribution of glaucoma cases and no glaucoma stratified by polygenic risk score (PRS) deciles.

Participants were divided into the bottom 50% and top 10% based on their glaucoma PRS, where the prevalence of glaucoma cases from (A) the bottom 50% (n=58,358) was 1.3% and from the top 10% (n=11,770) was 14.4%. (B) Box plot illustrating the distribution of participants with glaucoma (top) and no glaucoma (bottom) as a function of PRS decile. The blue line denotes participants at the bottom 50% of glaucoma PRS, the red line highlights the participants at the top decile of glaucoma PRS, and the red box represents the participants resilient to glaucoma despite high PRS.

Figure 5.. Interaction of three putative resilient metabolites (lactate, pyruvate, and citrate) and polygenic risk score (PRS) on glaucoma risk.

(A) The bar chart shows the interaction of resilient probit-transformed metabolite sum with glaucoma genetic predisposition in each PRS quartile. In each glaucoma PRS quartile, the lowest metabolic sum quartile (Q1) is the metabolite reference group used to calculate the odds ratios. Each color represents resilient-metabolite sum quartiles (red = second quartile; blue = third quartile; and green = fourth quartile). Error bars show 95% confidence interval (CI). The table under the bar chart shows the ranges for the PRS and metabolite sum value quartiles. (B) The table shows odds ratios for glaucoma by PRS, and putative resilient metabolite sum within various quartiles. The number of glaucoma cases within each resilient metabolite sum quartile and the number of glaucoma cases in the first quartile of resilient metabolite sum (Q1, labeled as glaucoma metabolite reference) are used to calculate the odd ratios. This analysis is adjusted for time since the last meal/drink (hours), age (years), age-squared (years-squared), sex, ethnicity (Asian, Black, White, and other), season, time of day of specimen collection (morning, afternoon, night), smoking status (never, past, and current smoker), alcohol intake, caffeine intake, physical activity (metabolic equivalent of task [MET] hours/week), body mass index (kg/m²), average systolic blood pressure (mm Hg), history of diabetes (yes or no), HbA1c (mmol/mol), history of coronary artery disease, systemic beta-blocker use, oral steroid use, and spherical equivalent refractive error (diopters).

Figure 6.. Interaction of the holistic metabolite risk score (n=168 metabolites) and polygenic risk score (PRS) on glaucoma risk.

(A) The bar chart plots the odds ratio of glaucoma as a function of holistic probit-transformed MRS quartile with further stratification by glaucoma PRS in each MRS bin. The lowest quartile of glaucoma PRS and MRS is the reference group (see dotted red line) for the entire population. Each color represents the MRS quartiles (red = first quartile; blue = second quartile; green = third quartile; and purple = fourth quartile). Error bars show the 95% confidence interval (CI). The table under the bar chart shows the ranges for the PRS and MRS quartiles. (B) Table showing odds ratios for glaucoma by polygenic risk score (PRS) and MRS within various quartiles. The number of glaucoma cases within each MRS and the number of glaucoma cases in PRS Q1 and MRS Q1 are used to calculate the odds ratios. This analysis is adjusted for time since the last meal/drink (hours), age (years), age-squared (years-squared), sex, ethnicity (Asian, Black, White, and other), season, time of day of specimen collection (morning, afternoon, night), smoking status (never, past, and current smoker), alcohol intake, caffeine intake, physical activity (metabolic equivalent of task [MET] hours/week), body mass index (kg/m²), average systolic blood pressure (mm Hg), history of diabetes (yes or no), HbA1c (mmol/mol), history of coronary artery disease, systemic beta-blocker use, oral steroid use, and spherical equivalent refractive error (diopters).

Figure 7.. Pyruvate treatment protects from intraocular pressure (IOP) elevation and glaucoma.

(A) Representative photos of eyes from mice of the indicated genotypes and treatments (Unt = untreated, Pyr = pyruvate treated). Lmx1b is expressed in the iris and cornea, so Lmx1b^V265D mutant eyes have primary abnormalities of the iris and cornea. This includes corneal haze, which is present before IOP elevation in many eyes and likely reflects a direct transcriptional role of LMX1B in collagen gene expression. Lmx1b^V265D mutant eyes also develop anterior chamber deepening (ACD), a sensitive indicator of IOP elevation in mice. The WT and pyruvate-treated mutant eyes have shallow anterior chambers, while the untreated mutant eye has a deepened chamber (arrowheads). (B) Distributions of ACD are based on a previously defined scoring system.³¹ Groups are compared by Fisher’s exact test. n > 40 eyes were examined in each group. (C) Boxplots of IOP (interquartile range and median line) in WT and mutant eyes. Pyruvate treatment significantly lessens IOP elevation in mutants compared to untreated mutant controls. Groups were compared by ANOVA followed by Tukey’s honestly significant difference. n > 30 eyes were examined in each Lmx1b^V265D mutant group, and n > 20 eyes were examined in WT groups. (D) Distributions of damage based on analysis of para-phenylenediamine (PPD)-stained optic nerve cross sections from 6-mo-old mice (Methods). Pyruvate treatment lessened the incidence of glaucoma (Fisher's exact test). No glaucoma was found in WT mice. Geno = genotype. n = 38-41 nerves examined per group. NOE = no glaucoma, MOD = moderate. SEV = severe, and V. SEV = very severe (see Methods).

Appendix 2—figure 1.. Interaction of the holistic metabolite risk score (n=168 metabolites) and polygenic risk score (PRS) on glaucoma risk.

(A) The bar chart shows the interaction of holistic probit-transformed metabolite risk score (MRS) with glaucoma genetic predisposition in each PRS quartile. In each glaucoma PRS quartile, the lowest metabolic sum quartile (Q1) is the metabolite reference group used to calculate the odds ratios. Each color represents the MRS quartiles (red = second quartile; blue = third quartile; and green = fourth quartile). Error bars show 95% confidence interval (CI). The table under the bar chart shows the ranges for the PRS and MRS quartiles. (B) Table showing odds ratios for glaucoma by polygenic risk score (PRS) and MRS within various quartiles. The number of glaucoma cases within each MRS quartile and the number of glaucoma cases in the first quartile of MRS (Q1), labeled as glaucoma metabolite reference, are used to calculate the odds ratios. This analysis is adjusted for time since the last meal/drink (hours), age (years), age-squared (years-squared), sex, ethnicity (Asian, Black, White, and other), season, time of day of specimen collection (morning, afternoon, night), smoking status (never, past, and current smoker), alcohol intake, caffeine intake, physical activity (metabolic equivalent of task [MET] hours/week), body mass index (kg/m²), average systolic blood pressure (mm Hg), history of diabetes (yes or no), HbA1c (mmol/mol), history of coronary artery disease, systemic beta-blocker use, oral steroid use, and spherical equivalent refractive error (diopters).

Appendix 2—figure 2.. Unadjusted levels of plasma metabolites lactate, pyruvate, and citrate in all UK Biobank participants (top row) and among the top 10% of glaucoma polygenic risk score (bottom row).

Author response image 1.