DOI: 10.5061/dryad.83bk3jb6x
title: Supplementary data from: Systems genomics reveals age- and sex-dependent metabolic dysregulation from Glo1 reduction in mice

description: Objectives: Glyoxalase 1 (Glo1) detoxifies reactive dicarbonyl compounds
 such as methylglyoxal, a precursor of advanced glycation end products
 (AGEs), which contribute to metabolic disorders. However, the contribution
 of AGE-independent mechanisms to Glo1-related metabolic dysfunction
 remains unclear. Methods: We conducted a longitudinal study in male and
 female Glo1 heterozygous knockdown (Glo1⁺/⁻) mice (~50% Glo1 expression).
 Metabolic phenotypes, including body weight, adiposity, glycemic control,
 and plasma lipid levels, were assessed over time. Atherosclerotic burden,
 AGE levels, and gene expression profiles in liver, adipose, muscle,
 kidney, and aorta were examined to identify pathway alterations and
 regulatory genes affected by Glo1 reduction. Results: Partial Glo1 loss
 resulted in obesity, hyperglycemia, dyslipidemia, and altered lipid
 metabolism in an age- and sex-dependent manner, with most phenotypes
 emerging after ~14 weeks. Glo1⁺/⁻ females exhibited impaired glycemic
 control and elevated triglycerides, along with perturbations in
 adipogenesis, PPARγ signaling, insulin signaling, and fatty acid
 metabolism in liver and adipose tissue. Glo1⁺/⁻ males displayed increased
 skeletal muscle mass and visceral adiposity with changes in lipid
 metabolic pathways. Methylglyoxal-derived AGE accumulation was altered
 only in male skeletal muscle and did not explain broader phenotypes.
 Transcriptomic analyses suggest altered glucose and lipid metabolism may
 be partially driven by alternative detoxification of methylglyoxal to
 metabolites such as pyruvate. Transcription factor analysis identified
 Hnf4a (across tissues) and Arntl (in aorta, liver, and kidney) as
 female-biased regulators altered by Glo1 deficiency. Conclusions: Glo1
 reduction disrupts metabolic health through sex- and age-dependent
 pathways largely independent of AGE accumulation, involving
 tissue-specific metabolic reprogramming and transcriptional regulation.
description: # Supplementary data from: Systems Genomics Reveals Age- and Sex-Dependent
 Metabolic Dysregulation from Glo1 Reduction in Mice Dataset DOI:
 [10.5061/dryad.83bk3jb6x](https://doi.org/10.5061/dryad.83bk3jb6x) ##
 Description of the data and file structure The data were collected from a
 mouse model of reduced glyoxalase 1 activity using heterozygous Glo1⁺/⁻
 knockdown mice and littermate controls. Animals were genotyped by PCR and
 followed longitudinally from weaning into adulthood with repeated
 metabolic phenotyping, including weekly body weight, biweekly NMR-based
 body composition, and serial intraperitoneal glucose tolerance tests;
 fasting plasma was collected at multiple ages for lipid, glucose, and
 insulin measurements. At study endpoints, key metabolic tissues (e.g.,
 liver, kidney, gonadal adipose, skeletal muscle, and aorta) were harvested
 for Glo1 enzyme activity assays, AGE quantification by ELISA (MGH1 and
 CEL), protein abundance by Western blot (e.g., Glo1 and RAGE), and
 targeted gene expression by qPCR. In parallel, atherosclerotic burden was
 assessed histologically by Oil Red O staining of aortic sinus sections,
 and a multi-tissue transcriptomic microarray dataset (Illumina MouseRef-8
 v2.0) was generated in 34-week-old female mice to identify differentially
 expressed genes for downstream pathway and integrative genomics analyses. 
 
 \- **Supplementary_Table_1.pdf** – qPCR primer sequences used in mouse
 genotyping and tissue gene-expression assays. *Legend:* **Supplementary
 Table 1.** List of qPCR mouse primers (forward and reverse) for Glo1/Gapdh
 and metabolic/AGE-related genes assayed by SYBR Green qPCR. \-
 **Supplementary_Table_2.pdf** – Results table for cross-species overlap
 analyses (mouse Glo1-regulated genes vs human sex-/age-biased programs).
 *Legend:* **Supplementary Table 2.** Overlap analysis between
 tissue-specific Glo1 differential expression signatures in mice and human
 sex-biased and age-associated gene sets from GTEx and GenAge. The table
 reports overlapping gene symbols, counts, Fisher’s exact test p-values,
 odds ratios, and background/comparator set sizes. \-
 **Supplementary_Figure_1.pdf** – Validation of the Glo1 knockdown
 (Glo1⁺/⁻) mouse model at the DNA and protein levels. *Legend:*
 **Supplementary Figure 1.** (A) PCR-based genotyping showing the internal
 control (Gapdh; top band) and the Glo1 knockdown-specific band (~220 bp;
 bottom band), with WT lacking the KD band. (B) Representative immunoblots
 showing Glo1 protein levels in male and female WT vs Glo1 KD mice with
 actin as a loading control (examples of KD/WT shown). \-
 **Supplementary_Figure_2.pdf** – Longitudinal monitoring of food and water
 intake by sex and genotype. *Legend:* **Supplementary Figure 2.** Weekly
 chow and water intake measurements in WT and Glo1 KD mice across
 development. (A–B) Female weekly chow (g) and water (mL) intake. (C–D)
 Male weekly chow (g) and water (mL) intake. Intake was recorded weekly to
 track caloric/fluid consumption across experimental groups. \-
 **Supplementary_Figure_3.pdf** – Glucose tolerance testing across ages in
 male mice. *Legend:* **Supplementary Figure 3.** Intraperitoneal glucose
 tolerance tests (IPGTT) in male WT vs Glo1 KD mice at multiple ages. (A)
 IPGTT area under the curve (AUC) at 5, 12, 23, and 33 weeks. (B–E) Glucose
 curves (0–120 min) following IP glucose challenge at the indicated ages,
 with reported p-values for genotype, time, and genotype×time effects. \-
 **Supplementary_Figure_4.pdf** – Fasting plasma glucose and insulin across
 ages, separated by sex. *Legend:* **Supplementary Figure 4.** Fasting
 plasma glucose and insulin concentrations in WT and Glo1 KD mice at 7, 12,
 and 28 weeks. (A–B) Female glucose (mg/dL) and insulin (pg/mL). (C–D) Male
 glucose (mg/dL) and insulin (pg/mL). Measurements were obtained from
 overnight-fasted animals. \- **Supplementary_Figure_5.pdf** – Additional
 fasting lipid measures across ages, separated by sex. *Legend:*
 **Supplementary Figure 5.** Fasting plasma lipid measurements in WT and
 Glo1 KD mice at 7, 12, and 28 weeks. (A–C) Female unesterified cholesterol
 (UC), free fatty acids (FFA), and LDL cholesterol. (D–F) Male UC, FFA, and
 LDL cholesterol. \- **Supplementary_Figure_6.pdf** – AGE/RAGE inflammatory
 signaling schematic and liver pathway-level expression changes. *Legend:*
 **Supplementary Figure 6.** (A) Schematic of AGE–RAGE signaling leading to
 ROS activation, NFKB signaling, and downstream inflammatory/apoptotic
 programs. (B) Heatmaps of liver gene expression changes (WT vs Glo1 KD)
 across functional categories, including ROS/NFKB signaling, apoptosis
 regulators, cell-cycle regulators, cytokines, chemokines, and adhesion
 molecules. \- **Supplementary_Figure_7.pdf** – Differential expression of
 key glucose metabolism enzymes in adipose and aorta. *Legend:*
 **Supplementary Figure 7.** Heatmap of log2 fold-change (Glo1 KD vs WT)
 for selected glycolysis/gluconeogenesis enzymes in adipose and aorta.
 Asterisks denote statistical significance (* p&lt;0.05, ** p&lt;0.01, ***
 p&lt;0.001).