Metabolic Profiling in Human Fibroblasts Enables Subtype Clustering in Glycogen Storage Disease

Abstract

Glycogen storage disease subtypes I and III (GSD I and GSD III) are monogenic inherited disorders of metabolism that disrupt glycogen metabolism. Unavailability of glucose in GSD I and induction of gluconeogenesis in GSD III modify energy sources and possibly, mitochondrial function. Abnormal mitochondrial structure and function were described in mice with GSD Ia, yet significantly less research is available in human cells and ketotic forms of the disease. We hypothesized that impaired glycogen storage results in distinct metabolic phenotypes in the extra- and intracellular compartments that may contribute to pathogenesis. Herein, we examined mitochondrial organization in live cells by spinning-disk confocal microscopy and profiled extra- and intracellular metabolites by targeted LC-MS/MS in cultured fibroblasts from healthy controls and from patients with GSD Ia, GSD Ib, and GSD III. Results from live imaging revealed that mitochondrial content and network morphology of GSD cells are comparable to that of healthy controls. Likewise, healthy controls and GSD cells exhibited comparable basal oxygen consumption rates. Targeted metabolomics followed by principal component analysis (PCA) and hierarchical clustering (HC) uncovered metabolically distinct poises of healthy controls and GSD subtypes. Assessment of individual metabolites recapitulated dysfunctional energy production (glycolysis, Krebs cycle, succinate), reduced creatinine export in GSD Ia and GSD III, and reduced antioxidant defense of the cysteine and glutathione systems. Our study serves as proof-of-concept that extra- and intracellular metabolite profiles distinguish glycogen storage disease subtypes from healthy controls. We posit that metabolite profiles provide hints to disease mechanisms as well as to nutritional and pharmacological elements that may optimize current treatment strategies.

Untargeted plasma metabolomics identifies broad metabolic perturbations in glycogen storage disease type I

“…The role of glutathione in the pathophysiology of GSDI, especially with regard to hepatic tumorgenesis is not clearly defined yet. Interestingly, decreased glutathione levels have been detected in human GSDI fibroblasts, although these cells do not represent the organs primarily affected by the metabolic defect and do not express glucose‐6‐phosphatase‐α 45 …”

Utilization of Human Samples for Assessment of Mitochondrial Bioenergetics: Gold Standards, Limitations, and Future Perspectives

“…Patient fibroblasts provide an important research tool to measure mitochondrial bioenergetics as well as a full characterization of mitochondria including, but not limited to, mitochondrial morphology, turnover, and in-depth analysis of mitochondrial metabolism [86,87]. For research purposes, commercially available fibroblasts can be obtained for different mitochondrial disorders and diseases of aging.…”

Micro-Meta App: an interactive software tool to facilitate the collection of microscopy metadata based on community-driven specifications

“…In response to significant interest we observed in the community and after beta testing, microscope custodians at several light microscopy facilities (Supplemental Table III) volunteered to serve as case studies on the use of Micro-Meta App to document both microscope instrumentation and example published image datasets produced in microscopy platforms [59][60][61][62][63][64][65][66][67][68][69][70][71][72][73][74][75][76][77][78] . The application was utilized by microscope custodians at 16 sites to document one of their microscopes alongside the settings utilized for published images based on the 4DN-BINA-OME model Core + Basic Extension 37,38,51 (Figure 5 and Supplemental Figures 2-16).…”