Glycogen dysregulation is a hallmark of aging, and aberrant glycogen drives metabolic reprogramming and pathogenesis in multiple diseases. However, glycogen heterogeneity in healthy and diseased tissues remains largely unknown. Herein, we describe a method to define spatial glycogen architecture in mouse and human tissues using matrix-assisted laser desorption/ionization mass spectrometry imaging. This assay provides robust and sensitive spatial glycogen quantification and architecture characterization in the brain, liver, kidney, testis, lung, bladder, and even the bone. Armed with this tool, we interrogated glycogen spatial distribution and architecture in different types of human cancers. We demonstrate that glycogen stores and architecture are heterogeneous among diseases. Additionally, we observe unique hyperphosphorylated glycogen accumulation in Ewing sarcoma, a pediatric bone cancer. Using preclinical models, we correct glycogen hyperphosphorylation in Ewing sarcoma through genetic and pharmacological interventions that ablate in vivo tumor growth, demonstrating the clinical therapeutic potential of targeting glycogen in Ewing sarcoma.

The brain metabolome directly connects to brain physiology and neuronal function. Brain glucose metabolism is highly heterogeneous among brain regions and continues postmortem. Therefore, challenges remain to capture an accurate snapshot of the physiological brain metabolome in healthy and diseased rodent models. To overcome this barrier, we employ a high-power focused microwave for the simultaneous euthanasia and fixation of mouse brain tissue to preserve metabolite pools prior to surgical removal and dissection of brain regions. We demonstrate exhaustion of glycogen and glucose and increase in lactate production during conventional rapid brain resection prior to preservation by liquid nitrogen that is not observed with microwave fixation. Next, microwave fixation was employed to define the impact of brain glucose metabolism in the mouse model of streptozotocin-induced type 1 diabetes. Using both total pool and isotope tracing analyses, we identified global glucose hypometabolism in multiple regions of the mouse brain, evidenced by reduced 13C enrichment into glycogen, glycolysis, and the TCA cycle. Reduced glucose metabolism correlated with a marked decrease in GLUT2 expression and several metabolic enzymes in unique brain regions. In conclusion, our study supports the incorporation of microwave fixation to study terminal brain metabolism in rodent models.

No abstract

The endocannabinoid system is a neuromodulatory system that is conserved among vertebrate species. Cannabidiol (CBD) is a modulator of cannabinoid (CB) receptor action both in the central nervous system and in the periphery. CB receptors are involved in inflammatory responses in peripheral organs, including lung tissue. CBD administration has been shown to reduce airway inflammation and fibrosis in vitro and in experimentally induced rodent models of asthma through modulation of the immune system. However, little is understood regarding the relationship between pulmonary disease and CBD modulation of general CB receptors in vivo. The African Green Monkey (Chlorocebus aethiops sabaeus; AGM) has bronchiolar tissue and tracheal branching remarkably similar to that in human lungs, therefore making it a translational model for the study of CBD treatment on pulmonary fibrosis‐related lung dysfunction. We hypothesize that administration of NCMB‐1, a hemp extract with CBD, will increase the tidal volume, inspiratory flow rate, and respiration rate of animals with previously induced pulmonary fibrosis. Baseline tidal volume, inspiration flow rate, and respiration rate of 16 male AGM with induced lung fibrosis were measured. Animals were divided into a vehicle and 3 treatment groups: 50 μL of undiluted, 5X dilution, and 25X dilution NCMB‐1 administered orally twice daily via banana sections for two weeks. Compared to control (n=4, 206.6 ± 36.4 mL), 5X NCMB‐1 treated animals increased tidal volume (n=4, 484.0 ± 54.6 mL; p<0.05) from baseline at two weeks of treatment. Inspiratory flow rate also increased in 5X treated animals (n=4, 412.0 ± 49.9 mL/s; p<0.05) compared to control (n=4, 212.3 ± 50.7 mL/s) after two weeks of administration. Respiration rates increased from baseline in 1X treated animals (n=4, 33.0 ± 4.5 breaths/min) and 5X treated animals (n=4, 30.8 ± 3.9 breaths/min) compared to control (n=4, 28.5 ± 4.1 breaths/min; p<0.05). Treatment with 25X dilution of NCMB‐1 did not alter pulmonary function at either one or two weeks of treatment compared to vehicle treated animals. Thus, administration of 5X diluted NCMB‐1 to AGM with induced pulmonary fibrosis increased tidal volume, inspiratory flow rate, and respiration rate from compared to vehicle treated AGM. These results indicate CBD as a potential modulator of CB receptors improving pulmonary function and suggest a potential for therapeutic value of CBD in the treatment of restrictive pulmonary disease. Future directions include identification of active CBD compounds within the extract responsible for changes in airway resistance, expression of specific pulmonary genes involved in reduction of inflammation, and immunohistochemistry of CB receptors in the hypothalamus of AGM with induced pulmonary fibrosis. CBD crossing the blood brain barrier in animals treated with NCMB‐1 may cause up or downregulation of CB1 and/or CB2 receptors that could positively impact lung function. Support or Funding Information Funding provided by Primates Plus, LLC.

Glucose Transporter 1 Deficiency Syndrome (G1D) is a rare genetic disorder characterized by impaired brain glucose metabolism caused by mutations in the SLC2A1 gene. Mutations in the SLC2A1 gene lead to reduced or loss of function in the glucose transporter protein type 1 (GLUT‐1), affecting its ability to deliver glucose across the blood brain barrier. This lack of glucose in the brain affects brain function and development, causing people with the disorder to suffer from seizures, learning disabilities, and struggle for independence. While decreased levels of glucose in G1D is recognized, glycogen levels in the brain have not been studied in human or animal models of the disease. We employed a mouse model of G1D syndrome characterized by a loss of function in the SLC2A1 gene to study glycogen levels. Wildtype and G1D animals of the same sex at 14 weeks old were utilized for this study. Immunohistochemical (IHC) staining with IV58B6 and an amyloglucosidase glycogen assay were used to determine regional and total glycogen levels, respectively. HALO software was employed to quantify IHC staining, and the enzymatic glycogen determination was measured with a spectrophotometric microplate reader. Through these techniques, it was determined that G1D mice had perturbed glycogen levels present in multiple regions of the brain. These data suggest that the G1D phenotype could be affected by perturbed levels of glycogen in the brain. Further research into the effect of glycogen levels in G1D may be important for determining disease modalities, defining disease progression, and understanding preclinical data and drug treatment.