Researchers discover that glycogen and N-linked glycans accumulate in fibrotic regions of the lung and may be important for therapy development.
Charlene Lancaster, PhD
Ramon Sun and his team use MALDI-MSI to investigate aberrant metabolism in diseases including pulmonary fibrosis.
CAMERON SHEDLOCK
Most people take breathing for granted, but patients with interstitial lung diseases (ILDs) remember its importance every time they struggle to take a breath. Many patients with ILDs develop pulmonary fibrosis, which is the gradual scarring of the lungs. This progressive and fatal process results from excessive extracellular matrix production and deposition by lung fibroblasts and myofibroblasts, where collagen, elastin, and N-linked glycans are the major components of the matrix.1 Ultimately, pulmonary fibrosis results in the thickening of the alveolar walls, which limits oxygen uptake and causes difficulty breathing. ILDs with pulmonary fibrosis can result from genetic mutations, an underlying disease, or environmental exposure to microbes, smoke, or radiation.2 If clinicians are unable to find the cause of the fibrosis, they diagnose the patient with idiopathic pulmonary fibrosis (IPF).
Currently, there are only two anti-fibrotic drugs endorsed by the Food and Drug Administration for the treatment of pulmonary fibrosis.3 Although both drugs slow the disease’s progression, neither treatment can completely stop or reverse the lung damage, highlighting the need for novel therapeutics. A recently published study in Nature Communications suggests that glycogen could be an important therapeutic target.4
Ramon Sun, a biochemist at the University of Florida and corresponding author of the paper, studies metabolic pathways involved in diseases such as Alzheimer’s disease, Ewing sarcoma, and lung cancer. When the covid pandemic shut down non-essential research, Sun was presented with the opportunity to study metabolic changes that occur during this infectious disease. “We actually got some covid-19 lungs from a person that ended up passing away,” recalled Sun.
These are no longer tools being developed in an engineering or chemistry lab somewhere that are still waiting to be perfected. These are ready to use … for biological research.
Ramon Sun, University of Florida
Complex carbohydrate metabolism is irregular in many human disorders, including neurodegeneration and some cancers, but scientists did not know if it was abnormal in patients with covid. Sun and his research team first scanned the lung tissue sections using matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry imaging (MALDI-MSI), which measured the metabolite abundance at individual pixels. They next used a technique called high dimensionality reduction and spatial clustering (HDR-SC) to sort pixels with similar metabolite levels into clusters. By comparing the clusters’ locations to pathological regions of the lung, the researchers observed that three clusters corresponded exactly to the early-, mid-, and end-stage fibrotic regions of the SARS-CoV-2 infected lungs, indicating that complex carbohydrate metabolism is aberrant in these areas. To further investigate this abnormality, they analyzed the abundance of complex carbohydrates and uncovered that N-linked glycans and glycogen accumulated within the lungs’ fibrotic regions.
Because patients with IPF have lung fibrosis, Sun wondered if complex carbohydrate metabolism was also altered in this condition. Using MALDI-MSI, they observed that glycogen- and N-linked glycan-rich areas corresponded to fibrotic regions, indicating that this carbohydrate signature could help clinicians identify fibrosis.
Ramon Sun and colleagues acquired MALDI mass spectrometry images like this one, which shows glucose 6-phosphate abundance in a genetically engineered mouse model of lung cancer.
HARRISON CLARK
The researchers next questioned whether glycogen metabolism is directly involved in scar formation, as glycogen breakdown in myofibroblast lysosomes could supply the cells with substrates for N-linked glycan production and deposition. They employed mouse models that lacked enzymes required for glycogen catabolism and induced lung injury through intratracheal administration of bleomycin, an antibiotic used to treat cancer, which causes inflammation that later progresses to fibrosis. Through MALDI-MSI analysis, the researchers found less N-linked glycan deposition and fibrosis in the lungs of mice unable to break down glycogen, which suggested that the degradation of glycogen was important for pulmonary fibrosis development.
“I think the ability to localize signals within a tissue and find these coordinated events across the tissue region and compare these in a disease, like pulmonary fibrosis where you have such regional heterogeneity, was very powerful,” said Nicholas Banovich, a genomic scientist at the Translational Genomics Research Institute, who was not involved in the study. “I want to see these authors, as well as others in the field, continue to develop these imaging mass spectrometry approaches, and to see these platforms mature in the same way that we are starting to see the spatial transcriptomic platforms mature.”
Sun is optimistic that this research could change the way that clinicians treat and diagnose pulmonary fibrosis. His team plans to find inhibitors targeting glycogen synthesis and degradation to develop into pulmonary fibrosis therapies. He also intends to create a spatial metabolomics-based protocol that could assist clinicians with pulmonary fibrosis diagnosis, saving them hours by pointing out regions that could be of interest. Additionally, Sun hopes that this paper inspires other researchers to employ spatial metabolomics techniques in their laboratories. “These are no longer tools being developed in an engineering or chemistry lab somewhere that are still waiting to be perfected. These are ready to use … for biological research,” Sun said.
Charlene Lancaster, PhD 
