Poster S2 - STRIDE-SRS and 2PEF Imaging of Glucose Uptake Under Insulin Receptor Regulation

Introduction: Type 2 diabetes mellitus (T2DM) is a widespread metabolic disorder characterized by reduced insulin sensitivity. Digestive proteases have been associated with T2DM due to their elevated activity following high-fat meals, which has been linked with a decrease in insulin sensitivity. However, the dynamics of protease-cleaved insulin receptor extracellular domain regeneration and the impact of lifestyle choices on this process remain unclear. To address this, we utilized Stimulated Raman Scattering (SRS) and Two-Photon Excitation Fluorescence (2PEF) microscopy to investigate the regenerative mechanism of proteolytic protease-cleaved insulin receptors at the single-cell level. Deuterium-labeled glucose was employed to enhance the SRS analysis, allowing for the visualization of carbon-deuterium bonds without interference from other biomolecular signals within the cell. Our results demonstrate the strength of Spectral Tracing of DEuterium with Stimulated Raman Scattering (STRIDE-SRS) microscopy in investigating the link between digestive proteases and insulin receptor function, and may aid in the development of preventive and control measures for T2DM.

Materials and

Methods: NIH 3T3 cells are cultured in complete DMEM with 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin until reaching 80% confluence. The cells are washed with 1x Phosphate Buffered Saline (PBS) without ions, and protease activity was stimulated by introducing 3 mL of 1x Trypsin for 5 minutes. After incubation, the cells are centrifuged and the supernatant is removed. The remaining cell pellet is diluted in culture media for seeding onto glass bottom dishes for live cell imaging. The one of each dish are allowed to recover for 2, 24, 36, and 48 hours, respectively, then incubated in DMEM culture media supplemented with 0.45% D7-glucose, 10% FBS, and 1% P/S, and 10 μg/mL insulin for 2 hours for D-glucose uptake. After uptake, the media is replaced with PBS for cell imaging at wavenumbers in the Raman spectrum corresponding to proteins, unsaturated lipids, saturated lipids, D7-glucose, and phosphorylated/unphosphorylated tyrosine. Optical redox ratio from NADH and flavin taken using our multimodal platform's multiphoton fluorescence capabilities for readily accessible comparison against chemical imaging data in a spatially resolved manner.

Results, Conclusions, and Discussions: The results indicate the enzyme treatment had significant impact on glucose transport behavior across the cell membrane as well as intracellular lipid dynamics. It was found that the D7-glucose uptake of the cells was the highest when given 48 hours to recover and lowest when given 2 hours. Along with the elevated glucose uptake, the results suggest that glycolysis was upregulated for cellular metabolism in response to this stress. Through the visualization of endogenous fluorophores with 2PEF, the optical redox ratio (NADH/Flavin) corroborated the increased levels of NADH due to glycolysis. Additionally, saturated lipid levels continued to increase following the treatment interval suggesting that the cells were in an especially metabolically active state following enzyme treatment. Furthermore, the phosphorylated tyrosine channel indicated an upregulation in the tyrosine phosphorylation relative to unphosphorylated tyrosine, suggesting a relative decrease in the insulin signaling pathway immediately following the treatment, with a steady recovery to baseline levels observed over a 48hr span.
Using STRIDE-SRS and 2PEF multimodal imaging platform, we have determined that the incubation of NIH 3T3 cells with the proteolytic enzyme trypsin results in significant deviation from the standard metabolic behavior of cells in situ due to cleavage of the extracellular domain of insulin receptors. As cell recovery period increases, the D7-glucose intake trends upwards, leveling out at 48-hours post-recovery. Greater D-7 glucose uptake promotes glycolysis and higher levels of NADH, saturated lipids, and phosphorylated tyrosine activity observed in single cells support these findings. The results support the claim that digestive enzymes may be generating an atypical cellular phenotype, and that applying STRIDE-SRS and multiphoton fluorescence in conjunction can yield rapid, in depth, spatially-resolved information about cellular metabolic dynamics optically.

Acknowledgements (Optional): We thank Drs. Lingyan Shi, Geert Schmid-Schonbein, Yajuan Li, Mr. Anthony A. Fung, Ms. Pegah Bagheri and the Shi Lab members for helpful discussions. We acknowledge support from UC San Diego Startup Funds, Hellman Fellowship, Sloan Research Fellowship, NIH U54CA132378, NIH 5R01NS111039, Shu Chien-Gene Lay Department of Bioengineering Undergraduate Travel Award, UC San Diego Undergraduate Research Hub Development Grant, and the Jacobs School of Engineering Student Travel Fund.