12 Nov 2021

Characterization of Metabolically Distinct and Unique Cellular Signaling Populations of Adipose Tissue Using FLIM Fluctuation Microscopy


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Seminar Abstract:

Abstract: Adipose tissue dysfunction, through energy overload or chronic inflammatory conditions, is associated with several metabolic complications, including obesity and diabetes. Development of a rapid and non-invasive label-free method for monitoring the metabolic state of adipocytes cells in vivo would provide a routine method for managing progress towards disease state(s). Furthermore, studies have revealed activation of different downstream signaling pathways that result from differences in the time course and location of ERK signaling, governed largely by distinct receptor trafficking pathways and mechanisms of feedback regulation. In this work we utilized NAD(P)H fluorescence lifetime imaging microscopy (FLIM) to identify metabolic signatures of adipocytes under various energetic states (e.g insulin resistance) and characterize unique localization of growth factor receptors. We identified distinct phasor trajectories for 3T3-L1 during maturation to adipocytes with the migration trajectory indicating a reduction in bound NAD(P)H and that mature adipocytes were primarily undergoing glycolytic metabolism. This reduction in oxidative phosphorylation within mitochondrial metabolism was confirmed via seahorse analysis. This corroboration in metabolic readouts between methods establishes NAD(P)H FLIM phasor as a sensitive marker for metabolic analysis. Stimulation with of mature adipocytes with TNFα, which was used to simulate insulin resistance under cell culture conditions, produced a metabolic signature that was unique and easily distinguished from healthy mature adipocytes while clearly showing a reduction in metabolic cytokines. Under different stimulation conditions, we were able to demonstrate alterations in the expression of growth factor receptors on the membrane surface and that these receptors, under disease state, translocate into the nucleus. These unique metabolic fingerprints provide a novel measurement of adipose energetic status and could be potentially adapted for a quantitative, non-invasive technique for assessing adipose tissue maintenance of energy balance.


Speaker Bio:

Dr. James received his Ph.D. in biophysics and biochemistry from the University of Vermont in 2009. After continuing his training in biophysics as a post-doc under David Jameson Ph.D. at UH Manoa, he became a junior P.I. and began to lead research on topics ranging from diabetes to cancer using fluorescence based applications. As a biophysicist, he is eager to apply fluorescent techniques to probe the mechanisms that drive Parkinson’s disease, Alzheimer’s disease, and Diabetes pathologies. Dr. James’ lab uses several tools for their research including spectrophotometers, fluorimeters, microscopes (including a 2-photon laser), and genetic engineering. He is passionate about science and committed to helping shape excellent scientists. Dr. James serves as a fellowship mentor for the Hawaiʻi Data Science Institute.

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