CC BY
0Marker-free diagnostics for assessing pancreas and islet quality
P. Ermakova1*, E. Vasilchikova1, A. Kashirina1, A. Bogomolova1, N. Naraliev1, D. Kuchin12, L. Lugovaya1, E. Zagaynova13, V. Zagainov14, A. Kashina1
1- Privolzhsky research medical university, Russia 2- Nizhny Novgorod Regional Clinical Hospital named after Semashko, Russia 3- Lopukhin federal research and clinical center of physical-chemical medicine, Russia 4- Nizhny Novgorod Regional Oncology Dispensary, Nizhny Novgorod, Russia
* bardina-polina@mail.ru
Insulin-deficient conditions present a significant global public health challenge. Cellular stress and dysfunction precede the gradual loss of cell mass in conditions such as type 1 and type 2 diabetes, or pancreatitis. Cell dysfunction arises from altered glucose metabolism as part of a conserved pro-survival signaling mechanism. In healthy cells, elevated glucose levels lead to increased adenosine triphosphate (ATP) production through oxidative phosphorylation (OxPhos), which in turn influences insulin secretion via membrane channels. In contrast, stressed cells exhibit increased glycolysis unrelated to OxPhos, resembling the Warburg effect seen in cancer cells.
Conventional methods like PCR, immunohistochemistry, and transcriptomic studies are typically used to study cell metabolism but may offer incomplete or conflicting insights into islets. Therefore, a single method for fast, non-staining assessment of islet quality in both whole tissues and post-isolation is lacking. Metabolic Fluorescence Lifetime Imaging Microscopy (FLIM) has emerged as a potent tool for non-invasive, label-free diagnostics of pancreatic islet quality.
To make FLIM more clinically applicable, new criteria must be established to evaluate islet quality and metabolism across various insulin-deficient conditions. FLIM can be used to assess islet quality in whole tissues affected by insulin deficiency diseases or to monitor islet status pre- and posttransplantation. This study utilized FLIM and intracellular metabolite NAD(P)H to develop a non-invasive, label-free method for assessing isolated islet cell and pancreatic islet quality.
Fluorescence lifetimes and the relative proportions of free and bound NAD(P)H forms were analyzed using advanced FLIM systems. The FLIM parameters of islet cells from pathological and normal pancreases differed, with an increased glycolytic phenotype observed in pancreases with type 1 and type 2 diabetes/chronic pancreatitis. Metabolic FLIM imaging proved effective in evaluating isolated islet cell metabolism and viability.
FLIM data showed an increase in bound NAD(P)H levels and a predominance of oxidative phosphorylation over glycolysis upon glucose stimulation, which correlated with elevated insulin production in isolated cells. This confirmed the functional activity and viability of isolated islets. Non-contrast FLIM diagnostics can aid in establishing new criteria for identifying islet cell quality and metabolism, offering a swift technique for assessing islet viability in transplantation settings.
Overall, FLIM is a valuable tool for assessing islet quality in both whole tissues and during isolation, enhancing its utility in transplantation technologies.
The study was supported by the Russian Science Foundation (project № 24-65-00044).
