Abstract
A cascade of highly regulated biochemical processes connects glucose stimulation to insulin secretion in specialized cells of mammalian pancreas, the β‐cells. Given the importance of this process for systemic glucose homeostasis, noninvasive and fast strategies capable to monitor the response to glucose in living cells are highly desirable. Here, we use the phasor‐based approach to Fluorescence Lifetime IMaging (FLIM) microscopy to quantify the ratio between protein‐bound and free Nicotinamide adenine dinucleotide (phosphate) species in their reduced form (NAD(P)H), and the Insulinoma cell line INS‐1E as a β‐like cellular model. Phasor‐FLIM analysis shows that the bound/free ratio of NAD(P)H species increases upon pulsed glucose stimulation. Such response is impaired by 48‐hours preincubation of cells under hyperglycemic conditions. Phasor‐FLIM concomitantly monitors the appearance of long‐lifetime species (LLS) as characteristic products of hyperglycemia‐induced oxidative stress.
Highlights
The biochemical process by which β-cells orchestrate Glucose-Stimulated Insulin Secretion is pivotal to the maintenance of glucose systemic homeostasis.[1]
NADH acts as a potent electron carrier during the mitochondrial oxidative phosphorylation, Abbreviations: adenosine triphosphate (ATP)/ADP, Adenine TriPhospate/Adenine DiPhosphate; ELISA, Enzyme-Linked Immunosorbent Assay; ETC, Electron Transport Chain; Fluorescence Lifetime IMaging (FLIM), Fluorescence Lifetime Imaging; GSIS, Glucose Stimulated Insulin Secretion; HEK 293, Human Embryonic Kidney 293 cells; INS-1E, Insulinoma cell line; long-lifetime species (LLS), Long Lifetime Species; mitochondrial glycerol phosphate dehydrogenase (mGPDH), mitochondrial Glycerol-3-Phosphate DeHydrogenase; mitochondrial malate dehydrogenase (mMDH), mitochondrial Malate DeHydrogenase; NAD(P)H, Nicotinamide Adenine Dinucleotide (Phosphate), reduced; reactive oxygen species (ROS), Reactive Oxygen Species, MCT1, MonoCarboxylate Transporter 1; RPMI, Roswell Park Memorial Institute
From NAD(P)H intensity analysis, FLIM has the potential to retrieve quantitative information about the ratio of NAD(P)H molecules in the ‘bound’ and ‘free’ form, with a few selected limitations, namely: (a) NADH and NAD(P)H species cannot be distinguished; (b) the metabolic response is reported in terms of bound/free ratio but changes can occur in both the numerator and denominator; (c) the approach, as used here, does not have native single-enzyme or single-pathway resolution (eg to study the respiratory chain with NAD(P)H-FLIM, specific respiratory-chain inhibitors need to be applied)
Summary
The biochemical process by which β-cells orchestrate Glucose-Stimulated Insulin Secretion (hereafter referred to as GSIS) is pivotal to the maintenance of glucose systemic homeostasis.[1]. Notwithstanding the undoubted importance of using intact islets, immortalized β-like cellular models have become a useful tool to get insights into the behavior of β-cells,[20,21] provided that the selected model recapitulates properly the main features of actual β-cells in response to different experimental conditions (eg glucose stimulation). In this regard, here we test Insulinoma 1E (INS-1E) β-like cells and their metabolic response in terms of bound/free NAD(P)H upon glucose stimulation by phasor-FLIM analysis. Phasor-FLIM analysis is able to monitor, within the same experiment, the appearance of characteristic long-lifetime species (LLS) (metabolic products of oxidative stress,22,23) in cells chronically exposed to hyperglycemic conditions
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