Abstract
β-cells convert glucose (input) resulting in the controlled release of insulin (output), which in turn has the role to maintain glucose homeostasis. β-cell function is regulated by a complex interplay between the metabolic processing of the input, its transformation into second-messenger signals, and final mobilization of insulin-containing granules towards secretion of the output. Failure at any level in this process marks β-cell dysfunction in diabetes, thus making β-cells obvious potential targets for therapeutic purposes. Addressing quantitatively β-cell (dys)function at the molecular level in living samples requires probing simultaneously the spatial and temporal dimensions at the proper resolution. To this aim, an increasing amount of research efforts are exploiting the potentiality of biophysical techniques. In particular, using excitation light in the visible/infrared range, a number of optical-microscopy-based approaches have been tailored to the study of β-cell-(dys)function at the molecular level, either in label-free mode (i.e., exploiting intrinsic autofluorescence of cells) or by the use of organic/genetically-encoded fluorescent probes. Here, relevant examples from the literature are reviewed and discussed. Based on this, new potential lines of development in the field are drawn.
Highlights
Optical Microscopy to Study Glucose Metabolism in B-CellsFor the aim of studying glucose metabolism in living cells, techniques capable of not altering the chemical identity and endogenous stoichiometry of the key molecular players are fundamental
A peculiarity of the β-cell is the tight coupling between glucose stimulation and insulin secretion exerted by means of a cascade of highly regulated biochemical processes [3,7,8,9]
Ca2+ influx promotes and sustains insulin secretion, starting from the mobilization of the insulin secretory granules (ISGs) of the readily releasable pool (RRP) located near the plasma membrane, and with the mobilization of ISGs located far from the plasma membrane using the microtubules as tracks
Summary
For the aim of studying glucose metabolism in living cells, techniques capable of not altering the chemical identity and endogenous stoichiometry of the key molecular players are fundamental. Given its biological importance, especially in the β-cell metabolic response to glucose, the possibility to monitor intrinsic NAD(P)H level in living cells could represent a valid marker for qualitative/quantitative assessments on cellular metabolic state In this sense, the pioneering studies of Chance et al [12,13] have laid the ground for future works based on NAD(P)H optical properties. Being label-free, FLIM on NAD(P)H does not yield artifacts due to the alteration of the molecular stoichiometry but still suffers from a few specific limitations, namely: (i) NADPH and NADH species cannot be distinguished; (ii) the metabolic response is reported in terms of bound/free ratio but changes can occur in both the numerator and denominator of the measured ratio; (iii) the approach does not have native single-enzyme or single-pathway resolution
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