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. 2021 May 19;4(1):594.
doi: 10.1038/s42003-021-02113-1.

Live-cell imaging of glucose-induced metabolic coupling of β and α cell metabolism in health and type 2 diabetes

Zhongying Wang  1   2 , Tatyana Gurlo  2 , Aleksey V Matveyenko  3 , David Elashoff  4 , Peiyu Wang  1   5   6 , Madeline Rosenberger  2 , Jason A Junge  1   5 , Raymond C Stevens  7 , Kate L White  1 , Scott E Fraser  8   9   10 , Peter C Butler  11
Affiliations

Live-cell imaging of glucose-induced metabolic coupling of β and α cell metabolism in health and type 2 diabetes

Zhongying Wang et al. Commun Biol. .

Abstract

Type 2 diabetes is characterized by β and α cell dysfunction. We used phasor-FLIM (Fluorescence Lifetime Imaging Microscopy) to monitor oxidative phosphorylation and glycolysis in living islet cells before and after glucose stimulation. In healthy cells, glucose enhanced oxidative phosphorylation in β cells and suppressed oxidative phosphorylation in α cells. In Type 2 diabetes, glucose increased glycolysis in β cells, and only partially suppressed oxidative phosphorylation in α cells. FLIM uncovers key perturbations in glucose induced metabolism in living islet cells and provides a sensitive tool for drug discovery in diabetes.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Validation of FLIM in a pancreatic β cell line.
INS-1E β cells were monitored by FLIM at low glucose (LG) concentration 1.1 mM and then after increasing the glucose concentration to 16.7 mM (high glucose, HG), a value known to stimulate insulin secretion in this cell line. The typical INS-1E adherent and flat morphology is seen in bright field (a) permitting a clear view of intracellular compartments. b 2-photon excitation of the same field yields a fluorescent intensity map gated for NAD(P)H emission. c The phasor plot of the fluorescence lifetime of the NAD(P)H signal offers a simple way to analyze and visualize FLIM data, as the emission from NAD(P)H will lie along the dashed line: the red triangle (lifetime = 3.43 ns) marks the phasor position of NAD(P)H bound to protein complexes, and the blue diamond (lifetime = 0.4 ns) marks the phasor position of free NAD(P)H, unbound to proteins. The position along this dashed line can be rendered by the depicted rainbow color code: red represents the longer lifetimes, corresponding to the high Bound/total NAD(P)H indicating oxidative phosphorylation (OxPhos); blue represents the shorter lifetimes corresponding to glycolysis. d, e The fluorescence lifetimes from NAD(P)H recorded in low glucose (LG) and high glucose (HG) conditions show the expected metabolic state, with the pseudocolored lifetimes of most cells moving to redder hues. f To analyze this metabolic state, the average Bound/total NAD(P)H value is plotted, revealing the expected increase as conditions are changed from LG to HG, reproducibly in 6 experiments. g The relative metabolic state between conditions (e.g., LG to HG) can be expressed as ΔBound/total NAD(P)H, which is positive for a relative increase in OxPhos, and negative for a relative increase glycolysis. Data are presented as mean ± SD from n = 6 independent experiments. Scale bar, 10 µm. *P < 0.05, two-tailed Wilcoxon test.
Fig. 2
Fig. 2. Metabolic trajectory of β and α cells in response to glucose.
a, b Mouse islets were dispersed into single cells, attached to a cover slip with imprinted grid, evaluated by FLIM and then fixed and immunostained for glucagon and insulin (a) so that the FLIM studies could be attributed to α cells and β cells, respectively. Green arrows, β cells; red arrows, α cells; white arrows, unknown because cells detached during staining. Scale bar, 10 µm. c Metabolic trajectory of dispersed Single primary β and α cells (n = 8 cells each) imaged at basal glucose (BG, 4 mM) and after exposure to high glucose (HG, 16 mM) for 30 min. The pseudocolored images suggest that the β cells become more OxPhos after glucose treatment and the α cells become more glycolytic. Scale bar 5 µm; dashed white line indicates cell outline and dotted line indicates the nucleus outline. d The basal glucose Bound/total NAD(P)H values for α and β cells are significantly different. e, f The opposite metabolic trajectories of dispersed α cells and β cells are revealed by plotting the Bound/total NAD(P)H values for each cell in BG and HG conditions, and calculating the ΔBound/total NAD(P)H of β and α cells. n = 8 for each cell type. g, h, i In 8 intact WT mouse islets, we examined the Bound/total NAD(P)H and ∆Bound/total NAD(P)H value of 47 α and 72 β individual cells at basal and high glucose. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, the two-tailed paired t test was used for comparison of responses to BG versus HG in dispersed mouse islet cells. The two-tailed Mann–Whitney test was used in comparison of responses between α versus β cells from dispersed mouse islet cells. Linear mixed effect models were used to compare responses between islets cells in whole islets.
Fig. 3
Fig. 3. Impact of protein misfolding stress characteristic of T2D on metabolic trajectory of β and α cells in response to glucose.
FLIM images depicting relative glycolysis and OxPhos in islets from a wild type (WT), (a) and human IAPP transgenic (hTG), (b) mouse islet at basal glucose (BG, 4 mM) and high glucose (HG, 16 mM). c, d Panels show Bound/total NAD(P)H value for β cells and α cells respectively in WT (open circle and square) and hTG (solid circle and square) mouse islets at BG and HG. e Relative increase (+) or decrease (−) in OxPhos (ΔBound/total NAD(P)H) in α and β cells from WT and hTG mouse islets after increase in glucose from BG to HG. Data are presented as mean ± SD, n = 11 for WT islets, 1–3 islets from 6 mice, n = 10 for hTG islets, 1–3 islets from 4 hTG mice. *P < 0.05, **P < 0.01, ***P < 0.001; the linear mixed effect models were used for statistical analysis. Scale bar, 10 µm.
Fig. 4
Fig. 4. Spatial temporal relationship of β cell metabolic responses to glucose in vitro and insulin and glucagon secretion in vivo in the presence and absence of misfolded protein stress.
a FLIM evaluation of metabolic trajectory from representative wild type (WT) and human IAPP transgenic (hTG) mouse islets with basal glucose (BG, 4 mM) and then at 30, 60 and 120 min after high glucose (HG, 16 mM). White solid line, hub β cells observed in WT islets; white dashed line, a cluster of adjacent β cells in hTG mice. Scale bar, 10 µm. Plasma glucose (b), insulin (c), C-peptide (d), and glucagon (e) concentrations at baseline (min-0) and following 1.5 g/kg body weight oral glucose by lavage in WT (n = 5) and HIP (n = 11) rats. Data presented as mean ± SEM.
Fig. 5
Fig. 5. Human islets.
3D projection of human islets from non-diabetic donors (ND) (a) and donors with T2D (b) exposed to basal glucose (BG, 4 mM) and high glucose (HG, 16 mM) concentration. c Quantification of changes in relative OxPhos levels of β cells in ND and T2D human islets presented as mean of each donor sample. n = 8 for T2D (2 donors, 4 islets each), n = 6 for ND (3 donors, 1-3 islets each). Scale bar, 20 µm. P = 0.1, linear mixed models were used for statistical analysis.
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