Spatiotemporal Correlation Spectroscopy Reveals a Protective Effect of Peptide-Based GLP-1 Receptor Agonism against Lipotoxicity on Insulin Granule Dynamics in Primary Human β-Cells

Abstract

Glucagon-like peptide-1 receptor (GLP-1R) agonists are being used for the treatment of type 2 diabetes (T2D) and may have beneficial effects on the pancreatic β-cells. Here, we evaluated the effects of GLP-1R agonism on insulin secretory granule (ISG) dynamics in primary β-cells isolated from human islets exposed to palmitate-induced lipotoxic stress. Islets cells were exposed for 48 h to 0.5 mM palmitate (hereafter, ‘Palm’) with or without the addition of a GLP-1 agonist, namely 10 nM exendin-4 (hereafter, ‘Ex-4′). Dissociated cells were first transfected with syncollin-EGFP in order to fluorescently mark the ISGs. Then, by applying a recently established spatiotemporal correlation spectroscopy technique, the average structural (i.e., size) and dynamic (i.e., the local diffusivity and mode of motion) properties of ISGs are extracted from a calculated imaging-derived Mean Square Displacement (iMSD) trace. Besides defining the structural/dynamic fingerprint of ISGs in human cells for the first time, iMSD analysis allowed to probe fingerprint variations under selected conditions: namely, it was shown that Palm affects ISGs dynamics in response to acute glucose stimulation by abolishing the ISGs mobilization typically imparted by glucose and, concomitantly, by reducing the extent of ISGs active/directed intracellular movement. By contrast, co-treatment with Ex-4 normalizes ISG dynamics, i.e., re-establish ISG mobilization and ability to perform active transport in response to glucose stimulation. These observations were correlated with standard glucose-stimulated insulin secretion (GSIS), which resulted in being reduced in cells exposed to Palm but preserved in cells concomitantly exposed to 10 nM Ex-4. Our data support the idea that GLP-1R agonism may exert its beneficial effect on human β-cells under metabolic stress by maintaining ISGs’ proper intracellular dynamics.

β-Cell Pathophysiology: A Review of Advanced Optical Microscopy Applications

“…This latter is able to normalize ISG dynamics, i.e., re-establish ISG mobilization and ability to perform active transport in response to glucose stimulation. These data support the idea that GLP-1R agonism may exert its beneficial effect on human β-cells under metabolic stress by maintaining ISGs’ proper intracellular dynamics [ 73 ].…”

“…In our approach, these values can be used to identify a unique point in a 3D parametric space, so that the clustering of single-cell data points depicts the overall structural and dynamic properties of the structure of interest [ 71 ]. The iMSD approach was recently applied by some of us to perform screening of ISGs labelled with different genetically-encoded fluorescent proteins both in INS-1E cells [ 71 , 72 ] and in cells disaggregated from human-derived islets [ 73 ] ( Figure 9 A,B). Concerning the application to INS-1E cells, a reference using cells expressing proinsulin fused to a FP (c-peptide EGFP) under basal culture conditions was created by iMSD analysis and then validated by testing well-established stimuli, such as glucose intake, cytoskeleton disruption, or cholesterol overload [ 71 ].…”

“…( D ) Comparison between syncollin-EGFP labelled granules in INS-1E cells (grey) and in dispersed human islet β cells (dHI). Adapted from Ferri et al [ 71 , 73 ].…”

Single-molecule analysis of intracellular insulin granule behavior and its application to analyzing cytoskeletal dependence and pathophysiological implications

“…Several live-cell imaging techniques have been used to evaluate mobilization processes prior to exocytosis, such as the dynamic movement of insulin secretory granules ( Pouli et al, 1998 ; Ivarsson et al, 2004 ; Tabei et al, 2013 ; Heaslip et al, 2014 ; Hoboth et al, 2015 ; Ferri et al, 2019 ; 2021 ). Specifically, total internal reflection fluorescence (TIRF) microscopy has made a major contribution to unveiling such dynamics including the dependence of cytoskeletal elements on the granule movement ( Heaslip et al, 2014 ).…”

“…Furthermore, it is difficult to precisely track individual secretory granule movements with these approaches because the dense fluorescent signals resulting from the visualization of all fluorescent molecules makes it difficult to resolve individual granules. Recently, an approach based on image correlation spectroscopy for analyzing the dynamics of insulin granules without tracking individual granules has been demonstrated ( Ferri et al, 2019 ; 2021 ). This approach is very useful for understanding the ensemble behavior of insulin granules in cells because it can extract structural and dynamic properties directly from images without single-particle techniques.…”