β‐Cell subcellular localization of glucose‐stimulated Mn uptake by X‐ray fluorescence microscopy: implications for pancreatic MRI

Abstract

Manganese (Mn) is a calcium (Ca) analog that has long been used as a magnetic resonance imaging (MRI) contrast agent for investigating cardiac tissue functionality, for brain mapping and for neuronal tract tracing studies. Recently, we have extended its use to investigate pancreatic β-cells and showed that, in the presence of MnCl2, glucose activated pancreatic islets yield significant signal enhancement in T1-weigheted MR images. In this study, we exploited for the first time the unique capabilities of X-ray fluorescence microscopy (XFM) to both visualize and quantify the metal in pancreatic β-cells at cellular and sub-cellular levels. MIN-6 insulinoma cells grown in standard tissue culture conditions had only a trace amount of Mn, 1.14 ± 0.03 ×10-11 μg/μm2, homogenously distributed across the cell. Exposure to 2mM glucose and 50 μM MnCl2 for 20 minutes resulted in non-glucose dependent Mn uptake and the overall cell concentration increased to 8.99 ± 2.69 ×10-11 μg/μm2. When cells were activated by incubation in 16mM glucose in the presence of 50 μM MnCl2, a significant increase in cytoplasmic Mn was measured reaching 2.57 ± 1.34 ×10-10 μg/μm2. A further rise in intracellular concentration was measured following KCl induced depolarization, with concentrations totaling 1.25 ± 0.33 ×10-9 and 4.02 ± 0.71 ×10-10 μg/μm2 in the cytoplasm and nuclei respectively. In both activated conditions Mn was prevalent in the cytoplasm and localized primarily in a perinuclear region, possibly corresponding to the Golgi apparatus and involving the secretory pathway. These data are consistent with our previous MRI findings confirming that Mn can be used as a functional imaging reporter of pancreatic β-cell activation and also provide a basis for understanding how subcellular localization of Mn will impact MRI contrast

Pancreatic magnetic resonance imaging after manganese injection distinguishes type 2 diabetic and normoglycemic patients

“…At least experimentally, MEMRI allows for the quantitative detection of individual islets, and their changes in a model of diabetes 13 . Third, in the context of type 2 diabetes, in which both the function and likely the mass of β cells is altered, MEMRI may provide information about both events, as manganese is uptaken by β cells as a function of their ability to release insulin in response to glucose 12 - 15 . The inverse correlation observed between BMI and the MEMRI signal of pancreas is consistent with this view, given that BMI relates to residual β cell function 27 .…”

“…One promising alternative is manganese-enhanced magnetic resonance imaging (MEMRI). Recent studies have shown that the intensity of the MRI pancreas signal can be enhanced by infusion of manganese, 10 - 13 a cation which mimics the behavior of Ca 2+ , an essential regulator of insulin secretion 14 , 15 …”

Identification of dopaminergic neurons of the substantia nigra pars compacta as a target of manganese accumulation

“…26 Distribution in the soma was also observed in pancreatic ß-cells following a 20 minute incubation in 50 μM MnCl 2 , 20 minute incubation in 4% paraformaldehyde, several washes, and air drying. 54 …”

Translational Molecular Imaging of Diabetes

“…It has been demonstrated that in the presence of MnCl2, glucoseactivated pancreatic islets yield significant signal enhancement in T1-weigheted MR images in vitro and in animal models of diabetes [16][17][18][19][20][21][22][23]. This approach has also been tested in preliminary studies in patients with T2D.…”