(T0930-05-31) Non-invasive In Vivo Imaging of β-Cell Function in the Rodent Pancreas Using a Zinc-Responsive Contrast Agent

Purpose: Hormone Insulin from pancreatic 𝛽-cells plays a key role in maintaining blood glucose in normal healthy tissue along with its opponent glucagon. A non-invasive real time imaging of rodent pancreas for recognizing 𝛽-cell function could be a valuable approach for investigating the functional loss of 𝛽-cell activity during the development of type 2 diabetes[1]. It is well known that two Zn²⁺ packed along with six insulin monomer in granules are released from 𝛽-cells when stimulated by high glucose levels, a process known as glucose-stimulated insulin and zinc secretion (GSZS). Upon secretion of these vesicles, this hexameric insulin complex converted back into active monomeric form and yields significant amount of free Zn²⁺ into extracellular milieu of 𝛽-cells. This makes a rise is extracellular Zn²⁺ level upto ~ 450µM around pancreatic islets. Considering the co-release of Zn²⁺ with insulin, a real time spatial MR imaging method can boost the pathophysiological understanding of T2DM development and disease progress along with the efficacy of treatment strategy. A gadolinium ion-based zinc-responsive agent (GdL₂) has an affinity for binding with extracellular Zn²⁺ released from 𝛽-cells after glucose stimulation. Briefly, upon Zn²⁺ and insulin co-release from 𝛽-cells, free Zn²⁺ ions interact with GdL₂ which in turn binds with HSA to form a GdL₂*Zn^2+*HSA ternary complex. This complex slowdowns the molecular tumbling rotation and increases the longitudinal relaxation (r1) which leads to the enhancement of image contrast in the area having high concentration of free Zn²⁺[2].

Methods: In the present study, Firstly we have investigated the ex-vivo glucose-stimulated insulin and zinc secretion (GSZS) using INS1 (Rat Insulinoma cell line) cells. The cell culture was stained with ZIMIR dye using standard protocol and confocal imaging was performed using Inverted LSM 880 Airyscan/ Confocal microscope at an excitation/detection wavelength 488 nm /490-600nm. For in vivo imaging, we infused the rats with cell-impermeant GdL₂ in continuous infusion along with an optimized 2D gradient echo-MRI sequence to investigate alterations in insulin secretion after Glucose administration. A series of axial images with fat saturation were collected to monitor the dynamic glucose response and Zn²⁺ release from the pancreas in response to the glucose stimulation. Further, the plasma insulin and Zn²⁺ level were estimated using ELISA and ICP-MS respectively.

Results: Sprague-Dawley (SD) rats of 9-12 weeks age (Normal healthy and body weight: ~ 400-500 grams) were used for pancreatic β-cells MR imaging and all experiments were performed under approved IACUC institutional ethical protocol. As hypothesized, increased signal intensity and so; image contrast enhancement was observed in the pancreas (Figure 1A) after glucose injection which confirms that GdL₂ non-invasively detects Zn²⁺ secretion from β-cells. The post glucose injection signal enhancement shown in figure 1B clearly represents the trend of biphasic insulin secretion under hyperglycemic condition. The mean intensity increase after glucose injection maybe only possible due to the interaction between secreted Zn²⁺ along with insulin and GdL₂ as it is well known that GdL₂ cannot pass the cell membrane [1, 3]. Also, there is no decrease in signal intensity of islets was observed after glucose injection till the end of experiment. This represents that continuous infusion of GdL₂ makes its concentration around islets constant and ruled out its loss due to filtration through kidney. Also, this finding correlates with the increased blood plasma insulin level (pre and post GdL₂ & Glucose administration blood insulin level was found to be 0.208 ± 0.13 ng/mL and 0.956 ± 0.63 ng/mL, respectively; **P < 0.01) in post glucose-injection samples (Figure 1C).

Conclusion: The presented study provides an improved method of non-invasive in vivo imaging of pancreatic β-cell function in rats using 2D MRI and the continuous infusion of Zn²⁺ sensor, GdL₂. This new 𝛽-cell function imaging protocol allows for the investigation of more physiological events, avoiding confounding issues related to the first-pass clearance of the contrast agent and temporal changes in agent concentration during the monitoring of glucose-stimulated insulin secretion (GSIS). Given that it is non-invasive, this method allows longitudinal studies for timely diagnosis of abnormalities in 𝛽-cell function due to the obesity or type II DM in rodents. Further, this method is useful for monitoring the efficacy of new drugs designed to stimulate β-cell function.

References: 1. Martins, A.F., et al., Imaging insulin secretion from mouse pancreas by MRI is improved by use of a zinc-responsive MRI sensor with lower affinity for Zn2+ ions. Journal of the American Chemical Society, 2018. 140(50): p. 17456-17464.
2. Thapa, B., et al., Imaging β-cell function using a zinc-responsive MRI contrast agent may identify first responder islets. Frontiers in Endocrinology, 2022. 12: p. 809867.
3. Lubag, A.J., et al., Noninvasive MRI of β-cell function using a Zn2+-responsive contrast agent. Proceedings of the National Academy of Sciences, 2011. 108(45): p. 18400-18405.

Acknowledgements: We would like to acknowledge the Preclinical Imaging & Research Core facility of University of North Texas Health Science Center in Fort Worth for supporting with the MRI equipment and Confocal Fluorescence microscope.