Accurate measurement of pancreatic islet beta-cell mass using a second-generation fluorescent exendin-4 analog

Abstract

The hallmark of type 1 diabetes is autoimmune destruction of the insulin-producing β-cells of the pancreatic islets. Autoimmune diabetes has been difficult to study or treat because it is not usually diagnosed until substantial β-cell loss has already occurred. Imaging agents that permit noninvasive visualization of changes in β-cell mass remain a high-priority goal. We report on the development and testing of a near-infrared fluorescent β-cell imaging agent. Based on the amino acid sequence of exendin-4, we created a neopeptide via introduction of an unnatural amino acid at the K(12) position, which could subsequently be conjugated to fluorophores via bioorthogonal copper-catalyzed click-chemistry. Cell assays confirmed that the resulting fluorescent probe (E4(×12)-VT750) had a high binding affinity (~3 nM). Its in vivo properties were evaluated using high-resolution intravital imaging, histology, whole-pancreas visualization, and endoscopic imaging. According to intravital microscopy, the probe rapidly bound to β-cells and, as demonstrated by confocal microscopy, it was internalized. Histology of the whole pancreas showed a close correspondence between fluorescence and insulin staining, and there was an excellent correlation between imaging signals and β-cell mass in mice treated with streptozotocin, a β-cell toxin. Individual islets could also be visualized by endoscopic imaging. In short, E4(×12)-VT750 showed strong and selective binding to glucose-like peptide-1 receptors and permitted accurate measurement of β-cell mass in both diabetic and nondiabetic mice. This near-infrared imaging probe, as well as future radioisotope-labeled versions of it, should prove to be important tools for monitoring diabetes, progression, and treatment in both experimental and clinical contexts.

Fig. 1.

Binding, affinity, and inhibition. (A) Cell-binding assays of different concentrations of E4_×12-VT750 against HEK/hGLP-1R (GLP-1R overexpressing), MIN6 (naturally expressing GLP-1R), and NIH 3T3 (GLP-1R–negative). (B) In vitro blocking experiments of E4_×12-VT750/GLP-1R binding (E4_×12-VT750 = 10 nM) with different concentrations of exendin-4. (C) Cell imaging experiments of HEK/hGLP-1R cells following incubation with E4_×12-VT750 (red) alone (Upper) or after preincubation with excess exendin-4 (Lower). Green, whole cell stain.

Fig. 2.

Pharmacokinetics. (A–D) Intravital confocal imaging of a pancreatic islet (arrows) in a live MIP-GFP mouse. Green, GFP signal from MIP-GFP–positive pancreatic β-cells; red, E4_×12-VT750 (0.2 nmol/g); blue, fluorescent vascular agent. All images were acquired with an objective with a magnification of 20× in anesthetized live mice. (E) Increase in islet signal following a dose of systemic E4_×12-VT750 injection. (F) Blood half-life measurement of E4_×12-VT750 (0.2 nmol/g) injected into a MIP-GFP mouse.

Fig. 3.

GFP and probe colocalization in E4_×12-VT750–injected MIP-GFP mice. (A and B) Fluorescence, histology of harvested pancreas (green, MIP-GFP; red, E4_×12-VT750). The arrow points to H&E staining of a single islet (C) and antiinsulin staining of the same islet (D) using adjacent sections. (E) Correlation of islet sizes estimated via the MIP-GFP reporter (488 nm) vs. the GLP-1R probe E4_×12-VT750 (750 nm). (F) Correlation of islet sizes observed with the MIP-GFP reporter (488 nm) vs. signal intensity observed at 750 nm in the same area.

Fig. 4.

E4_×12-VT750 and histological β-cell mass quantification. (A) Typical staining patterns observed for pancreata injected with E4_×12-VT750 (0.1 nmol/g, 40 min, mouse perfused and pancreas flushed) and imaged via surface reflectance microscopy. Note the very high target accumulation and very low background. (B) Higher magnification of a section from A, as indicated by the dashed box. The arrow points to a single islet. (C) Quantitative fluorescence signals (750 nm) were observed in pancreata of diabetic and nondiabetic mice injected with E4_×12-VT750 (0.1 nmol/g, 40 min, mouse perfused and pancreas flushed; P < 0.0001) [light gray, STZ-treated but nondiabetic mice (blood glucose levels <200 mg/dL); darker gray, overt diabetic mice (blood glucose levels >300 mg/dL)]. Blood glucose levels are indicated by ♢ symbols (P < 0.0001). (D) β-Cell mass, as estimated from histology of untreated mice and mice treated with different concentrations of STZ. (E) Correlation of β-cell mass quantification data from immunohistochemical staining and from E4_×12-VT750 fluorescence measurements (blue line, 99% confidence bands). BCM, β-cell mass.

Fig. 5.

Intravital pancreatic laparoscopy in a live MIP-GFP mouse. (A) Intravital imaging system. (B) Endoscopic imaging probe. (C) Higher magnification of the imaging probe head (outer diameter = 1.6 mm, probe head indicated in B by red box). (D) Field of view under white light conditions. (E) NIR fluorescence signal in islets (arrows) of the same area as in D. Note that the fiberoptic scope only has NIR channel fluorescence and white light capability. (F) Composite image. Arrows point to three distinct islets in this field of view.