Reporter islets in the eye reveal the plasticity of the endocrine pancreas

Abstract

The islets of Langerhans constitute the endocrine part of the pancreas and are responsible for maintenance of blood glucose homeostasis. They are deeply embedded in the exocrine pancreas, limiting their accessibility for functional studies. Understanding regulation of function and survival and assessing the clinical outcomes of individual treatment strategies for diabetes requires a monitoring system that continuously reports on the endocrine pancreas. We describe the application of a natural body window that successfully reports on the properties of in situ pancreatic islets. As proof of principle, we transplanted "reporter islets" into the anterior chamber of the eye of leptin-deficient mice. These islets displayed obesity-induced growth and vascularization patterns that were reversed by leptin treatment. Hence, reporter islets serve as optically accessible indicators of islet function in the pancreas, and also reflect the efficacy of specific treatment regimens aimed at regulating islet plasticity in vivo.

Keywords: beta cell mass; in vivo imaging; islet grafts; ob/ob mouse; pharmacologic treatment.

Fig. 1.

Increases in islet size in response to high insulin demand in the ob/ob mouse pancreas. (A) Representative morphological appearance of a 4-mo-old ob/ob mouse (Left) compared with a control littermate (Right), showing a strong obese phenotype. (B) Fasted body weight of ob/ob and control mice at different ages shows a rapid increase in body mass in the ob/ob mice. (C) Fasted blood glucose and plasma insulin levels in 3-mo-old ob/ob and control mice. (D and E) Image montages of 5-µm-thick sections of 8-mo-old ob/ob (D) and control littermate (E) pancreata, stained with H&E. Note the staining of islet sections in light gray. (Insets) Magnified views of typical islet dimensions and morphology. B and C show values for mixed males and females (n = 5 males + 4 females for ob/ob; n = 5 males + 5 males for control littermates). Values are average ± SEM. **P < 0.01; ***P < 0.001. (Scale bar: 1 mm; inset dimensions, 1 mm × 1 mm.)

Fig. 2.

In vivo longitudinal imaging of islet growth. (A) Islets from 4-wk-old mice were transplanted into the anterior chamber of the eye of control and ob/ob mice at the age of 4 wk. Photography of transplanted eyes at different time points shows that individual islets can be identified and followed longitudinally (see yellow dashed circle). (B) Magnified views of islet grafts (marked by red frames in A) show the clearly visible large and tortuous blood vessels in the islet engrafted onto the iris of ob/ob recipient. (C) In vivo imaging of single islets 1 mo after transplantation by confocal microscopy shows morphological differences between islet grafts in control versus ob/ob mice. Vascularization is visualized by i.v. injection of FITC-labeled dextran prior to imaging. Note differences in backscatter intensity and vessel diameters. (D) In vivo imaging of islet grafts at different time points after transplantation by confocal microscopy. (E) Quantification of islet volumes by analysis of backscatter images reveals a significantly increased growth in ob/ob (solid lines) compared with control (dashed lines). Gray lines represent average islet volumes in single mice; black lines represent averaged values obtained per genotype (n = 3). (F) Immunohistochemistry analysis shows a strong proliferation of beta cells in both ob/ob transplanted eye and pancreas, as seen by insulin and Ki67 staining. (G) Average beta cell area was quantified from insulin and DAPI staining, demonstrating that beta cells from ob/ob mice (solid lines) were significantly larger than those from their control littermates (dashed lines). This hypertrophy was similar and independent of whether the islets were located in situ in the pancreas or in the transplanted eye (no significant differences). All images are representative. Confocal images are displayed as maximum intensity projections (MIPs) of optical Z-stacks. Immunohistochemistry experiments were performed using 4-mo-old mice (n ≥ 3 per group). Error bars represent SEM. *P < 0.05; **P < 0.01. (Scale bars: 100 µm.)

Fig. 3.

Physiological effects of leptin treatment on ob/ob mice. (A) The ob/ob mice received daily i.p. injections of leptin between 3 and 4 mo of age. Body weight and blood glucose and plasma insulin levels were monitored before, during, and after the treatment; the beginning and end of treatment are represented by vertical dashed lines. (B) The i.p. glucose tolerance tests show impaired glucose handling in the ob/ob mice compared with control littermates at the age of 4 mo (Top), which is normalized by leptin treatment but not by sham treatment (Middle). (Bottom) Bar graph showing area under the curve (AUC) values from the above traces, demonstrating the beneficial effect of leptin on glucose handling. Values are average ± SEM. **P < 0.01; ***P < 0.001.

Fig. 4.

Leptin treatment reverses dysregulation of ob/ob islets. (A) Longitudinal in vivo imaging of islet grafts in ob/ob mice receiving leptin treatment (Upper) or sham treatment (Lower) between 3 and 4 mo of age. Vasculature was visualized by tail vein injection of dextran-FITC. (B) Islet volume analysis showing reversal of islet growth during leptin treatment (gray bars) compared with sham treatment (white bars). There was no difference in islet growth after the end of leptin or sham treatment. (C) Immunohistochemistry analysis of transplanted eye and pancreas samples from ob/ob mice at the end of the leptin treatment demonstrating the attenuated beta cell proliferation by immunostaining for insulin and Ki67 (compare with Fig. 2F). Confocal images are representative and shown as MIPs. Values are average ± SEM. ***P < 0.001. (Scale bars: 100 µm.)

Fig. 5.

Effect of leptin treatment on intra-islet vascularization. Islets were transplanted into the anterior chamber of the eye of 4-wk-old ob/ob mice. At the age of 3 mo, the mice received daily i.p. injections of leptin. (A) Leptin administration had a rapid effect on blood vessel diameters, as demonstrated by in vivo imaging of the same islet before treatment (Left) and after 1 wk of treatment (Right). The vasculature was visualized by tail vein injection of dextran-FITC. The same vessel segments could be identified and their diameters measured at different time points (red lines). Note that angiogenesis is evident after leptin administration (arrows). (B) Longitudinal analysis of individual vessel diameters showing vessel morphological changes before, during, and after leptin treatment. (Upper) Traces of 20 vessel segments from islet grafts in the ob/ob mouse over time. (Lower) Bar graph showing corresponding average monthly diameter increases. Confocal images are representative and shown as MIPs. Values are average ± SEM. ***P < 0.001; ****P < 0.0001. (Scale bars: 100 µm.)