Intra-islet lesions and lobular variations in β-cell mass expansion in ob/ob mice revealed by 3D imaging of intact pancreas

Abstract

The leptin deficient ob/ob mouse is a widely used model for studies on initial aspects of metabolic disturbances leading to type 2 diabetes, including insulin resistance and obesity. Although it is generally accepted that ob/ob mice display a dramatic increase in β-cell mass to compensate for increased insulin demand, the spatial and quantitative dynamics of β-cell mass distribution in this model has not been assessed by modern optical 3D imaging techniques. We applied optical projection tomography and ultramicroscopy imaging to extract information about individual islet β-cell volumes throughout the volume of ob/ob pancreas between 4 and 52 weeks of age. Our data show that cystic lesions constitute a significant volume of the hyperplastic ob/ob islets. We propose that these lesions are formed by a mechanism involving extravasation of red blood cells/plasma due to increased islet vessel blood flow and vessel instability. Further, our data indicate that the primary lobular compartments of the ob/ob pancreas have different potentials for expanding their β-cell population. Unawareness of the characteristics of β-cell expansion in ob/ob mice presented in this report may significantly influence ex vivo and in vivo assessments of this model in studies of β-cell adaptation and function.

Figure 1. Islet β-cell distribution in ob/ob and lean control mice between 4 and 52 weeks of age.

(A–J) Iso-surface rendered OPT images of representative splenic lobes of lean control (A–E) and ob/ob (F–J) pancreata. The islet β-cell volumes are reconstructed based on the signal from insulin specific antibody staining (red) and pancreas outline (gray) is based on the signal from tissue autofluorescense. In contrast to lean controls, the expected expansion in BCV is clearly observed in ob/ob pancreata. (K) Graph showing the average total β-cell volume in ob/ob and lean controls illustrating the progressive increase in ob/ob BCV (entire pancreas). (L) Graph showing the average total pancreas volume. (M) Percentage of the total pancreatic volume constituted by insulin positive cells. Data is shown as means ± SEM (n = 5) where *P < 0.05; **P < 0.01 and ***P < 0.001. Scale bar in (J) corresponds to 2 mm in (A–J).

Figure 2. Lobular pancreatic β-cell volumes in ob/ob and lean controls.

(A) Graph illustrating the total average (black lines) and lobular splenic (SL, red), duodenal (DL, green) and gastric (GL, yellow) β-cell volumes in lean control (broken lines) and ob/ob pancreata (intact lines). The BCV expansion is close to linear in all groups (r² scores are indicated). (B) Graph displaying the average percentual β-cell volume in the respective splenic, duodenal and gastric lobes of lean control (white bars) and ob/ob (black bars). Inset shows a photomicrograph of a gut segment including the stomach, duodenum, spleen and pancreas from a C57Bl/6 mouse at 8 weeks, illustrating the delineation of the lobes. Data is shown as means ± SEM (n = 5) where *P < 0.05; **P < 0.01 and ***P < 0.001. Abbreviations: SL, Splenic lobe; DL, Duodenal lobe; GL, Gastric lobe; St, Stomach; Spl, Spleen; Duo, Duodenum. Scale bar in inset is 2 mm.

Figure 3. ob/ob β-cell expansion by islet β-cell volume size categories.

(A–J) Iso-surface rendered OPT images of representative splenic lobes of lean control (A–E) and ob/ob (F–J) pancreata between 4 and 52 weeks of age. Individual islet β-cell volumes are reconstructed based on the signal from insulin-specific antibodies and have been pseudo colored to highlight the distribution of small (<1 × 10⁶ μm³ [white]), intermediate (1 × 10⁶ −5 × 10⁶μm³ [red]), and large (>5 × 10⁶ μm³ [yellow]) islets. (K) Histogram showing the average total β-cell volume constituted by each size category in lean control and ob/ob pancreata respectively. (L) Histogram showing the average total number of islets within each size category in lean control and ob/ob pancreata respectively. Data is shown as means ± SEM (n = 5) where *P < 0.05; **P < 0.01 and ***P < 0.001. Abbreviations: S, small; M, medium; L, large. Scale bar in (J) corresponds to 2 mm in (A–J).

Figure 4. Cystic lesions are frequently formed in hypertrophic ob/ob islets.

(A) Tomographic OPT section from a representative ob/ob pancreas at 26 weeks of age (splenic lobe). Insulin positive areas are pseudocolored red. (See also Movie S2). (B–D) Iso-surface rendered OPT images of the specimen in (A). The islet β-cell volumes are reconstructed based on the signal from insulin specific antibody staining (B, red) and the outlines of the cystic lesions were manually delineated in the tomographic sections (C, gray). The islet β-cell volumes and the areas occupied by lesions are merged together with the outline of the organ (dark gray) in (D). (E–H) Representative ultramicroscopy images of hypertrophic islets at 26 weeks (duodenal lobe) showing the surface morphology (E,F) and the same islets applying a digital section plane to visualize the internal lesions (G,H) Area marked by a broken line in (E,G) corresponds to high magnification images in (F,H). White arrow in (E) points to a cystic lesion that has generated an opening towards the surface of the islet. Abbreviations; SL, Splenic lobe; DL, Duodenal lobe; Tom. section, Tomographic section. Scale bar in (D) is 506 μm in (A) and 2 mm in (B–D). Scale bar in H is 291 μm in (E,G) and 100 μm in (F,G).

Figure 5. Histological analysis of ob/ob and lean control pancreata between 4 and 52 weeks of age.

(A–O) Hematoxylin/Eosin staining of representative sections from lean control (A,D,G,J,M) and ob/ob (B,C,E,F,H,I,K,L,N,O) at 4 weeks (A–C), 8 weeks (D–F), 17 weeks (G–I), 26 weeks (J–L) and 52 weeks (M–O). Abbreviations; RBCs, Red blood cells; Exo, Exocrine tissue. Scale bar in (O) is 50 μm in (A–O).

Figure 6. Immunohistochemical assessment of proteins involved in blood coagulation in ob/ob pancreata.

(A,B) Hematoxylin/Eosin staining of an islet from a lean control (A) and a ob/ob (B) pancreas at 52 weeks. Note the accumulation of RBCs (white arrows in (B). (C,D) Consecutive sections to (A,B) stained for Fibrinogen (green) and DAPI (Blue) indicating the presence of a fibrin mesh within the areas of the lesions (white arrows in (D) compare with (B)). (E–J) Photomicrographs of representative pancreatic cryosections from lean control (E,F) and ob/ob (G,H) pancreata at 52 weeks of age labeled for Fibronectin (Green E,G) and von Willenbrand Factor (Green, F,H) together with DAPI (blue). Areas enclosed by a broken line in (G,H) corresponds to (I,J) respectively. The areas in the lesions positive for Fibronection and von Willenbrand factor are not associated with any nucleated cells. Abbreviations; vWF, von Willenbrand Factor; Exo, Exocrine tisse. Scale bar in (D) is 50 μm in (A–D) and scale bar in (J) is 92 μm in (E–H) and 50 μm in (I,J).

Figure 7. Cystic lesions are formed in ob/ob islets by a mechanism involving extravasation of red blood cells.

Schematic illustration of a putative model for lesion formation in ob/ob islets (see text for details).