Altered islet morphology but normal islet secretory function in vitro in a mouse model with microvascular alterations in the pancreas

Abstract

Background: Our previous studies have shown that signal transducer and activator of transcription 3 (STAT3) signaling is important for the development of pancreatic microvasculature via its regulation of vascular endothelial growth factor-A (VEGF-A). Pancreas-specific STAT3-KO mice exhibit glucose intolerance and impaired insulin secretion in vivo, along with microvascular alterations in the pancreas. However, the specific role of STAT3 signaling in the regulation of pancreatic islet development and function is not entirely understood.

Methodology/principal findings: To investigate the role of STAT3 signaling in the formation and maintenance of pancreatic islets, we studied pancreas-specific STAT3-KO mice. Histological analysis showed that STAT3 deficiency affected pancreatic islet morphology. We found an increased proportion of small-sized islets and a reduced fraction of medium-sized islets, indicating abnormal islet development in STAT3-KO mice. Interestingly, the islet area relative to the whole pancreas area in transgenic and control mice was not significantly different. Immunohistochemical analysis on pancreatic cryosections revealed abnormalities in islet architecture in STAT3-KO mice: the pattern of peripheral distribution of glucagon-positive α-cells was altered. At the same time, islets belonging to different size categories isolated from STAT3-KO mice exhibited normal glucose-stimulated insulin secretion in perifusion experiments in vitro when compared to control mice.

Conclusions: Our data demonstrate that STAT3 signaling in the pancreas is required for normal islet formation and/or maintenance. Altered islet size distribution in the KO mice does not result in an impaired islet secretory function in vitro. Therefore, our current study supports that the glucose intolerance and in vivo insulin secretion defect in pancreas-specific STAT3-KO mice is due to altered microvasculature in the pancreas, and not intrinsic beta-cell function.

Figure 1. Pancreas-specific STAT3-KO (p-KO) mice exhibit altered pancreatic islet size distribution.

A. Hematoxylin and eosin (H&E)-stained histological sections from 8-week-old control and p-KO mice. Composite images were obtained from multiple overlapping single images of histological sections. Scale bar  = 400 μm for upper and 200 μm for lower panels. B. Islet area relative to the whole pancreatic area in p-KO (blue) and control (red) mice (N = 4 mice). Data are presented as means ± SEM. Ten composite images of each pancreas were used for analysis, four mice per group. C. Histogram showing islet distributions in p-KO (blue) and control (red) mice according to islet areas. D. Proportion of small, medium, and large islets in p-KO (blue bar) and control (red bar) mice. Data in C and D were obtained from 500–570 islets from pancreatic histological sections from four mice per genotype. The proportion of medium-sized islets was significantly lower in p-KO mice as compared with those in controls, while the number of small islets was increased. *p<0.05.

Figure 2. Pancreas-specific STAT3-KO (p-KO) mice exhibit altered pancreatic islet architecture.

Immunohistochemical analyses of insulin (green) and glucagon (red) expression were performed on 5-μm pancreatic cryosections of p-KO and control mice. Insulin-positive β-cells (green) were located in the core of islets in both mouse lines. Glucagon-positive α-cells (red) were evenly distributed along the periphery of islets in control mice (Ctrl.); in p-KO mice the pattern of peripheral distribution of α-cells was disrupted, and some α-cells were scattered throughout islet core (p-KO). Scale bar  = 50 µm.

Figure 3. p-KO mice exhibit normal insulin secretion per unit volume.

A. Groups of large and small isolated islets were separately examined for glucose-stimulated insulin secretion. Refer to the Results section for details on islet grouping. There was no difference in insulin secretion between p-KO and control islets of different sizes for the first phase or the entire stimulation period. Small islets secreted less insulin than large islets isolated from mice of the same genotype. B. Insulin secretion per unit volume. When insulin secretion data from A were normalized to islet size, there was no significant difference in insulin secretion between p-KO and control small islets as well as p-KO and control large islets during the first phase or the entire period of glucose-stimulated insulin release. Small p-KO islets secreted more insulin per unit of islet volume than large p-KO islets during the first phase (p<0.01) but not during the entire period of glucose-stimulated insulin release. C. Glucose-stimulated insulin secretion was measured in isolated islets from p-KO and control mice. Groups of 50-60 islets were incubated in KRH buffer containing 3 mM glucose (basal) before switching to 20 mM glucose (stimulatory). Net glucose-stimulated insulin secretion, were calculated as the sum of the insulin amount per islet in all fractions during the first 15 min (first phase) or the entire stimulation period (Total) after baseline subtraction. No difference was observed in the first phase or total insulin secretion between p-KO (grey bars, N = 3) and control (white bars, N = 3) mice. Data are presented as means ± SEM. D. Insulin content in pancreatic islets was assessed as the average pixel intensity of insulin positive signal per islet. Digital images were acquired with identical settings from 8–12 randomly selected pancreatic cryosections immunolabeled for insulin of each mouse. There was no difference in the intensity of insulin positive staining per islet in pancreatic sections obtained from control (white bar, N = 3) and p-KO mice (grey bar, N = 3).