Islet Regeneration and Pancreatic Duct Glands in Human and Experimental Diabetes

Abstract

Contrasting evidence is present regarding the contribution of stem/progenitor cell populations to pancreatic regeneration in diabetes. Interestingly, a cell compartment with stem/progenitor cell features has been identified in the pancreatic duct glands (PDGs). The aims of the present study were to evaluate pancreatic islet injury and regeneration, and the participation of the PDG compartment in type 2 diabetic mellitus (T2DM) and in an experimental model of diabetes. Human pancreata were obtained from normal (N = 5) or T2DM (N = 10) cadaveric organ donors. Experimental diabetes was generated in mice by intraperitoneal injection of 150 mg/kg of streptozotocin (STZ, N = 10); N = 10 STZ mice also received daily intraperitoneal injections of 100 µg of human recombinant PDX1 peptide (STZ + PDX1). Samples were examined by immunohistochemistry/immunofluorescence or RT-qPCR. Serum glucose and c-peptide levels were measured in mice. Islets in T2DM patients showed β-cell loss, signs of injury and proliferation, and a higher proportion of central islets. PDGs in T2DM patients had a higher percentage of proliferating and insulin⁺ or glucagon⁺ cells compared to controls; pancreatic islets could be observed within pancreatic duct walls of T2DM patients. STZ mice were characterized by reduced islet area compared to controls. PDX1 treatment increased islet area and the percentage of central islets compared to untreated STZ mice but did not revert diabetes. In conclusion, T2DM patients show signs of pancreatic islet regeneration and involvement of the PDG niche. PDX1 administration could support increased endocrine pancreatic regeneration in STZ. These findings contribute to defining the role and participation of stem/progenitor cell compartments within the pancreas.

Keywords: Pdx1; endocrine pancreas; insulin; stem/progenitor cells; streptozotocin.

FIGURE 1

Pancreatic islet histology and phenotype in normal (NR) human pancreas and in type 2 diabetes mellitus (T2DM) pancreas samples. (A) Hematoxylin and eosin (H&E) stain. T2DM patients showed higher islet area and islet size compared to normal samples. Dotted lines individuate pancreatic islets. Histograms show means and standard deviation (SD) for islet area and diameter. Scale bar: 150 μm. (B) Double immunofluorescence for insulin (green) and glucagon (red). Pancreatic islets in T2DM patients were characterized by a lower β-cell percentage and by a higher α-/β-cell ratio compared to normal pancreata. Histograms show means and SD for β-cell percentages and for the α-/β-cell ratio. Scale bar: 100 μm. Nuclei are displayed in blue (DAPI staining). (C) Immunohistochemistry for proliferating cell nuclear antigen (PCNA). T2DM patients showed increased percentage of proliferating PCNA + cells within islets compared to normal pancreata. Original magnification: 40x. Areas in the circle are magnifications of the images above. The histogram shows means and SD for the percentage of proliferating cells. (D) Immunohistochemistry for γH2A.x (upper panels) and cleaved caspase 3 (cCasp3, lower panels). Pancreatic islets in diabetic patients were characterized by a higher expression of senescence marker γH2A.x and apoptosis marker cCasp3 compared to normal pancreata. Histograms show means and standard deviation for the percentage of positive cells. Original magnification: 40x. (E) H&E stain on pancreata from T2DM patients. Pancreata from T2DM patients were characterized by a higher percentage of central islets compared to normal ones. Arrowheads indicate pancreatic ducts. The area in the box is magnified on the right; dotted line individuates a pancreatic duct branch surrounding an islet. Scale bar: 75 μm. The histogram shows means and SD for the percentage of central islets and the average distance between islets and neighboring ducts. * = p< 0.05 versus T2DM.

FIGURE 2

Pancreatic duct glands (PDGs) in normal (NR) human pancreas and in type 2 diabetes mellitus (T2DM) pancreas samples. (A) Hematoxylin and eosin (H&E) stain (upper panels) and immunohistochemistry for proliferating cell nuclear antigen (PCNA, lower panels). Pancreatic ducts in T2DM patients were characterized by a higher PDG mass and by a higher expression of PCNA within PDGs compared to normal ducts. Histograms show means and standard deviation (SD) for the percentage of duct wall area occupied by PDGs and for the percentage of PCNA + PDG cells. Scale bar for H&E: 100 μm. Original magnification for PCNA: 40x. (B) Immunohistochemistry for insulin (upper panels) and glucagon (lower panels). T2DM patients showed a higher percentage of insulin+ and glucagon + cells within PDGs compared to normal ones. Histograms show means and SD for the percentage of positive cells. Scale bar: 75 μm (insulin) and 50 μm (glucagon). (C) H&E stain on T2DM samples shows pancreatic ducts with dysplastic lesions of surface epithelium and PDGs and the presence of pancreatic islets among PDGs (circle). Seriated sections of the same area show that islets in pancreatic ducts express insulin, glucagon and are vascularized. Scale bar: 100 μm * = p< 0.05 versus T2DM.

FIGURE 3

Pancreatic islets and pancreatic duct glands (PDGs) in control mice, streptozotocin (STZ)-treated mice and STZ mice treated with PDX1. (A) Hematoxylin and eosin (H&E) stain on pancreas samples. STZ treated mice were characterized by a lower pancreatic islet area compared to controls; STZ + PDX1 mice showed a higher islet area compared to STZ, without significant differences compared to controls. Dotted lines individuate pancreatic islets. Histogram shows means and standard deviation (SD) for pancreatic area percentage. Scale bar: 150 μm. (B) H&E stain (upper panels) and double immunofluorescence for insulin (ins, in green) and cytokeratin 19 (CK19, in red) on pancreas samples. STZ-treated mice show a higher percentage of central islets compared to controls; moreover, STZ + PDX1 mice showed a significantly higher percentage of central islets compared to STZ and control mice. Scale bar in H&E: 75 μm. In immunofluorescence image, arrowheads indicate small CK19 + cells within islets. Nuclei are displayed in blue (DAPI staining). Original magnification: 40x. Histograms show means and SD for the percentage of central islets and islets containing CK19 + cells. (C) H&E stain and immunohistochemistry for proliferating cell nuclear antigen (PCNA) on pancreatic ducts. STZ- and STZ + PDX1-treated mice show a higher PDG mass and percentage of PCNA + cells within PDGs compared to controls. Histograms show means and SD for the percentage of duct wall area occupied by PDGs and for the percentage of PCNA + PDG cells. Scale bar: 100 μm (H&E) and 50 μm (PCNA). * = p< 0.05 versus other groups; ^ = p< 0.05 versus STZ group.

FIGURE 4

Pancreatic islet phenotype and glycemic profile in control mice, streptozotocin (STZ)-treated mice and STZ mice treated with PDX1. (A) Graph shows individual values for serum glucose levels in STZ and STZ + PDX1 mice. Dotted line indicates the threshold for diabetes diagnosis (300 mg/dl). (B) Double immunofluorescence for insulin (ins, in green) and glucagon (glu, in red). (C) Immunohistochemistry for insulin (upper panels) and for glucagon (lower panels). STZ- and STZ + PDX1-treated mice showed a lower percentage of β-cells and a higher percentage of α-cells compared to controls. Histograms show means and standard deviation (SD) for the percentage of α-/β-cells. Original magnification: 40x. (D) Histograms show means and standard deviation for the RT-qPCR expression of NGN3, PDX1, MaFA and Insulin genes. Data are expressed as means and standard deviation (SD). GOI: gene of interest. ND: not detectable. (D) Immunofluorescence for NGN3 confirmed the higher expression of NGN3 in STZ + PDX1-treated mice compared to STZ group. NGN3 was mostly expressed by pancreatic duct cells (arrows). Separate channels were provided. Original Magnification: 40x. * = p< 0.05 versus other groups; ^ = p< 0.05 versus STZ group.