Automated Assessment of β-Cell Area and Density per Islet and Patient Using TMEM27 and BACE2 Immunofluorescence Staining in Human Pancreatic β-Cells

In this study we aimed to establish an unbiased automatic quantification pipeline to assess islet specific features such as β-cell area and density per islet based on immunofluorescence stainings. To determine these parameters, the in vivo protein expression levels of TMEM27 and BACE2 in pancreatic islets of 32 patients with type 2 diabetes (T2D) and in 28 non-diabetic individuals (ND) were used as input for the automated pipeline. The output of the automated pipeline was first compared to a previously developed manual area scoring system which takes into account the intensity of the staining as well as the percentage of cells which are stained within an islet. The median TMEM27 and BACE2 area scores of all islets investigated per patient correlated significantly with the manual scoring and with the median area score of insulin. Furthermore, the median area scores of TMEM27, BACE2 and insulin calculated from all T2D were significantly lower compared to the one of all ND. TMEM27, BACE2, and insulin area scores correlated as well in each individual tissue specimen. Moreover, islet size determined by costaining of glucagon and either TMEM27 or BACE2 and β-cell density based either on TMEM27 or BACE2 positive cells correlated significantly. Finally, the TMEM27 area score showed a positive correlation with BMI in ND and an inverse pattern in T2D. In summary, automated quantification outperforms manual scoring by reducing time and individual bias. The simultaneous changes of TMEM27, BACE2, and insulin in the majority of the β–cells suggest that these proteins reflect the total number of functional insulin producing β–cells. Additionally, β–cell subpopulations may be identified which are positive for TMEM27, BACE2 or insulin only. Thus, the cumulative assessment of all three markers may provide further information about the real β–cell number per islet.

[1]  Joachim M. Buhmann,et al.  Computational Pathology Analysis of Tissue Microarrays Predicts Survival of Renal Clear Cell Carcinoma Patients , 2008, MICCAI.

[2]  W. Oyen,et al.  Non-invasive quantification of the beta cell mass by SPECT with 111In-labelled exendin , 2014, Diabetologia.

[3]  L. Rosenberg,et al.  {beta}-Cell mass dynamics and islet cell plasticity in human type 2 diabetes. , 2010, Endocrinology.

[4]  J. Holst,et al.  Insulin Resistance Alters Islet Morphology in Nondiabetic Humans , 2014, Diabetes.

[5]  R. Rizza,et al.  β-Cell Mass and Turnover in Humans , 2012, Diabetes Care.

[6]  Jan Krützfeldt,et al.  Tmem27: a cleaved and shed plasma membrane protein that stimulates pancreatic beta cell proliferation. , 2005, Cell metabolism.

[7]  R. Gomis,et al.  The role of transmembrane protein 27 (TMEM27) in islet physiology and its potential use as a beta cell mass biomarker , 2010, Diabetologia.

[8]  Frank J. Gonzalez,et al.  Loss of ARNT/HIF1β Mediates Altered Gene Expression and Pancreatic-Islet Dysfunction in Human Type 2 Diabetes , 2005, Cell.

[9]  M. Hebrok,et al.  Diabetic β Cells: To Be or Not To Be? , 2012, Cell.

[10]  S. Bonner-Weir Islet growth and development in the adult. , 2000, Journal of molecular endocrinology.

[11]  Joachim M. Buhmann,et al.  Graph-Based Pancreatic Islet Segmentation for Early Type 2 Diabetes Mellitus on Histopathological Tissue , 2009, MICCAI.

[12]  Robert A. Rizza,et al.  β-Cell Deficit and Increased β-Cell Apoptosis in Humans With Type 2 Diabetes , 2003, Diabetes.

[13]  Robert A Rizza,et al.  Beta-cell deficit and increased beta-cell apoptosis in humans with type 2 diabetes. , 2003, Diabetes.

[14]  Jenny E Gunton,et al.  Loss of ARNT/HIF1beta mediates altered gene expression and pancreatic-islet dysfunction in human type 2 diabetes. , 2005, Cell.

[15]  J. Ahnfelt-Rønne,et al.  A new view of the beta cell , 2012, Diabetologia.

[16]  R. Aebersold,et al.  Bace2 is a β cell-enriched protease that regulates pancreatic β cell function and mass. , 2011, Cell metabolism.

[17]  K. Polonsky,et al.  Role of apoptosis in failure of beta-cell mass compensation for insulin resistance and beta-cell defects in the male Zucker diabetic fatty rat. , 1998, Diabetes.

[18]  J. Ripoll,et al.  Multimodal imaging of pancreatic beta cells in vivo by targeting transmembrane protein 27 (TMEM27) , 2012, Diabetologia.

[19]  C. Sempoux,et al.  Pancreatic β‐cell mass in European subjects with type 2 diabetes , 2008, Diabetes, obesity & metabolism.

[20]  H. Kasai,et al.  The HNF-1 target collectrin controls insulin exocytosis by SNARE complex formation. , 2005, Cell metabolism.

[21]  C. Talchai,et al.  Pancreatic β Cell Dedifferentiation as a Mechanism of Diabetic β Cell Failure , 2012, Cell.