Increased ectodomain shedding of cell adhesion molecule 1 from pancreatic islets in type 2 diabetic pancreata: correlation with hemoglobin A1c levels

Abstract

Pulmonary emphysema and type 2 diabetes mellitus (T2DM), both caused by lifestyle factors, frequently concur. Respectively, the diseases affect lung alveolar and pancreatic islet cells, which express cell adhesion molecule 1 (CADM1), an immunoglobulin superfamily member. Protease-mediated ectodomain shedding of full-length CADM1 produces C-terminal fragments (CTFs) with proapoptotic activity. In emphysematous lungs, the CADM1 shedding rate and thus the level of CTFs in alveolar cells increase. In this study, CADM1 expression in islet cells was examined by western blotting. Protein was extracted from formalin-fixed, paraffin-embedded sections of pancreata isolated from patients with T2DM (n = 12) or from patients without pancreatic disease (n = 8) at autopsy. After adjusting for the number of islet cells present in the adjacent section, we found that full-length CADM1 decreased in T2DM islets, while ectodomain shedding increased. Hemoglobin A1c levels, measured when patients were alive, correlated inversely with full-length CADM1 levels (P = 0.041) and positively with ectodomain shedding rates (P = 0.001). In immunofluorescence images of T2DM islet cells, CADM1 was detected in the cytoplasm, but not on the cell membrane. Consistently, when MIN6-m9 mouse beta cells were treated with phorbol ester and trypsin to induce shedding, CADM1 immunostaining was diffuse in the cytoplasm. When a form of CTFs was exogenously expressed in MIN6-m9 cells, it localized diffusely in the cytoplasm and increased the number of apoptotic cells. These results suggest that increased CADM1 ectodomain shedding contributes to blood glucose dysregulation in T2DM by decreasing full-length CADM1 and producing CTFs that accumulate in the cytoplasm and promote apoptosis of beta cells. Thus, this study has identified a molecular alteration shared by pulmonary emphysema and T2DM.

Figure 1. Increased ectodomain shedding of CADM1 in T2DM pancreata.

(a) Western blot analysis of CADM1 expression in control and T2DM pancreata. Cases are numbered as in Table S1. Arrowheads indicate bands corresponding to the full-length, αCTF, and βCTF forms of CADM1. The blot was reprobed with an anti-β-actin antibody to show protein loading. (b) CADM1 expression per islet cell in each patient sample. For each lane in a, the intensities of the full-length CADM1, αCTF, βCTF, and β-actin bands were quantified, and CADM1 levels were expressed relative to β-actin and the islet cell count (/cm² of tissue). Statistical significance was analyzed with the Mann-Whitney U-test. P-values are shown. (c) CADM1 ectodomain shedding rates (relative amounts of CTFs to the full-length CADM1). Statistical significance was analyzed with the Mann-Whitney U-test. P-values are shown. (d) Correlation of HbA1c levels with full-length CADM1 expression per islet cell (left) or the CADM1 shedding rate (αCTF/full-length; right). In each graph, the dot distribution approximated a linear function (dotted lines). Correlations and statistical significance were analyzed with Spearman’s rank test. R² and P-values are shown.

Figure 2. Representative photomicrographs of CADM1 immunofluorescence in pancreatic islets.

Pancreatic sections from control (case 7; left) and T2DM (case 3; right) patients were double stained with antibodies against CADM1 (red) and glucagon (green; top) or insulin (green; bottom) and then counterstained with DAPI (blue). Merged images are shown. Bar  = 50 µm.

Figure 3. Subcellular localization of endogenous and exogenous αCTF in MIN6-m9 cells.

MIN6-m9 cells were untreated (−), treated with PMA (200 nM) and trypsin (0.025% w/v), or transfected with pCX4bsr-SP-αCTF. After 45 min of treatment and 2 days of transfection, CADM1 levels were assessed in western blot (a) and immunofluorescence (b) analyses using a CADM1 antibody. In a, arrowheads indicate full-length CADM and αCTF. The blot was reprobed with an anti-β-actin antibody to show the protein loading. In b, cells were double stained with CADM1 antibody (green; top) and MitoTracker (red; middle). Merged images are also shown (bottom). Data are representative of three independent experiments. Bar  = 10 µm.

Figure 4. Functional analyses of αCTF in MIN6-m9 cells.

MIN6-m9 cells were untreated (−), or transfected with the empty pCX4bsr vector, or the vector expressing either αCTF or αCTFmut. After 2 days of transfection, CADM1 levels were assessed in a western blot using a CADM1 antibody (a). Arrowheads indicate full-length CADM, αCTF, and αCTFmut. The blot was reprobed with an anti-β-actin antibody to show the protein loading. (b) Glucose-induced insulin secretion assay. The mean ratio (fold increase) relative to the baseline (t = 0) and normalized to the cell volume is plotted with a bar indicating SE. There was no difference between any pair at each timepoint; the smallest P-values by t-test are shown. (c) TUNEL assay. TUNEL and DAPI fluorescent stains are colored green and blue, respectively. TUNEL-positive cells are recognized by bluish white nuclei where the two stains merge (indicated by arrows). Mean ± SE of the percentage of TUNEL-positive cells and P-values by t-test are shown below the photomicrographs. Data in b and c are representative of three independent assays, respectively. Bar  = 50 µm.