Fig 1.
CFTR is expressed in human islets.
A-C. Immunohistochemistry (A) and immunofluorescence (B-C) images of human pancreas (normal tissue from an 11-month-old control) immunostained by using CFF-217 antibody (Table 1). The islet shown in A, encircled by a dashed red trace, contains endocrine cells stained by the antibody, which is shown in the magnified red square in the right bottom corner of the figure. CFTR-positive pancreatic duct cells are shown within the white squares. Shown in B is CFTR immunoreactivity in human exocrine and endocrine cells. The edges of all cells were immunolabeled by using E-cadherin antibodies (Table 1). The islet is encircled by a dashed trace and the square represents the area magnified and shown in C. D-H. In situ hybridization of normal human pancreas tissue by using fluorophore-labeled RNA probes directed against CFTR (green, D), insulin (INS, white, E) and glucagon (GLC, red, F) transcripts. Red and white arrows in H, a magnification of G, indicate specific CFTR labeling on acinar and endocrine cells, respectively. Bars in A and B, C, D, and in H correspond to 50μm and 10μm, respectively.
Fig 2.
A-D. Shown are microscopy images of human pancreas from a normal donor (A, B, 11-month-old control) or homozygous for the CFTR mutation F508del (C, D, 6-month-old CF patient) immunostained using CFF-217 (A, C) or CFF 412 (B, D). Note the expected apical and intracellular immunolabeling of CFTR in normal and mutant pancreas, respectively. The insets indicate higher magnification. E, F. Microscopy images of human pancreas from a 1 yr. old CF patient homozygous for CFTRG542X using the antibodies CFF-217 (E) and CFF-412 (F). Shown is absence of immunostaining with both antibodies. G-I. Microscopy images of pancreas (G) and intestine (H) of CftrKO mice immunolabeled using CFF-217. Shown are the expected patterns of immunostaining for specific antibodies. For comparison, shown are magnified images of CftrWT pancreas islets using CFTR and insulin antibodies (top and bottom right corners in G, respectively). I. Omission of the CFTR antibody did not generate immunostaining in the intestine. Bars in A-I represents 50μm. J-L. Pancreatic tissue from CftrG542X mice co-immunolabeled by using the indicated mouse CFTR-specific antibody acl-006 (J), insulin (K, Ins) and glucagon (L, Glc). M-N. Overlay image of J-L and magnified squared area shown in N. O-P. Control images obtained from CftrG542X mice in the absence of primary (O) or secondary antibodies (P). DAPI was used to counterstain nuclei in P.
Table 1.
List of primary antibodies raised against the indicated antigens are identified by their clone/catalogue number from vendors/suppliers.
Table 2.
Human islet donor demographics.
Table 3.
List of primer-sets used in RT-PCR experiments named after the target transcript followed by numbers indicating amplicon sizes in base pairs (bp).
Fig 3.
Cftr is expressed in endocrine cells of the mouse islet.
A-J. Immunofluorescence images of WT mouse (CftrWT) pancreas immunostained with the Cftr antibody acl-006 [(Table 1), A and F] in combination with antibodies against insulin (Ins, B), glucagon (Glc, C), somatostatin (Stt, H) and pancreatic polypeptide (PP, G). Magnified overlay images of D and I are shown in E and J, respectively, wherein Cftr immunoreactivity in α-, β-, δ- and PP-cells of the mouse islet is indicated by arrows. K-L. Immunohistochemistry images of normal human pancreas immunostained with acl-006. The islet encircled by a dashed red trace in K is magnified in L to show CFTR immunoreactivity. Bars in A-G represent 25μm.
Fig 4.
CFTR expression in human and mouse islets and in MIN6 β-cells.
A, B. Immunoblots of protein extracts obtained from purified human islets, the β-cell line MIN6 and mouse pancreas as positive control (A) and primary mouse islets (B). Bands of expected size are shown for human/mouse Cftr (C band: ~170kDa, B band: ~150kDa) detected using Cftr antibody 24–1. Actin was used as loading control. C-E. Representative original RT-PCR experiments showing Cftr transcripts of expected sizes (Table 3) amplified from total RNA obtained from primary human (n = 5 donors), mouse islets (n = 3 preparations) (C and D, respectively) and MIN6 β-cells (n = 4 preparations) (E). F. Partial sequence chromatograms of purified human CFTR amplicons, as indicated in C.
Fig 5.
Cftr contributes to the insulin secretory response in mouse, rat and human islets and the MIN6 β-cell line.
A. Proof of specificity for CFTRinh-172 (0–10μM) on the secretory response using islets obtained from transgenic mice lacking Cftr (CftrKO) and CftrWT mice, in response to 5.5mM and 12.5mM glucose (n = 8, *p<0.05). B, C. Effect of 5μM CFTRinh-172 on insulin secretion of mouse CftrKO and CftrWT islets (B, n = 5, *p<0.05) and rat islets (C, n = 3, *p<0.05) in response to 5.5mM and 12.5mM glucose. D. Basal (5.5mM glucose) and stimulated (12.5mM glucose) insulin secretory response of freshly isolated primary human islets in the presence of vehicle (DMSO) or 5μM CFTRinh-172 (n = 5 donors, *p<0.05). E. Dose-response curve of basal (5.5mM glucose) and stimulated (12.5mM glucose) insulin secretion from human islets (n = 4 donors, *p<0.05) treated with the indicated concentrations of CFTRinh-172. F. Basal (5.5mM glucose) and stimulated (12.5mM glucose) insulin secretory response of MIN6 β-cells incubated with vehicle (DMSO) or 5μM Inh172 (n = 3, *p<0.05).
Fig 6.
Cftr is functionally detected in rat β-cells.
A. Representative Cl−currents recorded using rat primary pancreatic β-cells pre-incubated for 1h in 5.5 or 12.5mM glucose before (control, black trace), after forskolin addition (10μM, blue trace) alone or 5 min after adding CFTRinh-172 (5μM, red trace). B, C. Current density-voltage relationships of β-cell Cl−currents recorded in 5.5mM (n = 10) glucose (B) or 12.5mM (n = 8) glucose (C) in the presence of forskolin (10μM, blue squares) alone or plus CFTR-inh-172 (5μM, red squares). Insets in B and C denote peak currents at +100mV (n = 8, for all conditions, *p<0.05). D. Current density-voltage relationship of β-cell Cl−currents in 5.5mM or 12.5 mM glucose after addition of forskolin (10μM). Traces show β-cell responders (blue circles/squares, n = 10 and n = 8 for 5.5mM and 12.5 mM respectively) and non-responders to forskolin in 5.5mM or 12.5mM glucose (open triangles, n = 19 and n = 41, respectively). E. Numerical proportion of the latter observation.