Characterization of Antibodies to Products of Proinsulin Processing Using Immunofluorescence Staining of Pancreas in Multiple Species

Abstract

The efficient processing of proinsulin into mature insulin and C-peptide is often compromised under conditions of beta cell stress, including diabetes. Impaired proinsulin processing has been challenging to examine by immunofluorescence staining in pancreas tissue because the characterization of antibodies specific for proinsulin, proinsulin intermediates, processed insulin and C-peptide has been limited. This study aimed to identify and characterize antibodies that can be used to detect products of proinsulin processing by immunofluorescence staining in pancreata from different species (mice, rats, dog, pig and human). We took advantage of several knockout mouse lines that lack either an enzyme involved in proinsulin processing or an insulin gene. Briefly, we report antibodies that are specific for several proinsulin processing products, including: a) insulin or proinsulin that has been appropriately processed at the B-C junction; b) proinsulin with a non-processed B-C junction; c) proinsulin with a non-processed A-C junction; d) rodent-specific C-peptide 1; e) rodent-specific C-peptide 2; and f) human-specific C-peptide or proinsulin. In addition, we also describe two 'pan-insulin' antibodies that react with all forms of insulin and proinsulin intermediates, regardless of the species. These antibodies are valuable tools for studying proinsulin processing by immunofluorescence staining and distinguishing between proinsulin products in different species.

Keywords: beta cells; diabetes; immunofluorescence staining; islets; proinsulin processing.

Figure 1.

Proinsulin amino acid sequence. (A) Schematic of the complete human proinsulin sequence. Blue boxes represent A-chain amino acids; green boxes represent B-chain amino acids, and white boxes represent C-peptide. One and three letter abbreviations are provided for each amino acid. Variations in the amino acid sequence are also illustrated for mouse proinsulin 1 (purple line) and mouse proinsulin 2 (pink line). (B) A general proinsulin sequence is shown to illustrate the site of cleavage for proinsulin processing enzymes. Yellow scissors indicate cleavage sites for prohormone convertase 1/3 (PC1/3) and PC2. Red triangles indicate cleavage sites for carboxypeptidase E (CPE). Yellow prisms represent di-sulphide bonds (S-S). (C) Proinsulin amino acid sequence of five different species, including human, dog, pig, mouse and rat. Grey boxes indicate regions with significant sequence variability among the species (amino acids that differ from the human proinsulin sequence are shown in purple within these regions).

Figure 2.

Schematic diagram representing the sequence of events required for processing proinsulin into mature insulin and C-peptide. The pathway on the right (indicated by solid arrows) is thought to be the predominant route of proinsulin processing, with cleavage first at the B-C junction by prohormone convertase 1/3 (PC1/3) to generate split-32,33 proinsulin, followed by removal of the two basic amino acids to generate des-31,32 proinsulin. Finally, PC2 and CPE act on the A-C junction to generate the mature C-peptide and insulin. The pathway on the left (indicated by dashed arrows), in which the A-C junction is processed first and the B-C junction second, is less dominant and thus the split-65,66 proinsulin, des-64,65 proinsulin, and diarginyl proinsulin intermediates are not typical products of proinsulin processing.

Figure 3.

Double immunofluorescence staining for β-Galactosidase (β-Gal; red) and insulin^(I8510), insulin^(MAb1), proinsulin^(GS9A8), or proinsulin^(82-PIN) (all green) in pancreas sections of mice lacking both insulin genes (Ins1^-/-;Ins2^-/-). Single immunofluorescence staining for insulin^(C27C9), C-peptide1, or C-peptide2 (green) was performed on adjacent serial sections. Two different islets are shown for β-Gal as a positive control; lack of immunoreactivity for each insulin-related antibody is shown from one of these two islet regions. All images are merged with DAPI nuclear staining (grey). Scale, 100 μm.

Figure 4.

Double immunofluorescence staining for insulin^(C27C9) (pan-insulin antibody; red) and insulin^(MAb1) (green) in pancreas sections of (A) various mouse strains, including wild type C57/BL6, PC1/3^-/-, PC2^-/-, CPE^-/-, Ins1^-/-;Ins2^+/+ and Ins1^+/+;Ins2^-/-, and (B) various species, including rat, human, pig and dog. Individual channels, as well as merged overlay images are shown with DAPI nuclear staining (grey). (C) Predicted epitope for the insulin^(MAb1) antibody is shown in orange. Scale, 100 μm.

Figure 5.

Double immunofluorescence staining for insulin^(C27C9) (pan-insulin antibody; red) and proinsulin^(GS9A8) (green) in pancreas sections of (A) various mouse strains, including wild type C57/BL6, PC1/3^-/-, CPE^-/-, Ins1^-/-;Ins2^+/+ and Ins1^+/+;Ins2^-/-, and (B) various species, including rat, human, pig and dog. Individual channels, as well as merged overlay images, are shown with DAPI nuclear staining (grey). (C) Predicted epitope for the proinsulin^(GS9A8) antibody is shown in orange. Scale, 100 μm.

Figure 6.

Double immunofluorescence staining for insulin^(C27C9) (pan-insulin antibody; red) and proinsulin^(82-PIN) (green) in pancreas sections of (A) various mouse strains, including wild type C57/BL6, PC2^-/-, Ins1^-/-;Ins2^+/+ and Ins1^+/+;Ins2^-/-, and (B) various species, including rat, human, pig and dog. Individual channels, as well as merged overlay images are shown with DAPI nuclear staining (grey). (C) Predicted epitope for the proinsulin^(82-PIN) antibody is shown in orange. Scale, 100 μm.

Figure 7.

Double immunofluorescence staining for insulin^(I8510) (pan-insulin antibody; red) and C-peptide1 (green) in pancreas sections of (A) various mouse strains, including wild type C57/BL6, Ins1^-/-;Ins2^+/+ and Ins1^+/+;Ins2^-/-, and (B) various species, including rat, human, pig and dog. Individual channels, as well as merged overlay images are shown with DAPI nuclear staining (grey). (C) Predicted epitope for the C-peptide1 antibody is shown in orange. Scale, 100 μm.

Figure 8.

Double immunofluorescence staining for insulin^(I8510) (pan-insulin antibody; red) and C-peptide2 (green) in pancreas sections of (A) various mouse strains, including wildtype C57/BL6, Ins1^-/-;Ins2^+/+ and Ins1^+/+;Ins2^-/-, and (B) various species, including rat, human, pig and dog. Individual channels, as well as merged overlay images are shown with DAPI nuclear staining (grey). (C) Predicted epitope for the C-peptide2 antibody is shown in orange. Scale, 100 μm.

Figure 9.

Double immunofluorescence staining for (A) insulin^(C27C9) (pan-insulin antibody; red) and proinsulin^(GNID4) (green) in pancreas sections of wild type C57/BL6 mouse, rat, human, pig and dog. Individual channels, as well as merged overlay images are shown with DAPI nuclear staining (grey). (B) Predicted epitope for the proinsulin^(GNID4) antibody is shown in orange. Scale, 100 μm.

Figure 10.

(A) Double immunofluorescence staining for insulin^(C27C9) (pan-insulin antibody; red) and C-peptide^(Abcam) (green) in pancreas sections of wild type C57/BL6 mouse, rat, human, pig and dog. Individual channels, as well as merged overlay images are shown with DAPI nuclear staining (grey). (B) Predicted epitope for the C-peptide^(Abcam) antibody is shown in orange. Scale, 100 μm.