Background

In recent years, we and others have developed methods to detect non-native proinsulin in cells expressing recombinant proinsulin, pancreatic β-cell lines expressing recombinant or endogenous proinsulin, and β-cells within isolated pancreatic islets. A significant advance emerged from a simple immunoblotting technique that identified intermolecular disulfide-linked proinsulin complexes in the islets of the leptin receptor defective type-2 diabetes mouse model, db/db (LepR^db/db) [1]. When the proinsulin folding was analyzed as a function of blood glucose using this approach, it was observed that proinsulin participated in misfolding and disulfide-linked complex formation much before the onset of hyperglycemia (see Figure 1, normoglycemic db/db, glucose 101 mg/dL), indicating that proinsulin misfolding is a prelude to type-2 diabetes [1]. The immunoblotting technique to detect misfolded proinsulin in beta cells has been widely used in subsequent studies by us and others [2–6]. However, we soon realized that the original immunoblotting approach faced challenges in accurately quantifying proinsulin misfolding due to variations in the biophysical properties of different proinsulin complexes and differences in antibody affinity for these diverse folded states (Figure 1). To address these limitations, we have made key methodological refinements to enhance the accuracy in measuring the abundance of different proinsulin folded forms as separated by nonreducing SDS-PAGE. This methodology empowers any diabetes research laboratory worldwide to reliably quantify proinsulin misfolding in various contexts, including healthy and diseased human pancreas, as well as β-cell models. While techniques like differential scanning fluorimetry (DSF) can indicate changes in thermal stability potentially linked to misfolding, and circular dichroism (CD) spectroscopy can reveal alterations in secondary and tertiary structure, neither directly distinguishes between native and non-native disulfide bond arrangements or quantifies oligomeric states with the clarity offered by nonreducing SDS-PAGE. This direct visualization of disulfide-linked species makes the refined immunoblotting approach advantageous for studying the specific misfolding mechanisms of proinsulin.

Figure 1. Detection of misfolded proinsulin in LepR^db/db mouse islets using non-reducing SDS-PAGE followed by proinsulin immunoblotting. Islet lysates from LepR^db/+ heterozygote (control, random blood glucose: 138 mg/dL) or LepR^db/db mice (random blood glucose: 101 mg/dL) were analyzed by nonreducing or reducing SDS-PAGE and immunoblotting with mAb anti-proinsulin (CCI-17, above black line; molecular mass markers are noted) or guinea pig anti-insulin (below black line). The nonreducing gel (left) exhibited two key issues. First, an apparent overrepresentation of disulfide-linked proinsulin complexes and a near absence of the monomer band. Second, the red double-headed arrow highlights a significant discrepancy: the limited total proinsulin detected under reducing conditions (right) contrasted sharply with the abundant disulfide-linked complexes observed in the nonreducing gel (left). (Modified from Arunagiri et al. [1], eLife 2019 Jun 11: 8: e44532.)

Refinement of proinsulin misfolding assay: The initial assay showed potential to identify proinsulin disulfide-linked complexes (Figure 1), but i) failed to accurately detect proinsulin monomers under nonreducing conditions, and ii) overrepresented the non-native disulfide-linked complexes, which appeared to exceed the total proinsulin recovered under reducing conditions. A revised protocol was developed to address these key limitations. A schematic comparing the traditional and the new protocol is illustrated below (Figure 2). A detailed description of the protocol is presented later in this manuscript.

Figure 2. Schematic of different immunoblotting methods to detect proinsulin misfolding. Left: Conventional immunoblotting after nonreducing SDS-PAGE is super-sensitive to misfolded disulfide-linked complexes of proinsulin. Red arrow pathway: Treatment of samples with DTT + heating after nonreducing SDS-PAGE converts all proinsulin contained within the gel to reduced monomers. Using a fixed percentage of acrylamide ensures even transfer efficiency across the gel. This method enhances detection of proinsulin monomers (particularly native monomers) after nonreducing SDS-PAGE and provides a more quantitative estimate of the distribution of differently-folded proinsulin species. (Source: Arunagiri et al. [7], Protein Science, 33(4), e4949.)