Figure 5. Pdia1 deletion in HFD fed mice increases islet steady state proinsulin to insulin ratio with accumulation of high molecular weight (HMW) proinsulin complexes.

(A, C) Western blotting was performed for murine islets isolated after HFD for 30 wks. After overnight recovery, islets were lysed and analyzed under reducing (A) or non-reducing (C, D) conditions by SDS-PAGE and Western blotting. Image exposed for a different time for reduced proinsulin (CCI-17) in (A) was used to represent the total proinsulin levels in (D). Six independent mice were analyzed and technical duplicates are indicated as’ on the top of gel. (B). Quantification of indicated proteins was performed under reducing conditions (A). Each value was normalized to vinculin. Proinsulin/insulin ratios were calculated based on the quantification of proinsulin and insulin species under reducing conditions (A). (C) Five samples from A were analyzed under non-reducing conditions on a 4–12% Bis-Tris SDS gel. The proinsulin blot under reducing conditions is under the red line. Left side. Quantification of HMW proinsulin complexes under non-reducing conditions is indicated. (C), left side, upper) The ratio of 49–198 kDa (a) to 14–49 kDa (b) proinsulin complexes is shown. (C), left side, lower) The ratio of 14–49 kDa (b) proinsulin complexes to proinsulin monomer under reducing conditions is shown. Mean value is indicated in graph. (D) The ten samples in A were analyzed under non-reducing conditions after the gel was incubated in 25 mM DTT for 10 min at RT prior to transfer. To control for variable transfer from a gradient gel, we used a fixed percentage gel (12% Bis-Tris SDS). Right side. Quantification is shown for HMW proinsulin complexes under non-reducing conditions. (D), right side, upper) The ratio of 49–198 kDa (a) to 14–49 kDa (b) proinsulin complexes is shown. (D), right side, lower) The ratio of 14–49 kDa (b) proinsulin complexes to proinsulin monomer under reducing conditions is shown. (A–D) genetic controls; n = 3, KO; n = 3 mice. Mean ± SEM, p<0.05*, p<0.01**, p<0.001***. (E) WT murine islets were treated with or without NEM and lysates were analyzed under non-reducing conditions. Equal numbers of islets were divided into two tubes. Left side islets were rinsed with cold-PBS and lysed on ice. Right side islets were rinsed with cold-PBS containing 20 mM NEM and lysed in lysis buffer containing 2 mM NEM. Samples were prepared alongside and lysates were boiled for 5 min. Equal amounts of lysates were loaded and analyzed with or without gel incubation in 25 mM DTT for 10 min at RT prior to transfer. Two different exposure time images (short, long) are shown after DTT incubation. (F) WT murine islets were treated with increasing concentrations of DTT for 20 min in culture at room temperature and then analyzed by non-reducing and reducing SDS-PAGE and Western blotting with proinsulin antibody (CCI-17). The range of oligomeric proinsulin species are identified by an open arrowhead and monomeric proinsulin is indicated by black arrowhead.

Figure 5—figure supplement 1. Pdia1 deletion increases accumulation of HMW proinsulin complexes under regular diet.

(A) Islets were isolated from female mice at 14 wks after Tam injection, as described in Figure 1C, and analyzed by Western blotting under non-reducing conditions. The longer exposed image of reduced proinsulin (CCI-17) in Figure 1C is shown to represent the total proinsulin levels. (B) The same amounts of lysates from A were analyzed under non-reducing conditions after the gel was incubated in 25 mM DTT for 10 min at RT prior to transfer. To avoid unequal transfer from a gradient gel, we used a fixed percentage gel (12% Bis-Tris SDS). The proinsulin blot under reducing conditions is located under the red line. (D) The quantification of disulfide-linked proinsulin complexes (B) is shown. The left side of (C–D) shows the ratio of 49–198 kDa (a) to 14–49 kDa (b) proinsulin complexes. The right side of (C–D) shows the ratio of 14–49 kDa (b) proinsulin complexes to proinsulin monomer under reducing conditions.

Figure 5—figure supplement 2. Inhibition of ER to Golgi trafficking increases proinsulin disulfide linked HMW complex formation.

After overnight recovery, WT islets were incubated in media containing brefeldin A (BFA, 5 µg/ml) and/or cycloheximide (CHX, 100 μg/ml) for 1 hr at 37°C and then analyzed by non-reducing and reducing SDS-PAGE and Western blotting with proinsulin antibody (CCI-17).

Figure 5—figure supplement 3. PDIA1 overexpression reduces proinsulin.

Adenoviruses that express human proinsulin (Ad-hProins) or human PDIA1 (Ad-hPDI) or catalytically inactive PDIA1 mutant (Ad-hPDImut) were infected into WT MEFs. After 48 hr, cells were treated with DTT (2.5, 5 mM) for 20 min or Menadione (50, 100 μM) for 1 hr. Lysates were analyzed by non-reducing and reducing SDS-PAGE and Western blotting with anti-human proinsulin antibody (1B24). Unfortunately, this antibody for human proinsulin does not recognize HMW proinsulin aggregates.