Proinsulin misfolding is an early event in the progression to type 2 diabetes
- University of Michigan Medical School, United States
- Sanford Burnham Prebys Medical Discovery Institute, United States
- Konkuk University, Republic of Korea
- Columbia University, United States
- University of Adelaide, Australia
- University of Michigan, United States
- Tianjin Medical University, China
Figures
Figure 1 with 1 supplement
Detection of improperly folded proinsulin.
(A) Non-diabetic human pancreatic islets were lysed in RIPA buffer and divided into three equal parts, one of which was (partially) pre-reduced by boiling in the presence of 2 mM DTT (lane 3). This …
Figure 1—figure supplement 1
Proper and improper disulfide bond formation in proinsulin.
(A) Secreted proinsulin does not exhibit available free thiols. Media from INS1E cells incubated overnight without or with PERK inhibitor (‘+') were collected, divided in half, and either not …
Figure 2 with 1 supplement
Formation of proinsulin disulfide-linked complexes.
(A) INS1E cells were incubated for 20 hr in culture medium; the last 0, 5, 10, or 20 hr of this incubation included PERK inhibitor as indicated. At the end of the 20 hr, the media were collected and …
Figure 2—figure supplement 1
Proinsulin disulfide-linked complexes in human islets.
Lysates of human islets from two different donors were analyzed by nonreducing or reducing SDS-PAGE and processed as in panel C using mAb anti-human proinsulin (20G11).
Figure 3 with 1 supplement
Misfolding of proinsulin in human (and rodent) pancreatic islets.
(A) Islets from humans not known to be diabetic (Prodo Labs) were treated overnight with vehicle or PERK inhibitor before lysis, reducing or nonreducing SDS-PAGE, electrotransfer to nitrocellulose, …
Figure 3—figure supplement 1
Proinsulin misfoding induced by perturbation of the ER folding environment.
(A) Inhibition of PERK promotes formation of proinsulin disulfide-linked complexes in human islets. Live islets from a human donor were incubated overnight in the absence of presence of PERK …
Figure 4 with 1 supplement
Improper proinsulin folding from pharmacological or physiological alteration of the ß-cell ER folding environment.
(A) INS1E cells were incubated for 24 hr ± 20 µM GRP94 inhibitor (PU-WS13). During the last 3 hr, SubAB was added where indicated. Cell lysates were analyzed by immunoblotting for BiP (top panel), …
Figure 4—figure supplement 1
Intracellular proinsulin distribution in the LepRdb/db mouse.
Sections of homozygous diabetic LepRdb/db or heterozygous LepRdb/+ (control) mouse pancreas from conditions of Figure 4C (fed ad lib or fasted overnight) were immunostained with mAb anti-proinsulin …
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Figure 4—figure supplement 1—source data 1
- https://doi.org/10.7554/eLife.44532.011
Figure 5 with 1 supplement
Accumulation of improperly folded proinsulin and detection of ER stress response are early events in the development of diabetes in leptin receptor-deficient mice.
(A) Islet lysates from LepRdb/+ heterozygote (control) or LepRdb/db mice at different stages of diabetes progression (random blood glucose values shown above) were immunblotted for p58ipk and …
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Figure 5—source data 1
- https://doi.org/10.7554/eLife.44532.014
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Figure 5—source data 2
- https://doi.org/10.7554/eLife.44532.015
Figure 5—figure supplement 1
Intracellular proinsulin distribution in the LepRdb/db mouse.
Left side: Double immunofluorescence with mAb anti-proinsulin (red) and rabbit anti-calnexin (green) in islets of heterozygous LepRdb/+ (control) and homozygous LepRdb/db mice with various levels of …
Figure 6 with 1 supplement
Proinsulin Cys residues that contribute to covalent complex formation.
(A) INS832/13 were transfected to express myc-tagged recombinant human proinsulin ‘keep-A6/A11’, ‘keep-B7/A7’, or ‘keep-B19/A20’ constructs. At 48 hr post-transfection, cells were treated with …
Figure 6—figure supplement 1
Free thiols in recombinant proinsulin mutants.
293 T cells transiently transfected to express recombinant myc-tagged proinsulin mutants known as keep-B7/A7, keep-B19/A20, or keep-A6/A11 were metabolically labeled with 35S-amino acids for 30 min, …
Figure 7 with 1 supplement
Proinsulin intermolecular disulfide crosslinking is promoted by Cys(B19).
(A) 293 T cells were transfected to individually express six distinct human proinsulin mutants bearing only one cysteine. At 28 hr post-transfection, cell lysates were analyzed either by nonreducing …
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Figure 7—source data 1
- https://doi.org/10.7554/eLife.44532.020
Figure 7—figure supplement 1
Native and non-native proinsulin disulfide pairing.
(A) Native intramolecular disulfide bonding of proinsulin. Native disulfide bonds of proinsulin are indicated schematically as green lines connecting cysteine residues of the insulin B-chain (in …
Figure 8
Islet dysfunction during the natural history of diabetes in the LepRdb/db mouse, as a model.
A schematic is shown, indicating progression of early islet dysfunction during the natural history of diabetes in the LepRdb/db mouse. In the first stage of postnatal life, random blood glucose is …
Author response image 1
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Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.44532.022
