Journal: Cell reports

Article Title: Amino acids-Rab1A-mTORC1 signaling controls whole-body glucose homeostasis

doi: 10.1016/j.celrep.2021.108830

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-Proinsulin (253627) , Novus biologicals , Cat# MAB13361; RRID: AB_2126534.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Calcium Assay, esiRNA, Real-time Polymerase Chain Reaction, shRNA, Software, Hybridization, Modification

Journal: The Journal of Biological Chemistry

Article Title: Nutrient-dependent regulation of β-cell proinsulin content

doi: 10.1016/j.jbc.2023.104836

Figure Lengend Snippet: Nutrient-dependent fluctuation in proinsulin pool size in INS1E β-cells. Cells grown in RPMI-1640 medium were seeded and grown in tissue culture plates for 2 days, fed every 24 h (100 μl per cm 2 surface area). A , a set of cells were lysed at the “zero time” (24 h after the last feeding), and the other sets were refed complete RPMI medium spiked to 25 mM glucose at (100 μl/cm 2 ) before lysis at different times as indicated. Lysates were resolved by reducing SDS-PAGE and analyzed by immunoblotting with guinea pig anti-insulin ( upper panel ), mouse mAb anti-rodent proinsulin ( middle panel ), or tubulin as a loading control ( lower panel ). B , a protocol similar to panel A was used with complete medium at the usual RPMI level of 11.1 mM glucose; samples run as biological duplicates. Lysates were resolved by reducing SDS-PAGE and analyzed by immunoblotting with guinea pig anti-insulin ( upper panel ), rabbit polyclonal anti-phospho-eIF2α ( middle panel ), or tubulin as a loading control ( lower panel ). Molecular weight markers (kDa) are indicated.

Article Snippet: Antibodies included guinea pig anti-insulin (Covance, RRID: AB_10013624 ), mouse mAb anti-rodent proinsulin (Novus Biologicals, RRID: AB_1107982 ), mouse mAb anti-human proinsulin B-C junction sequence KTRREAEDLQ (Abmart, RRID: AB_2921300 ), a custom-made rabbit polyclonal antibody against mouse C-peptide-2 (C-PQVAQLELGGGPGAGDLQT, Abmart, RRID: AB_2921302 ), mouse mAb anti-α-tubulin (Sigma-Aldrich, RRID: AB_477579 ), mouse mAb anti-beta actin (Proteintech, RRID: AB_2919667 ), rabbit mAb anti-HSP90 (Cell Signaling, RRID:AB_2233307), and rabbit anti-phospho-eIF2α (Ser51) (Cell Signaling, RRID: AB_330951 ).

Techniques: Lysis, SDS Page, Western Blot, Control, Molecular Weight

Journal: The Journal of Biological Chemistry

Article Title: Nutrient-dependent regulation of β-cell proinsulin content

doi: 10.1016/j.jbc.2023.104836

Figure Lengend Snippet: Contribution of glucose and amino acids to the regulation of proinsulin pool size in INS1E cells. Cells were seeded and initially fed as in Figure 1 . A , using a protocol similar to that in Figure 1 with timepoints as indicated: cell lysates (biological duplicates) were analyzed by immunoblotting with guinea pig anti-insulin ( upper panel ), mouse mAb anti-proinsulin ( middle panel ), rabbit polyclonal anti-phospho-eIF2α (third panel), or tubulin as a loading control ( bottom panel ). B and C , at the start of the experiment, biological duplicates either remained unfed in their 24 h-old medium or were fed selectively with fresh medium components as indicated, and the cells lysed thereafter at 30 min ( B ) or 4 h ( C ). Cell lysates were resolved by reducing SDS-PAGE and analyzed by immunoblotting with mouse mAb anti-rodent proinsulin, rabbit polyclonal anti-phospho-eIF2α, or tubulin as a loading control. Molecular weight markers (kDa) are indicated. D , quantitation (mean ± SD) of proinsulin and phospho-eIF2α protein levels relative to that obtained in the unfed control (Ctrl) sample; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001.

Article Snippet: Antibodies included guinea pig anti-insulin (Covance, RRID: AB_10013624 ), mouse mAb anti-rodent proinsulin (Novus Biologicals, RRID: AB_1107982 ), mouse mAb anti-human proinsulin B-C junction sequence KTRREAEDLQ (Abmart, RRID: AB_2921300 ), a custom-made rabbit polyclonal antibody against mouse C-peptide-2 (C-PQVAQLELGGGPGAGDLQT, Abmart, RRID: AB_2921302 ), mouse mAb anti-α-tubulin (Sigma-Aldrich, RRID: AB_477579 ), mouse mAb anti-beta actin (Proteintech, RRID: AB_2919667 ), rabbit mAb anti-HSP90 (Cell Signaling, RRID:AB_2233307), and rabbit anti-phospho-eIF2α (Ser51) (Cell Signaling, RRID: AB_330951 ).

Techniques: Western Blot, Control, SDS Page, Molecular Weight, Quantitation Assay

Journal: The Journal of Biological Chemistry

Article Title: Nutrient-dependent regulation of β-cell proinsulin content

doi: 10.1016/j.jbc.2023.104836

Figure Lengend Snippet: Nutrient-dependent fluctuation in proinsulin pool size in Min6 β-cells. Cells grown in DMEM medium (containing 25 mM glucose) were seeded and grown in tissue culture plates for 2 days, fed every 24 h (100 μl per cm 2 surface area). A and B , a set of cells were lysed at the “zero time” (24 h after the last feeding), and other sets (biological duplicates) were refed complete DMEM (100 μl/cm 2 ) before lysis at different times after feeding. Cell lysates were resolved by reducing SDS-PAGE and analyzed by immunoblotting with mouse mAb anti-rodent proinsulin ( upper panel ), guinea pig anti-insulin (B-chain, second panel), anti-phospho-eIF2α (third panel; there is a gel artifact between the duplicates at 12 h), or tubulin as a loading control ( lower panel ). A , cell lysed every 3 h during a 15 h time course. B , cells lysed every 30 min during a 90 min time course. The first two lanes are from the same gel and membrane, but the image has been spliced and is separated by a vertical line. Molecular weight markers (kDa) are indicated. DMEM, Dulbecco's modified Eagle's medium.

Article Snippet: Antibodies included guinea pig anti-insulin (Covance, RRID: AB_10013624 ), mouse mAb anti-rodent proinsulin (Novus Biologicals, RRID: AB_1107982 ), mouse mAb anti-human proinsulin B-C junction sequence KTRREAEDLQ (Abmart, RRID: AB_2921300 ), a custom-made rabbit polyclonal antibody against mouse C-peptide-2 (C-PQVAQLELGGGPGAGDLQT, Abmart, RRID: AB_2921302 ), mouse mAb anti-α-tubulin (Sigma-Aldrich, RRID: AB_477579 ), mouse mAb anti-beta actin (Proteintech, RRID: AB_2919667 ), rabbit mAb anti-HSP90 (Cell Signaling, RRID:AB_2233307), and rabbit anti-phospho-eIF2α (Ser51) (Cell Signaling, RRID: AB_330951 ).

Techniques: Lysis, SDS Page, Western Blot, Control, Membrane, Molecular Weight, Modification

Journal: The Journal of Biological Chemistry

Article Title: Nutrient-dependent regulation of β-cell proinsulin content

doi: 10.1016/j.jbc.2023.104836

Figure Lengend Snippet: The effect of nutrient availability on proinsulin disappearance and appearance in pancreatic β-cells. A , Min6 cells were seeded and fed exactly as in Figure 3 . One set of cells was then lysed (“zero time”). In a second set of samples, old medium was removed, quickly spiked with cycloheximide (100 µg/ml final concentration) and added back to the same cells with subsequent lysis at different times thereafter, as indicated. In a third set of samples, cells were fed new complete medium containing cycloheximide before lysis at different times thereafter, as indicated. Cell lysates were resolved by reducing SDS-PAGE and immunoblotting with mouse mAb anti-rodent proinsulin ( middle panel ; a long exposure is shown to increase detection of proinsulin); tubulin was a loading control ( lower panel ). B , quantitation of fraction of proinsulin (normalized to tubulin) remaining at different times after cycloheximide addition (mean ± SD; error bars are included but underlie the data shown), with no significant differences between old and new medium at any time point (n = 5 independent experiments; the 5-h time point was included for only two independent experiments). C , INS1E cells grown in RPMI-1640 medium (11.1 mM glucose) were seeded and grown in tissue culture plates for 2 days, fed every 24 h (100 μl per cm 2 surface area). One set of cells was lysed (“zero time;” 24 h after the last feeding), and the remaining cells were fed either at 180 μl per 2 cm 2 surface area or 500 μl per 2 cm 2 surface area of fresh complete RPMI. D , Min6 cells grown in DMEM medium (25 mM glucose) were seeded and grown in tissue culture plates for 2 days, fed every 24 h (100 μl per cm 2 surface area). In contrast with Figure 3 A , cells were fed with the larger volume of 500 μl per 2 cm 2 surface area of fresh complete DMEM. In both ( C and D ), the cells were lysed at various time points as indicated. Lysates were resolved by reducing SDS-PAGE and immunoblotting with mouse mAb anti-rodent proinsulin ( upper panel ), anti-phospho-eIF2α ( middle panel ), or tubulin as a loading control ( lower panel ). Molecular weight markers (kDa) are indicated. DMEM, Dulbecco's modified Eagle's medium.

Article Snippet: Antibodies included guinea pig anti-insulin (Covance, RRID: AB_10013624 ), mouse mAb anti-rodent proinsulin (Novus Biologicals, RRID: AB_1107982 ), mouse mAb anti-human proinsulin B-C junction sequence KTRREAEDLQ (Abmart, RRID: AB_2921300 ), a custom-made rabbit polyclonal antibody against mouse C-peptide-2 (C-PQVAQLELGGGPGAGDLQT, Abmart, RRID: AB_2921302 ), mouse mAb anti-α-tubulin (Sigma-Aldrich, RRID: AB_477579 ), mouse mAb anti-beta actin (Proteintech, RRID: AB_2919667 ), rabbit mAb anti-HSP90 (Cell Signaling, RRID:AB_2233307), and rabbit anti-phospho-eIF2α (Ser51) (Cell Signaling, RRID: AB_330951 ).

Techniques: Concentration Assay, Lysis, SDS Page, Western Blot, Control, Quantitation Assay, Molecular Weight, Modification

Journal: The Journal of Biological Chemistry

Article Title: Nutrient-dependent regulation of β-cell proinsulin content

doi: 10.1016/j.jbc.2023.104836

Figure Lengend Snippet: The time course of proinsulin, insulin, and phospho-eIF2α in pancreatic β-cells. A , INS1E cells grown in RPMI-1640 medium were seeded and grown in tissue culture plates for 2 days, fed every 24 h (100 μl per cm 2 surface area). A set of cells were lysed at the “zero time” (24 h after the last feeding), and the other sets were refed complete RPMI medium (100 μl/cm 2 ) before lysis at different times over a 24 h time course. Lysates were resolved by reducing SDS-PAGE and analyzed by immunoblotting with mouse mAb anti-rodent proinsulin ( upper panel ), anti-phospho-eIF2α ( second panel ), or tubulin as a loading control ( bottom panel ), or resolved by nonreducing SDS-PAGE for immunoblotting with guinea pig anti-insulin (third panel). Molecular weight markers (kDa) are indicated. B , using the protocol described in panel A, quantitation (mean ± SD) of phospho-eIF2α and proinsulin protein levels (normalized to tubulin) from both INS1E cells (RPMI medium with 11.1 mM glucose, solid dots ) and Min6 cells in (DMEM with 25 mM glucose, open dots ) are shown. ∗ p < 0.05 compared to the zero-time point. DMEM, Dulbecco's modified Eagle's medium.

Article Snippet: Antibodies included guinea pig anti-insulin (Covance, RRID: AB_10013624 ), mouse mAb anti-rodent proinsulin (Novus Biologicals, RRID: AB_1107982 ), mouse mAb anti-human proinsulin B-C junction sequence KTRREAEDLQ (Abmart, RRID: AB_2921300 ), a custom-made rabbit polyclonal antibody against mouse C-peptide-2 (C-PQVAQLELGGGPGAGDLQT, Abmart, RRID: AB_2921302 ), mouse mAb anti-α-tubulin (Sigma-Aldrich, RRID: AB_477579 ), mouse mAb anti-beta actin (Proteintech, RRID: AB_2919667 ), rabbit mAb anti-HSP90 (Cell Signaling, RRID:AB_2233307), and rabbit anti-phospho-eIF2α (Ser51) (Cell Signaling, RRID: AB_330951 ).

Techniques: Lysis, SDS Page, Western Blot, Control, Molecular Weight, Quantitation Assay, Modification

Journal: The Journal of Biological Chemistry

Article Title: Nutrient-dependent regulation of β-cell proinsulin content

doi: 10.1016/j.jbc.2023.104836

Figure Lengend Snippet: Nutrient-depleted upregulation of phospho-eIF2α in β-cells. A and B , INS1E cells grown in RPMI-1640 medium were seeded and grown in tissue culture plates for 2 days, fed every 24 h (100 μl per cm 2 surface area). A , At 8 h after the last refeeding, either vehicle (DMSO) or ISRIB (100 nM) was swirled directly into the existing medium, and the cells lysed at the 24 h time point. B , At 4 h after the last refeeding, either vehicle (DMSO) or ISRIB (100 nM) was swirled directly into the existing medium, and the cells lysed at the time points indicated. Lysates were resolved by reducing SDS-PAGE and analyzed by immunoblotting with mouse mAb anti-rodent proinsulin ( upper panel ), anti-phospho-eIF2α ( middle panel ), or tubulin as a loading control ( bottom panel ). C , Min6 cells grown in complete DMEM medium (25 mM glucose) were seeded and grown in tissue culture plates for 2 days, fed every 24 h (100 μl per cm 2 surface area). At 8 h after the last refeeding, either vehicle or GCN2 inhibitor (GCN2i, 5 μM) was swirled directly into the existing medium and the incubation continued for another 2 h before cell lysis. At left : Lysates were resolved by reducing SDS-PAGE and analyzed by immunoblotting with mouse mAb anti-rodent proinsulin ( upper panel ) and anti-phospho-eIF2α (second panel); HSP90 ( bottom panel ) is a loading control. Molecular weight markers (kDa) are indicated. At right: Quantitation (mean ± SD) of the levels of proinsulin and phospho-eIF2α (normalized to HSP90), respectively. ∗ p < 0.05 compared to vehicle alone. DMEM, Dulbecco's modified Eagle's medium; GCN2, general control nonderepressible 2.

Article Snippet: Antibodies included guinea pig anti-insulin (Covance, RRID: AB_10013624 ), mouse mAb anti-rodent proinsulin (Novus Biologicals, RRID: AB_1107982 ), mouse mAb anti-human proinsulin B-C junction sequence KTRREAEDLQ (Abmart, RRID: AB_2921300 ), a custom-made rabbit polyclonal antibody against mouse C-peptide-2 (C-PQVAQLELGGGPGAGDLQT, Abmart, RRID: AB_2921302 ), mouse mAb anti-α-tubulin (Sigma-Aldrich, RRID: AB_477579 ), mouse mAb anti-beta actin (Proteintech, RRID: AB_2919667 ), rabbit mAb anti-HSP90 (Cell Signaling, RRID:AB_2233307), and rabbit anti-phospho-eIF2α (Ser51) (Cell Signaling, RRID: AB_330951 ).

Techniques: SDS Page, Western Blot, Control, Incubation, Lysis, Molecular Weight, Quantitation Assay, Modification

Journal: The Journal of Biological Chemistry

Article Title: Nutrient-dependent regulation of β-cell proinsulin content

doi: 10.1016/j.jbc.2023.104836

Figure Lengend Snippet: Direct evidence that nutrient-dependent upregulation of the proinsulin pool is biosynthetic. A and B , Min6 cells grown in complete DMEM medium (25 mM glucose) were seeded and grown in tissue culture plates for 2 days, fed every 24 h (100 μl per cm 2 surface area) before initiating this experiment by refeeding the cells (100 μl/cm 2 ) for 4 h. A , mycolactone (200 nM) was added for the time points indicated (up to 30 min), and the cells lysed, resolved by reducing SDS-PAGE, and analyzed by immunoblotting with anti-rodent proinsulin; HSP90 is a loading control. B , Min6 cells were treated as in Figure 3 A , but during the last 30 min before each time point, the cells were treated with either vehicle alone, mycolactone (200 nM), or mycolactone plus MG132 (10 μM). Lysates were resolved by reducing SDS-PAGE and analyzed by immunoblotting with mAb anti-rodent proinsulin that favors the Ins1 gene product and polyclonal anti-rodent proinsulin that favors the Ins2 gene product; these antibodies also recognize preproinsulin as indicated. HSP90 ( middle panel ) is a loading control. C , murine islets were incubated in a limiting volume (2 μl/islet) of complete RPMI-1640 (11.1 mM glucose) for 24 h and then the medium spiked for 30 min with mycolactone (100 nM) plus MG132 (10 μM) (‘zero time point’), or islets were refed fresh medium for the times indicated, and during the last 30 min before each time point, the islets were treated with mycolactone plus MG132. Cell lysates were resolved by reducing SDS-PAGE and immunoblotting with rabbit polyclonal anti-proinsulin as in panel B; actin ( bottom panel ) is a loading control. D , quantitation of preproinsulin as a function of time after feeding (normalized to actin; the preproinsulin level at the zero-time point was set to 1.0 in each of three independent experiments; mean ± SD; ∗ p < 0.05). E , human islets (from a nondiabetic donor) were incubated in a limiting volume (2 μl/islet) of Prodo medium (5.6 mM glucose) for 24 h. One set of islet samples was then either untreated (vehicle alone) or treated with mycolactone (100 nM) + MG132 (10 μM) for 30 min before lysis (“zero time”, lanes 5 + 6). In a second set of islet samples, the old medium was removed and returned back to the same islets for 3 h plus 30 min in the presence of either vehicle or mycolactone + MG132 (lanes 3 + 4). In a third set of islet samples, the old medium was removed and replaced with new fresh medium for 3 h plus 30 min in the presence of either vehicle or mycolactone+MG132 (lanes 1 + 2). Islet lysates were resolved by reducing SDS-PAGE and analyzed by immunoblotting with anti-human-proinsulin (also recognizing preproinsulin as indicated); actin is a loading control. Molecular weight markers (kDa) are indicated. DMEM, Dulbecco's modified Eagle's medium.

Article Snippet: Antibodies included guinea pig anti-insulin (Covance, RRID: AB_10013624 ), mouse mAb anti-rodent proinsulin (Novus Biologicals, RRID: AB_1107982 ), mouse mAb anti-human proinsulin B-C junction sequence KTRREAEDLQ (Abmart, RRID: AB_2921300 ), a custom-made rabbit polyclonal antibody against mouse C-peptide-2 (C-PQVAQLELGGGPGAGDLQT, Abmart, RRID: AB_2921302 ), mouse mAb anti-α-tubulin (Sigma-Aldrich, RRID: AB_477579 ), mouse mAb anti-beta actin (Proteintech, RRID: AB_2919667 ), rabbit mAb anti-HSP90 (Cell Signaling, RRID:AB_2233307), and rabbit anti-phospho-eIF2α (Ser51) (Cell Signaling, RRID: AB_330951 ).

Techniques: SDS Page, Western Blot, Control, Incubation, Quantitation Assay, Lysis, Molecular Weight, Modification

Journal: Diabetes

Article Title: The Role of TRAPγ/SSR3 in Preproinsulin Translocation Into the Endoplasmic Reticulum.

doi: 10.2337/db21-0638

Figure Lengend Snippet: Figure 1—The TRAPg/SSR3 subunit of the TRAP/SSR complex contributes to the efficiency of recombinant proinsulin biosynthesis. A: Schematic of the TRAP/SSR complex highlights the predominant cytosolic exposure of the TRAPg/SSR3 subunit, including a hypothetical contact with a signal peptide N-terminus during polypeptide translocation (red line). B: Control (Ctrl) (wild-type [WT] 293T cells) and SSR3- KO 293T cells were transfected with untagged human preproinsulin plasmids. At 48 h posttransfection, cell lysates were analyzed by reducing SDS-PAGE gel and immunoblotting with antiproinsulin and anti-SSR1–4 antibodies as indicated. C: Quantitation (mean ± SD) of preproinsulin, proinsulin, and TRAP/SSR subunit protein levels from four independent experiments like that shown in B (normalized to tubulin). *P < 0.05 compared with Ctrl. D: Ctrl and SSR3-KO 293T cells were transfected with human preproinsulin plasmids. At 48 h post- transfection, the cells were pulse labeled with 35S-Met/Cys for the times indicated; this is a representative pulse labeling from three identi- cal experiments. Cell lysates (normalized to trichloroacetic acid–precipitable counts) were subjected to immunoprecipitation with anti- insulin and analyzed by reducing SDS-PAGE and phosphorimaging; both the absolute and relative amounts of recovered preproinsulin and proinsulin are shown in the graph.

Article Snippet: Mouse anti-rat proinsulin (CCI-17) and rabbit polyclonal anti-SSR1 antibodies (NB100-73013 and NBP1-86912, respectively) were from Novus Biologicals (Littleton, CO).

Techniques: Recombinant, Translocation Assay, Control, Transfection, SDS Page, Western Blot, Quantitation Assay, Labeling, Immunoprecipitation

Journal: Frontiers in endocrinology

Article Title: Pharmacological Inhibition of Inositol-Requiring Enzyme 1α RNase Activity Protects Pancreatic Beta Cell and Improves Diabetic Condition in Insulin Mutation-Induced Diabetes.

doi: 10.3389/fendo.2021.749879

Figure Lengend Snippet: FIGURE 5 | STF improves Akita islet b-cell function. (A) Glucose-stimulated insulin secretion of 20 islets isolated from Akita mice treated with STF or vehicle and incubated with 2.5 mM and 16.7 mM glucose. Secreted insulin was measured by ELISA. The data was presented as fold change and normalized with total protein concentration, with the amount of insulin secreted in response to 2.5 mM glucose from vehicle-treated group set to 1.0. (B–E) mRNA levels for indicated genes were analyzed in islets isolated from Akita mice treated with STF or vehicle by qRT-PCR. The results are expressed as fold change and are representative of 3 independent experiments. (F) Proinsulin content measurement by ELISA as detailed in Methods and Materials. (G, H) Proinsulin content and secretion measurement. 20 islets isolated from Akita mice treated with STF or vehicle were incubated with 2.5 mM and 16.7 mM glucose. Secreted proinsulin was measured by ELISA. Proinsulin content measurement by ELISA as detailed in Methods and Materials. The data was presented as fold change and normalized with total protein concentration, with the amount of proinsulin in response to 2.5 mM glucose from vehicle-treated group set to 1.0. (I) Proinsulin misfolding detection by Western blotting under nonreducing condition. Islets were treated with compounds at indicated concentrations for 16 hours. The data is representative of 3 independent experiments. (J–L) Ultrastructure of b-cells in islets isolated from Akita treated with STF as in by transmission electron microscopy. Images at the top panel and the bottom panel were taken at 3,000x and 10,000x, respectively. Arrows point to dark mature insulin granules. *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: The nonreducing gels were incubated in 25 mM dithiothreitol (DTT) solution for 10 min at room temperature before being transferred to PVDFmembranes for 25 min at 4°C at 40 V. Membranes were probed with anti-proinsulin antibody (CCI-17, NOVUS) and HRP-conjugated secondary antibodies (1:3000; Santa Cruz Biotechnology, CA, USA).

Techniques: Cell Function Assay, Isolation, Incubation, Enzyme-linked Immunosorbent Assay, Protein Concentration, Quantitative RT-PCR, Western Blot, Transmission Assay, Electron Microscopy