Journal: iScience

Article Title: Islet cell stress induced by insulin-degrading enzyme deficiency promotes regeneration and protection from autoimmune diabetes

doi: 10.1016/j.isci.2024.109929

Figure Lengend Snippet: UPR effector mRNA and global protein expression by I de −/ − islet cells (A–H) mRNA expression levels, as measured by RT-qPCR, of genes linked to the UPR ( Atf6 , Hspa5 , Xbp1s , Ddit3 ), to regeneration ( Reg2 ) or to proliferation ( Pcna , Mki67 ) in steady state islets from Ide +/+ and Ide −/− C57BL/6 and NOD mice of different ages, as indicated. Results are expressed as fold change, i.e., the ratio of expression in Ide −/− relative to Ide +/+ islets. Islets were either analyzed immediately after isolation or after overnight culture in RPMI medium, as indicated. Each dot represents islets from one mouse. Data in A–H were evaluated by Mann-Whitney tests with Bonferroni correction for multiple comparisons. p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. (I) Ide +/+ or control Ide −/− NOD mice aged 10 weeks were injected for 2 weeks with carfilzomib or for 2 days with tunicamycin with or without addition of rapamycin before isolation of islets and quantification of Ide mRNA expression by RT-qPCR. Data were evaluated by two-way ANOVA with Dunnett’s multiple comparison correction. ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. (J) Proteomic analysis of islet proteins from 3 Ide +/+ and 3 Ide −/− mice aged 10 weeks. Left: volcano plot showing the proteins up- (in green) and down- (in red) regulated in Ide −/− NOD islets. The horizontal dashed line represents the significance threshold ( p -value <0.05) (for details see Table S1 ). Right: Barplot representing the Gene Ontology Biological Process (GO BP) terms enriched using enrichR software and the GO Biological Process 2021 database. In green are the TOP10 GO BP terms enriched using the up-regulated proteins in Ide −/− and in red the TOP10 GO BP terms enriched using the up-regulated proteins in Ide +/+ islets for analysis. A log-transformed adjusted p -value was used for TOP10 ranking (for details see Table S2 ).

Article Snippet: Recombinant mouse REG2 (R&D Systems) and human insulin (Roche) were digested with recombinant WT or protease-dead (cf-E111Q) human IDE enzyme in 20 mM Hepes pH 7.2, 50 mM NaCl buffer at a ratio IDE : INS ratio (w/w) of 1:1, or a ratio IDE : REG2 of 1:10.

Techniques: Expressing, Quantitative RT-PCR, Isolation, MANN-WHITNEY, Control, Injection, Comparison, Software, Transformation Assay

Journal: iScience

Article Title: Islet cell stress induced by insulin-degrading enzyme deficiency promotes regeneration and protection from autoimmune diabetes

doi: 10.1016/j.isci.2024.109929

Figure Lengend Snippet:

Article Snippet: Recombinant mouse REG2 (R&D Systems) and human insulin (Roche) were digested with recombinant WT or protease-dead (cf-E111Q) human IDE enzyme in 20 mM Hepes pH 7.2, 50 mM NaCl buffer at a ratio IDE : INS ratio (w/w) of 1:1, or a ratio IDE : REG2 of 1:10.

Techniques: Recombinant, Lysis, Protease Inhibitor, Staining, Plasmid Preparation, Blocking Assay, Reverse Transcription, SYBR Green Assay, Imaging, Mass Spectrometry, Enzyme-linked Immunosorbent Assay, Software, Transmission Assay, Microscopy

Journal: Redox biology

Article Title: Role and mechanism of REG2 depletion in insulin secretion augmented by glutathione peroxidase-1 overproduction.

doi: 10.1016/j.redox.2022.102457

Figure Lengend Snippet: Fig. 1. Depletion of REG2 mRNA, transcript, and protein in pancreatic islets of mice by GPX1 over production Relative mRNA levels of Ins1, Pdx1, Foxa2, Reg1, and Reg2 in pancreatic islets (70 per sample) of OE mice to those of WT (as 1) mice (2-month old, male, n = 6) after normalization with the respective beta-actin mRNA (A), depletion of Reg2 mRNA in pancreatic islets (70 per sample) of OE mice across three ages compared with the WT (as 1) mice (male, n = 6) after normalization with the respective beta-actin mRNA levels (B), comparisons of Reg1 and Reg2 transcript numbers/ng cDNA in pancreatic islets (70 per sam ple) between OE and WT mice (6-month old, male, n = 6) (C), depletion of REG2 protein in pancreatic islets (75 μg protein per sample) of OE mice compared with WT mice (6-month old, male, n = 4) shown by immunoblotting (upper panel, a represen tative of four independent experiments), and relative densities (lower panel) of bands were quantified with Image J (the WT as 100) after normalization with beta-actin protein density. Data are mean ± SE, *, p < 0.05 for differences between genotypes.

Article Snippet: The binding complex was incubated with REG2 antibody (1 μg, R&D Systems, MAB2098) or rat IgG (Thermo Fisher, 31933, Supplemental Table 2) for 8 h and then with protein G beads (Santa Cruz, Dallas, TX) for 1 h at 4 ◦C on rotation wheel.

Techniques: Western Blot

Journal: Redox biology

Article Title: Role and mechanism of REG2 depletion in insulin secretion augmented by glutathione peroxidase-1 overproduction.

doi: 10.1016/j.redox.2022.102457

Figure Lengend Snippet: Fig. 2. Inhibition of glucose-stimulated insulin secretion by exogenous REG2. Effects of REG2 (1 μg/mL) on insulin secretion of in static cultured islets (70 per sample) of OE mice (2-month old, male, n = 4) at 2.8 mmol glucose (2.8 G) for 0.5 h or 30 mmol glucose/l (30 G) for 1 h (A), an illustration of designed mutations in the C-type lectin binding domain (REG2 M1) and the acidic residues (REG2 M2) based on predicted three-dimensional protein structure of REG2 (B), effects of REG2 (C), REG2 M1 (D) and REG2 M2 (E) (1 μg/mL) on GSIS of perifused (1 ml/min) islets (70 per sample) of OE mice (2-months old, male, n = 4 per treatment) in comparison with those of Krebs buffer. In all panels (except for B), data are mean ± SE, *, p < 0.05 for differences between REG2 or REG2 M2 verse buffer or PBS controls. In panel A, **, p < 0.05 for difference between 2.8 G and 30 G in insulin secretion. In panels C, D, and E, values on the top are areas under curve (AUC) of insulin secretion stimulated by glucose or KCl for the perifusion with buffer or REG2 and mutants, *p < 0.05 for difference between the buffer and treatments (REG2 or REG2 M2); G: glucose; mM, mmol/l; and the gradient bar above the trace: glucose ramp from 0 to 50 mmol/l.

Article Snippet: The binding complex was incubated with REG2 antibody (1 μg, R&D Systems, MAB2098) or rat IgG (Thermo Fisher, 31933, Supplemental Table 2) for 8 h and then with protein G beads (Santa Cruz, Dallas, TX) for 1 h at 4 ◦C on rotation wheel.

Techniques: Inhibition, Cell Culture, Binding Assay, Comparison

Journal: Redox biology

Article Title: Role and mechanism of REG2 depletion in insulin secretion augmented by glutathione peroxidase-1 overproduction.

doi: 10.1016/j.redox.2022.102457

Figure Lengend Snippet: Fig. 3. Evidences for a ligand-receptor binding between REG2 and CaV1.2. Western blot analysis of CaV1.2 protein produced by beta TC3 cells (50 μg protein per lane) transfected with pcDNA vector and pcDNA-CaV1.2 plasmid (A), co- immunoprecipitation of CaV1.2 protein with REG2: the lysates (500 μg protein per sample) of beta-TC3 cells transfected with pcDNA-CaV1.2 plasmid were incu bated with REG2 protein (2 μg) and REG2 antibody (1 μg) and the precipitates were collected by protein G beads and immunoblotted against CaV1.2 antibody (B), co- immunoprecipitation of REG2 with glycosylated verse deglycosylated CaV1.2 in lysates of Min6 cells transfected with pcDNA-CaV1.2 plasmid: cell lysates (1 mg protein per sample) were treated with or without PNGase F (20 μl, 10,000 units) and incubated with REG2 protein (2 μg) and REG2 antibody (1 μg); the precipitates were collected by protein G beads and immunoblotted against CaV1.2 antibody; and the CaV1.2 α1c subunit was detected only in the precipitated cell lysates without deglycosylation (C), the putative ligand-receptor binding of alkaline phosphatase (AP)-conjugated REG2 (AP-REG2) vs. AP-REG2 M1 and AP (1 μg/ml) overexpressed and secreted by HEK293T cells to CaV1.2 on the surface of beta TC3 cells transfected with pcDNA vector or pcDNA-CaV1.2 plasmid in the presence or absence of CaV1.2 antibody (1 μg/ml) or REG2 (1 μg/ml), and the extent of binding was shown by the retained AP activity on cell surface and quantitated (the right box) by the shading/dark pixels using software Image J (D), co-immunofluorescence of green fluorescence protein (GFP)-conjugated REG2 (GFP-REG2) vs. GFP-REG2 and GFP (1 μg/ml) overexpressed and secreted by HEK293T cells with mCherry-CaV1.2 on the surface of beta TC3 cells transfected with pmCherry vector or pcmCherry-CaV1.2 plasmid for 48 h, and the relative percentage of yellow fluorescence area (% of GFP positive cells in mCherry positive cells) was scored using MetaMorph image analysis software (E). In panels A, B, and C, the images were representative of 4 independent experiments. In panels of D and E, scale bar = 10 μm; values are mean ± SE from four independent experiments; p < 0.05 for differences between REG2 and respective controls. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The binding complex was incubated with REG2 antibody (1 μg, R&D Systems, MAB2098) or rat IgG (Thermo Fisher, 31933, Supplemental Table 2) for 8 h and then with protein G beads (Santa Cruz, Dallas, TX) for 1 h at 4 ◦C on rotation wheel.

Techniques: Binding Assay, Western Blot, Produced, Transfection, Plasmid Preparation, Immunoprecipitation, Incubation, Activity Assay, Software, Immunofluorescence, Fluorescence

Journal: Redox biology

Article Title: Role and mechanism of REG2 depletion in insulin secretion augmented by glutathione peroxidase-1 overproduction.

doi: 10.1016/j.redox.2022.102457

Figure Lengend Snippet: Fig. 5. In vivo effects of exogenous REG2 admin istration to OE mice. Effects of an intraperitoneal (ip) injection of REG2 (2 μg/g body weight), in comparison with an equal volume (10 μL/g body weight) of phosphate-buffed saline (PBS), to male OE mice (6 months old, n = 10) at 15 min before a glucose challenge (ip, 1 g/kg body weight) on plasma concentrations of insulin (A) and glucose (B) over 60 min. Data are mean ± SE, *, p < 0.05 for area under curve (AUC) of REG2 verse PBS.

Article Snippet: The binding complex was incubated with REG2 antibody (1 μg, R&D Systems, MAB2098) or rat IgG (Thermo Fisher, 31933, Supplemental Table 2) for 8 h and then with protein G beads (Santa Cruz, Dallas, TX) for 1 h at 4 ◦C on rotation wheel.

Techniques: In Vivo, Injection, Comparison, Saline, Clinical Proteomics

Journal: Redox biology

Article Title: Role and mechanism of REG2 depletion in insulin secretion augmented by glutathione peroxidase-1 overproduction.

doi: 10.1016/j.redox.2022.102457

Figure Lengend Snippet: Fig. 4. Inhibition of Ca2+ influx in pancreatic islets of OE and WT mice by exogenous REG2 Effects of REG2 (1 μg/ml) vs. PBS on Ca2+ influx, measured by fluorescence of Fluo 4-AM (2 μmol/l, with the peak excitation wavelength at 490 nm and the peak emission wavelength at 516 nm), in islets (50 per sample) of OE mice (6-month old, male) before and after (5 min) stimulations of glucose (2.8, 16.7, and 30 mmol/l) and KCl (30 mmol/l) (A), effects of a continuous perifusion of REG2 (1 μg/ml, labeled in red verse buffer, labeled in black) on the Ca2+ influx, measured by fluorescence ratio of Fura 2-AM (2 μmol/l) at excitation wavelengths of 340 (Ca-bound) to 380 (free anion) with emission wavelength of 520, in single islets (n = 4) of WT mice (6-month old, male) perifused with glucose (3 and 16.7 mmol/l) and KCl (30 mmol/l) (B), rapid and reversible inhibition of the Ca2+ influx by REG2 (1 μg/mL), measured as in B, in single-perifused islets (n = 9) of WT mice (the same as in B) after the glucose ramp reached 16.7 mmol/l and the loss of inhibition after the removal of REG2 from the perifusate (C). In panel A, fluorescence was imaged using an Axiovert 200 M Fluorescent Microscope (Zeiss, Filter set 09). In panels B and C, an inverted Zeiss AxioVision microscope with Attofluor ration vision software was used to record the time course of intracellular Ca concentration changes. The overall inhibition (B) by REG2 vs buffer and the reversible inhibition of REG2 vs before and after the removal of REG2 at the same glucose concentration (16.7 mmol/l) (C) were expressed as AUC (area under curve), *p < 0.05 vs control. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The binding complex was incubated with REG2 antibody (1 μg, R&D Systems, MAB2098) or rat IgG (Thermo Fisher, 31933, Supplemental Table 2) for 8 h and then with protein G beads (Santa Cruz, Dallas, TX) for 1 h at 4 ◦C on rotation wheel.

Techniques: Inhibition, Fluorescence, Labeling, Microscopy, Software, Concentration Assay, Control