Journal: Life Science Alliance

Article Title: Dysfunctional β-cell longevity in diabetes relies on energy conservation and positive epistasis

doi: 10.26508/lsa.202402743

Figure Lengend Snippet: (A) Scheme of PFKFB3-dependent loss of β-cell function and locked CFC impeding functional β-cell regeneration. Protein aggregates lead to up-regulation of the HIF1α-PFKFB3 pathway that delinks glucose sensing from mitochondrial respiration and insulin secretion. PFKFB3 expression promotes “loser” β-cell survival by CFC deactivation. Targeting PFKFB3 reactivates CFC and clearance of “loser” β-cells. (B) Genomic pipeline to reveal the transcriptional profile of “loser” β-cells and PFKFB3-expressing dysfunctional human β-cells across health, prediabetes (AAB), T1D, and T2D using HPAP scRNA-seq database and the spatial transcriptomics from nPOD T2D pancreata, respectively. (C) Diagrammatic presentation of the anticipated effect of PFKFB3 targeting on “loser” β-cells in healthy versus diabetic islets. “Loser” (L) and “winner” (W) β-cells are part of CFC, and CFC is active in health. However, in diabetes, “loser” β-cells that express high PFKFB3 (P+L) are no more CFC-elective. When PFKFB3 is targeted, CFC becomes specifically unlocked in P+L cells, whereas β-cells expressing low PFKFB3 (P) are not affected in the absence of L signatures. (D) Dot plot showing the coverage of Reactome enriched pathways of DEGs for AAB HPAP , T2D HPAP , and T2D PFKFB3 . (E) Overlap of enriched pathways across Control HPAP , prediabetes (AAB HPAP ), T2D HPAP , and T2D PFKFB3 . Top ten shared and enriched pathways across the Control HPAP , prediabetes (AAB HPAP ), T2D HPAP , and T2D PFKFB3 are shown as a list on the left bottom corner.

Article Snippet: The following antibodies were used: guinea pig anti-insulin (ab195956, 1:400; Abcam); mouse anti-glucagon (G2654, 1:1,000; Sigma-Aldrich); mouse anti-c-Myc (9E10, sc-40, 1:100; Santa Cruz Biotechnology Inc.); and mouse anti-HIF1α (NB100-105, 1:50; Novus Biologicals), anti-MAIP1 (D-12, 1:50; Santa Cruz Biotechnology), and anti-AGAP1 (MBS5312069, 1:50; MyBioSource Inc.).The following secondary antibodies were used: F(ab’)2 conjugates with fluorescein isothiocyanate donkey anti-guinea pig immunoglobulin G (IgG) (heavy and light, H + L) (706-096-148, 1:200 for intrinsic factor [IF]; Jackson ImmunoResearch) and F(ab’)2 conjugates with Cy3 donkey anti-mouse IgG (H + L) (711-165-151, 1:200 for IF; Jackson ImmunoResearch).

Techniques: Cell Function Assay, Functional Assay, Expressing, Control

Journal: Life Science Alliance

Article Title: Dysfunctional β-cell longevity in diabetes relies on energy conservation and positive epistasis

doi: 10.26508/lsa.202402743

Figure Lengend Snippet: (A) PFKFB3 rs 1983890 -SNP interaction analysis with the DEGs in PFKFB3-positive relative to PFKFB3-negative β-cells revealed by spatial transcriptomics yielded significant interactions with collective SNPs of AGAP1 and MAIP1 (SNPs 115). Diagrammatic reconstruction of the results indicates a proposed mechanism of PFKFB3-mediated survival of injured “loser” β-cells. MAIP1 is an anti-apoptotic gene that controls mitochondrial Ca 2+ via MCU uniporter. MCU is composed of channel-forming subunits, EMRE, and a gatekeeper subunit MICU. The gating of the MCU complex requires an association between EMRE and MICU. MAIP1 may protect EMRE from i-AAA–mediated degradation preventing constitutive activation of MCU and cell death. (B) Quantitative representation of insulin-positive and glucagon-positive (bihormonal) injured cells (% of all β-, α-, and bihormonal cells). (C) Quantitative representation of c-Myc–positive injured β-cells (% of all β-cells). (D) Quantitative representation of HIF1α-positive injured β-cells (% of all β-cells). (E, F, G) Representative immunofluorescence images of islets, from wild-type mice exposed to high-fat diet, HFD (WT), mice under diabetogenic stress treated with vehicle (IAPP+HFD = DS; DS + vehicle), mice with conditional PFKFB3 knockout exposed to diabetogenic stress for 6 wk (PFKFB3 βKO DS), and mice under diabetogenic stress receiving the PFKFB3 inhibitor AZ67 for 6 wk (DS + iPFKFB3); (E) double immunostained for insulin and glucagon (to reveal bihormonal cells), (F) immunostained for cytoplasmic c-Myc, and (G) HIF1α (all markers in red), insulin (green), and nuclei (blue) showing eradication of marker-positive injured β-cells in PFKFB3 βKO DS and DS + iPFKFB3 mice. The sectioning was performed across the whole pancreas. For quantification of the markers’ frequency, the whole pancreas section per mouse from the treatment group was used. We have counted all the pancreatic islets from each whole pancreas section (150–200 islets per treatment group) (n = 3). Scale bar: 50 μm.

Article Snippet: The following antibodies were used: guinea pig anti-insulin (ab195956, 1:400; Abcam); mouse anti-glucagon (G2654, 1:1,000; Sigma-Aldrich); mouse anti-c-Myc (9E10, sc-40, 1:100; Santa Cruz Biotechnology Inc.); and mouse anti-HIF1α (NB100-105, 1:50; Novus Biologicals), anti-MAIP1 (D-12, 1:50; Santa Cruz Biotechnology), and anti-AGAP1 (MBS5312069, 1:50; MyBioSource Inc.).The following secondary antibodies were used: F(ab’)2 conjugates with fluorescein isothiocyanate donkey anti-guinea pig immunoglobulin G (IgG) (heavy and light, H + L) (706-096-148, 1:200 for intrinsic factor [IF]; Jackson ImmunoResearch) and F(ab’)2 conjugates with Cy3 donkey anti-mouse IgG (H + L) (711-165-151, 1:200 for IF; Jackson ImmunoResearch).

Techniques: Activation Assay, Immunofluorescence, Knock-Out, Marker

Journal: Life Science Alliance

Article Title: Dysfunctional β-cell longevity in diabetes relies on energy conservation and positive epistasis

doi: 10.26508/lsa.202402743

Figure Lengend Snippet: (A, B, C, D) Representative individual and merged immunofluorescence images of islets from (A) wild-type + high-fat diet, that is, WT, (B) DS + vehicle, (C) PFKFB3 βKO DS, and (D) DS + iPFKFB3 mice, immunostained for HIF1α (red), insulin (green), and nuclei (blue) showing eradication of HIF1α-positive injured β-cells in PFKFB3 βKO DS and DS + iPFKFB3 mice. For quantification of injured marker frequency, we have counted all the pancreatic islets from each whole pancreas section, that is, 150–200 islets per mouse treatment group (n = 3). Scale bar: 25 μm.

Article Snippet: The following antibodies were used: guinea pig anti-insulin (ab195956, 1:400; Abcam); mouse anti-glucagon (G2654, 1:1,000; Sigma-Aldrich); mouse anti-c-Myc (9E10, sc-40, 1:100; Santa Cruz Biotechnology Inc.); and mouse anti-HIF1α (NB100-105, 1:50; Novus Biologicals), anti-MAIP1 (D-12, 1:50; Santa Cruz Biotechnology), and anti-AGAP1 (MBS5312069, 1:50; MyBioSource Inc.).The following secondary antibodies were used: F(ab’)2 conjugates with fluorescein isothiocyanate donkey anti-guinea pig immunoglobulin G (IgG) (heavy and light, H + L) (706-096-148, 1:200 for intrinsic factor [IF]; Jackson ImmunoResearch) and F(ab’)2 conjugates with Cy3 donkey anti-mouse IgG (H + L) (711-165-151, 1:200 for IF; Jackson ImmunoResearch).

Techniques: Immunofluorescence, Marker

Journal: International Journal of Molecular Sciences

Article Title: Smooth Muscle Silent Information Regulator 1 Contributes to Colitis in Mice

doi: 10.3390/ijms26051807

Figure Lengend Snippet: The conditioned medium (CM) of Sirt1 -Tg CSMCs inhibits the proliferation of Caco-2 cells in vitro. ( A ) qRT-PCR of cZFP609 expression in CSMCs from WT or Sirt1 -Tg mice treated with TNF-α for 24 h. ( B ) qRT-PCR of cZFP609 expression in Caco-2 cells incubated with the TNF-α-induced CSMC conditioned medium (CM) for 24 h and exposed to hypoxia. ( C ) Ratio of cell counting of Caco-2 cells incubated with the TNF-α-induced CSMC (CM) for 24 h and exposed to hypoxia. ( D , E ) Migration of Caco-2 cells was assessed using a scratch wound assay. Caco-2 cells were incubated with the TNF-α-induced CSMC exosomes (CM) for 24 h and exposed to hypoxia treated with TNF-α. (Scale bar: 200 µm.) Data are presented as mean ± SEM. ( F ) Immunofluorescent confocal microscopy of HIF1α nuclear translocation in the Caco-2 cells, Scale bars: 25 μm. Bar graphs show mean ± SEM. Student’s t -test or one-way ANOVA was used. ** p < 0.01 versus the corresponding control.

Article Snippet: The cells were then incubated with mouse anti- HIF1α (1:100, GTX640664, GeneTex, Irvine, CA, USA) antibody at 4 °C overnight.

Techniques: In Vitro, Quantitative RT-PCR, Expressing, Incubation, Cell Counting, Migration, Scratch Wound Assay Assay, Confocal Microscopy, Translocation Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Smooth Muscle Silent Information Regulator 1 Contributes to Colitis in Mice

doi: 10.3390/ijms26051807

Figure Lengend Snippet: cZFP609 inhibits proliferation in Caco-2 cells via inhibiting HIF1α nuclear translocation. ( A ) qRT-PCR of cZFP609 expression in Caco-2 cells treated with vector or cZFP609. ( B ) Ratio of cell counting of Caco-2 cells treated with vector or cZFP609. ( C , D ) Migration of Caco-2 cells was assessed using a scratch wound assay. Caco-2 cells treated with vector of cZFP609. (Scale bar: 200 µm). Data are presented as mean ± SEM. ( E ) Immunofluorescent confocal microscopy of HIF1α nuclear translocation in the Caco-2 cells. (Scale bars: 25 μm). Bar graphs show mean ± SEM. Student’s t -test or one-way ANOVA was used. ** p < 0.01 versus the corresponding control.

Article Snippet: The cells were then incubated with mouse anti- HIF1α (1:100, GTX640664, GeneTex, Irvine, CA, USA) antibody at 4 °C overnight.

Techniques: Translocation Assay, Quantitative RT-PCR, Expressing, Plasmid Preparation, Cell Counting, Migration, Scratch Wound Assay Assay, Confocal Microscopy, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: ER Stress and Mitochondrial Perturbations Regulate Cell Death in Retinal Detachment: Exploring the Role of HIF1α

doi: 10.1167/iovs.65.11.39

Figure Lengend Snippet: HIF1α is associated with mitophagy induction after RD in retinal cells. ( A ) Representative immunoblots of HIF1α protein from 661W cells subjected to 8 hours, 18 hours, and 24 hours of hypoxia. Blots are representative of three independent experiments in each condition; α-tubulin was used as the loading control. ( B ) Representative immunoblots of HIF1α protein from retinas of C57BL/6J mice at 1 dprd and 3 dprd. Blots are representative of three independent animals in each condition; α-tubulin was used as the loading control. ( C ) Relative mRNA expression of Hspa5 obtained from retinas of Rho/Cre + and HIF1αΔrod mice at 3 dprd compared to the contralateral eye (fellow). Pum-1 was used as a housekeeping control gene for normalization. ( D ) Relative mRNA expression of Ddit3 obtained from retinas of Rho/Cre + and HIF1αΔrod mice at 3 dprd compared to the contralateral eye (fellow). Pum-1 was used as a housekeeping control gene for normalization. ( E ) Relative mRNA expression of Pgc1a and Tfam obtained from retinas of Rho/Cre + and HIF1αΔrod mice at 3 dprd compared to the contralateral eye (fellow). Pum-1 was used as a housekeeping control gene for normalization. ( F ) Representative immunoblots of Parkin and FUNDC1 proteins from retinas of Rho/Cre + and HIF1αΔrod mice at 3 dprd compared to the contralateral eye (fellow). GAPDH and α-tubulin were used as the loading control. ( G ) Relative mRNA expression of Prkn obtained from retinas of Rho/Cre+ and HIF1αΔrod mice at 3 dprd compared to the contralateral eye (fellow). Pum-1 was used as a housekeeping control gene for normalization. ( H ) Relative mRNA expression of Pink obtained from retinas of Rho/Cre + and HIF1αΔrod mice at 3 dprd compared to the contralateral eye (fellow). Pum-1 was used as a housekeeping control gene for normalization. Bar graphs represent mean ± SEM. Statistical analysis was performed using two-way ANOVA with repeated measures followed by Tukey's test. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The following antibodies were used: anti-mouse CHOP (#2895, 1:1000; Cell Signaling Technology, Danvers, MA, USA); anti-rabbit pIRE1α (#NB100-2323SS, 1:1000; Novus Biologicals, Littleton, CO, USA); anti-mouse BiP/Grp78 (#610979, 1:1000; BD Biosciences, Franklin Lakes, NJ, USA); anti-mouse mono-and polyubiquitinylated conjugates (#BML-PW8810, 1:1000; Enzo Life Sciences, Long Island, NY, USA); anti-mouse caspase 3 (#NB100-56113, 1:1000; Novus Biologicals); anti-mouse HIF1α (#MAB1536, 1:1000; R&D Systems, Minneapolis, MN, USA), anti-rabbit FUNDC1 (#NBP1-81063, 1:1000; Novus Biologicals); anti-rabbit PINK1 (#NB100-493, 1:1000; Novus Biologicals); anti-rabbit Parkin (#NBP2-67017, 1:1000; Novus Biologicals); and anti-mouse PGC-1α (#sc-517380, 1:500; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Western Blot, Control, Expressing