Journal: Metabolism: clinical and experimental

Article Title: The MafA-target gene PPP1R1A regulates GLP1R-mediated amplification of glucose-stimulated insulin secretion in β-cells.

doi: 10.1016/j.metabol.2021.154734

Figure Lengend Snippet: Fig. 1. Regulation of PPP1R1A expression in MafA mutant β-cells. (A) Expression of β-cell genes in 8 months old wild type (WT) and MafA−/−islets. Heat maps were generated using Log- CPM values. Color gradient (bottom) represents expression of each gene/sample (red = highest expression (Max); deep blue = lowest expression (Min); white = intermediate expression). Last row in heatmap shows MafA expression. (B) Box plot of PPP1R1A expression in WT and MafA−/−islets. N = 4 mice/genotype. (C) Immunostaining of PPP1R1A (red), Insulin (green) and Glucagon (blue) in MafAfl/fland MafARIP pancreatic sections (scale bar = 50 μm). (D) Immunoblot of PPP1R1A and MafA in INS1 (832/13) β-cells 72 h after transfection with scramble and MAFA siRNA (upper panel) and densitometric analysis for PPP1R1A protein levels in control and MAFA KD INS1 (832/13) β-cells cells (bottom panel). (E) Spearman's rank correlation analysis of PPP1R1A and MAFA (r = 0.38, p < 0.0001, n = 189) (F) or MAFB (r = 0.67, p < 0.0001, n = 190) mRNA levels in human islets. * p < 0.05 by unpaired student's t-test.

Article Snippet: Primary antibodies: PPP1R1A (1:1000, Origene), Insulin (1:2000; Dako), Glucagon (1:2000; Sigma), MafA (1:1000, Bethyl laboratories).

Techniques: Expressing, Mutagenesis, Generated, Immunostaining, Western Blot, Transfection, Control

Journal: Metabolism: clinical and experimental

Article Title: The MafA-target gene PPP1R1A regulates GLP1R-mediated amplification of glucose-stimulated insulin secretion in β-cells.

doi: 10.1016/j.metabol.2021.154734

Figure Lengend Snippet: Fig. 2. Correlation between PPP1R1A, MAFA and MAFB mRNA levels and metabolic parameters. (A) Immunostaining of PPP1R1A in pancreatic islets from type 2 diabetic and normoglycemic donors (bar = 20 μm). (B) PPP1R1A mRNA levels in type 2 diabetic (n = 31) and non-diabetic (ND, n = 160) donor islets, ****p < 0.0001. (C\\D) Rank-Spearman correlation analysis of PPP1R1A mRNA levels with: (C) HbA1c (mmol/mol) (r = −0.43, p < 0.0001, n = 173), (D) BMI (r = −0.191, p = 0.008, n = 191). (E) PPP1R1A mRNA levels in islets from lean (BMI < 27, n = 107) and overweight/obese (BMI > 27, n = 51) ND donors. **p = 0.0017. (F) PPP1R1A mRNA levels in human islets stratified by donor age, *p < 0.05. (G) MAFA mRNA levels in islets from ND and T2D donors and in (H) lean (BMI < 27, n = 107) and overweight/obese (BMI > 27, n = 51) ND donors. (I-J) Rank-Spearman correlation analysis of MAFA mRNA levels with (I) BMI and (J) HbA1c. (K) MAFA mRNA levels in human islets stratified by donor age. (L) MAFB mRNA levels in islets from ND and T2D donors and in (M) lean (BMI < 27, n = 107) and overweight/obese (BMI > 27, n = 51) ND donors. (N\\O) Rank-Spearman correlation analysis of MAFB mRNA levels with (N) BMI and (O) HbA1c. (P) MAFB mRNA levels in human islets stratified by donor age. *p < 0.05, **p < 0.01. Data are mean ± SEM. (B, F, G, H, K, L, M, P) Student's t-test followed by a Mann-Whitney post hoc test. (F, K, P) one-way ANOVA.

Article Snippet: Primary antibodies: PPP1R1A (1:1000, Origene), Insulin (1:2000; Dako), Glucagon (1:2000; Sigma), MafA (1:1000, Bethyl laboratories).

Techniques: Immunostaining, MANN-WHITNEY

Journal: Metabolism: clinical and experimental

Article Title: The MafA-target gene PPP1R1A regulates GLP1R-mediated amplification of glucose-stimulated insulin secretion in β-cells.

doi: 10.1016/j.metabol.2021.154734

Figure Lengend Snippet: Fig. 3. Correlations between PPP1R1A mRNA levels and insulin secretion. PPP1R1A mRNA levels correlated to (A) Stimulatory index (r = 0.235, p = 0.0013, n = 191); (B) insulin secretion in response to glucose (r = 0.187, p = 0.022, n = 149), (C) glucose+GIP (r = 0.246, p = 0.003, n = 142), and (D) glucose+GLP1 (r = 0.255, p = 0.002, n = 138). (E) PPP1R1A mRNA expression levels measured by qPCR in human islets transduced with mock or PPP1R1A shRNA5 lentivirus (MOI = 2). Graph bars represent mean ± SEM of n = 3 independent experiments. *p < 0.05 with student's t-test. (F) Insulin secretion of human islets transduced with mock or PPP1R1A shRNA5 lentivirus was stimulated with low glucose (2.8 mM), high (16.7), high glucose+Exendin-4 (100 nM) or high glucose+Forskolin (10 μM), n = 4.

Article Snippet: Primary antibodies: PPP1R1A (1:1000, Origene), Insulin (1:2000; Dako), Glucagon (1:2000; Sigma), MafA (1:1000, Bethyl laboratories).

Techniques: Expressing, Transduction

Journal: Metabolism: clinical and experimental

Article Title: The MafA-target gene PPP1R1A regulates GLP1R-mediated amplification of glucose-stimulated insulin secretion in β-cells.

doi: 10.1016/j.metabol.2021.154734

Figure Lengend Snippet: Fig. 4. PPP1R1A contributes to IBMX-promoted insulin secretion and PKA-targets phosphorylation in INS1 (832/13) β-cells. INS1 (832/13) β-cells transfected with scramble or PPP1R1A siRNA. (A) Insulin secretion was stimulated for 1 h with low (2.8 mM), high (16.7) or high glucose+IBMX (100 μM). (B) In INS1 (832/13) β-cells under the same conditions as in (A) immunoblot of PPP1R1A (top) and PKA-phosphorylated proteins (bottom) after 30 min of stimulation with low (2.8 mM), high (16.7) and high glucose+IBMX (100 μM) w/wo PKA inhibitor H89, (C\\D) Densitometric analysis for (C) PPP1R1A and (D) total PKA-phosphorylated proteins. Graph bars represent mean ± SEM of n = 11 (A), n = 5 (C), n = 7 (D) independent experiments. Within group comparison; #p < 0.001. Between groups comparison (control vs. KD) *p < 0.05; **p < 0.01 using a Two-way ANOVA.

Article Snippet: Primary antibodies: PPP1R1A (1:1000, Origene), Insulin (1:2000; Dako), Glucagon (1:2000; Sigma), MafA (1:1000, Bethyl laboratories).

Techniques: Phospho-proteomics, Transfection, Western Blot, Comparison, Control

Journal: Metabolism: clinical and experimental

Article Title: The MafA-target gene PPP1R1A regulates GLP1R-mediated amplification of glucose-stimulated insulin secretion in β-cells.

doi: 10.1016/j.metabol.2021.154734

Figure Lengend Snippet: Fig. 5. PPP1R1A contributes to IBMX-promoted mitochondrial coupling efficiency in INS1 (832/13) β-cells. Mitochondrial Oxygen Consumption Rate (OCR) was measured by Seahorse (XF24) in INS1 (832/13) β-cells transfected with scramble or PPP1R1A siRNA (10 nM). (A) Average OCR traces in basal glucose (2.8 mM), glucose (16.7 mM) + IBMX (100 μM) or vehicle (DMSO), Oligomycin (4 μM), FCCP (4 μM) and Antimycin A + Rotenone (1 μM) conditions (traces represent average FC to basal OCR at 2.8 mM glucose). Mitochondrial activity parameters; (B) Acute Response OCR, (C) ATP-mediated OCR, (D) Proton-Leak OCR and, (E) Coupling-Efficiency. Data are mean ± SEM of n = 9 independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 with student t-test.

Article Snippet: Primary antibodies: PPP1R1A (1:1000, Origene), Insulin (1:2000; Dako), Glucagon (1:2000; Sigma), MafA (1:1000, Bethyl laboratories).

Techniques: Transfection, Activity Assay

Journal: Metabolism: clinical and experimental

Article Title: The MafA-target gene PPP1R1A regulates GLP1R-mediated amplification of glucose-stimulated insulin secretion in β-cells.

doi: 10.1016/j.metabol.2021.154734

Figure Lengend Snippet: Fig. 6. Reduced PPP1R1A expression causes β-cell dysfunction under normoglycemic and glucotoxic conditions. (A) PPP11R1A expression levels in INS1 (832/13) β-cells transfected with scramble or PPP1R1A siRNA and cultured under normoglycemic (NG) or hyperglycemic (HG) conditions. (B–D) Insulin secretion of NG (B) and HG (C) cells after 1 h incubation with low (2.8 mM), high (16.7) or high glucose+IBMX (100 μM). (D) Comparison of insulin secretion of PPP1R1A-KD cells cultured in NG and HG conditions. (E) Cell viability of PPP1R1A-KD cells in NG and HG. (F-M) qPCR expression analysis of glucose sensing (F–I) and β-cell transcription factors (J-M) of PPP1R1A-KD cells in NG and HG. (N) Immunostaining for PPP1R1A, MafA and Pdx1 protein of PPP1R1A-KD cells in NG and HG. Graph bars represent mean ± SEM of n = 3–6 independent experiments. *p < 0.05; **p < 0.01 with student's t-test.

Article Snippet: Primary antibodies: PPP1R1A (1:1000, Origene), Insulin (1:2000; Dako), Glucagon (1:2000; Sigma), MafA (1:1000, Bethyl laboratories).

Techniques: Expressing, Transfection, Cell Culture, Incubation, Comparison, Immunostaining

Journal: Antioxidants (Basel, Switzerland)

Article Title: Nrf2 Plays a Key Role in Erythropoiesis during Aging.

doi: 10.3390/antiox13040454

Figure Lengend Snippet: Figure 4. Sorted Nrf2−/−mouse erythroblasts display overactivation of the system and impaired autophagy with caspase-3 pro-apoptotic pathway activation. (a) Wb analysis with specific anti- phospho-NF-kB (p-NF-kB) and NF-kB in sorted erythroid precursors from bone marrow of 12-month- old wild-type (WT) and Nrf2−/−mice. Actin was used as protein loading control. One representative gel of the other four is shown (upper panel). Densitometric analysis of immunoblots is shown in the lower panel. Data are presented as means ± SD (n = 4); * p < 0.05 compared to WT mice. (b) Wb analysis with specific anti-HSP70, ATF6 and GADD34 in sorted erythroid precursors as in a. Densitometric analysis of immunoblots is shown in the lower panel. Data are presented as means ± SD (n = 4); * p < 0.05 compared to WT mice. (c) Caspase 3 activity determined by cleavage of a fluorescent substrate in sorted erythroid precursors from bone marrow of WT and Nrf2−/−mice. Data are presented as means ± SD * p < 0.05 compared to WT. (d) Wb analysis with specific anti-LC3 I/II, Atg4, Atg5, Rab5 and p62 in sorted erythroid precursors from bone marrow of 12-month-old WT and Nrf2−/−mice. Actin was used as protein loading control. Densitometric analysis of immunoblots is shown in the right panel. Data are presented as means ± SD (n = 4); * p < 0.05 compared to WT mice. (e) Rab5 immunostaining of sorted erythroid precursors from bone marrow of 12-month-old WT and Nrf2−/−mice. DAPI was used to stain nuclei. Large clusters of positive cells were measured using ImageJ. At least 40 cells were analyzed in 8 different fields of acquisition. Data are presented as median and minimum/maximum, with boxes indicating 25–75th percentiles; * p < 0.05 compared to WT mice.

Article Snippet: The following specific antibodies were used: anti-NFkB-phospho-S536 (3033 Cell Signaling Technology, Leiden, NL, USA); anti-NFkB p65 and anti-Atg5 (8242, 12994 Cell Signaling Technology, Leiden, NL, USA); anti-Nrf2-phospho-S40 (ab76026 Clone EP1809Y, Abcam, Cambridge, UK); anti-Nrf2 (ab62352 Abcam, Cambridge, UK); anti Gadd34, anti-Lamp-1, anti-SQSTM1/P62, anti-Rab5 and anti-LC3A/B (ab9869, ab24170, ab109012, ab109534, ab62721 Abcam, Cambridge, UK); anti-APG7 (Atg7) (3615 ProSci, Poway, CA, USA); anti-ATF6 (70B1413.1 Novus Biologicals, Centennial, CO, USA); antiCHOP (ma1-250 Thermo Fisher Scientific, Waltham, MA, USA); anti-Actin (SAB4301137 clone BIII-136; Sigma-Aldrich, Saint Louis, MO, USA) and anti-GAPDH (HPA040067 SigmaAldrich, Saint Louis, MO, USA) as loading controls.

Techniques: Activation Assay, Control, Western Blot, Activity Assay, Immunostaining, Staining

Journal: Antioxidants (Basel, Switzerland)

Article Title: Nrf2 Plays a Key Role in Erythropoiesis during Aging.

doi: 10.3390/antiox13040454

Figure Lengend Snippet: Figure 7. Schematic diagram of the role of Nrf2 in erythropoiesis during aging and the protective effects of astaxanthin PLGA nanoparticles. Aging is associated with increased ROS production, which is limited by the activation of Nrf2. This results in the upregulation of ARE genes encoding for antioxidants and cytoprotective systems as well as by the activation of adaptative mechanisms such as the UPR system to face ER stress and autophagy to clear damaged proteins. The absence of Nrf2 (Nrf2−/−mice) negatively affects the antioxidant cell machinery, resulting in severe and sustained oxidation. Nrf2−/−mouse red cells display severe membrane oxidation, exposition of phosphatidylserine, membrane binding of hemichromes and reduced expression of antioxidants and cytoprotective systems such as Prdx2. Red cell membrane protein oxidation favors band 3 protein clusterization, which is recognized by the naturally occurring anti-band 3 IgG antibodies. Both mechanisms drive Nrf2−/−mouse red cells towards erythrophagocytosis by splenic macrophages. In erythroblasts lacking Nrf2, the prolonged and severe oxidation due to the downregulation of antioxidants and cytoprotective systems promotes intense ER stress with overactivation of the UPR system and autophagy. Although the persistence of oxidative stress promotes compensatory activation of NF-kB, this is insufficient to prevent the overwhelming of proteostasis with impairment autophagy and accumulation of Rab5. This drives Nrf2−/−erythroblasts towards apoptosis via the caspase-3 pathway, resulting in ineffective erythropoiesis. ATS-NPs act as efficient antioxidants preventing the deleterious effects of the absence of Nrf2 on erythropoiesis and red cells during aging. PS: phosphatidylserine; ER: endoplasmic reticulum; UPR: unfolded protein response; ARE-: antioxidant-related element; ROS: reactive oxygen species; Prdx2: peroxiredoxin-2; Atg: autophagy- related protein; GADD34: growth arrest and DNA damage-inducible protein 34; PLGA: poly(lactic- co-glycolic acid).

Article Snippet: The following specific antibodies were used: anti-NFkB-phospho-S536 (3033 Cell Signaling Technology, Leiden, NL, USA); anti-NFkB p65 and anti-Atg5 (8242, 12994 Cell Signaling Technology, Leiden, NL, USA); anti-Nrf2-phospho-S40 (ab76026 Clone EP1809Y, Abcam, Cambridge, UK); anti-Nrf2 (ab62352 Abcam, Cambridge, UK); anti Gadd34, anti-Lamp-1, anti-SQSTM1/P62, anti-Rab5 and anti-LC3A/B (ab9869, ab24170, ab109012, ab109534, ab62721 Abcam, Cambridge, UK); anti-APG7 (Atg7) (3615 ProSci, Poway, CA, USA); anti-ATF6 (70B1413.1 Novus Biologicals, Centennial, CO, USA); antiCHOP (ma1-250 Thermo Fisher Scientific, Waltham, MA, USA); anti-Actin (SAB4301137 clone BIII-136; Sigma-Aldrich, Saint Louis, MO, USA) and anti-GAPDH (HPA040067 SigmaAldrich, Saint Louis, MO, USA) as loading controls.

Techniques: Activation Assay, Membrane, Binding Assay, Expressing