Journal: Pharmaceutics

Article Title: Amino Acid Nanofibers Improve Glycemia and Confer Cognitive Therapeutic Efficacy to Bound Insulin

doi: 10.3390/pharmaceutics14010081

Figure Lengend Snippet: AAC2-hINS increases glucose uptake in 3T3-L1 preadipocytes and human BBB endothelial cells via GLUT4 and GLUT1. ( a ) FD-glucose uptake in mouse 3T3-L1 preadipocytes ( n = 24 per treatment) treated with vehicle (Veh; PBS, open bar) or hINS (1.7 µM, red bar) or AAC2 (0.03 and 3 µM black bar) or AAC2-hINS (purple bar) with the respective concentration of AAC2 and a constant hINS concentration. After 2 h 15 min starvation, FD glucose and treatment reagents were added for an additional 85 min of incubation. Data (%) were normalized to FD-glucose uptake in control (Veh) 3T3-L1 cells (100%). Student’s independent t -test for all tests in figure. ( b ) AAC2 dose-dependent uptake FD-glucose in 3T3-L1 preadipocytes. Data are shown as % of increase compared to non-stimulated cells. ( c ) Non-differentiated NIH-3T3 cells were transiently transfected with GLUT4-GFP. Translocation of GLUT4-GFP (white, yellow arrow) cytosol to the membrane after stimulation with Veh (PBS), hINS (10 µg/mL), AAC2 (10 µM), or AAC2-hINS was demonstrated using confocal microscopy (60× magnification). ( d ) FD-glucose uptake in human brain endothelial cells ( n = 7 per treatment) treated with vehicle (Veh; PBS, open bar) or hINS (1.7 µM, red bar) or AAC2 (0.1 µM black bar), or AAC2-hINS (purple bar) in the presence and absence of GLUT1 inhibitor (BAY-876; 10 nM in DMSO). Cells were pre-treated with or without BAY-876 for 40 min and then FD glucose and treatment reagents were added for an additional 50 min of incubation. Data (%) were normalized to FD-glucose uptake in control (Veh without inhibitor, 100%).

Article Snippet: GLUT1 inhibitor (BAY-876, Selleckchem, Houston, TX S8452, USA)

Techniques: Concentration Assay, Incubation, Control, Transfection, Translocation Assay, Membrane, Confocal Microscopy

Journal: Journal of Diabetes Investigation

Article Title: FOSL2 activates TGF ‐β1‐mediated GLUT1 / mTOR signaling to promote diabetic kidney disease

doi: 10.1111/jdi.14360

Figure Lengend Snippet: FOSL2‐mediated TGF‐β1 activates the GLUT1/mTOR signaling pathway. The DKD mice were treated with sh‐NC + oe‐NC, sh‐NC + TGF‐β1‐oe, sh‐FOSL2 + oe‐NC, or sh‐FOSL2 + TGF‐β1‐oe. (a) Detection of urine albumin‐to‐creatinine ratio and blood glucose concentration in mice. (b) The expression of FOSL2 and TGF‐β1 in kidney tissues of DKD mice was measured by RT‐qPCR. (c) The GLUT1 and mTOR protein expression in kidney tissues of DKD mice was measured using immunoblotting assays. (d) The GLUT1 and mTOR protein expression in kidney tissues of DKD mice was measured using immunohistochemistry (scale bars: 50 μm). (e) The detection of ECM expansion and basement membrane thickening (indicated by black arrows) in kidney tissues of DKD mice was viewed using PAS staining (scale bars: 50 μm). (f) ECM stromal dilatation and thickened basement membranes were viewed using Masson's staining (scale bars: 100 μm for the upper lane and 50 μm for the lower lane). Data were expressed as means ± SD, n = 6. One‐way anova was used for the comparison among multiple groups. * P < 0.05 vs the sh‐NC + oe‐NC group; # P < 0.05 vs the sh‐FOSL2 + oe‐NC group.

Article Snippet: FOSL2‐oe or oe‐NC‐treated MCs were further treated with the mTOR inhibitor Rapamycin at 100 nM for 48 h or GLUT1 inhibitor BAY‐876 (99.67%, HY‐100017, MedChemExpress) at 50 nM for 24 h , which were used to construct the oe‐NC + DMSO, FOSL2‐oe + DMSO, oe‐NC + Rapamycin, oe‐NC + BAY‐876, FOSL2‐oe + Rapamycin, and FOSL2‐oe + BAY‐876 groups.

Techniques: Concentration Assay, Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Membrane, Staining, Comparison

Journal: Journal of Diabetes Investigation

Article Title: FOSL2 activates TGF ‐β1‐mediated GLUT1 / mTOR signaling to promote diabetic kidney disease

doi: 10.1111/jdi.14360

Figure Lengend Snippet: Blocking the GLUT1/mTOR signaling pathway reverses the HG‐MCs activity induced by FOSL2‐oe. NG‐MCs/HG‐MCs were treated with oe‐NC + DMSO, oe‐NC + Rapamycin, FOSL2‐oe + DMSO, FOSL2‐oe + Rapamycin, oe‐NC + BAY‐876, and FOSL2‐oe + BAY‐876. (a) The expression of FOSL2 and TGF‐β1 in NG‐MCs/HG‐MCs infected with oe‐NC or FOSL2‐oe detected by RT‐qPCR. (b) The FOSL2, TGF‐β1, GLUT1, and mTOR protein expression in NG‐MCs/HG‐MCs were measured using immunoblotting assays. (c) Proliferative capacity of NG‐MCs/HG‐MCs detected by CCK‐8 assay. (d) The proliferation rate in NG‐MCs/HG‐MCs was detected by EdU staining. (e) Detection of Collagen VI expression in NG‐MCs/HG‐MCs measured using immunoblotting assays. Scale bars: 50 μm. Data were expressed as means ± SD, n = 3. An unpaired t‐test was used for the comparison of two groups (a), and one‐way anova was used for the comparison among multiple groups (b, d, e). The comparison of data at different time points was analyzed using two‐way anova (c). * p < 0.05 vs the oe‐NC + DMSO group; # p < 0.05 vs the oe‐NC + Rapamycin group; & p < 0.05 vs the oe‐NC + BAY‐876 group.

Article Snippet: FOSL2‐oe or oe‐NC‐treated MCs were further treated with the mTOR inhibitor Rapamycin at 100 nM for 48 h or GLUT1 inhibitor BAY‐876 (99.67%, HY‐100017, MedChemExpress) at 50 nM for 24 h , which were used to construct the oe‐NC + DMSO, FOSL2‐oe + DMSO, oe‐NC + Rapamycin, oe‐NC + BAY‐876, FOSL2‐oe + Rapamycin, and FOSL2‐oe + BAY‐876 groups.

Techniques: Blocking Assay, Activity Assay, Expressing, Infection, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Staining, Comparison

Journal: Journal of Diabetes Investigation

Article Title: FOSL2 activates TGF ‐β1‐mediated GLUT1 / mTOR signaling to promote diabetic kidney disease

doi: 10.1111/jdi.14360

Figure Lengend Snippet: Inhibition of mTOR signaling relieves DKD‐associated kidney injury accelerated by overexpression of FOSL2. The DKD mice were treated with oe‐NC + DMSO, oe‐NC + Rapamycin, FOSL2‐oe + DMSO, and FOSL2‐oe + Rapamycin. (a) Detection of urine albumin‐to‐creatinine ratio and blood glucose concentration in mice. (b) The FOSL2, TGF‐β1, GLUT1, and mTOR protein expression in kidney tissues of DKD mice was measured using immunohistochemistry (scale bars: 50 μm). (c) The detection of ECM expansion and basement membrane thickening (indicated by black arrows) in kidney tissues of DKD mice was viewed using PAS staining (scale bars: 50 μm). (d) ECM stromal dilatation and thickened basement membranes were viewed using Masson's staining (scale bars: 100 μm for the upper lane and 50 μm for the lower lane). Data were expressed as means ± SD, n = 6. One‐way anova was used for the comparison among multiple groups. * P < 0.05 vs the oe‐NC + DMSO group; # P < 0.05 vs the oe‐NC + Rapamycin group.

Article Snippet: FOSL2‐oe or oe‐NC‐treated MCs were further treated with the mTOR inhibitor Rapamycin at 100 nM for 48 h or GLUT1 inhibitor BAY‐876 (99.67%, HY‐100017, MedChemExpress) at 50 nM for 24 h , which were used to construct the oe‐NC + DMSO, FOSL2‐oe + DMSO, oe‐NC + Rapamycin, oe‐NC + BAY‐876, FOSL2‐oe + Rapamycin, and FOSL2‐oe + BAY‐876 groups.

Techniques: Inhibition, Over Expression, Concentration Assay, Expressing, Immunohistochemistry, Membrane, Staining, Comparison

Journal: Journal of Diabetes Investigation

Article Title: FOSL2 activates TGF ‐β1‐mediated GLUT1 / mTOR signaling to promote diabetic kidney disease

doi: 10.1111/jdi.14360

Figure Lengend Snippet: Schematic diagram of the proposed role of FOSL2/TGF‐β1/GLUT1/mTOR axis in DKD. Abnormally high expression of FOSL2 activates the expression of TGF‐β1 to induce the downstream GLUT1/mTOR signaling pathway, which can drive renal injury in mice, ultimately leading to the development of DKD.

Article Snippet: FOSL2‐oe or oe‐NC‐treated MCs were further treated with the mTOR inhibitor Rapamycin at 100 nM for 48 h or GLUT1 inhibitor BAY‐876 (99.67%, HY‐100017, MedChemExpress) at 50 nM for 24 h , which were used to construct the oe‐NC + DMSO, FOSL2‐oe + DMSO, oe‐NC + Rapamycin, oe‐NC + BAY‐876, FOSL2‐oe + Rapamycin, and FOSL2‐oe + BAY‐876 groups.

Techniques: Expressing

Journal: Nutrition & Diabetes

Article Title: Detrimental effects of branched-chain amino acids in glucose tolerance can be attributed to valine induced glucotoxicity in skeletal muscle

doi: 10.1038/s41387-022-00200-8

Figure Lengend Snippet: A qPCR of glucose transporters and stress markers in C2C12 mouse myotubes after treatment with 2 mM Val or 3-HIB in presence of 250 µM PA for 24-h normalized to BSA control with B2m as reference gene ( n = 4). B Representative western blot of insulin stimulated (15 min) AKT phosphorylation in differentiated C2C12 mouse myotubes after treatment with 2 mM valine (Val) or 3-HIB for 48-h normalized to BSA control ( n = 4). C Relative basal glucose uptake in differentiated C2C12 mouse myotubes after treatment with 2 mM valine (Val) or 3-HIB for 48-h normalized to PA ( n = 8). D Relative insulin stimulated (30 min) glucose uptake in differentiated C2C12 mouse myotubes after treatment with 2 mM valine (Val) or 3-HIB for 48-h normalized to PA ( n = 8). E – G qPCR of glucose transporter and stress markers in C2C12 mouse myotubes after treatment with 2 mM Val or 3-HIB in presence of 250 µM PA and 25 nM BAY-876 (Bay) for 24 h normalized to PA-Ctl with B2m as reference gene ( n = 4). H Representative western blot of insulin-stimulated (15 min) AKT phosphorylation in differentiated C2C12 mouse myotubes after treatment with 2 mM valine (Val) or 3-HIB in presence of 25 nM BAY-876 normalized to PA control ( n = 4). Fold change in Western blots ( B , H ) refers to insulin-induced change normalized to BSA control ( B ) or PA control ( H ). Data are shown as mean + SEM except for western blot quantification where only mean of fold change is indicated below representative blots. * p < 0.05; ** p < 0.01; *** p < 0.001 compared to respective control and. # p < 0.05 comparing Ctl to Bay treated groups. Different letters represent significant difference between groups in Western blots. BSA bovine serum albumin, PA palmitic acid, Bay BAY-876.

Article Snippet: Glucose Uptake-Glo Assay (Promega, J1341) was used to determine basal and insulin stimulated glucose uptake after 48 h. GLUT1 inhibitor studies were performed at indicated time points with BAY-876 (R&D, 6199).

Techniques: Western Blot

Journal: Diabetes

Article Title: Glucose Transporters Are Key Components of the Human Glucostat

doi: 10.2337/db23-0508

Figure Lengend Snippet: GLUT1 and GLUT2 expression in human primary islets and SC-islets. A : Top: Differentiation protocol schematic of hESC into SC-islets cells. DE, definitive endoderm; ESC, embyonic stem cells; PP1 and PP2, pancreatic progenitor stage 1 and 2. Bottom: Representative flow cytometry plot of SC-islets differentiation efficiency on day 35 of the protocol, measuring expression of β-cell markers C-peptide (C-PEP) and NKX6.1, and of endocrine hormones C-PEP, GCG, and somatostatin (SST). B : Representative immunostaining images of isolated SC-islets and adult human islets. Scale bars represent 50 μm. C and D : Quantification of GLUT1 and GLUT2 expressing INS + cells in SC-islets ( C ) and adult human islets ( D ). Quantifications were based on images of multiple SC-islets and human adult islets from n = 6 independent differentiations or n = 6 different human donors, respectively. E : Representative immunostaining of human fetal (top) and adult (bottom) pancreas sections. Specimens were obtained from four human donors for both adult and fetal pancreas. Individual channel images are the same magnification as the larger merged images. The white box marks areas of interest. Scale bars represent 50 μm. F and G : Quantification of GLUT1 and GLUT2 expressing INS + cells in panel E . H : Summary of data presented in C , D , F , and G of GLUT1/2 expression distribution into different subpopulations in INS + β-cells.

Article Snippet: Each buffer was prepared with and without 2 nmol/L final concentration GLUT1 inhibitor BAY-876 (Millipore Sigma, SML1774-5MG).

Techniques: Expressing, Flow Cytometry, Immunostaining, Isolation

Journal: bioRxiv

Article Title: Candida albicans enhances melanoma cell aggressiveness through p38-MAPK and HIF-1α pathways and metabolic reprogramming

doi: 10.1101/2025.01.17.633543

Figure Lengend Snippet: a Extracellular acidification rate (ECAR) of melanoma cells in controls and in cell in contact with C. albicans for six hours. Data shown as the mean of the fold change (FC) of initial fluorescence values in three independent experiments. b VEGF cytokine release induced by six hours of stimulation with C. albicans alone or treated with glucose transporter 1 (GLUT1) inhibitor (BAY-876; 25 nM) and hexokinase (HK) inhibitor (2-DG; 5 mM). Gene expression of aerobic glycolysis related genes ( Eno2 , Hk2 and Slc2a1 ) by six hours of stimulation with C. albicans alone or treated with c , d , e p38 inhibitor (SB 203580, 10 µM) or f , g , h HIF-1α inhibitor (LW6, 10 μM). For all data, individual values and mean ± SEM are shown. a ( n = 4 biologically independent samples); b , c , d , e , f and g ( n = 3 biologically independent samples). # and #### denotes P < 0.05 and P < 0.001 respect to control without C. albicans , respectively; * and *** denote P < 0.05 and P < 0.005 of each inhibitor compared to inhibitor with C. albicans , respectively (two-tailed, unpaired, t-student test).

Article Snippet: The inhibitors of hexokinase (2-deoxyglucose, 2-DG; 5 mM), GLUT1 (BAY 876; 25 nM), SYK (Piceatannol, 30 µM), MyD88 (TJ-M2010-5, 30 µM), EGFR (PD153035, 1 µM), EphA2 (NVP-BHG712, 1 µM), ERK1/2 (PD-0325901; 1 µM), JNK (SP600125, 10 µM), p38a (SB 203580, 10 µM), NF-κB (BAY 11-7082, 2 µM), c-Fos/AP-1 (T-5224, 10 µM) and HIF-1α (LW6, 10 μM) were obtained from MedChemExpress.

Techniques: Fluorescence, Expressing, Control, Two Tailed Test