Journal: International Journal of Molecular Sciences

Article Title: Dapagliflozin Prevents NOX- and SGLT2-Dependent Oxidative Stress in Lens Cells Exposed to Fructose-Induced Diabetes Mellitus

doi: 10.3390/ijms20184357

Figure Lengend Snippet: Glucose transporters (GLUTs) may act through the receptor for advanced glycation end products (RAGE) and induce Ras-related C3 botulinum toxin substrate 1 (RAC1) expression and superoxide generation in the lens tissue of cataract patients with diabetes mellitus (DM). ( A ) Representative fluorescence images of sodium/glucose cotransporter 2 (SGLT2)- and 3-NT-positive cells (green) and SGLT2-positive cells (red) in lens epithelial tissue from cataract patients with and without DM. Cell nuclei are counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). ( B , C ) Representative fluorescence images of RAGE- and GLUT1-positive cells (green) and RAC1- and GLUT5-positive cells (red) in lens epithelial tissue from cataract patients with and without DM. Cell nuclei are counterstained with DAPI (blue). Scale bar: 20 μm. Quantified values (right) are the means ± SEMs ( n = 10 per group, separate experimental groups in each figure). * p < 0.05.

Article Snippet: Antibodies directed against glucose transporter 1 (GLUT1), receptor for advanced glycation end products (RAGE), Ras-related C3 botulinum toxin substrate 1 (RAC1), and sodium/glucose cotransporter 2 (SGLT2) were purchased from Abcam (Abcam, Cambridge, MA, USA; ab115730, ab3611, ab65965, and ab37296, respectively), against nicotinamide adenine dinucleotide phosphate reduced form (NADPH) oxidase 4 (NOX4) from Novus Biologicals (NB110-58849, Englewood, CO, USA), against p67-phox from Millipore (07-502, Billerica, MA, USA), and those against GLUT5 from GeneTex (GTX12098, Englewood, CO, USA).

Techniques: Expressing, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Dapagliflozin Prevents NOX- and SGLT2-Dependent Oxidative Stress in Lens Cells Exposed to Fructose-Induced Diabetes Mellitus

doi: 10.3390/ijms20184357

Figure Lengend Snippet: Glucose transporter (GLUT)-induced superoxide production in lenses of rats with fructose-induced type 2 diabetes mellitus (DM). ( A ) Lenses from the control (a) and fructose (b) groups. Rats exhibited fructose-induced type 2 DM after eight weeks (8 W). ( B ) Representative images of dihydroethidium-treated lens epithelial cells. Sections from rats with fructose-induced type 2 DM displayed significantly higher dihydroethidium fluorescence compared to the control group, whereas treatment with the sodium-glucose cotransporter 2 (SGLT2) inhibitor (dapagliflozin, 1.2 mg/kg/day) prevented this effect in fructose-fed rats. ( C – D ) Real-time polymerase chain reaction (RT-PCR) depicting SGLT2, GLUT1, and GLUT5 mRNA expression in the lens epithelium or fibers from animals with fructose-induced type 2 DM with or without dapagliflozin administration. Scale bar: 20 μm. Data are presented as the means ± SEM ( n = 6 per group, separate experimental groups). * p < 0.05 vs. control; # p < 0.05 vs. Fructose 8 W.

Article Snippet: Antibodies directed against glucose transporter 1 (GLUT1), receptor for advanced glycation end products (RAGE), Ras-related C3 botulinum toxin substrate 1 (RAC1), and sodium/glucose cotransporter 2 (SGLT2) were purchased from Abcam (Abcam, Cambridge, MA, USA; ab115730, ab3611, ab65965, and ab37296, respectively), against nicotinamide adenine dinucleotide phosphate reduced form (NADPH) oxidase 4 (NOX4) from Novus Biologicals (NB110-58849, Englewood, CO, USA), against p67-phox from Millipore (07-502, Billerica, MA, USA), and those against GLUT5 from GeneTex (GTX12098, Englewood, CO, USA).

Techniques: Control, Fluorescence, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Dapagliflozin Prevents NOX- and SGLT2-Dependent Oxidative Stress in Lens Cells Exposed to Fructose-Induced Diabetes Mellitus

doi: 10.3390/ijms20184357

Figure Lengend Snippet: Dapagliflozin reduces glucose transporter (GLUT)-induced expression of the receptor for advanced glycation end products (RAGE), nicotinamide adenine dinucleotide phosphate reduced form oxidase 4 (NOX4) and nicotinamide adenine dinucleotide phosphate reduced form (NADPH) oxidase subunit (p67-phox) levels in the lenses of rats with fructose-induced type 2 diabetes mellitus (DM). ( A – B ) Representative fluorescence images of GLUT5- and RAGE-expressing cells (green) and p7-phox and NOX4-expressing cells (red) in the lens with or without a systemic administration of fructose or dapagliflozin. Cell nuclei are counterstained with DAPI (blue). ( C – E ) Quantitative immunoblotting analysis demonstrating that the GLUT1, GLUT5, and p67-phox levels in the lenses of rats with fructose-induced type 2 DM were significantly decreased by dapagliflozin administration. Values are presented as the means ± SEMs ( n = 6 per group, separate experimental groups in each figure). Scale bar: 20 μm. * p < 0.05; # p < 0.05 vs. Fructose 8 W or 12 W.

Article Snippet: Antibodies directed against glucose transporter 1 (GLUT1), receptor for advanced glycation end products (RAGE), Ras-related C3 botulinum toxin substrate 1 (RAC1), and sodium/glucose cotransporter 2 (SGLT2) were purchased from Abcam (Abcam, Cambridge, MA, USA; ab115730, ab3611, ab65965, and ab37296, respectively), against nicotinamide adenine dinucleotide phosphate reduced form (NADPH) oxidase 4 (NOX4) from Novus Biologicals (NB110-58849, Englewood, CO, USA), against p67-phox from Millipore (07-502, Billerica, MA, USA), and those against GLUT5 from GeneTex (GTX12098, Englewood, CO, USA).

Techniques: Expressing, Fluorescence, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Dapagliflozin Prevents NOX- and SGLT2-Dependent Oxidative Stress in Lens Cells Exposed to Fructose-Induced Diabetes Mellitus

doi: 10.3390/ijms20184357

Figure Lengend Snippet: Dapagliflozin blocked the sodium-glucose cotransporter 2 (SGLT2)-induced production of the NADPH Oxidase Subunits and the receptor for advanced glycation end products (RAGE) in lens epithelial sections of type 2 diabetes mellitus (DM) rats. ( A – B ) Representative fluorescence images of 3-nitrotyrosine- (3-NT) and glucose transporter (GLUT)5-expressing cells (green) and SGLT2- and GLUT1-expressing cells (red) in the lens of rats with or without the systemic administration of fructose or dapagliflozin. Cell nuclei are counterstained with DAPI (blue). The presented values are the means ± SEMs ( n = 6 per group, separate experimental groups in each figure). ( C – D ) Quantitative immunoblotting analysis demonstrating that the expression levels of SGLT2, GLUT1, GLUT5, RAGE, NOX2 and p67-phox in the lenses of rats with fructose-induced type 2 DM were decreased by dapagliflozin administration. Scale bar: 20 μm. The presented values are the means ± SEMs ( n = 6 per group, separate experimental groups in each figure). * p < 0.05 vs. control; # p < 0.05 vs. Fructose 8 W.

Article Snippet: Antibodies directed against glucose transporter 1 (GLUT1), receptor for advanced glycation end products (RAGE), Ras-related C3 botulinum toxin substrate 1 (RAC1), and sodium/glucose cotransporter 2 (SGLT2) were purchased from Abcam (Abcam, Cambridge, MA, USA; ab115730, ab3611, ab65965, and ab37296, respectively), against nicotinamide adenine dinucleotide phosphate reduced form (NADPH) oxidase 4 (NOX4) from Novus Biologicals (NB110-58849, Englewood, CO, USA), against p67-phox from Millipore (07-502, Billerica, MA, USA), and those against GLUT5 from GeneTex (GTX12098, Englewood, CO, USA).

Techniques: Fluorescence, Expressing, Western Blot, Control

Journal: Stem cells (Dayton, Ohio)

Article Title: Fructose Potentiates Bone Loss and Marrow Adipose Tissue Accumulation by Inhibiting Adenosine 5'-Monophosphate-Activated Protein Kinase in Mesenchymal Stem Cells.

doi: 10.1093/stmcls/sxae001

Figure Lengend Snippet: Figure 5. Fructose altered BMMSCs differentiation fate by AMPK pathway. (A) The gene expression of slc2a1, slc2a2, slc2a3, slc2a4, slc2a5, and slc2a8. (B) Higher FBP level in glucose and fructose groups. Fructose stimulation is induced to generate the highest level of FBP. (C) WB indicated the descending trend in PGC-1α and phosphorating AMPK (pAMPK) expression in glucose and fructose, more significant in the latter one. PPARγ expression was on the contrary. (D) Glucose and fructose suppressed osteogenic induction while fueled adipogenic activity of BMMSCs by AMPK pathway. (E) WB showed a decrease in RUNX2 and pAMPK expression in glucose and fructose, more significant in the latter one. (F) Extracellular acidification rate (ECAR) of 3 groups in line chart. The glycolysis rate assay showed basal and compensated glycolytic capacity. Nu referred to nuclear. Data were from 3 independent experiments. Values are means ± SD. *P < .05, **P < .01, ***P < .001, ****P < .0001.

Article Snippet: Small-interfering RNA (siRNA) targeting mouse GLUT1, GLUT5 and scramble siRNA (Santa Cruz Biotechnology, USA) as a negative control were used in gene knockdown experiments.

Techniques: Gene Expression, Expressing, Activity Assay

Journal: Stem cells (Dayton, Ohio)

Article Title: Fructose Potentiates Bone Loss and Marrow Adipose Tissue Accumulation by Inhibiting Adenosine 5'-Monophosphate-Activated Protein Kinase in Mesenchymal Stem Cells.

doi: 10.1093/stmcls/sxae001

Figure Lengend Snippet: Figure 6. GLUT1 or GLUT5 knockdown mitigated the glucose or fructose function on osteogenesis, respectively. (A) WB determined the transfection efficacy of siGLUT1 and siGLUT5 in BMMSCs, respectively. (B) Alpl, Runx2, Sp7, and Bglap expression in scramble siRNA groups. (C) WB illustrated the lower expression of COL-1, RUNX2, and OCN in scramble glucose and scramble fructose groups and the more significant reduction in scramble fructose group. (D) Alizarin S red staining. (E) 562 nm OD value of stained calcium deposits showed the corresponding results with RT- PCR and WB. (F) Alpl, Runx2, Sp7, and Bglap expression in siGLUT1 groups. (G-I) WB, Alizarin S red staining and 562 nm OD value vindicated the RT-PCR results. (J) Alpl, Runx2, Sp7, and Bglap expression in siGLUT5 groups. (K) Fructose lost its effects after the GLUT5 blocking, higher expression of the COL-1, RUNX2, and OCN than siGLUT5 glucose group. (L, M) Alizarin S red staining and 562 nm OD value demonstrated the weakened inhibition in fructose after GLUT5 knockdown. Nu referred to nuclear. Data were from 3 independent experiments. Values are means ± SD. *P < .05, **P < .01, ***P < .001, ****P < .0001.

Article Snippet: Small-interfering RNA (siRNA) targeting mouse GLUT1, GLUT5 and scramble siRNA (Santa Cruz Biotechnology, USA) as a negative control were used in gene knockdown experiments.

Techniques: Knockdown, Transfection, Expressing, Staining, Reverse Transcription Polymerase Chain Reaction, Blocking Assay, Inhibition

Journal: Stem cells (Dayton, Ohio)

Article Title: Fructose Potentiates Bone Loss and Marrow Adipose Tissue Accumulation by Inhibiting Adenosine 5'-Monophosphate-Activated Protein Kinase in Mesenchymal Stem Cells.

doi: 10.1093/stmcls/sxae001

Figure Lengend Snippet: Figure 7. GLUT1 or GLUT5 knockdown mitigated the glucose or fructose function on adipogenesis and pAMPK inhibition, respectively. (A) The gene expression of adipogenic-related genes regarding Pparg, Fabp4, and Lpl in scramble siRNA groups. (B) PPARγ expression in WB also increased in scramble glucose and scramble fructose and the latter one to the largest extent. (C) PGC-1α and pAMPK inhibition were examined by WB. (D, E) Oil red staining and OD value assisted with the adipogenesis assay. (F) The expression of Pparg, Fabp4, and Lpl in siGLUT1 groups. (G) PPARγ expression in WB supported the results in (F). (H) WB showed the promoted expression of PGC-1α and pAMPK in siGLUT1 glucose group. (I, J) Oil red staining and OD value were consistent with (H) and (G). (K) The expression of Pparg, Fabp4, and Lpl in siGLUT5 groups. Pparg, Fabp4, and Lpl gene expressions were downregulated in 25 mM fructose compared with 25 mM glucose. (L) WB of PPARγ expression confirmed the RT-PCR results. (M) PGC-1α and pAMPK expression enhanced in 25 mM fructose than 25 mM glucose, but were less than control group. (N, O) Oil red staining and OD value. Data were from 3 independent experiments. Values are means ± SD. *P < .05, **P < .01, ***P < .001, ****P < .0001.

Article Snippet: Small-interfering RNA (siRNA) targeting mouse GLUT1, GLUT5 and scramble siRNA (Santa Cruz Biotechnology, USA) as a negative control were used in gene knockdown experiments.

Techniques: Knockdown, Inhibition, Gene Expression, Expressing, Staining, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Avicenna Journal of Medical Biotechnology

Article Title: Antidiabetic Activity of Momordica charantia Extracts Through Incretin Pathway in Streptozotocin-Nicotinamide Induced Diabetic Rat Depends on Dose Differences

doi: 10.18502/ajmb.v17i1.17677

Figure Lengend Snippet: Alpha glucosidase levels

Article Snippet: Examination of markers GLP-1, DPP 4, alpha glucosidase, and GLUT5 using Elisa kit reagents from BT Lab®.

Techniques:

Journal: Avicenna Journal of Medical Biotechnology

Article Title: Antidiabetic Activity of Momordica charantia Extracts Through Incretin Pathway in Streptozotocin-Nicotinamide Induced Diabetic Rat Depends on Dose Differences

doi: 10.18502/ajmb.v17i1.17677

Figure Lengend Snippet: Anova test for alpha glucosidase levels

Article Snippet: Examination of markers GLP-1, DPP 4, alpha glucosidase, and GLUT5 using Elisa kit reagents from BT Lab®.

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: Loss of the fructose transporter SLC2A5 inhibits cancer cell migration

doi: 10.3389/fcell.2022.896297

Figure Lengend Snippet: Attenuation of the SLC2A5 gene expression in fibrosarcoma cells. (A) . Images of scratch test (left) and quantitative analysis of closure (right) as a function of time for HT1080tdT and HT1080tdT-ε2A5 fibrosarcoma cells (n = 3). Scale bar 10 µm. (B) . Images of colonies formed by HT1080tdT and HT1080tdT-ε2A5 cells and analysis of colony number (500 cells/well plated at time 0 h). * p = 0.007; n = 3. Colonies were fixed with 70% ethanol and visualized by staining with 1% trypan blue. (C) . Proliferation assay of HT1080tdT and HT1080tdT-ε2A5 cells. HT1080tdT and HT1080tdT-ε2A5 cells were plated at a density of 5.0 × 10 3 cells/well of a 96 well plates. Fructose was added to the media as indicated in the Figure. Cells were cultured for 48 h followed by MTS assay (n = 3). All data in the Figure is representative of more than 3 experiments with 3 biological replicates.

Article Snippet: Anti-SLC2A5 antibodies were purchased from Boster (Cat# PA 2064) and used at 1:500 dilution.

Techniques: Gene Expression, Staining, Proliferation Assay, Cell Culture, MTS Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Loss of the fructose transporter SLC2A5 inhibits cancer cell migration

doi: 10.3389/fcell.2022.896297

Figure Lengend Snippet: Editing of the SLC2A5 gene. (A) . Inactivation of the SLC2A5 gene in human MIA-PaCa-2 and HT1080tdT cells using CRISPR/Cas9-mediated in situ gene editing. Guide RNAs were targeted to Exon 3. Genomic DNA of edited cells was sequenced, and positive clones were identified as having a 62 base pair disruption, leading to an early stop codon 14 amino acids downstream of the disruption point. (B) . qPCR analysis of SLC2A5 mRNA abundance in MIA-PaCa-2 and MIA-PaCa-ε2A5 cells (clones B3 and F11) (n = 3). DNA primers were designed to amplify Exon 12 of the SLC2A5 gene. Immunoblot analysis was carried out with antibodies directed at the C-terminus of SLC2A5. Anti-β-tubulin or anti-GAPDH antibodies were used to assess amounts of loaded protein samples. (C) . qPCR analysis of SLC2A5 mRNA abundance in HT1080tdT and HT1080tdT-ε2A5 cells. DNA primers and control antibodies were as described above. (D) . qPCR analyses of SLC2A1, SLC2A2, and SLC2A4 mRNA abundance in MIA-PaCa-2 and MIA-PaCa-ε2A5 cells (n = 3). (E) . qPCR analyses of SLC2A1, SLC2A2, and SLC2A4 mRNA abundance in HT1080tdT and HT1080tdT-ε2A5 cells (n = 3). All data in the Figure is representative of more than 3 biological replicates. NS, not significant.

Article Snippet: Anti-SLC2A5 antibodies were purchased from Boster (Cat# PA 2064) and used at 1:500 dilution.

Techniques: CRISPR, In Situ, Clone Assay, Disruption, Western Blot, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Loss of the fructose transporter SLC2A5 inhibits cancer cell migration

doi: 10.3389/fcell.2022.896297

Figure Lengend Snippet: Attenuation of the SLC2A5 gene expression in pancreatic cancer cells. (A) . Images of scratch test (left panel) and quantitative analysis of closure (right panel) for MIA-PaCa-2 and MIA-PaCa-ε2A5 cells (clone B3; ** p = 0.002, n = 3). Scale bar 10 µm. (B) . Scratch test analysis for MIA-PaCa-2 and MIA-PaCa-ε2A5 cell lines clone F11 (** p = 0.001, n = 3). Scale bar 10 µm. (C) . Colony formation images and colony number for MIA-PaCa-ε2A5 cells, clone B3 (left panel; 1000 cells/well plated at time 0 h; * p = 0.0001; n = 3) and clone F11 (right panel; 500 cells/well plated at time 0 h; * p = 0.001; n = 3). (D) . Fructose-dependent proliferation of the MIA-PaCa-2 and MIA-PaCa-ε2A5 cells. MIA-PaCa-2 and MIA-PaCa-ε2A5 (clones B3 and F11) cells were plated at a density of 1.0 × 10 4 cells/well of a 96 well plates. Fructose was added to the culture medium as indicated in the figure. The absorbance at 490 nm (OD 490 ) of the culture medium after MTS assay at time 0 and 48 h in culture are shown. Experiments were performed a minimum of three times in triplicates. * p = 0.0002; n = 3.

Article Snippet: Anti-SLC2A5 antibodies were purchased from Boster (Cat# PA 2064) and used at 1:500 dilution.

Techniques: Gene Expression, Clone Assay, MTS Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Loss of the fructose transporter SLC2A5 inhibits cancer cell migration

doi: 10.3389/fcell.2022.896297

Figure Lengend Snippet: Restoration of SLC2A5 function via SLC2A5 transgene expression. Proliferation assay (A) and scratch test assay (B) of MIA-PaCa-2, MIA-PaCa-ε2A5 (clone F11) and MIA-PaCa-ε2A5 cells expressing wild-type SLC2A5 or the non-functional SLC2A5-E401A mutant (* p = 0.002; n = 3). In (A) , fold change in cell number is shown at day 0 to day 3. In (B) , the percentage of open scratch is shown at time 0 and 48 h. Proliferation assay (C) and scratch test assay (D) of HT1080tdT, HT1080tdT-ε2A5 cells transfected with expression vectors encoding wild-type SLC2A5 or the non-functional SLC2A5-E401A mutant (* p = 0.001, n = 3). In (C) , the absorbance at 490 nm (OD 490 ) of the culture medium after MTS assay from time 0 and 7 h in culture is shown. In (D) , the percentage of open scratch is shown at time 0 and 7 h. NS, not significant. All data in the Figure is representative of more than 3 experiments with 3 replicates.

Article Snippet: Anti-SLC2A5 antibodies were purchased from Boster (Cat# PA 2064) and used at 1:500 dilution.

Techniques: Expressing, Proliferation Assay, Wound Healing Assay, Functional Assay, Mutagenesis, Transfection, MTS Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Loss of the fructose transporter SLC2A5 inhibits cancer cell migration

doi: 10.3389/fcell.2022.896297

Figure Lengend Snippet: Mitochondria localization and dynamics in SLC2A5-deficient cancer cells. (A) . Electron microscopy analysis of MIA-PaCa-2 cells shows that mitochondria were localized in the center of these cells in the perinuclear space (encircled). In MIA-PaCa-ε2A5 cells, mitochondria were spread throughout the cell towards the cell periphery, and appeared elongated (arrows) (n = 3). (B) . and (C) . Mitochondria area (B) and length (C) analyses in MIA-PaCa-2 and MIA-PaCa-ε2A5 cells. * p < 0.0006; ** p < 0.0001 (n = 3). (D) . Confocal images of mitochondria stained with MitoTracker ® Green in red fluorescent protein-labelled HT1080tdT cells,HT1080tdT-ε2A5 cells or HT1080tdT-ε2A5 cells expressing wild-type SLC2A5. Time-lapse video is shown in the (arrowhead). (E) . Mitochondria number and (F) . mitochondria length in fibrosarcoma HT1080tdT cells, HT1080tdT-ε2A5 or HT1080tdT-ε2A5 cells expressing wild-type SLC2A5. *** p < 0.0002 (n = 3).

Article Snippet: Anti-SLC2A5 antibodies were purchased from Boster (Cat# PA 2064) and used at 1:500 dilution.

Techniques: Electron Microscopy, Staining, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Loss of the fructose transporter SLC2A5 inhibits cancer cell migration

doi: 10.3389/fcell.2022.896297

Figure Lengend Snippet: SLC2A5 function is necessary for polarized localization of mitochondria in HT1080tdT human fibrosarcoma cells in vitro and in vivo . (A) mitochondria (green) localization in HT1080tdT or HT1080tdT-ε2A5 (vasculature, grey; blue, DAPI) cells that are extravasating out of the chicken CAM vasculature ( in vivo ). Right panels show all three channels: red fluorescent protein (red), mitochondria (green), nuclei (blue) and vasculature (grey). Time-lapse video is shown in the . (B) . Fluorescent channel intensity along the line scans (dashed arrows in (A) (right panel) indicate the direction of the scan). Green line depicts mitochondria localization. The short arrows in (A) and (B) indicate vascular membrane breaches.

Article Snippet: Anti-SLC2A5 antibodies were purchased from Boster (Cat# PA 2064) and used at 1:500 dilution.

Techniques: In Vitro, In Vivo, Membrane