Journal: PLoS ONE

Article Title: Activation of Wnt/β-Catenin Signaling Increases Insulin Sensitivity through a Reciprocal Regulation of Wnt10b and SREBP-1c in Skeletal Muscle Cells

doi: 10.1371/journal.pone.0008509

Figure Lengend Snippet: ( A ) Myotubes cultured in 5 mM glucose (G5) or in 25 mM glucose (G25) for 48 hours were transfected with a mouse Wnt10b cDNA or treated with 1 µM BIO. 2-deoxyglucose (2-DG) uptake was then measured in the presence or absence of 10 nM insulin for 30 minutes as described in . Data are expressed as mean±SE from 5 independent experiments performed in triplicate. Significant difference from G5, (***) p<0.0001; (**) p<0.02; Significant difference from G25, (###) p<0.0001; (##) p<0.001. ( B ) BIO induced GLUT4 translocation to the plasma membrane. Myotubes were cultured in 5 mM (G5) or 25 mM glucose (G25) for 48 hours in the presence or absence of 1 µM BIO. Myotubes were treated or not with 10 nM insulin for 30 minutes, then plasma membranes were isolated. Western blot analysis showed that insulin and BIO induced GLUT4 translocation to the plasma membrane, whereas GLUT1 was unaffected. ( C ) Quantification of GLUT4 and GLUT1 translocation. Data are expressed as mean±SE from 4 independent experiments. Significant difference from G5, (***) p<0.0001; (**) p<0.01; (*) p<0.05.

Article Snippet: Polyclonal antibodies against MyoD, GAPDH, Wnt10b, GLUT4 and β-tubulin were from Santa-Cruz Biotechnology.

Techniques: Cell Culture, Transfection, Translocation Assay, Clinical Proteomics, Membrane, Isolation, Western Blot

Journal: PLoS ONE

Article Title: Activation of Wnt/β-Catenin Signaling Increases Insulin Sensitivity through a Reciprocal Regulation of Wnt10b and SREBP-1c in Skeletal Muscle Cells

doi: 10.1371/journal.pone.0008509

Figure Lengend Snippet: Wnt10b ( a ) and BIO ( b ) activate the Wnt/β-catenin pathway through the inactivation of GSK-3β Y216 phosphorylation, which results in the nuclear translocation of active β-catenin, stimulation of myogenic genes transcription such as myoD, and inhibition of Srebp-1c transcription ( f ). Insulin-induced Srebp-1c transcription is mediated by the MAPK pathway in muscle cells. BIO inhibits the MAPK pathway, which could explain the down-regulation of Srebp-1c gene expression ( c ). In parallel, inactivation of GSK-3β Y216 is followed by autophosphorylation of PDK1 S241 which phosphorylates Akt1 T308 (but not Akt2 S473 ), then the subsequent phosphorylation of AS160 S588 induces GLUT4 translocation ( e ). In contrast, insulin stimulates GLUT4 translocation through the PI3K/Akt2 S473 /AS160 pathway ( d ). BIO activates the AMP kinase pathway by phosphorylating AMPK-α1 S485 , which also induces GLUT4 translocation ( e ). These results strongly suggest that Wnt signaling, in contrast to insulin signaling, increases glucose transport in both insulin-sensitive and insulin-resistant myotubes through the activation of AMPK-α1 and Akt2/AS160 pathways.

Article Snippet: Polyclonal antibodies against MyoD, GAPDH, Wnt10b, GLUT4 and β-tubulin were from Santa-Cruz Biotechnology.

Techniques: Phospho-proteomics, Translocation Assay, Inhibition, Gene Expression, Activation Assay

Journal: Diabetologia

Article Title: Chronic hyperglycaemia promotes lipogenesis and triacylglycerol accumulation in human skeletal muscle cells.

doi: 10.1007/s00125-004-1465-9

Figure Lengend Snippet: Fig. 5. Expression of GLUT1 and GLUT4 mRNAs after 4 days of hyperglycaemia (HG). Skeletal muscle cells were treated with HG medium (20 mmol/l glucose) from day 4 to 8 during differentiation. At day 8, cells were harvested and total mRNA was isolated by RNeasy Mini kit. Reverse-transcription and real-time PCR were performed. GLUT1 and GLUT4 were quantified relative to the housekeeping control β-actin. Values are ratios between HG and control cells from five separate ex- periments

Article Snippet: DGAT-1, acyl-CoA:1,2-diacylglycerol acyltransfease; F, forward primer; R, reverse primer Signaling Technology, Beverly, Mass., USA) and a rabbit polyclonal antibody raised against a recombinant protein corresponding to amino acids 230-290 within human GLUT4 (H-61, Santa Cruz Biotechnology, Santa Cruz, Calif., USA).

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

Journal: bioRxiv

Article Title: Microtubule-mediated GLUT4 trafficking is disrupted in insulin resistant skeletal muscle

doi: 10.1101/2022.09.19.508621

Figure Lengend Snippet: A-B) Structured illumination microscopy (SIM) imaging of endogenous α-tubulin and GLUT4 (left panel) and 3D reconstruction of GLUT4 (green) and α-Tubulin (white) (right panel) in mouse flexor digitorum brevis (FDB) muscle (A) and human vastus lateralis muscle (B). Arrows indicate GLUT4 at microtubule nucleation sites, arrowheads mark GLUT4 vesicles along the microtubule filaments. C) Overview of workflow for live-imaging of fluorescently conjugated proteins in adult mouse FDB muscle fibers. D) Live-imaging of FDB expressing GLUT4-GFP and mCherry-Tubulin. Yellow projection of mCherry-Tubulin outlines the microtubule filaments (top panel left). Movement of GLUT4-GFP was visualized by color coded projection (first image cyan, last image red, top panel right). The merged projection (bottom), demonstrated movement of GLUT4-GFP along the mCherry-Tubulin containing microtubule filaments indicated by color-coded projections on top of the microtubule filaments. The movement of GLUT4-GFP is shown in Movie 1. A-B). Images are representative of >5 fibers from ≥3 different mice in A+D and 3 different fibers from 3 different subjects in B. Scale bar = 5 µm (A, B, D) and 2 µm (inserts in B, D).

Article Snippet: The following antibodies raised in rabbit: GLUT4 (PA5-23052, Invitrogen), Detyrosinated α-tubulin (AB48389, Abcam), rabbit Syntaxin6 (110 062, Synaptic Systems), or in mouse: GLUT4 (MAB8654, R&D Systems), α-tubulin (T9026, Merck).

Techniques: Microscopy, Imaging, Expressing

Journal: bioRxiv

Article Title: Microtubule-mediated GLUT4 trafficking is disrupted in insulin resistant skeletal muscle

doi: 10.1101/2022.09.19.508621

Figure Lengend Snippet: A) Confocal imaging of α-tubulin (top panel), detyrosinated tubulin (De-Tubulin, bottom panel) and GLUT4-GFP in flexor digitorum brevis (FDB) fibers from muscles expressing GLUT4-GFP. B) Structured illumination microscopy (SIM) imaging of FDB fiber expressing GLUT4-GFP and mCherry-Tubulin. In A and B, arrows mark GLUT4-GFP localized at microtubule nucleation sites and arrowheads indicate GLUT4-GFP on the microtubule filaments. C) Color-coded projection (first image blue, last image white as indicated) from 120 second live-imaging of FDB fiber expressing GLUT4-GFP. D) Live-imaging indicating spherical and tubular GLUT4-GFP vesicles/structures undergoing fusion and fission. E) Time-montage of GLUT4-GFP and mCherry-Tubulin from the recording shown in . White arrowheads mark GLUT4-GFP moving on the microtubule filament incorporated with mCherry-Tubulin. Red arrowheads mark static GLUT4-GFP localized away from the microtubule filament. F) Color-coded projection (first image blue, last image white as indicated) from 120 seconds live-imaging of GLUT4-GFP-enriched structures at the microtubule nucleation sites in FDB fiber. Directionality of the GLUT4 trafficking is visible in movie 2. G) Quantified graph (left) and time series images (right) of GLUT4-GFP fluorescence in region surrounding microtubule nucleation sites (outside red outline), before and after photobleaching of the surrounding region to estimate plus-end-directed GLUT4 transport. Yellow arrowheads indicate presence of new GLUT4 structures. H) Quantified graph (left) and time series images (right) of GLUT4-GFP fluorescence at microtubule nucleation site (encircled in red), before and after photobleaching of the central nucleation site region to estimate minus-end-directed GLUT4 transport. Time series images shown are representative of fibers from 3 different mice. Images are representative of at least 5 fibers from ≥3 different mice. Scale bar = 5 µm (A-C) and 2 µm (D-H and inserts in B). Figure 1 – figure supplement 2: GLUT4 travelled on microtubules Movie of live FDB fibers electroporated with GLUT4-GFP (green) and mCherry-Tubulin (magenta) 6 days earlier. The movie is played in 10 frames per second with one frame representing 1 second. White arrows mark areas for frequent GLUT4-travelling. Scale bar = 5 μm. Figure 1 – figure supplement 3: GLUT4 travelled to and from GLUT4-enriched regions at microtubule nucleation sites. Movie of GLUT4-GFP at GLUT4 enriched/large structure in live FDB fibers showing spherical and tubular GLUT4-GFP structures that fuse and bud off from the GLUT4-enriched area. To facilitate visualization, the movie was generated so moving GLUT4 appears green-red flashing, whereas static GLUT4 appears yellow as described in the methods section. The movie is played in 10 frames per second with one frame representing 4 seconds. Scale bar = 5 μm.

Article Snippet: The following antibodies raised in rabbit: GLUT4 (PA5-23052, Invitrogen), Detyrosinated α-tubulin (AB48389, Abcam), rabbit Syntaxin6 (110 062, Synaptic Systems), or in mouse: GLUT4 (MAB8654, R&D Systems), α-tubulin (T9026, Merck).

Techniques: Imaging, Muscles, Expressing, Microscopy, Fluorescence, Generated

Journal: bioRxiv

Article Title: Microtubule-mediated GLUT4 trafficking is disrupted in insulin resistant skeletal muscle

doi: 10.1101/2022.09.19.508621

Figure Lengend Snippet: Phosphorylation of Akt Thr308 in GLUT4-GFP expressing flexor digitorum brevis (FDB) fibers treated ± 30 nM insulin (INS) for 15 minutes. B) Polymerized microtubules in GLUT4-GFP expressing fibers treated ± Nocodazole (Noco, 13 µM) for 4 hours and stained with α-Tubulin. C) Fibers as in B stained for detyrosinated tubulin (De-Tubulin). D) Microtubule-based GLUT4-GFP trafficking quantified as the travelled distance and the lateral displacement of particles within the dynamic fraction in muscle fibers treated ± Noco (13 µM) for 4 hours and ± INS (30 nM) for 15-30 minutes. E) Identification of GLUT4-containing structures in GLUT4-GFP expressing FDB fibers. Black/white arrows mark large (>4 µm 2 ) structures indicative of GLUT4 clusters at the microtubule nucleation sites. Red arrow marks a structure sized between 0.4 and 4 µm 2 and categorized as a medium size structure. Red arrowhead marks a structure of <0.4 µm 2 ) which is categorized as a small structure. F) Bar graphs showing the relative prevalence and area in the different membrane size categories in fibers ± microtubule network disruption by addition of Noco (13 µM) 15 hours prior to imaging or 9 hours after Noco removal. G) Total number and area of vesicles in fibers as in F. H) GLUT4-EOS and Syntaxin6 (Stx6) in mouse FDB fibers. For A, n=3 mice, for B-C n ≥ 8 muscle fibers from 3 different mice. For D, n ≥ 14 muscle fibers from 5 different mice. For F-G, n = 9-11 muscle fibers from 3 different mice. For H, n = 6-7 fibers from 2 different mice. Data are presented as mean with individual data points. */*** p<0.05/0.001 different from Basal/DMSO. ##/### p<0.01/0.001 different from INS. ¤¤/¤¤¤ p<0.01/0.001 ANOVA effect Figure 2 – figure supplement 2: GLUT4 movement was disrupted by nocodazole treatment Movie of live FDB fibers electroporated with GLUT4-GFP 6 days earlier. To facilitate visualization, the movie was generated so moving GLUT4 appears green-red flashing while static GLUT4 appears yellow, as described in methods section. The left panel shows fiber in the basal state, the middle panel shows fiber treated with insulin for 15 min and the right panel shows fiber treated with insulin for 15 min after a prior treatment with 4 µg/ml nocodazole for four hours. The movie is played in 10 frames per second with one frame representing 4 seconds. Scale bar = 5 μm. Figure 2 – source data 1: Data used for quantification of GLUT4 trafficking and localization Figure 2 – figure supplement 1 - source data 1: Data used for quantification of GLUT4 trafficking and localization

Article Snippet: The following antibodies raised in rabbit: GLUT4 (PA5-23052, Invitrogen), Detyrosinated α-tubulin (AB48389, Abcam), rabbit Syntaxin6 (110 062, Synaptic Systems), or in mouse: GLUT4 (MAB8654, R&D Systems), α-tubulin (T9026, Merck).

Techniques: Phospho-proteomics, Expressing, Staining, Membrane, Disruption, Imaging, Generated

Journal: bioRxiv

Article Title: Microtubule-mediated GLUT4 trafficking is disrupted in insulin resistant skeletal muscle

doi: 10.1101/2022.09.19.508621

Figure Lengend Snippet: A) Representative time-lapse traces of GLUT4-GFP vesicle tracking in muscle fibers ± insulin (INS, 30 nM) for 15-30 minutes with or without microtubule network disruption by Nocodazole (Noco, 13 µM) for 4 hours prior to insulin. The dynamics of GLUT4-GFP in the different conditions are also exemplified in movie 3. B) Quantified microtubule-based GLUT4 trafficking. C) Quantification of microtubule-based GLUT4 trafficking in basal and insulin-stimulated fibers pooled from 4 independent experiments. D) Representative images of muscle fibers ± pre-treatment with Noco (13 µM, for 15 hours, followed by recovery in Noco-free medium for 9 hours. E) Quantification of GLUT4 distribution between the microtubule nucleation sites (structures sized > 4 µm2), intermediate sized structures (0.4-4 µm2) and the smallest resolvable structures (<0.4 µm2) in fibers treated as in D. Compartment identification is described in figure S2 E. F) GLUT4 and Syntaxin6 (Stx6) in muscle fiber from human vastus lateralis muscle. G) GLUT4-Stx6 overlap in mouse flexor digitorum brevis muscle fibers in DMSO medium with and without Noco (13 µM) treatment. For A-B, n ≥ 14 muscle fibers from 5 different mice. For D-E, n = 9-11 muscle fibers from 3 different mice. For F, n= 3 subjects. Data are presented as mean with individual data points. *** p<0.001 different from basal, ### p<0.001 different from INS, ## p<0.01 different from Noco recovery. ¤¤¤ p<0.001 ANOVA effect Scale bar = 5 µm (A-D) and 2 µm (F).

Article Snippet: The following antibodies raised in rabbit: GLUT4 (PA5-23052, Invitrogen), Detyrosinated α-tubulin (AB48389, Abcam), rabbit Syntaxin6 (110 062, Synaptic Systems), or in mouse: GLUT4 (MAB8654, R&D Systems), α-tubulin (T9026, Merck).

Techniques: Disruption

Journal: bioRxiv

Article Title: Microtubule-mediated GLUT4 trafficking is disrupted in insulin resistant skeletal muscle

doi: 10.1101/2022.09.19.508621

Figure Lengend Snippet: A) 2-Deoxyglucose (2-DG) transport in basal and insulin-stimulated mouse soleus and extensor digitorum longus (EDL) muscles pretreated with Nocodazole (Noco, 13 µM) for the indicated time. B) Insulin-stimulated 2-DG transport (insulin minus basal) from muscles shown in A. C) Experimental setup for muscle on a chip system with glucose sensor. D) Microtubules imaged with α-tubulin in GLUT4-GFP expressing mouse flexor digitorum brevis (FDB) fibers treated ± Noco (13 µM) for 5 min or 2h. E) 180 sec. measurements of glucose concentration in perifusate from basal and insulin-treated FDB muscle fibers in muscle chips pre-incubated with DMSO, Noco (13 µM, 5min or 2h) or colchicine (25 µM, 2h). F) Insulin-stimulated glucose uptake into FDB muscle fibers in muscle chips calculated from the last 20 seconds of the concentration curves in E. G) Representative GLUT4 images from isolated mouse FDB muscle fibers treated ± Noco (13 µM) for 5 min. or 2h. H) Quantification of GLUT4 in large, intermediate and small sized membrane structures in FDB fibers treated ± Noco (13 µM) for 5 min. or 2h. The membrane compartment division by size is shown in figure S2 E. For A-B n = 6-7 muscles from 6-7 mice. For D and G-H, n =8-10 muscle fibers from 3 different mice. For E-F, n ≥ 3 muscle chips from 3-4 mice. Data are presented as mean with individual data points. Paired observations from the same mouse are indicated by a connecting line. */**/*** p<0.05/0.01/0.001 different from basal/DMSO, #/## p<0.05/0.01 different from DMSO. ¤/¤¤/¤¤¤ p<0.05/0.01/0.001 ANOVA effect. Scale bar = 5 µm.

Article Snippet: The following antibodies raised in rabbit: GLUT4 (PA5-23052, Invitrogen), Detyrosinated α-tubulin (AB48389, Abcam), rabbit Syntaxin6 (110 062, Synaptic Systems), or in mouse: GLUT4 (MAB8654, R&D Systems), α-tubulin (T9026, Merck).

Techniques: Muscles, Expressing, Concentration Assay, Incubation, Isolation, Membrane

Journal: bioRxiv

Article Title: Microtubule-mediated GLUT4 trafficking is disrupted in insulin resistant skeletal muscle

doi: 10.1101/2022.09.19.508621

Figure Lengend Snippet: A-B) Quantification of protein expression in soleus (A) and EDL (B) muscles in the basal and insulin-stimulated state ± Nocodazole (Noco, 13 µM) as indicated. C) Representative immunoblots of the proteins quantified in A-B. D) Deposition of poly-(m-phenylenediamine) (m-PD) film as exclusion membrane to improve sensor selectivity demonstrated by abolished anodic signal in the voltammograms (scanning rate (SR)= 100 mV s -1 ) performed at the clean Pt surface in 1 mM FcMeOH after m-PD deposition. E) Glucose oxidase presence validation by current (nA) change upon sensor transfer to and from a glucose solution (5mM). F) Current change and calculated area over the curve (AOC) in perifusate from mouse flexor digitorum brevis (FDB) fibers before and after insulin stimulation. G) Typical calibration trace (Q = 1 μl s -1 ) when exposing glucose sensor to stepwise 1.1 mM glucose increments. Sensitivity of the sensing was calculated as 3*SD / Δ current * Δ glucose. SD was calculated from 10 baseline data points, Δ current was calculated when switching from 0 to 1.1 mM glucose as indicated by the red bracket. H) Associated calibration curve, fit with a Michaelis–Menten model. I) Current fluctuations in perifusate from FDB fibers ± Noco (13 µM). ME = main effect. For A-C n = 6-7 muscles from 6-7 mice. Data are presented as mean with individual data points. Paired observations from the same mouse are indicated by a connecting line. F and I, graphs indicate mean ± standard deviation. */*** p<0.05/0.001 different from DMSO. ¤/¤¤¤ p<0.05/0.001 ANOVA effect. Figure 3 – source data 1: Data used for quantification of glucose uptake, polymerized microtubules and GLUT4 localization in figure 3 Figure 3 – figure supplement 1 - source data 1: Data used for quantification of protein expression and electrochemical glucose sensing in figure 3 – figure supplement 1

Article Snippet: The following antibodies raised in rabbit: GLUT4 (PA5-23052, Invitrogen), Detyrosinated α-tubulin (AB48389, Abcam), rabbit Syntaxin6 (110 062, Synaptic Systems), or in mouse: GLUT4 (MAB8654, R&D Systems), α-tubulin (T9026, Merck).

Techniques: Expressing, Muscles, Western Blot, Membrane, Biomarker Discovery, Standard Deviation

Journal: bioRxiv

Article Title: Microtubule-mediated GLUT4 trafficking is disrupted in insulin resistant skeletal muscle

doi: 10.1101/2022.09.19.508621

Figure Lengend Snippet: A) Schematic overview of L6 muscle cell system to assess GLUT4 surface content. B) Imaging (left) of α-tubulin (magenta) and GLUT4 (cyan) and quantification (right) of perinuclear GLUT4 in L6 myoblasts ± kinesore treatment (2h, 50 µM). C) Imaging (left) of GLUT4 and quantification (right) of GLUT4 at the microtubule nucleation sites in mouse flexor digitorum brevis (FDB) muscle fibers treated ± kinesore (2h). D-E) Exofacial GLUT4 signal at the surface membrane in L6 myoblast (D) or 7 day differentiated myotubes (E) serum starved for 4h and treated ± kinesore (50 µM) for the last 2h before 15 min ± insulin (100 nM). ANOVA main effect of insulin (¤¤¤) and Kinesore (¤¤¤) and interaction (¤¤). F) Exofacial GLUT4 signal in serum starved (4h) basal and insulin-stimulated (100 nM, 15 min) L6 myoblasts (left) and insulin-response (insulin minus basal, right) in GLUT4 surface content. L6 myoblasts were transfected with short hairpin scramble RNA (shScramble) or shRNA targeting KIF5B 72h prior to the experiment. ANOVA main effect of insulin (¤¤¤) and shKIF5B (¤¤¤) and interaction (¤). B, n = 6-7 individual samples pooled from 2 independent experiments. C, n=23-24 muscle fibers from 2 different mice. D-F, each data point represents the average of 3 replicates and originate from at least 3 independent experiments. Data are presented as mean with individual data points. **/*** p<0.01/0.001 effect of insulin. ##/### p<0.01/0.001 different from DMSO/Scramble. ¤/¤¤/¤¤¤ p<0.05/0.01/0.001 ANOVA effect. Scale bar = 5 µm.

Article Snippet: The following antibodies raised in rabbit: GLUT4 (PA5-23052, Invitrogen), Detyrosinated α-tubulin (AB48389, Abcam), rabbit Syntaxin6 (110 062, Synaptic Systems), or in mouse: GLUT4 (MAB8654, R&D Systems), α-tubulin (T9026, Merck).

Techniques: Imaging, Membrane, Transfection, shRNA

Journal: bioRxiv

Article Title: Microtubule-mediated GLUT4 trafficking is disrupted in insulin resistant skeletal muscle

doi: 10.1101/2022.09.19.508621

Figure Lengend Snippet: A) Exofacial GLUT4 signal at the surface membrane of L6 myoblast serum starved for 4h and treated ± kinesore at indicated concentration for the last 2h before cell fixation. ANOVA effect (¤¤¤). B) Quantification and representative immunoblots showing protein expression and phosphorylation in basal and insulin stimulated (100 nM, 15 min) L6 myoblasts serum starved for 4h and treated ± kinesore for 2h. ANOVA main effect of insulin (Akt Thr308+Ser473 = ¤¤¤, ACC Ser222 = ¤), ANOVA main effect of Kinesore (ACC Ser222 = ¤, Akt Ser473+AMPK Thr172 p=0.07), interaction (Akt Thr308 = ¤¤, Akt Ser473 p=0.06). C) 2-deoxyglucose (2-DG) transport in L6 myoblast serum starved for 4h and treated ± kinesore (50 µM) for 2h before 20 min ± insulin (100 nM) and 5 min 2-DG accumulation. ANOVA main effect of insulin (¤¤¤) and Kinesore (¤¤¤) and interaction (¤¤). D) Quantification of KIF5B protein expression in L6 myoblasts transfected with shScramble RNA or shRNA targeting Kif5b for 72h. E) Representative immunoblots showing protein expression in L6 myoblasts as in D. A, n = 6 replicates from 2 independent experiments. B, n = 3 independent experiment. C, n = 5-6 replicates from 2 independent experiments. D-E, n = 6 replicates from 2 independent experiments. *** p<0.001 effect of insulin/shKif5b. #/##/### p<0.05/0.01/0.001 different from DMSO. ¤/¤¤/¤¤¤ p<0.05/0.01/0.001 ANOVA effect. Figure 4 – source data 1: Data used for quantification of GLUT4 localization and GLUT4 surface content in figure 4 Figure 4 – figure supplement 1 - source data 1: Data used for quantification of GLUT4 surface content, protein expression and glucose uptake in figure 4 – figure supplement 1

Article Snippet: The following antibodies raised in rabbit: GLUT4 (PA5-23052, Invitrogen), Detyrosinated α-tubulin (AB48389, Abcam), rabbit Syntaxin6 (110 062, Synaptic Systems), or in mouse: GLUT4 (MAB8654, R&D Systems), α-tubulin (T9026, Merck).

Techniques: Membrane, Concentration Assay, Western Blot, Expressing, Phospho-proteomics, Transfection, shRNA

Journal: bioRxiv

Article Title: Microtubule-mediated GLUT4 trafficking is disrupted in insulin resistant skeletal muscle

doi: 10.1101/2022.09.19.508621

Figure Lengend Snippet: A) Overview of in vitro and in vivo insulin resistance models used. B) Quantified microtubule-based GLUT4 trafficking in basal, insulin (INS, 30 nM) and insulin + C2 Ceramide (C2) (INS+C2, 30 nM+50 µM) treated flexor digitorum brevis (FDB) muscle fibers. C) Quantified microtubule-based GLUT4 trafficking in basal or INS (30 nM) treated FDB fibers from chow or high fat diet (HFD) fed mice. D) Representative images of polymerizing microtubules in EB3-GFP expressing FDB muscle fibers treated ± C2 (50 µM), paclitaxel (Taxol, 10 µM) for 2 hours prior to 15-30 minutes of INS (30 nM) stimulation. Red circles highlight microtubule tip-bound EB3-GFP. E) Quantification of polymerizing microtubules based on EB3-GFP in FDB fibers treated as in D. F) Quantification of polymerizing microtubules based on EB3-GFP in FDB fibers isolated from chow or 60% HFD fed mice and treated ± INS (30 nM) for 15-30 minutes. For B-F, n ≥ 13 muscle fibers from 3-4 mice. Taxol-treated muscle fibers were only used as a control and not included in the statistical analysis. NA = not statistically analysed. Data are presented as mean with individual data points. #/##/### p<0.05/0.01/0.001 different from INS (B) or different from corresponding group in chow fed mice (C) or control fibers (E). ¤/¤¤/¤¤¤ p<0.05/0.01/0.001 main effect (ME) of diet/C2.

Article Snippet: The following antibodies raised in rabbit: GLUT4 (PA5-23052, Invitrogen), Detyrosinated α-tubulin (AB48389, Abcam), rabbit Syntaxin6 (110 062, Synaptic Systems), or in mouse: GLUT4 (MAB8654, R&D Systems), α-tubulin (T9026, Merck).

Techniques: In Vitro, In Vivo, Expressing, Isolation, Control

Journal: bioRxiv

Article Title: Microtubule-mediated GLUT4 trafficking is disrupted in insulin resistant skeletal muscle

doi: 10.1101/2022.09.19.508621

Figure Lengend Snippet: A) Phosphorylation of Akt Thr308 in GLUT4-GFP expressing flexor digitorum brevis (FDB) muscle fibers cultured ± 0.5 mM palmitic acid (PA) for 24 hours or 50 µM C2 ceramide (C2) for 2 hours prior to 15 minutes ± 30 nM insulin (INS) stimulation. B) Quantified microtubule-based GLUT4 trafficking assessed as the travelled distance and the displacement within the dynamic fraction of the GLUT4-GFP in basal, insulin (INS, 30 nM) or insulin and C2 Ceramide (INS+C2, 30 nM+50 µM)-treated FDB muscle fibers. C-D) Glucose and insulin tolerance tests of mice fed a control chow or a high fat diet (HFD) after intraperitoneal injection of glucose (2 g*kg -1 body weight) or insulin (0.5 U*kg -1 body weight). AUC/AOC = area under/over the curve. ANOVA main effect of diet (¤¤¤/¤, GTT/ITT), and time (¤¤¤) and interaction (¤¤¤/¤¤, GTT/ITT). E) Phosphorylation of Akt Thr308 and TBC1D4 Thr642 in isolated FDB fibers from muscles from chow or HFD fed mice treated ± INS (30 nM) for 15 min. F) Quantified microtubule-based GLUT4 trafficking assessed as the travelled distance and the displacement within the dynamic fraction of the GLUT4-GFP in basal or INS(30 nM)-stimulated FDB muscle fibers isolated from chow or HFD-fed mice. G) Color coded projection ((first image cyan, last image red as indicated by color code bar) of 60 sec. live-imaging of FDB fibers expressing EB3-GFP and treated without (CTRL) or with 10 µM paclitaxel (Taxol) for 2 hours. Scale bar = 5 µm. EB3-GFP dynamics are also illustrated in movie 4. H) Quantification of the total and average polymerizing distance of EB3-GFP containing microtubules in fibers incubated ± 50 µM C2 ceramide (C2) or 10 µM Taxol for 2 hours prior to 15-30 minutes of INS (30 nM) stimulation. I) Quantification of the total and average polymerizing distance of EB3-GFP containing microtubules in fibers isolated from chow or HFD fed mice and stimulated ± INS (30 nM) for 15 minutes. J) Quantification of microtubule polymerization directionality based on EB3-GFP dynamics recording as in I. For A, C-E n=3-4 mice, for B, F and H-J n ≥ 13 muscle fibers from 3-4 mice. G) representative of >10 fibers from 2 different mice. Taxol treated muscle fibers were only used as a reference and not included in the statistical analysis. NA = not analysed. Data are presented as mean with individual data points or mean±SD. **/*** p<0.01/0.001 different from basal, #/##/### p<0.05/0.01/0.001 different from insulin (A-B) or different from corresponding group in chow fed mice (C-F, H). ¤/¤¤/¤¤¤ p<0.05/0.01/0.001 ANOVA main effect (ME). Figure 5 – figure supplement 2: Polymerizing microtubules in muscle fibers Movie of polymerizing microtubule (MT) tips detected by EB3-GFP in live FDB fibers electroporated with EB3-GFP 6 days earlier. To facilitate visualization, the movie was generated so moving MT tips (EB3 dots) appear green-red flashing whereas static EB3 appear yellow, as described in methods section. The movie is played in 10 frames per second with one frame representing 4 seconds. Scale bar = 5 μm. Figure 5 – source data 1: Data used for quantification of GLUT4 trafficking and microtubule polymerization in figure 5 Figure 5 – figure supplement 1 - source data 1: Data used for quantification of protein expression, GLUT4 trafficking, glucose and insulin tolerance, microtubule polymerization and polymerization directionality in figure 5 – figure supplement 1

Article Snippet: The following antibodies raised in rabbit: GLUT4 (PA5-23052, Invitrogen), Detyrosinated α-tubulin (AB48389, Abcam), rabbit Syntaxin6 (110 062, Synaptic Systems), or in mouse: GLUT4 (MAB8654, R&D Systems), α-tubulin (T9026, Merck).

Techniques: Phospho-proteomics, Expressing, Cell Culture, Control, Injection, Isolation, Muscles, Imaging, Incubation, Generated

Journal: Frontiers in Aging Neuroscience

Article Title: α-Lipoic Acid Maintains Brain Glucose Metabolism via BDNF/TrkB/HIF-1α Signaling Pathway in P301S Mice

doi: 10.3389/fnagi.2020.00262

Figure Lengend Snippet: α-Lipoic acid (LA) improved glucose metabolism deficiency in P301S mice. (A) Representative western blots showed the expression levels of glucose transporter 1 (GLUT1), GLUT3, GLUT4, hexokinase-1 (HK-1), and HK2. (B–F) Quantified results of GLUT1, GLUT3, GLUT4, HK1, and HK2 levels between wild type (WT) and P301S mice. β-actin served as an internal loading control. (G) HK activity between WT and P301S mice. (H–L) Quantified results of the levels of GLUT1, GLUT3, GLUT4, HK-1, and HK2 among vehicle, LA 3 mg/kg, and 10 mg/kg groups. β-actin served as an internal loading control. (M) HK activity among vehicles, LA 3 mg/kg, and 10 mg/kg groups. (N–Q) Representative western blots and quantified results of the levels of heme oxygenase-1 (HO-1), vascular endothelial growth factor (VEGF), and proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). β-actin served as an internal loading control. (R–U) mRNA level of GLUT1, GLUT3, GLUT4, and VEGF. Values are represented as the means ± SEM ( n = 7). * p < 0.05, ** p < 0.01.

Article Snippet: Membranes were blocked with 5% skim milk and incubated overnight at 4°C with the follwing primary antibodies: rabbit anti-HIF-1α (1:2,000; Thermo Fisher Scientific, PA316521, USA), rabbit anti-BDNF (1:1,000; Santa Cruz Biotechnology, sc-546, USA), rabbit anti-p-TrkA/B (1:2,000; Thermo Fisher Scientific, MA5-14926, USA), rabbit anti-TrkB (1:2,000; Cell Signaling Technology, 4638T, USA), rabbit anti-GLUT1 (1:2,000, BIOSS, bs-20173R, China), mouse anti-GLUT3 (1:1,000; Santa Cruz Biotechnology, sc-74497, USA), rabbit anti-GLUT4 (1:2,000, BIOSS, bs-0384R, China), rabbit anti-hexokinase II (1:2,000; Cell Signaling Technology, C35C4, USA), rabbit anti-hexokinase II (1:2,000; Cell Signaling Technology, C64G5, USA), rabbit anti-HO-1 (1:2,000; Thermo Fisher Scientific, PA5-27338, USA), rabbit anti-VEGF (1:2,000; Cell Signaling Technology, 2463S, USA), rabbit anti-PGC-1α (1:1,000; Cell Signaling Technology, 2178S, USA), mouse anti-MUTYH (1:1,000; Santa Cruz Biotechnology, sc-374571, USA), mouse anti-OGG1/2 (1:1,000; Santa Cruz Biotechnology, sc-376935, USA), rabbit anti-MTH1 (1:1,000; Santa Cruz Biotechnology, sc-67291, USA), mouse anti-β-actin (1:10,000; Sigma–Aldrich, A1978, USA).

Techniques: Western Blot, Expressing, Activity Assay

Journal: Frontiers in Aging Neuroscience

Article Title: α-Lipoic Acid Maintains Brain Glucose Metabolism via BDNF/TrkB/HIF-1α Signaling Pathway in P301S Mice

doi: 10.3389/fnagi.2020.00262

Figure Lengend Snippet: Schematic model of the possible mechanisms of BDNF/TrkB/HIF-1α involvement in maintaining brain glucose metabolism in P301S mice. Chronic LA treatment induces increased BDNF expression level in neurons and astrocytes in P301S mice brain, then BDNF binding to TrkB-FL induces the activation of TrkB-FL (autophosphorylation of tyrosine sites), BDNF and p-TrkB-FL complex translocate from the cellular membrane through forming endosomes. The complex upregulates HIF-1α protein level and contributes to HIF-1α nucleus translocation, inducing the downstream target genes expression, such as GLUT3, GLUT4, and HO-1, VEGF. The increased glucose transporters and vascular endothelium reinstate glucose metabolism in P301S mice. Also, LA administration upregulates PGC-1α expression and promotes the stabilization of HIF-1α protein level, couple with HIF-1α to induce the downstream target genes.

Article Snippet: Membranes were blocked with 5% skim milk and incubated overnight at 4°C with the follwing primary antibodies: rabbit anti-HIF-1α (1:2,000; Thermo Fisher Scientific, PA316521, USA), rabbit anti-BDNF (1:1,000; Santa Cruz Biotechnology, sc-546, USA), rabbit anti-p-TrkA/B (1:2,000; Thermo Fisher Scientific, MA5-14926, USA), rabbit anti-TrkB (1:2,000; Cell Signaling Technology, 4638T, USA), rabbit anti-GLUT1 (1:2,000, BIOSS, bs-20173R, China), mouse anti-GLUT3 (1:1,000; Santa Cruz Biotechnology, sc-74497, USA), rabbit anti-GLUT4 (1:2,000, BIOSS, bs-0384R, China), rabbit anti-hexokinase II (1:2,000; Cell Signaling Technology, C35C4, USA), rabbit anti-hexokinase II (1:2,000; Cell Signaling Technology, C64G5, USA), rabbit anti-HO-1 (1:2,000; Thermo Fisher Scientific, PA5-27338, USA), rabbit anti-VEGF (1:2,000; Cell Signaling Technology, 2463S, USA), rabbit anti-PGC-1α (1:1,000; Cell Signaling Technology, 2178S, USA), mouse anti-MUTYH (1:1,000; Santa Cruz Biotechnology, sc-374571, USA), mouse anti-OGG1/2 (1:1,000; Santa Cruz Biotechnology, sc-376935, USA), rabbit anti-MTH1 (1:1,000; Santa Cruz Biotechnology, sc-67291, USA), mouse anti-β-actin (1:10,000; Sigma–Aldrich, A1978, USA).

Techniques: Expressing, Binding Assay, Activation Assay, Translocation Assay

Journal: Frontiers in Physiology

Article Title: Glial Growth Factor 2 Regulates Glucose Transport in Healthy Cardiac Myocytes and During Myocardial Infarction via an Akt-Dependent Pathway

doi: 10.3389/fphys.2019.00189

Figure Lengend Snippet: Acute treatment with GGF2 stimulates the regulation of glucose transport via the ErbB receptors in healthy adult cardiac myocytes. (A) GGF2 treatment stimulates GLUT4 trafficking to the cell surface. Top panel: representative Western blot. Bottom Panel: Mean ± SE of cell surface GLUT4 protein content; values normalized to basal ( n = 3-8/group); # P < 0.05 vs. basal. Methods: Photolabeled biotinylated assay in isolated rat ventricular myocytes incubated without (i.e., basal, −) or with insulin (+) or incremental dose of GGF2 (i.e., 1, 10, and 100 ng/ml). L, Labeled (cell surface fraction); UL, unlabeled (intracellular fraction). (B) ErbB receptor blockade (afatinib) blunts GGF2-stimulated GLUT4 translocation. Top panel: representative Western blot. Bottom Panel: Mean ± SE of cell surface GLUT4 protein content; values normalized to basal ( n = 3–8/group); # P < 0.05 vs. basal; ∗ P < 0.05 vs. GGF2. Methods: Photolabeled biotinylated assay in isolated rat ventricular myocytes incubated without (i.e., basal, −) or with (+) afatinib for 1 h prior to incubation with insulin or GGF2 (100 ng/ml) for 1 h. (C) GGF2 increases total GLUT4 expression. Top panel: representative Western blot. Bottom panel: Mean ± SE of protein expression (values expressed relative to basal; n = 11–15/group); # P < 0.05 vs. basal. Methods: Western blotting from total lysate of isolated rat ventricular myocytes incubated without (i.e., basal) or with insulin or incremental dose of GGF2. Calsequestrin was used as a loading control. RU, relative units.

Article Snippet: After blocking (1–5% non-fat dry milk or 2% goat serum), membranes were incubated with optimally diluted primary antibodies overnight (polyclonal rabbit anti-human GLUT4, 1:750, AbD Serotec 4670–1704; monoclonal rabbit anti-mouse total Akt, 1:1000, Cell Signaling 4061; monoclonal rabbit anti-human phosphorylated Akt s473, 1:1000, Cell Signaling 4060; monoclonal rabbit anti-mouse phosphorylated Akt Th308, 1:1000, Cell Signaling 2965; monoclonal rabbit anti-human total AS160, 1:1000, Cell Signaling 2670 and polyclonal rabbit anti-human phosphorylated AS160, 1:1000, Cell Signaling 9611; polyclonal rabbit anti-human PDK-1, 1:1000, Cell Signaling 3062; Monoclonal rabbit anti-human phosphorylated PDK-1 S241, 1:1000, Cell Signaling 3438; Monoclonal Rabbit IgG anti-human PKCζ Th410, 1:500, Cell Signaling 2060; polyclonal rabbit anti-human PKCζ, 1:1000, Cell Signaling 9372); washed for 10 min with TPBS (twice), 5 min with PBS, followed by a 1 h incubation of appropriate secondary antibodies conjugated to horseradish peroxidase.

Techniques: Western Blot, Isolation, Incubation, Labeling, Translocation Assay, Expressing, Control

Journal: Frontiers in Physiology

Article Title: Glial Growth Factor 2 Regulates Glucose Transport in Healthy Cardiac Myocytes and During Myocardial Infarction via an Akt-Dependent Pathway

doi: 10.3389/fphys.2019.00189

Figure Lengend Snippet: GGF2 treatment partially rescues impaired GLUT trafficking via an AS160 dependent pathway during MI. (A) Top panels: representative Western blot. Bottom Panels: Mean ± SE of cell surface GLUT4 protein content in myocytes from MI and age-matched control rats; values normalized to control basal ( n = 2–4/group); # P < 0.05 vs. control basal, ∗ P < 0.05 vs. MI Basal, † P < 0.05 vs. same treatment in Controls. Methods: Photolabeled biotinylated assay in isolated rat ventricular myocytes incubated without (i.e., basal, −) or with (+) insulin or GGF2 (100 ng/ml). L, labeled (cell surface fraction); UL, unlabeled (intracellular fraction); Con, control. (B) GLUT4 trafficking to the cell surface significantly correlates with AS160 activation in myocytes from healthy and MI rats following incubation with insulin or GGF2. Scatterplot and linear regression of myocardial cell surface GLUT4 content (dependent variable) and AS160 phosphorylation (independent variable) in myocytes of control and MI rats ( n = 2–4/group) under basal conditions or after in vitro insulin or GGF2 treatment; P < 0.0001; R 2 = 0.5482; Y = 0.6401 X + 0.5165. RU, relative units.

Article Snippet: After blocking (1–5% non-fat dry milk or 2% goat serum), membranes were incubated with optimally diluted primary antibodies overnight (polyclonal rabbit anti-human GLUT4, 1:750, AbD Serotec 4670–1704; monoclonal rabbit anti-mouse total Akt, 1:1000, Cell Signaling 4061; monoclonal rabbit anti-human phosphorylated Akt s473, 1:1000, Cell Signaling 4060; monoclonal rabbit anti-mouse phosphorylated Akt Th308, 1:1000, Cell Signaling 2965; monoclonal rabbit anti-human total AS160, 1:1000, Cell Signaling 2670 and polyclonal rabbit anti-human phosphorylated AS160, 1:1000, Cell Signaling 9611; polyclonal rabbit anti-human PDK-1, 1:1000, Cell Signaling 3062; Monoclonal rabbit anti-human phosphorylated PDK-1 S241, 1:1000, Cell Signaling 3438; Monoclonal Rabbit IgG anti-human PKCζ Th410, 1:500, Cell Signaling 2060; polyclonal rabbit anti-human PKCζ, 1:1000, Cell Signaling 9372); washed for 10 min with TPBS (twice), 5 min with PBS, followed by a 1 h incubation of appropriate secondary antibodies conjugated to horseradish peroxidase.

Techniques: Western Blot, Control, Isolation, Incubation, Labeling, Activation Assay, Phospho-proteomics, In Vitro