Journal: Journal of Biological Chemistry

Article Title: Insulin Regulates the Membrane Arrival, Fusion, and C-terminal Unmasking of Glucose Transporter-4 via Distinct Phosphoinositides

doi: 10.1074/jbc.m500501200

Figure Lengend Snippet: FIG. 1. Distinction of immunofluorescent signals of endoge- nous GLUT4 and GLUT4myc C termini through different detec- tor gains. Wild-type L6 myotubes or L6-GLUT4myc myotubes were grown on coverslips were starved of serum 4 h, then treated without or with 100 nM insulin for 10 min. Plasma membrane lawns were prepared and labeled with monoclonal anti-myc or polyclonal anti-C-terminal GLUT4 antibody, followed by incubation with goat anti-mouse A488, goat anti-rabbit Cy3 antibody, respectively, as described under “Exper- imental Procedures.” Immunolabeled lawns were processed and ana- lyzed by confocal fluorescence microscopy. Signals were acquired at low and high detector gain levels. At the detector gain used to acquire the C terminus signal of L6-GLUT4myc myotubes (low gain), there was no detectable signal of the C terminus of wild-type GLUT4 myotubes. The endogenous GLUT4 C terminus of wild-type myotubes was only detect- able at the high gain. The results illustrate that the C-terminal signal of the endogenous GLUT4 cannot be detected under the conditions used to acquire the C-terminal signal from the transfected GLUT4myc. Bar, 10 m

Article Snippet: Monoclonal (9E10) and polyclonal (A-14) anti-c-myc antibodies, polyclonal (H-61) antibody against the intracellular loop region (amino acids 230–290) of GLUT4, and polyclonal (N-20) antibody sc1606 against the first exofacial loop region of GLUT4 were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Clinical Proteomics, Membrane, Labeling, Incubation, Immunolabeling, Fluorescence, Microscopy, Transfection

Journal: Journal of Biological Chemistry

Article Title: Insulin Regulates the Membrane Arrival, Fusion, and C-terminal Unmasking of Glucose Transporter-4 via Distinct Phosphoinositides

doi: 10.1074/jbc.m500501200

Figure Lengend Snippet: FIG. 2. Wortmannin and LY294002 arrest GLUT4myc vesicle fusion with the membrane. L6-GLUT4myc myotubes were treated with vehicle (Me2SO) or 100 nM wortmannin (Wm) (A) or 25 M LY 294002 (B) for 20 min as indicated followed by incubation without (basal) or with 100 nM insulin for 10 min. Plasma membrane lawns were prepared and processed for detection of myc epitope, C-terminal GLUT4, or caveolin by confocal fluorescence microscopy. The fluores- cence intensity was quantified from at least 50 plasma membrane lawn pieces for each condition per experiment as described under “Experi- mental Procedures” and is illustrated as the mean -fold change per lawn over the basal ( S.E.) obtained from at least five independent exper- iments. *, p 0.005 and #, p 0.05 relative to the corresponding basal values (ANOVA).

Article Snippet: Monoclonal (9E10) and polyclonal (A-14) anti-c-myc antibodies, polyclonal (H-61) antibody against the intracellular loop region (amino acids 230–290) of GLUT4, and polyclonal (N-20) antibody sc1606 against the first exofacial loop region of GLUT4 were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Membrane, Incubation, Clinical Proteomics, Fluorescence, Microscopy

Journal: Journal of Biological Chemistry

Article Title: Insulin Regulates the Membrane Arrival, Fusion, and C-terminal Unmasking of Glucose Transporter-4 via Distinct Phosphoinositides

doi: 10.1074/jbc.m500501200

Figure Lengend Snippet: FIG. 4. Immunoelectron microscopy reveals GLUT4 vesicles on plasma membrane lawns from L6-myotubes treated with wortmannin prior to insulin stimulation. Myotubes are treated with vehicle (Me2SO), 100 nM wortmannin for 30 min, 100 nM insulin for 10 min, or 100 nM wortmannin for 20 min prior to 100 nM insulin for 10 min. Plasma membrane lawns were generated, labeled with polyclonal anti-C terminus antibody followed by goat polyclonal anti-rabbit anti- body conjugated with 6 nm-gold particles, and processed for electron microscopy as described under “Experimental Procedures.” Gold parti- cles in non-vesicular areas are indicated by arrows. Bar, 100 nm.

Article Snippet: Monoclonal (9E10) and polyclonal (A-14) anti-c-myc antibodies, polyclonal (H-61) antibody against the intracellular loop region (amino acids 230–290) of GLUT4, and polyclonal (N-20) antibody sc1606 against the first exofacial loop region of GLUT4 were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Immuno-Electron Microscopy, Clinical Proteomics, Membrane, Generated, Labeling, Electron Microscopy

Journal: Journal of Biological Chemistry

Article Title: Insulin Regulates the Membrane Arrival, Fusion, and C-terminal Unmasking of Glucose Transporter-4 via Distinct Phosphoinositides

doi: 10.1074/jbc.m500501200

Figure Lengend Snippet: FIG. 5. High salt treatment of plasma membrane lawns in- creases GLUT4 C terminus antigenicity in the basal state. L6- GLUT4myc myotubes were incubated without or with insulin (100 nM for 10 min), as indicated. Membrane lawns were generated then incubated with PBS without or with an additional 150 or 500 mM NaCl for 7 min on ice prior to fixation, epitope labeling, and confocal fluorescence microscopy analysis. Results are the -fold change per unit area over the basal (mean S.E.) obtained from at least four independent experiments. *, p 0.005; #, p 0.05 relative to the corresponding basal values (ANOVA).

Article Snippet: Monoclonal (9E10) and polyclonal (A-14) anti-c-myc antibodies, polyclonal (H-61) antibody against the intracellular loop region (amino acids 230–290) of GLUT4, and polyclonal (N-20) antibody sc1606 against the first exofacial loop region of GLUT4 were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Clinical Proteomics, Membrane, Incubation, Generated, Labeling, Fluorescence, Microscopy

Journal: Journal of Biological Chemistry

Article Title: Insulin Regulates the Membrane Arrival, Fusion, and C-terminal Unmasking of Glucose Transporter-4 via Distinct Phosphoinositides

doi: 10.1074/jbc.m500501200

Figure Lengend Snippet: FIG. 6. PI(3,4,5)P3 causes a gain in myc epitope on membrane lawns; the gain caused by PI3P is revealed upon treatment with Triton X-100. L6-GLUT4myc myotubes were incubated without or with insulin (100 nM for 10 min), carrier alone (10 M and 20 min), or carrier plus PI(3,4,5)P3 or PI3P or PI(4,5)P2 (10 M, 20 min), as indi- cated. Plasma membrane lawns were generated and fixed, labeled with monoclonal anti-myc antibody followed by goat anti-mouse A488 anti- body (A) or polyclonal anti-C terminus of GLUT4 antibody followed by goat anti-rabbit Cy3 (B) and processed for confocal fluorescence micros- copy. Where indicated, lawns were treated with 0.1% Triton X-100 after fixation (solid bars). The fluorescence intensity per lawn was quantified from at least 50 plasma membrane lawn pieces for each condition per experiment as described under “Experimental Procedures.” Results are the mean -fold change per unit area over the basal ( S.E.) obtained from at least five independent experiments. *, p 0.005; #, p 0.05 relative to the corresponding basal values (ANOVA).

Article Snippet: Monoclonal (9E10) and polyclonal (A-14) anti-c-myc antibodies, polyclonal (H-61) antibody against the intracellular loop region (amino acids 230–290) of GLUT4, and polyclonal (N-20) antibody sc1606 against the first exofacial loop region of GLUT4 were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Membrane, Incubation, Clinical Proteomics, Generated, Labeling, Fluorescence

Journal: Journal of Biological Chemistry

Article Title: Insulin Regulates the Membrane Arrival, Fusion, and C-terminal Unmasking of Glucose Transporter-4 via Distinct Phosphoinositides

doi: 10.1074/jbc.m500501200

Figure Lengend Snippet: FIG. 7. High salt treatment of plasma membrane lawns in- creases the C terminus antigenicity of GLUT4 mobilized by PI(3,4,5)P3. L6-GLUT4myc myotubes were incubated without (basal) or with insulin (100 nM for 10 min), carrier plus PI(3,4,5)P3 or PI(4,5)P2 (10 M each, for 20 min), as indicated. Plasma membrane lawns were generated and incubated with PBS without or with additional 500 mM NaCl for 7 min (solid bars) on ice prior to fixation, labeling with monoclonal anti-myc antibody followed by goat anti-mouse A488 anti- body or polyclonal anti-C terminus of GLUT4 antibody followed by goat anti-rabbit Cy3 and processing for confocal fluorescence microscopy. The fluorescence intensity per lawn was quantified from at least 50 plasma membrane lawn pieces for each condition per experiment as described under “Experimental Procedures.” Results are the mean -fold change per unit area over the basal ( S.E.) obtained from at least five independent experiments. *, p 0.005; #, p 0.05 (ANOVA).

Article Snippet: Monoclonal (9E10) and polyclonal (A-14) anti-c-myc antibodies, polyclonal (H-61) antibody against the intracellular loop region (amino acids 230–290) of GLUT4, and polyclonal (N-20) antibody sc1606 against the first exofacial loop region of GLUT4 were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Clinical Proteomics, Membrane, Incubation, Generated, Labeling, Fluorescence, Microscopy

Journal: Journal of Biological Chemistry

Article Title: Insulin Regulates the Membrane Arrival, Fusion, and C-terminal Unmasking of Glucose Transporter-4 via Distinct Phosphoinositides

doi: 10.1074/jbc.m500501200

Figure Lengend Snippet: FIG. 8. GLUT4myc arrival, fusion, and unmasking in round- ed-up L6-myoblasts. L6-GLUT4myc myoblasts were pretreated with wortmannin (Wm) where indicated, then detached from the substratum as described under “Experimental Procedures.” During re-attachment, cells were incubated without (basal) or with insulin (100 nM for 10 min), wortmannin (100 nM), wortmannin (100 nM) plus insulin (100 nM, 10 min; 100 nM Wm plus insulin); or 10 M PI(3,4,5)P3 or PI3P, or PI(4,5)P2, each with carrier. myc-epitope detection with monoclonal anti-myc antibody in intact cells was performed immediately following fixation, without cell permeabilization (A). Cells were permeabilized with 0.1% Triton X-100 treatment (15 min) following fixation but prior to labeling with monoclonal anti-myc or polyclonal anti-GLUT4 C ter- minus as described under “Experimental Procedures” (B). Shown are representative images obtained by confocal fluorescence microscopy from five independent experiments. Bar, 10 m. A, at least 300 round- ed-up cells for each condition as indicated were counted and scored blindly for positive or negative peripheral signal of myc or C terminus epitopes, as described under “Experimental Procedures” (C). The per- centage of cells positive for myc or C terminus peripheral signal was calculated from three independent experiments (mean S.E.).

Article Snippet: Monoclonal (9E10) and polyclonal (A-14) anti-c-myc antibodies, polyclonal (H-61) antibody against the intracellular loop region (amino acids 230–290) of GLUT4, and polyclonal (N-20) antibody sc1606 against the first exofacial loop region of GLUT4 were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Incubation, Labeling, Fluorescence, Microscopy

Journal: Journal of Biological Chemistry

Article Title: Insulin Regulates the Membrane Arrival, Fusion, and C-terminal Unmasking of Glucose Transporter-4 via Distinct Phosphoinositides

doi: 10.1074/jbc.m500501200

Figure Lengend Snippet: FIG. 9. Proposed model of GLUT4 arrival, fusion, with the plasma membrane (PM) and C terminus unmasking regulated by insulin or distinct phosphoinositides. Insulin-promoted GLUT4 arrival at the PM is partly inhibited by 100 nM wortmannin (Wm). This arrival is also promoted by carrier delivery of the PI(3,4,5)P3 or PI3P. Insulin-induced GLUT4 fusion with the PM is prevented by 100 nM Wm, and carrier delivery of the PI(3,4,5)P3 but not PI3P causes GLUT4 fusion. Unmasking of the C terminus of GLUT4 (through a putative GLUT4-binding protein) is effected by insulin or carrier delivery of PI3P but not by PI(3,4,5)P3. The insulin-dependent C-terminal un- masking is partly inhibited by 100 nM Wm. The specific location at which unmasking occurs remains unknown.

Article Snippet: Monoclonal (9E10) and polyclonal (A-14) anti-c-myc antibodies, polyclonal (H-61) antibody against the intracellular loop region (amino acids 230–290) of GLUT4, and polyclonal (N-20) antibody sc1606 against the first exofacial loop region of GLUT4 were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Clinical Proteomics, Membrane, Binding Assay

Journal: The Journal of biological chemistry

Article Title: Reconstitution of insulin-sensitive glucose transport in fibroblasts requires expression of both PPARgamma and C/EBPalpha.

doi: 10.1074/jbc.274.12.7946

Figure Lengend Snippet: FIG. 3. A comparison of protein ex- pression during adipose conver- sion of 3T3-L1 preadipocytes versus PPARg- expressing fibroblasts. On the indicated days post-induction, whole cell extracts were prepared from differen- tiating 3T3-L1 (A), Swiss-Pg (B), BALB/ c-Pg (C), and NIH-Pg (D) cells. Equal amounts of protein (100 mg; 500 mg for GLUT4 and caveolin) were electrophore- sed and Western blotted for PI3-kinase, IRAP, GLUT4, GLUT1, and caveolin-1. These data are representative of two experiments.

Article Snippet: Antibodies—Monoclonal anti-GLUT4 antibody 1F8 (25), polyclonal antibodies against GLUT1 (a kind gift from Dr. C. Carter-Su) (26), PI3-kinase (Upstate Biotechnology, Lake Placid, NY), C/EBPa (Santa Cruz Biotechnology, Santa Cruz, CA), a monoclonal anti-caveolin-1 antibody (Transduction Laboratories, Lexington, KY), and anti-insulinresponsive aminopeptidase (IRAP) serum (27).

Techniques: Comparison, Expressing, Western Blot

Journal: The Journal of biological chemistry

Article Title: Reconstitution of insulin-sensitive glucose transport in fibroblasts requires expression of both PPARgamma and C/EBPalpha.

doi: 10.1074/jbc.274.12.7946

Figure Lengend Snippet: FIG. 5. Ectopic expression of C/EBPa in NIH-3T3 fibroblasts con- verts them to the adipocyte pheno- type, induces PPARg, and confers in- sulin-sensitive glucose uptake. NIH 3T3 cells expressing retroviral C/EBPa were differentiated in the presence or ab- sence of 5 mM troglitazone (Trog). Seven days later (day 7), cells were used for ex- perimentation. A, cells were fixed, stained with Oil Red O, and photographed as de- scribed; B, Northern blot analysis of PPARg and C/EBPa mRNAs in NIH-C/Ea cells; C, Western blot analysis of GLUT4, GLUT1, and IRAP protein levels in NIH- C/Ea cells; and D, insulin-responsive 2-deoxyglucose uptake assay of NIH-C/Ea cells performed as described for Fig. 4. These data are representative of two ex- periments. L1, 3T3-L1 (day 7) adipocyte control.

Article Snippet: Antibodies—Monoclonal anti-GLUT4 antibody 1F8 (25), polyclonal antibodies against GLUT1 (a kind gift from Dr. C. Carter-Su) (26), PI3-kinase (Upstate Biotechnology, Lake Placid, NY), C/EBPa (Santa Cruz Biotechnology, Santa Cruz, CA), a monoclonal anti-caveolin-1 antibody (Transduction Laboratories, Lexington, KY), and anti-insulinresponsive aminopeptidase (IRAP) serum (27).

Techniques: Expressing, Retroviral, Staining, Northern Blot, Western Blot, Control

Journal: eLife

Article Title: Deep transcriptome annotation enables the discovery and functional characterization of cryptic small proteins

doi: 10.7554/eLife.27860

Figure Lengend Snippet: ( a ) AltDDIT3 5’ coding sequence is located in exons 1 and 2 or the DDIT3/CHOP/GADD153 gene and in the 5’UTR of the canonical mRNA (RefSeq NM_004083.5).+2 and+1 indicate reading frames. AltDDIT3 amino acid sequence is also shown. ( b ) Confocal microscopy analyses of HeLa cells co-transfected with altDDIT3 GFP (green channel) and DDIT3 mCherry (red channel). Scale bar, 10 µm. ( c, d ) Colocalization analysis of the images shown in ( b ) performed using the JACoP plugin (Just Another Co-localization Plugin) implemented in Image J software (two independent biological replicates). ( c ) Scatterplot representing 50% of green and red pixel intensities showing that altDDIT3 GFP and DDIT3 mCherry signal highly correlate (with Pearson correlation coefficient of 0.92 [p-value<0.0001]). ( d ) Binary version of the image shown in ( b ) after Costes’ automatic threshold. White pixels represent colocalization events (p-value<0.001, based on 1000 rounds of Costes’ randomization colocalization analysis). The associated Manders Correlation Coefficient, M 1 and M 2 , are shown in the right upper corner. M 1 is the proportion of altDDIT3 GFP signal overlapping DDIT3 mCherry signal and M 2 is the proportion of DDIT3 mCherry signal overlapping altDDIT3 GFP . ( e ) Representative immunoblot of co-immunoprecipitation with GFP-Trap agarose beads performed on HeLa lysates co-expressing DDIT3 mcherry and altDDIT3 GFP or DDIT3 mcherry with pcDNA3.1 GFP empty vector (two independent experiments).

Article Snippet: Human altDDIT3 mCherry was cloned into pcDNA3.1 by Gibson assembly using coding sequence from transcript variant 1 (NM_004083.5) and mCherry coding sequence from pLenti-myc-GLUT4-mCherry (Addgene plasmid # 64049).

Techniques: Sequencing, Confocal Microscopy, Transfection, Software, Western Blot, Immunoprecipitation, Expressing, Plasmid Preparation

Journal: Nature Communications

Article Title: Histone H4 lysine 16 acetylation controls central carbon metabolism and diet-induced obesity in mice

doi: 10.1038/s41467-021-26277-w

Figure Lengend Snippet: a Immunofluorescence images of WAT after 26 weeks on HFD (SD controls are shown in Supplementary Fig. ). DAPI (blue) and neutral lipid staining by BODIPY-493 (green). Highlighted regions show recidual fat storage. Scale bar: 50 µm, enlarged view: 5 µm. b Violin-plot showing the MFI quantification of ( a ). Error bars indicate ±SEM. Statistical analysis was performed by one-way ANOVA followed by Kruskal–Wallis test, * p = 0.04, **** p = 0.0001. n = 3 biological replicates. Dotted lines inside the violin plots show the quartiles and dashed lines depict the medians. Dotted horizontal line marks the zero. c Violin-plot showing total quantification of adipocytes per section ( n = 3, 2–3 sections per animal). Normality was scored as in ( b ). Statistical analysis using Mann–Whitney test showed no significance. Dotted lines show the quartiles and dashed lines depict the median. d Barplot showing enrichment test for target GO terms and DEG in WAT. The black box represents differentially regulated genes in WAT of Mof +/− animals on SD. Statistical test was performed using the absolute numbers of genes and p values were scored by the two-sided Fisher’s exact test. The red bar highlights the only significantly enriched pathway. See also: Supplementary Fig. . e Left: Representative immunofluorescence images showing GLUT4 (red) expression in WAT of animals on HFD. Scale bar: 50 µm. Right: MFI quantification of GLUT4 signal. Statistical analysis was performed by two-sided Mann–Whitney test, * p = 0.048. n = 3 biological replicates. f Representative immunofluorescence images showing neutral lipids (BODIPY-493; green) in adipocytes differentiated from mesenchymal stromal cells (MSC). Scale bar: 20 µm. g Boxplot showing the quantification of overall lipid droplet area per droplet (µm 2 ) in logarithmic scale (right). Statistical analysis was performed by two-sided ANOVA followed by Tukey post test, * p = 0.03, *** p = 0.0007. n = 3 biological replicates. h Graphical scheme illustrating the pre-adipocyte (iAdipo) in vitro differentiation and induction of Mof knockout ( Mof -iKO) by treatment with 4-hydroxy-tamoxifen (4-OHT). See also: Supplementary Fig. . i RT-qPCR analyses of iAdipo cells with (+) or without (−) insulin/glucose challenge. Violin plot shows average Glut4 mRNA expression relative to Hprt of biological replicates ( n = 5). Statistical analysis was performed by two-way ANOVA followed by Holm–Sidak’s comparison test, * p = 0.05. Dotted lines show the quartiles and dashed lines depict the medians. j Representative histogram showing glucose uptake capacity after insulin/glucose challenge (left). Gray histogram represents the untreated cells, control (black), and Mof -iKO (purple). Floating plots showing the 2-NBD ratio uptake (treated MFI/untreated MFI). “+” indicates the treatment employed and the dashed horizontal line marks the basal glucose uptake. Statistical analysis was performed by one-way ANOVA followed by Tukey’s comparison test, * p = 0.013, *** p = 0.0001. n = 5 biological replicates. k Left panel: Line-plots showing the percentage of extracellular acidification rate (%ECAR) of control (black) or Mof -iKO (purple) iAdipo cells without (open circles) or upon insulin challenge (filled circles). Arrows indicate the addition of inhibitors. Statistical analysis was performed by two-sided two-way ANOVA followed by Holm–Sidak’s comparison test. Right panel: Violin plot showing quantification overall glycolysis. “+” indicates insulin treatment. Biological replicates ( n = 3). Statistical analysis was performed by two-sided two-way ANOVA followed by Holm–Sidak’s comparison test, ** p = 0.01, ns = not significant. Dotted lines show the quartiles and dashed lines depict the medians. See also: Supplementary Fig. .

Article Snippet: The Glut4 coding sequence was sub-cloned from Addgene plasmid number 52872 and Mof coding sequence was cloned into a pcDNATM5/pCMV vector.

Techniques: Immunofluorescence, Staining, MANN-WHITNEY, Expressing, In Vitro, Knock-Out, Quantitative RT-PCR, Comparison, Control

Journal: Nature Communications

Article Title: Histone H4 lysine 16 acetylation controls central carbon metabolism and diet-induced obesity in mice

doi: 10.1038/s41467-021-26277-w

Figure Lengend Snippet: a , b , d , f Left: representative images of neutral lipid staining (BODIPY-493; green). Scale bars: 5 µm. Right: Boxplots showing quantification of overall lipid droplet area per droplet (µm 2 ) in logarithmic scale with the boxes depicting interquartile range. Number of biological replicates per condition: n = 3. Total number of droplets used for quantification are indicated in the figure. Dashed horizontal lines mark the mean area of controls. a Control, Mof -iAdipo or Mg149-treated (50 nM) control cells at baseline (“-”, upper panel) or after 15 min of insulin/glucose challenge (“+”, lower panel). Statistical analysis was performed using the raw data and one-way ANOVA followed by Kruskal–Wallis comparison test, **** p = 10 −16 . b Control and Mof -iAdipo cells treated with Ex-527 (200 nM) after insulin/glucose challenge. Statistical analysis was performed using the raw data and one-way ANOVA followed by Kruskal–Wallis comparison test, *** p = 0.006. See Supplementary Fig. for baseline images. c Scatter-plot depicting the H4K16ac MFI in control and Mof- iKO iAdipo with (open circles) or without (filled circles) insulin and glucose challenge. Each dot represents a single iAdipo. Statistical analysis was performed using the raw data and two-sided one-way ANOVA followed by Kruskal–Wallis comparison test, ** p = 0.014, **** p = 10 −16 . Number of biological replicates n = 4. d Control and Mof -iAdipo cells at baseline, following overexpression of Glut4 , or following ectopic expression of wild-type Mof (wt- Mof ) or Mof catalytic mutant ( Mof -E350Q) challenged with insulin/glucose (+Ins/Gluc). Statistical analysis was performed using the raw data and two-sided one-way ANOVA followed by Dunn’s multiple comparison test, *** p = 0.001. Note that the data for untreated wild type (n = 1299) and Mof -iKO (n = 4644) is identical to the data depicted in ( a ). e Heatmap showing the H4K16ac levels from wild type and Mof -iKO upon ectopic expression of Glut4 , wt- Mof or Mof -E350Q with or without glucose and insulin treatment. Statistical analysis was performed using the raw data and two-sided two-way ANOVA followed by Sidak multiple comparison post test, **** p = 10 −16 . f Control and Mof -iAdipo cells treated with chloroquine at baseline or upon insulin/glucose challenge. Statistical analysis was performed using the raw data and two-sided one-way ANOVA followed by Dunn’s multiple comparison test, *** p = 0.0001, **** p = 10 −16 . g RT-qPCR analyses of visceral WAT. Violin plots show the mRNA expression of Glut4 , Pparg, Pcg1α and Mef2c as an average of biological replicates ( Mof +/+ n = 5; Mof +/− n = 5 for Glut4 , Pparg, Pcg1α and Mof +/+ n = 3; Mof +/− n = 3 for Mef2c ). Statistical analysis was performed by two-sided Mann–Whitney test, * p = 0.026. Dotted lines show the quartiles and solid lines depict the medians. h ChIP-qPCR analyses of MOF at promoters of Pparg , Glut4 , Pcg1α and Mef2c promoters in visceral WAT. The data are expressed as fold change over H3. Violin plots show the average of biological replicates ( Mof +/+ n = 6 and Mof +/− n = 5). Statistical analysis was performed by two-sided Mann–Whitney test, * p = 0.015. Dotted lines show the quartiles and dashed lines depict the medians. i ChIP-qPCR analyses of MOF (left) and H4K16ac (right) at promoters of Pparg in the presence or absence of Glu/Ins in wild-type and Mof -iKO adipocytes. Dotted lines inside the violin plots show the quartiles and dashed lines depict the medians.

Article Snippet: The Glut4 coding sequence was sub-cloned from Addgene plasmid number 52872 and Mof coding sequence was cloned into a pcDNATM5/pCMV vector.

Techniques: Staining, Control, Comparison, Over Expression, Expressing, Mutagenesis, Quantitative RT-PCR, MANN-WHITNEY, ChIP-qPCR

Journal: Nature Communications

Article Title: Histone H4 lysine 16 acetylation controls central carbon metabolism and diet-induced obesity in mice

doi: 10.1038/s41467-021-26277-w

Figure Lengend Snippet: a Left: representative images of neutral lipid staining (BODIPY-493; green). Control or Mof -iAdipo treated with thiazolidinedione (TZD)(10 −4 mol/L) at baseline (upper panel) or upon insulin/glucose challenge (bottom panel). Scale bar: 5 µm. Right: boxplot showing quantification of overall lipid droplet area per droplet (µm 2 ) in logarithmic scale with boxes showing the interquartile range. Number of biological replicates per condition: n = 3. Total number of droplets used for quantification are indicated on the figure. Dashed horizontal lines mark the mean area of controls. Statistical analysis was performed using the raw data and one-way ANOVA followed by Kruskal–Wallis comparison test, * p = 0.039. b , c RT-qPCR analyses of control or Mof -iAdipo cells treated with TZD at baseline or upon insulin/glucose challenge. Violin plots show the average Pgc1α ( b ) and Glut4 ( c ) mRNA expression of biological replicates ( n = 3). Dotted lines show the quartiles and solid lines depict the medians. d Heatmap showing H4K16ac levels after TZD treatment at different conditions. Statistical analysis was performed using the raw data and two-sided two-way ANOVA followed by Sidak multiple comparison post test, **** p = 10 −16 . e Representative images of neutral lipid staining (BODIPY-493; green). Control or Mof -iAdipo treated with Glut4 siRNA in the presence or absence of thiazolidinedione (TZD)(10 −4 mol/L) at baseline or upon insulin/glucose challenge (+Ins/Gluc). Scale bar: 5 µm. f – h Boxplots showing quantification of overall lipid droplet area per droplet (µm 2 ) in logarithmic scale. Dashed horizontal lines mark the mean area of controls. Number of biological replicates per condition: n = 3. The “Control siRNA ” data is identical in ( f ), ( g ) and ( h ). ( i ) Schematic representation of proposed MOF-mediated regulation of the Glut4 transcription network. See also: Supplementary Fig. .

Article Snippet: The Glut4 coding sequence was sub-cloned from Addgene plasmid number 52872 and Mof coding sequence was cloned into a pcDNATM5/pCMV vector.

Techniques: Staining, Control, Comparison, Quantitative RT-PCR, Expressing

Journal: Nature Communications

Article Title: Histone H4 lysine 16 acetylation controls central carbon metabolism and diet-induced obesity in mice

doi: 10.1038/s41467-021-26277-w

Figure Lengend Snippet: Schematic overview describing the proposed mechanism by which chronic reduction of MOF results in global remodeling of metabolism toward diabetic predisposition. In wild-type cells, insulin binds to the insulin receptor, which will trigger a cascade of phosphorylation events culminating in the phosphorylation of GLUT4 inside storage vesicles. These will be transported to the cytoplasmic membrane to regulate glucose import. In parallel, insulin triggers the transcription of Glut4 predominantly through the transcriptional network downstream of PPARγ and PGC1α. In healthy adipose tissue and skeletal muscle (SKM), MOF mediates glucose uptake by positively regulating gene expression of the nuclear hormone receptor Pparg , thereby triggering the downstream transcriptional network resulting in increased levels of the major insulin-dependent glucose transporter GLUT4. On the other hand, Mof haploinsufficient animals show impaired insulin secretion upon glucose challenge, leading to severe destablization of glucose homeostasis. Whilst insulin signaling and GLUT4 vesicle transport in Mof- depleted cells are functional, the transcriptional network regulating Glut4 expression is impaired, resulting in reduced glucose import. Limitation of intracellular glucose is responsible for impaired neutral lipid storage in Mof +/− adipocytes. This study identifies the epigenetic regulator MOF as the first histone acetyltransferase to regulate the onset of diet-induced obesity.

Article Snippet: The Glut4 coding sequence was sub-cloned from Addgene plasmid number 52872 and Mof coding sequence was cloned into a pcDNATM5/pCMV vector.

Techniques: Phospho-proteomics, Membrane, Gene Expression, Functional Assay, Expressing

Journal: Brain communications

Article Title: Differential regulation of insulin signalling by monomeric and oligomeric amyloid beta-peptide.

doi: 10.1093/braincomms/fcac243

Figure Lengend Snippet: Figure 4 mAβ1-40 induces GLUT4 membrane translocation. (A) Scheme of the intracellular effects of IR activation. Insulin binds the IR inducing its dimerization and activating the receptor to cause its autophosphorylation. This produces the binding and phosphorylation of the signalling adapter protein IRS. IRS activates several downstream pathways, but here we focus on PI3K/Akt system. The activation of PI3K leads to the phosphorylation of Akt, which produces the GLUT4 translocation to the membrane, allowing glucose uptake by cells. (B, C) mAβ1-40induces GLUT4 translocation. Cells were treated for 10 min with 100 nM insulin or 150 nM mAβ1-40, afterwards cells were fixed. Extracellular expressed GLUT4 was labelled with an anti-GLUT4 Ab and nuclei were stained with DAPI. (B) Representative images of cells used in (B). Bars represent 20 nm. (C) GLUT4 fluorescence intensity at 555 nm was quantified. Data are the mean ± SEM of 4 independent experiments. ** P < 0.01 compared with untreated controls by one-way ANOVA plus Student–Newman–Keuls as post hoc test. (D) Cells were treated as in (B, C). Glucose uptake was measured and expressed regarding untreated controls. Data are the mean ± SEM of five independent experiments. ***P < 0.01, *P < 0.05 compared with controls by one-way ANOVA plus Student–Newman–Keuls as post hoc test.

Article Snippet: This procedure was performed using 1:100 rabbit anti-GLUT4 Ab (NBP1-49533; Novus Biologicals).

Techniques: Membrane, Translocation Assay, Activation Assay, Binding Assay, Phospho-proteomics, Staining, Fluorescence

Journal: Brain communications

Article Title: Differential regulation of insulin signalling by monomeric and oligomeric amyloid beta-peptide.

doi: 10.1093/braincomms/fcac243

Figure Lengend Snippet: Figure 8 oAβ1-40blocks IR activation. (A) oAβ1-40binds to IR impairing its autophosphorylation. Human neuroblastoma cells were pre-treated with increasing concentrations of oAβ1-40for 30 min and then treated in the presence/absence of insulin (100 nM) for 10 min. The upper panel shows a representative WB with anti-p-IR, anti-p-Thr308-Akt and anti-GAPDH Abs. (B) Quantification of the p-IR bands and normalized by GAPDH obtained by WB as described in (A). Data are the mean ± SEM of 4–5 independent experiments. *P < 0.05 compared with untreated control cells by one-way ANOVA plus Student–Newman–Keuls as post hoc test. (C) Quantification of the p-Thr308-Akt bands and normalized by GAPDH obtained by WB as described in (A). Data are the mean ± SEM of 4–5 independent experiments. *P < 0.05 regarding untreated control cells by one-way ANOVA plus Student–Newman–Keuls as post hoc test. (D) oAβ1-40do not induce the translocation of GLUT4. Representative images of cells treated with 100 nM insulin and 150 nM oAβ1-40 for 10 and 30 min, respectively. Extracellular GLUT4 was labelled with an anti-GLUT4 Ab and nuclei are stained with DAPI. Bars represent 20 nm. (E) oAβ1-40 does not induce glucose uptake. Cells were treated as in (A) and glucose uptake was measured as indicated in the M&M section and expressed regarding untreated controls. Data are the mean ± SEM from five independent experiments. ** P < 0.01, n.s., non-significant compared with untreated cells by one-way ANOVA plus Student–Newman– Keuls as post hoc test.

Article Snippet: This procedure was performed using 1:100 rabbit anti-GLUT4 Ab (NBP1-49533; Novus Biologicals).

Techniques: Activation Assay, Control, Translocation Assay, Staining

Journal: Scientific Reports

Article Title: The Parkinson’s disease-linked Leucine-rich repeat kinase 2 (LRRK2) is required for insulin-stimulated translocation of GLUT4

doi: 10.1038/s41598-019-40808-y

Figure Lengend Snippet: GLUT4 translocation in Lrrk2 deficient fibroblasts. ( a ) GLUT4 immunostaining on the cell surface of fibroblasts from 6 months old Lrrk2 deficient and wild-type rats without (w/o) insulin and 10 and 30 min after insulin addition and ( c ) the corresponding quantification of the GLUT4 signal intensity at different time-points (0, 10 and 30 min) after stimulation (mean and SEM). ( b ) GLUT4 immunostaining on plasma membrane of fibroblasts from 22 months old Lrrk2 deficient and wild-type rats without insulin and 10 and 30 min after stimulation and ( d ) the corresponding quantification of the GLUT4 signal intensity (mean and SEM). Red: GLUT4 immunostaining; green: wheat germ agglutinin (=plasma membrane) staining; blue: DAPI.

Article Snippet: Primary antibodies used: Akt #9272 (1:1000), p-Akt (Ser473) # 4058 (1:1000), p-Akt (Thr308) #2965, Rab8A #6975 (1:1000), Rab10 #8127 (1:1000), AS160 #2670 (1:1000), p-AS160 (Thr642) #4288 (1:1000), IR beta #3025 (1:1000), p-IGF-IR beta (Tyr1131)/IR beta (Tyr1146) #3021 (1:1000) (all from Cell Signalling); LRRK2 (3514-1 (MJFF2) Epitomics); tubulin (ab6160, abcam); SLC2A4 (ARP43785_P050, avivasysbio, 1:100); GLUT4 (MAB1262, R&D Systems, 1:1000); Alexa Fluor 488 Mouse anti-β-Tubulin, Class III Clone TUJ1 #560338 (BD Pharmingen).

Techniques: Translocation Assay, Immunostaining, Clinical Proteomics, Membrane, Staining

Journal: Scientific Reports

Article Title: The Parkinson’s disease-linked Leucine-rich repeat kinase 2 (LRRK2) is required for insulin-stimulated translocation of GLUT4

doi: 10.1038/s41598-019-40808-y

Figure Lengend Snippet: Insulin signalling in Lrrk2 deficient animals. ( a ) Insulin-triggered phosphorylation of IRβ and AS160 (Thr642) in fibroblasts from 22 months old Lrrk2 deficient rats at different time-points after stimulation and the corresponding quantification of P-IRβ ( b , n = 7, normalized to IR-β) and P-AS160 Thr642 ( c , n = 10, normalized to AS160) signal intensity (mean ± SEM). ( d ) Western blot analysis (fibroblasts from 22 months old rats as example) and quantification of total GLUT4, AS160 and Rab10 expression in fibroblasts from 6 months ( e ) and 22 months old ( f ) Lrrk2 deficient and wild-type rats (normalized to tubulin, mean ± SEM). # are numbers of animals/cell lines. The difference in GLUT4 and AS160 signal intensity between 6 months und 22 months old sample-groups results from differences in experimental procedure and does not reflect the absolute quantity of GLUT4 or rather AS160 in these age groups. ( g ) Investigation of Rab10 phosphorylation by Mn 2+ Phos-tag SDS-PAGE in fibroblasts from 6 months old Lrrk2 deficient and wild-type rats at different time points (0-10-30-40 min) after insulin addition and ( h ) corresponding quantification of P-Rab10 signal intensity in wild-type cells at different time points after stimulation (normalized to Rab10, n = 7, mean and SEM).

Article Snippet: Primary antibodies used: Akt #9272 (1:1000), p-Akt (Ser473) # 4058 (1:1000), p-Akt (Thr308) #2965, Rab8A #6975 (1:1000), Rab10 #8127 (1:1000), AS160 #2670 (1:1000), p-AS160 (Thr642) #4288 (1:1000), IR beta #3025 (1:1000), p-IGF-IR beta (Tyr1131)/IR beta (Tyr1146) #3021 (1:1000) (all from Cell Signalling); LRRK2 (3514-1 (MJFF2) Epitomics); tubulin (ab6160, abcam); SLC2A4 (ARP43785_P050, avivasysbio, 1:100); GLUT4 (MAB1262, R&D Systems, 1:1000); Alexa Fluor 488 Mouse anti-β-Tubulin, Class III Clone TUJ1 #560338 (BD Pharmingen).

Techniques: Phospho-proteomics, Western Blot, Expressing, SDS Page

Journal: Scientific Reports

Article Title: The Parkinson’s disease-linked Leucine-rich repeat kinase 2 (LRRK2) is required for insulin-stimulated translocation of GLUT4

doi: 10.1038/s41598-019-40808-y

Figure Lengend Snippet: Insulin signalling in human fibroblasts from Parkinson’s patients with G2019S mutated LRRK2. ( a ) Example: Western blot analysis of protein extracts from fibroblasts of one PD patient with G2019S mutation (top) and one healthy control individual (button) and ( b ) the corresponding quantification of P-Akt Thr308 (top) and P-Akt Ser473 (button) signal intensity (normalized to Akt, n = 7 independent experiments, fibroblasts from 3 different healthy controls and 3 PD patients with G2019S mutation in LRRK2; mean ± SEM). ( c ) GLUT4 immunostaining on the plasma membrane of human fibroblasts derived from PD patients with G2019S mutation and healthy control individuals (red: GLUT4 immunostaining; green: wheat germ agglutinin (=plasma membrane) staining; blue: DAPI) and ( d ) the corresponding quantification of GLUT4 signal intensity (mean ± SEM). ( e ) Quantification of total GLUT4, AS160 and Rab10 protein expression in human fibroblasts (normalized to tubulin) analysed by Western blot (mean and SEM).

Article Snippet: Primary antibodies used: Akt #9272 (1:1000), p-Akt (Ser473) # 4058 (1:1000), p-Akt (Thr308) #2965, Rab8A #6975 (1:1000), Rab10 #8127 (1:1000), AS160 #2670 (1:1000), p-AS160 (Thr642) #4288 (1:1000), IR beta #3025 (1:1000), p-IGF-IR beta (Tyr1131)/IR beta (Tyr1146) #3021 (1:1000) (all from Cell Signalling); LRRK2 (3514-1 (MJFF2) Epitomics); tubulin (ab6160, abcam); SLC2A4 (ARP43785_P050, avivasysbio, 1:100); GLUT4 (MAB1262, R&D Systems, 1:1000); Alexa Fluor 488 Mouse anti-β-Tubulin, Class III Clone TUJ1 #560338 (BD Pharmingen).

Techniques: Western Blot, Mutagenesis, Control, Immunostaining, Clinical Proteomics, Membrane, Derivative Assay, Staining, Expressing

Journal: Scientific Reports

Article Title: The Parkinson’s disease-linked Leucine-rich repeat kinase 2 (LRRK2) is required for insulin-stimulated translocation of GLUT4

doi: 10.1038/s41598-019-40808-y

Figure Lengend Snippet: The role of LRRK2 for insulin signal transduction. ( a ) In wild-type, GDP-bound Rab10 is tightly bound by guanine dissociation inhibitor (GDI) in the cytosol. LRRK2 promotes the insertion of Rab10 in GLUT4 storage vesicles by phosphorylation. Insulin addition activates an insulin-dependent Phosphatase X with following dephosphorylation of Rab10 (accompanied by GDP-GTP exchange), necessary for efficient GLUT4 translocation. ( b ) In LRRK2 deficient situation, the phosphorylation of Rab10 by LRRK2 and the following insertion in GLUT4 storage vesicles fail. As consequence, GDP-bound Rab10 – GDI complexes accumulate in the cytosol. By insulin addition activated PI3K/Akt/AS160 signalling cascade did not reach the GLUT4 storage vesicles and the GLUT4 translocation fails.

Article Snippet: Primary antibodies used: Akt #9272 (1:1000), p-Akt (Ser473) # 4058 (1:1000), p-Akt (Thr308) #2965, Rab8A #6975 (1:1000), Rab10 #8127 (1:1000), AS160 #2670 (1:1000), p-AS160 (Thr642) #4288 (1:1000), IR beta #3025 (1:1000), p-IGF-IR beta (Tyr1131)/IR beta (Tyr1146) #3021 (1:1000) (all from Cell Signalling); LRRK2 (3514-1 (MJFF2) Epitomics); tubulin (ab6160, abcam); SLC2A4 (ARP43785_P050, avivasysbio, 1:100); GLUT4 (MAB1262, R&D Systems, 1:1000); Alexa Fluor 488 Mouse anti-β-Tubulin, Class III Clone TUJ1 #560338 (BD Pharmingen).

Techniques: Transduction, Phospho-proteomics, De-Phosphorylation Assay, Translocation Assay