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: 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

Journal: Journal of Pure and Applied Microbiology

Article Title: Antidiabetic Activity of Methanolic Extract of Artabotrys suaveolens Leaves in 3T3-L1 Cell Line

doi: 10.22207/jpam.14.1.59

Figure Lengend Snippet: Fig. 6. Overlaid expression of GLUT4 in untreated 3T3L1 cells (black colour line) & standard drug treated cells (metformin 100um) (red colour line) and test compound treated cells (cyan colour line).

Article Snippet: DMEM High Glucose (#AL219A, Himedia), DMEM without glucose (#AL186,Himedia), Fetal Bovine Serum (#RM10432,Himedia), D-PBS (#TL1006, Himedia), 2-NBDG (Invitrogen: Cat no. 13195), Metformin (#PHR 1084, Sigma), Acarbose (#A8980, Sigma), Anti-Mouse Glut4 –Alexa Fluor 488 Antibody (#NBPI-49533F, Novus Biologicals)), MT T Reagent (# 4060,Himedia) , DMSO (#PHR1309,Sigma), FACS Calibur (BD-Biosciences, USA), Microplate reader(#EC800,Biotek).

Techniques: Expressing

Journal: The American journal of clinical nutrition

Article Title: Resveratrol regulates human adipocyte number and function in a Sirt1-dependent manner.

doi: 10.3945/ajcn.2009.28435

Figure Lengend Snippet: FIGURE 2. Resveratrol inhibits adipogenic differentiation of Simpson-Golabi-Behmel syndrome (SGBS) preadipocytes. SGBS preadipocytes were treated with vehicle or increasing doses of resveratrol for the first 4 d of adipogenic differentiation. A: On day 14, cultures were stained with oil red O. One representative experiment of 3 performed is shown. B: Rate of adipogenic differentiation was determined as described in Materials and Methods. Data are expressed as means 6 SDs (n = 3 independent experiments each performed in triplicate). *P , 0.001 (resveratrol compared with vehicle). C: Lipid content was determined on day 10 with Nile Red. Data are expressed as means 6 SDs (n = 3 independent experiments each performed in triplicate). *P , 0.01 (resveratrol compared with vehicle). D: Total RNAwas prepared on day 10 and reverse transcribed. Quantitative polymerase chain reaction was performed by using specific primer pairs for the peroxisome proliferator-activated receptor (PPARc), glucose transporter-4 (Glut-4), and fatty acid synthase (FASN), and results were standardized to glyceraldehyde 3-phosphate dehydrogenase. Data are expressed as means 6 SDs (n = 3 independent experiments). *P , 0.05 (resveratrol compared with vehicle). E: Protein lysates were prepared on day 10 and subjected to Western blot analysis by using specific antibodies for PPARc, Glut-4, and FASN. Expression of a-tubulin was used as an equal protein loading control. One representative experiment of 3 performed is shown. kDa, kiloDalton.

Article Snippet: Western blot analysis was performed as described previously (23) by using rabbit PPARc monoclonal antibody (1:1000, New England Biolabs, Frankfurt, Germany), rabbit FASN monoclonal antibody (1:1000, New England Biolabs), mouse GLUT4 monoclonal antibody (1:200; R&D Systems, Wiesbaden, Germany), mouse Sirt1 monoclonal antibody (1:500; Upstate, Lake Placid, NY), and mouse anti-b-actin monoclonal antibody (1:5000; Sigma, Steinheim, Germany) followed by goat antimouse IgG or goat anti-rabbit IgG conjugated to horseradish peroxidase (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Staining, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Control

Journal: The American journal of clinical nutrition

Article Title: Resveratrol regulates human adipocyte number and function in a Sirt1-dependent manner.

doi: 10.3945/ajcn.2009.28435

Figure Lengend Snippet: FIGURE 3. Resveratrol inhibits de novo lipogenesis in Simpson-Golabi-Behmel syndrome (SGBS) adipocytes. SGBS adipocytes were incubated for 24 h with vehicle or 10 or 50 lmol/L resveratrol. A: Basal and insulin-stimulated (ins; 1026 mol/L) glucose uptake was measured by incorporation of 2-deoxy-D- [14C]-glucose after stimulation with insulin for 30 min. Data are expressed as means 6 SDs (n = 3 independent experiments each performed in triplicate). *P , 0.01 (insulin compared with vehicle control); **P , 0.02 (resveratrol compared with vehicle control). B: Basal and insulin-stimulated (1028 mol/L) de novo lipogenesis was measured by incorporation of D-[14C]-glucose into cellular lipids on a b-counter. Data are expressed as means 6 SDs (n = 3 independent experiments each performed in triplicate). *P , 0.002 (resveratrol compared with vehicle). C: Total RNA was prepared, and glucose transporter-4 (Glut-4), fatty acid synthase (FASN), and acetyl-CoA carboxylase (ACC) lightcycler reverse transcriptase–polymerase chain reaction was performed. Data are expressed relative to glyceraldehyde 3-phosphate dehydrogenase as means 6 SDs (n = 3). *P , 0.02 (resveratrol compared with vehicle). cpm, counts per minute.

Article Snippet: Western blot analysis was performed as described previously (23) by using rabbit PPARc monoclonal antibody (1:1000, New England Biolabs, Frankfurt, Germany), rabbit FASN monoclonal antibody (1:1000, New England Biolabs), mouse GLUT4 monoclonal antibody (1:200; R&D Systems, Wiesbaden, Germany), mouse Sirt1 monoclonal antibody (1:500; Upstate, Lake Placid, NY), and mouse anti-b-actin monoclonal antibody (1:5000; Sigma, Steinheim, Germany) followed by goat antimouse IgG or goat anti-rabbit IgG conjugated to horseradish peroxidase (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Incubation, Control, Reverse Transcription, Polymerase Chain Reaction