Journal: Biofactors (Oxford, England)

Article Title: Diverse Inhibitors of De Novo Purine Synthesis Promote AICAR ‐Induced AMPK Activation and Glucose Uptake in L6 Myotubes

doi: 10.1002/biof.70037

Figure Lengend Snippet: Assessment of sensitivity of L6 cells to inhibitors of purine metabolism. A: Purine metabolism and AMPK. The purine precursor ZMP is an AMPK activator. Methotrexate was shown to promote fatty acid oxidation (FAO) and glucose uptake via activation of AMPK in skeletal muscle tissue or cells [ , ]. Intermediates : 5,10‐CH 2 ‐THF, N 5 ,N 10 ‐methylene THF; 10‐CHO‐THF, N 10 ‐Formyl‐THF; AMP, adenosine monophosphate; DHF, dihydrofolate; dUMP, deoxyuridine monophosphate; dTMP, deoxythymidine monophosphate; FGAR, formylglycinamide ribonucleotide; GAR, glycinamide ribonucleotide; GMP, guanosine monophosphate; Hx, hypoxanthine; IMP, inosine monophosphate; PRA, phosphoribosylamine; PRPP, 5‐phosphoribosyl‐1‐pyrophosphate; SAICAR, N‐succinyl‐5‐aminoimidazole‐4‐carboxamide ribonucleotide; THF, tetrahydrofolate; Xan, xanthine; ZMP, 5‐aminoimidazole‐4‐carboxamide ribonucleotide. Enzymes : ACC, acetyl‐coenzyme A carboxylase; ADSL, adenylosuccinate lyase; ADSS, adenylosuccinate synthetase; AMPK, AMP‐activated protein kinase; ATIC, 5‐aminoimidazole‐4‐carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase; DHFR, dihydrofolate reductase; GART, glycinamide ribonucleotide formyltransferase; GMPS, GMP synthetase; GPAT, glutamine phosphoribosylpyrophosphate amidotransferase; IMPDH, IMP dehydrogenase; TS, thymidylate synthetase; XDH, xanthine oxidase. Inhibitors : ALA, alanosine; ALO, allopurinol; FAO, fatty acid oxidation; MMF, mycophenolate mofetil; MP, mercaptopurine; MTX, methotrexate; TMP, trimethoprim; TMX, trimetrexate. “P” on AMPK and ACC indicates phosphorylation. (B, C) L6 Myotubes express enzymes of nucleotide and folate metabolism targeted by MTX, ALA, MMF, MP, TMX, and ALO. L6 cells were grown for 2 days in MEMα with nucleosides and 10% serum and then differentiated for 7 days in MEMα with nucleosides and 2% serum and for an additional day in MEMα without nucleosides and serum. Cells were then analyzed for expression of Gart , Atic , Adss1 , Adss2 , Adsl , Impdh1 , Impdh2 , Tyms , Dhfr , and Xdh genes (B). Expression of target genes was normalized to expression of Actin beta gene ( Actb ). Graphs show means with SD ( n = 2). Tyms : Thymidylate synthetase. In addition, cells were analyzed for protein expression of GART, ATIC, ADSS, IMPDH2, DHFR, and XDH on day 2 (myoblasts; MB), day 9 (myotubes after 7 days in MEMα with nucleosides and 2% serum; MT+) and day 10 (myotubes after 7 days in MEMα with nucleosides and 2% serum and 1 day in MEMα without nucleosides and serum; MT‐) of culture (C). Numbers next to blots indicate position and molecular weight (in kDa) of molecular weight markers. (D–J) Effect of MMF, ALA, MP, TMP, sulfamethoxazole (SMX), TMX and MTX on proliferation of L6 myoblasts in absence or presence of nucleosides. L6 myoblasts were grown in absence of nucleosides for 24 h and then treated with MMF (0.1–10 μM) (D), ALA (0.1–10 μM) (E), MP (1–100 μM) (F), TMP (1–100 μM) (G), SMX (10–1000 μM) (H), TMX (0.1–10 μM) (I), MTX (0.1–10 μM) (J) or vehicle (control, C) in absence or in presence of nucleosides for 48 h. Cell cultures before and after the treatment were analyzed for DNA content with Hoechst assay. Hoechst fluorescence (Hoechst FL) after the treatment was expressed relative to Hoechst fluorescence before the treatment (0 h). Graphs show means with SD ( n = 4–8). * p < 0.05 versus respective (without or with nucleosides) control, two‐way ANOVA with Dunnett's test.

Article Snippet: Rat skeletal muscle cell line L6 was from American Type Culture Collection (ATCC, #CRL‐1458).

Techniques: Activation Assay, Phospho-proteomics, Expressing, Molecular Weight, Control, Fluorescence

Journal: Biology Open

Article Title: Live-cell GLUT4 translocation assay reveals Per3 as a novel regulator of circadian insulin sensitivity in skeletal muscle cells

doi: 10.1242/bio.061941

Figure Lengend Snippet: Circadian rhythm of GLUT4 translocation. The circadian patterns of GLUT4 translocation in L6 skeletal muscle cells across varying insulin concentrations, analysed over 60 h. Data plotted using R (v4.2.1) with a harmonic regression script. Statistics are presented in Table 2 with n =3.

Article Snippet: Our choice of L6 rat skeletal muscle cells (ATCC, CRL-1458) was guided by their high insulin sensitivity, making them particularly suitable for insulin sensitivity assays ( ).

Techniques: Translocation Assay

Journal: Biology Open

Article Title: Live-cell GLUT4 translocation assay reveals Per3 as a novel regulator of circadian insulin sensitivity in skeletal muscle cells

doi: 10.1242/bio.061941

Figure Lengend Snippet: Effect of target gene knockdown on GLUT4 translocation. (A) Validation of siRNA-mediated knockdown efficiency for Per3 , Tssk6 , Arntl , and Hoxb5 genes in L6 cells. Relative gene expression was quantified using qPCR and normalised to control (untreated cells). Data are presented as mean±s.d. ( n =3 biological replicates). Unpaired t -tests with Welch's correction were performed for each gene ( n =3 per group). A significant knockdown was observed for Per3 , Tssk6 , and Arntl compared to control ( P <0.01). (B) Quantification of GLUT4 translocation in response to 30 nM insulin stimulation following siRNA knockdown of Per3 , Tssk6 , and Arntl. Translocation was measured as the relative change in luminescence compared to control cells. Ordinary one-way ANOVA with Dunnett's post hoc test comparing each siRNA-treated group to Scramble control ( n =3/group). P <0.05 was considered significant. Knockdown of Per3 and Arntl significantly reduced GLUT4 translocation, while Tssk6 knockdown caused a moderate but statistically non-significant reduction ( P >0.05). Data are presented as mean±s.d. ( n =3 biological replicates).

Article Snippet: Our choice of L6 rat skeletal muscle cells (ATCC, CRL-1458) was guided by their high insulin sensitivity, making them particularly suitable for insulin sensitivity assays ( ).

Techniques: Knockdown, Translocation Assay, Biomarker Discovery, Gene Expression, Control

Journal: Biology Open

Article Title: Live-cell GLUT4 translocation assay reveals Per3 as a novel regulator of circadian insulin sensitivity in skeletal muscle cells

doi: 10.1242/bio.061941

Figure Lengend Snippet: Role of Per3 in regulating GLUT4 translocation and glucose uptake. (A) Systematic representation of siRNA-mediated knockdown in L6 cells using Lipofectamine RNAiMAX. (B) Time-course analysis of GLUT4 translocation under 30 nM insulin stimulation in Per3 knockdown and scrambled control cells. Data represent the percentage change in luminescence over time, with fitted curves illustrating the distinct patterns of GLUT4 response between Per3 knockdown (red) and control (black). Statistical significance was determined using a JTK analysis with Per3 knockdown losing the rhythm. (C) Glucose uptake in human cells with PER3 knockdown compared to non-targeting control cells. Cells were treated with or without 30 nM insulin, and glucose uptake was measured as relative luminescence units (RLU). Knockdown of PER3 significantly reduced glucose uptake under insulin-stimulated conditions. Data are presented as mean±s.d. ( n =3 biological replicates). Statistical analysis was performed using two-way ANOVA. Statistics were performed using GraphPad Prism (10.4.1).

Article Snippet: Our choice of L6 rat skeletal muscle cells (ATCC, CRL-1458) was guided by their high insulin sensitivity, making them particularly suitable for insulin sensitivity assays ( ).

Techniques: Translocation Assay, Knockdown, Control