l6 cells  (ATCC)

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