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First published online 23 March 2004
doi: 10.1242/jcs.01069

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Glucose induces de novo lipogenesis in rat muscle satellite cells through a sterol-regulatory-element-binding-protein-1c-dependent pathway

Département d'Endocrinologie, Institut Cochin, Institut National de la Santé et de la Recherche Médicale (INSERM) U567, Centre National de la Recherche Scientifique (CNRS), Unité Mixte de Recherche 8104, Université René Descartes, Paris, France

View larger version (22K): [in a new window]   Fig. 1. Glucose increases glucose uptake in contracting myotubes. After 48 hours in serum-free medium containing 5 mM glucose, 2-deoxyglucose (2-DG) uptake was measured after a 30 minute incubation of the cells with 5 mM glucose (G5), 25 mM glucose (G25) or 25 mM mannitol. Results are the means±s.e.m of 10-13 independent experiments. *P<0.05 versus G5.

View larger version (32K): [in a new window]   Fig. 2. Time course of the effect of 25 mM glucose on lipogenic enzymes in myotubes. After 48 hours in serum-free medium containing 5 mM glucose, contracting myotubes were incubated for the indicated times with 25 mM glucose. Total protein content was then extracted, separated by SDS-PAGE and analysed for ACC2, FAS, HKII and CPT2 protein levels, and for the phosphorylation state of ACC protein (A). (B) ACC activity, deduced from the ratio between ACC2 and phosphorylated ACC protein level. Western blots represent three independent experiments.

View larger version (34K): [in a new window]   Fig. 3. Glucose induces de novo lipogenesis and lipid accumulation in contracting myotubes. (A) After 48 hours in serum-free medium containing 5 mM glucose (G5), cells were treated or not with 100 nM insulin for the last 24 hours. The lipogenesis rate from [2-14C]acetate (5 mM) was measured in the presence or absence of 25 mM glucose (G25) and/or 5 µM TOFA during the last 3 hours of culture. Results are the means±s.e.m. of 3-13 independent experiments. *P<0.05 compared with G5; **P<0.01 compared with the respective controls. (B) After a 24-hour treatment with 100 nM insulin or 25 mM glucose (G25), myotubes were stained with Oil Red-O to detect the presence of intracellular lipid droplets. Nuclei were counterstained with DAPI. Scale bar, 15 µm.

View larger version (77K): [in a new window]   Fig. 4. Glucose induces the processing of SREBP-1c precursor (p) and the nuclear translocation of the mature form (m). Contracting myotubes were cultured in 5 mM glucose without serum for 48 hours. (A) Cytoplasmic (top) and nuclear (bottom) extracts were prepared at 0 minutes, 15 minutes, 30 minutes or 60 minutes after the addition of 25 mM glucose. The western blots represent three separate experiments. (B,C) Detection of the precursor and nuclear forms of SREBP-1c by immunofluorescence in contracting myotubes. In absence of 25 mM glucose, SREBP-1c precursor (green) was observed in the cytoplasm (a,b,g,h). Following the addition of 25 mM glucose, nuclear translocation of SREBP-1c was detected in myotubes after 30 minutes (c,d,i,j) and remained up to 60 minutes (e,f,k,l). Nuclei were counterstained with DAPI. Scale bars, 20 µm (B), 5 µm (C).

View larger version (40K): [in a new window]   Fig. 5. Silencing of SREBP-1 protein in contracting myotubes. After 48 hours in serum-free medium containing 5 mM glucose, myotubes were transfected with a SREBP-1 siRNA duplex. After 48 hours, total protein extracts were prepared at 0 hours, 1 hour, 2 hours or 3 hours after the addition of 25 mM glucose (G25). Western blotting was performed to detect SREBP-1c precursor (p), FAS, ACC2 and CPT2 proteins. Representative western blots of three independent experiments are shown.

View larger version (56K): [in a new window]   Fig. 6. Processing of SREBP-1c by glucose depends on the phosphorylation of STAT3. Contracting myotubes were cultured for 48 hours in 5 mM glucose without serum. (A) Cytoplasmic and nuclear extracts were prepared at 0 minutes, 15 minutes, 30 minutes, 60 minutes and 180 minutes after the addition of 25 mM glucose (G25). The western blots were probed using antibodies against SREBP-1c precursor (p) and mature (m) forms, Tyr705-phosphorylated STAT3, Tyr701-phosphorylated STAT1 or phosphorylated Erk1/Erk2 proteins. (B) Contracting myotubes were incubated in 5 mM (G5) or 25 mM (G25) glucose for 30 minutes in the absence or presence of AG490 (a specific inhibitor of Jak2 phosphorylation) or PD98059 (an inhibitor of MAPK phosphorylation). Cytoplasmic and nuclear extracts were prepared, analysed by SDS-PAGE and immunoblotted using antibodies against SREBP-1c and phosphorylated-STAT3. Western blots are representative of three separate experiments.