Journal: Journal of Nutrition and Metabolism

Article Title: Leucine Modulates Mitochondrial Biogenesis and SIRT1-AMPK Signaling in C2C12 Myotubes

doi: 10.1155/2014/239750

Figure Lengend Snippet: Leucine-induced phosphorylation of AMPK and ACC requires SIRT1 in C2C12 myotubes. (a) C2C12 myotubes were serum starved overnight and treated with leucine (0.5 mM), alanine (0.5 mM), valine (0.5 mM), and DMSO for 6 hours. The cell lysates were assessed by western blotting analysis with specific antibodies against phosphor-AMPK α (Thr 172), phosphor-ACC (Ser 79), total AMPK α (Thr 172), and beta-actin. Integrated density values for the p-AMPK and p-ACC were normalized to total-AMPK band density and represented as dark or gray bars. (b) C2C12 myotubes were treated with 0.2% FBS medium overnight and then treated with leucine (0.5 mM), resveratrol (100 nM), and leucine plus EX527 (25 μ M) for 6 hours. Whole cell lysates were prepared and detected by western blotting with specific antibodies against phosphor-AMPK α , AMPK α , and beta-actin. Integrated density value for phosphor-AMPK was normalized to total-AMPK. *Significantly different from controls with P < 0.05.

Article Snippet: Primary antibodies for total AMPK, phospho-AMPK α (Thr172), total ACC (Acetyl-CoA Carboxylase), and phospho-ACC (Ser79) were obtained from Cell Signaling Technology Inc. (Danvers, MA, USA).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Glucose and Fatty Acids Synergize to Promote B-Cell Apoptosis through Activation of Glycogen Synthase Kinase 3β Independent of JNK Activation

doi: 10.1371/journal.pone.0018146

Figure Lengend Snippet: ( A ) MIN6 cells were treated with either control 0.5% BSA or 400 µM palmitate+0.5% BSA at a concentration of 5, 10, 15, 25 mM glucose for 18-h. Nuclear fractions were extracted from the cells and were subjected to Western blot analyses using anti-SREBP1 and anti-Lamin antibodies. 25 µg of nuclear protein was loaded in each lane. The upper band normalized over Lamin was used to do the quantification (the lower band is nonspecific). The relative ratio of nuclear SREBP1 over Lamin calculated by densitometries was summarized as means ± S.E.M. in the graph respectively. The representative results of three experiments are shown, and graphically illustrated, * p<0.05. ( B ) MIN6 cells were treated with either control 0.5% BSA or 400 µM palmitate+0.5% BSA at a concentration of either 5 or 25 mM glucose for 24-h. Total cell lysates were subjected to Western blot analysis using anti-acetyl CoA carboxylase (ACC) and anti-α-Tubulin antibodies. The representative results of two individual experiments are shown. ( C ) Cells were treated with either 500 µg/ml NaCl or 500 µg/ml TUDCA 15-h prior to beginning of palmitate treatment. Cells were co-treated with either 0.5% BSA or 400 µM palmitate+0.5% BSA with 25 mM glucose and NaCl or TUDCA for 18-h. Nuclear fractions were extracted from the cells and were subjected to Western blot analyses using anti-SREBP1 and anti-Lamin antibodies. The upper band normalized over Lamin was used to do the quantification (the lower band is nonspecific). 25 µg of nuclear extracts were loaded in each lane. The representative results of three individual experiments are shown. The relative ratio of nuclear SREBP1 over Lamin calculated by densitometries was summarized as means ± S.E.M. in the graph respectively **p<0.01.

Article Snippet: Antibodies used were anti-total Akt, anti-phospho-Akt (S473), anti-cleaved Caspase3, anti-phospho-PERK (980Thr), anti-phospho-eIF2α(51Ser), anti-total JNK1/2, anti-phospho-JNK, anti-phospho-AMPK, total AMPK, anti-phospho-ACC, anti-total ACC from Cell Signaling Technology (Beverley, MA), anti-SREBP1 from Neo Markers (Fremont, CA), anti-IRS1, anti-IRS2 from Upstate (Billerica, MA), anti-ATF3, anti-Insig1, anti-Lamin from Santa Cruz (Santa Cruz, CA) and anti-α-Tubulin and from Sigma (Saint Louis, MO).

Techniques: Concentration Assay, Western Blot

Journal: PLoS ONE

Article Title: Glucose and Fatty Acids Synergize to Promote B-Cell Apoptosis through Activation of Glycogen Synthase Kinase 3β Independent of JNK Activation

doi: 10.1371/journal.pone.0018146

Figure Lengend Snippet: Islets from 14 weeks of age C57BL/6 male mice were isolated as described in Methods and were treated with either control 0.5% BSA or 400 µM palmitate+0.5% BSA in RPMI medium containing either 11 mM or 30 mM glucose, 10% FBS for 72-h. Total cell lysates were extracted from the islets and subjected to Western blot analysis using ( A ) anti-cleaved Caspase3 and anti-β-Actin, ( B ) anti-IRS2 antibodies, ( C ) anti-phospho-JNK, anti-total JNK, anti-GRP78, anti-CHOP, anti-α-Tubulin antibodies, ( D ) anti-ATF3 antibodies, ( E ) anti-Acetyl CoA Carboxylase (ACC), anti-SREBP1, anti-α-Tubulin antibodies, ( F ) anti-Pdx1, anti-phospho-Gsk3β, anti-total Gsk3β, anti-phospho-Akt (S473), anti-phospho-cJun, anti-α-Tubulin antibodies. The blots shown are representative of 3 individual islet experiments. The relative ratio of indicated protein over β-Actin or α-Tubulin as a loading control calculated by densitometries was summarized as means ± S.E.M. in the graph respectively *p<0.05, **p<0.01.

Article Snippet: Antibodies used were anti-total Akt, anti-phospho-Akt (S473), anti-cleaved Caspase3, anti-phospho-PERK (980Thr), anti-phospho-eIF2α(51Ser), anti-total JNK1/2, anti-phospho-JNK, anti-phospho-AMPK, total AMPK, anti-phospho-ACC, anti-total ACC from Cell Signaling Technology (Beverley, MA), anti-SREBP1 from Neo Markers (Fremont, CA), anti-IRS1, anti-IRS2 from Upstate (Billerica, MA), anti-ATF3, anti-Insig1, anti-Lamin from Santa Cruz (Santa Cruz, CA) and anti-α-Tubulin and from Sigma (Saint Louis, MO).

Techniques: Isolation, Western Blot

Journal: PLoS ONE

Article Title: Glucose and Fatty Acids Synergize to Promote B-Cell Apoptosis through Activation of Glycogen Synthase Kinase 3β Independent of JNK Activation

doi: 10.1371/journal.pone.0018146

Figure Lengend Snippet: High glucose and FFA together result in a vicious negative cycle that ultimately promotes β-cell death. As suggested by our findings, high glucose addition to FFA treated β-cells results in much more activation of SREBP1 than glucose alone. SREBP1 enhances ACC expression with generation of malonyl-CoA which impairs FFA oxidation. This in turn leads to augmented ER stress with further activation of ER-localized SREBP1 as a result of degradation of the anchoring protein Insig1. The excess non-metabolized FFA due to more impairment of FFA oxidation would partition in ER membranes compounding ER stress. In addition to SREBP1, ER stress activates ATF3. Both nuclear SREBP1 and ATF3 result in inhibition of IRS2, with concomitant impairment of insulin signaling, activation of Gsk3β and reduction of Pdx1 leading to apoptosis.

Article Snippet: Antibodies used were anti-total Akt, anti-phospho-Akt (S473), anti-cleaved Caspase3, anti-phospho-PERK (980Thr), anti-phospho-eIF2α(51Ser), anti-total JNK1/2, anti-phospho-JNK, anti-phospho-AMPK, total AMPK, anti-phospho-ACC, anti-total ACC from Cell Signaling Technology (Beverley, MA), anti-SREBP1 from Neo Markers (Fremont, CA), anti-IRS1, anti-IRS2 from Upstate (Billerica, MA), anti-ATF3, anti-Insig1, anti-Lamin from Santa Cruz (Santa Cruz, CA) and anti-α-Tubulin and from Sigma (Saint Louis, MO).

Techniques: Activation Assay, Expressing, Inhibition