BMTs activate AMPK through CaMKK and not through intracellular Ca²⁺ changes.

<p>Serum-starved L6 myotubes were treated with either vehicle (V) alone, BMT-17 at 0.1 µM, 1 µM or 10 µM for 30 min before lysis; 10 µg of lysate was then analysed by Western blot (<b>A</b>) and quantified by densitometry (<b>B</b>). A representative blot is shown. Serum-starved L6 myotubes were pre-treated with either 150 µM EGTA-AM (+) or diluent (-) for 15 min before then being treated with 10 µM BMT-17 or vehicle for a further 30 min before lysis. AMPK was then isolated from 50 µg lysate by pan-AMPKβ immunoprecipitation and assessed by AMPK <i>in vitro</i> kinase assay (<b>C</b>). Data are means relative to untreated vehicle control (V) ± SEM from 5 independent experiments. *p<0.05, **p<0.01 to V by one-way ANOVA.</p

BMTs do not directly activate AMPK in isolated AMPK complexes.

<p>AMPK complexes were isolated from untreated 18 hr serum-starved L6 myotube lysates by immunoprecipitation using a pan-AMPKβ antibody. The isolated AMPK was then incubated <i>in vitro</i> with either vehicle (V) alone, 10 µM BMT-17, BMT-1 or Abbott compound (A-769662) in the presence or absence of 200 µM AMP in AMPK assay buffer for 10 min at 30°C. The <i>in vitro</i> kinase assay was then initiated with the addition of 200 µM ³²P-Mg.ATP and assayed for a further 10 min at 30°C. Incorporation of the ³²P into the substrate peptide was then assessed by beta scintillation counting. Data are means ± SEM for 5 separate experiments. **p<0.01 for compound effect, ^##p<0.01 for AMP effects by two-way ANOVA. No interaction between treatment groups was apparent.</p

Activation of AMPK involves CaMKKβ and not LKB1.

<p>Serum-starved HeLa cells (<b>A–C</b>) or L6 myotubes (<b>D–F</b>) were pre-treated ±1 µM STO-609 for 15 min before being incubated in either vehicle (0.1% DMSO), 2 mM AICAR, or 10 µM BMTs 17 or 1, for 30 min; or 1 µM ionomycin (Iono) for 10 min before lysis. Whole cell lysates (10 µg) were analysed by Western blot analysis (<b>A</b> and <b>D</b>) and quantified by densitometry (<b>B</b> and <b>E</b>). AMPK complexes were isolated by immunoprecipitation with a pan-AMPKβ antibody and assayed by <i>in vitro</i> AMPK kinase assay (<b>C</b> and <b>F</b>). Incorporation of ³²P into the AMARA substrate peptide was then assessed by β–scintillation counting. Data are means ± SEM (n = 4–8 per group). *p<0.05, **p<0.01 for compound effects by two-way ANOVA; ^##p<0.01 for STO-609 effects, ^†p<0.05, ^††p<0.01 for interactions between treatment groups.</p

Incubation of EDL with 25mM glucose diminishes AMPK Thr¹⁷² phosphorylation, ACC Ser⁷⁹ phosphorylation, and α2 AMPK activity, and increases AMPK Ser^485/491 phosphorylation.

<p>EDL muscles were incubated in Krebs-Henseleit solution containing 25 mM glucose for 30, 60 and 120 min. Phosphorylation of AMPK Thr^<b>172</b> (A), ACC Ser^<b>79</b> (B), AMPK Ser^{<b>485/491</b>} (C) were measured by western blot and AMPK activity (D) was determined using the SAMS peptide assay. Results are means <u>+</u> SE (n = 6). *P < 0.05 relative to 30 min incubation with 5.5 mM glucose.</p

Correction: Insulin and diet-induced changes in the ubiquitin-modified proteome of rat liver

<p>Correction: Insulin and diet-induced changes in the ubiquitin-modified proteome of rat liver</p

Insulin and diet-induced changes in the ubiquitin-modified proteome of rat liver

<div><p>Ubiquitin is a crucial post-translational modification regulating numerous cellular processes, but its role in metabolic disease is not well characterized. In this study, we identified the <i>in vivo</i> ubiquitin-modified proteome in rat liver and determined changes in this ubiquitome under acute insulin stimulation and high-fat and sucrose diet-induced insulin resistance. We identified 1267 ubiquitinated proteins in rat liver across diet and insulin-stimulated conditions, with 882 proteins common to all conditions. KEGG pathway analysis of these proteins identified enrichment of metabolic pathways, TCA cycle, glycolysis/gluconeogenesis, fatty acid metabolism, and carbon metabolism, with similar pathways altered by diet and insulin resistance. Thus, the rat liver ubiquitome is sensitive to diet and insulin stimulation and this is perturbed in insulin resistance.</p></div

Effects of high glucose on lactate and pyruvate.

<p>Data are means ± SEM (n+4–5/group). Lactate and pyruvate are expressed as μmol/mg muscle.</p><p>*P < 0.05 relative to 30 min incubation with 5.5mM glucose</p><p>Effects of high glucose on lactate and pyruvate.</p

Incubation of muscle in 25mM diminishes SIRT1 protein abundance, NAMPT activity, and NAD/NADH ratio after 1 or 2h.

<p>EDL were incubated with 25 mM glucose for 30, 60 or 120 min. Western blot analysis and quantification of representative blots are shown. SIRT1 protein expression (A) NAMPT activity (B) and NAD/NADH (C) were determined as described in the methods section. Results are means <u>+</u> SE (n = 6). *P < 0.05 compared to 30 min incubation with 5.5 mM glucose.</p

Rat liver ubiquitome.

<p><b>(A)</b> Experimental design of the ubiquitomic analysis rat liver. <b>(B)</b> Immunoblot analysis of protein ubiquitination in representative samples of rat liver lysates. (<b>C)</b> Venn diagram of previously identified protein in this study and that in the known rat ubiquitome. <b>(D)</b> Ontology analysis of identified ubiquitinylated proteins. <b>(E)</b> Gluconeogenesis/Glycolysis biochemical pathway with ubiquitinylated enzymes marked in red.</p

High-fat, high-sucrose diet alters the ubiquitome of the rat liver.

<p><b>(A)</b> Venn diagram of identified proteins ubiquitinated in HFSD compared to HFSD + Insulin. <b>(B)</b> Ontology analysis of identified ubiquitinylated proteins in HFSD compared to HFSD + Insulin. <b>(C)</b> STRING analysis of differentially ubiquitinated proteins. <b>(D)</b>. Ontology analysis of identified ubiquitinated proteins responsive to insulin in Chow but not in HFSD.</p