H55N polymorphism is associated with low citrate synthase activity which regulates lipid metabolism in mouse muscle cells

Funding: This work was supported, in whole or in part, by European Social Fund under the Global Grant measure Grant VP1-3.1-ŠMM-07-K-02-057 (to A.L.), European Foundation for the Study of Diabetes grant (to T.V.), NHS Grampian Endowment grant (to A.R. and S.R.G.), Kuwait Ministry of Health grant (to M.A.), Saudi Ministry of Higher Education grant (to Y.A.,) as well as Saltire scholarship, Wenner-Gren Foundation Postdoctoral Fellowship, Albert Renold Travel Fellowship and a Novo Nordisk Foundation Challenge Grant (to B.G.).Peer reviewedPublisher PD

Cell signalling proteins in Con shRNA and Cs shRNA myotubes.

<p>The protein levels were was assayed by immunoblotting and the blots were analyzed using ImageJ software. The ratios of phosphorylated to total protein were calculated for AMPK (A) and ACC (B), MAPK p38 (C) and mTOR (D) for Con shRNA cells (n = 8) and Cs shRNA (n = 8) cells. Values are means ± SEM.</p

Cellular metabolism in Con shRNA and Cs shRNA myotubes.

<p>A, Oxygen consumption rate and (B) proton production rate were assessed using Seahorse Bioscience XF24-3 Extracellular Flux Analyser; C, Basal respiration, maximal respiration and spare respiratory capacity; D, Proton leak, aerobic ATP production and non-mitochondrial respiration. Values are means ± SEM (n = 10 each); *P < 0.05, ** <i>p</i> < 0.01 different between Con shRNA and Cs shRNA myotubes.</p

Palmitate metabolism in C2C12 muscle cells.

<p>The cells were treated with Con shRNA or Cs shRNA and then incubated in the differentiation media containing 5.5 mM glucose (G) and/or 0.8 mM palmitate for 2 h (P). A, palmitate oxidation was assessed by adding [1-¹⁴C]palmitate to the media and measuring its incorporation into CO₂ (n = 6); B, palmitate incorporation was measured as from the amount of [1-¹⁴C]palmitate cell lysates generated from the cells after the experiments with palmitate oxidation (n = 6). Results are means ± SEM; * <i>p</i> < 0.05 between Cs shRNA and Con shRNA cells.</p

Citrate synthase (CS) activity, mitochondrial markers and proliferation of Con shRNA and Cs shRNA cells.

<p>A, citrate synthase (CS) activity; B, CS protein levels; C, HAD activity; D, cytochrome C oxidase (COX) activity; E, proliferation rate as reflected in cell impedance index of growing cells. CS and HAD activity (n = 9 each) was measured using a spectrophotometric assays. COX activity (n = 6) was assessed using COX assay kit; F, Citrate accumulation was measured using the standard assay kit. Values for CS, HAD, COX, and citrate are shown as mean ± SEM. Cell impedance index was assessed in cells incubated in growth media and differentiation media (n = 4). Cells were grown in 6 well plates and the media was changed every day.</p

Citrate synthase (CS) activity in mouse tissues.

<p>Data are shown for the heart (A), liver (B) and gastrocnemius (C) muscle of BALB, C57BL/6J (B6), offspring of the cross between B6 and congenic B6.A-(rs3676616-D10Utsw1)/KjnB6 (B6/B6.A) mice, congenic B6.A and A/J mice, respectively. CS activity and protein levels were measured using spectrophotometric assays. Data are shown as mean ± S.E. Numbers of samples are indicated below in brackets. ** <i>p</i> < 0.01, *** <i>p</i> < 0.001 are different to BALB and B6 strains, respectively; ## <i>p</i> < 0.01 different to B6/B6.A mice, respectively.</p