primary human skeletal myoblasts  (PromoCell)

Journal: Science Advances

Article Title: Reducing the mitochondrial oxidative burden alleviates lipid-induced muscle insulin resistance in humans

doi: 10.1126/sciadv.adq4461

Figure Lengend Snippet: ( A ) Schematic overview of the major signaling events modulating insulin-stimulated glucose uptake in human skeletal muscle. ( B ) Proximal insulin signaling, as determined by phosphorylation of Akt2 on Thr308 and Ser473 in whole-muscle homogenates. ( C and D ) Distal insulin signaling, as determined by phosphorylation of the Akt substrates GSK3β on Ser9 (C) and TBC1D4 on Thr642 (D) in whole-muscle homogenates. ( E ) GLUT4 translocation, as determined by GLUT4 protein abundance in plasma membrane protein fractions. In-gel stain-free technology was used as a loading control. Lipid, n = 8 (Pre-clamp) and n = 9 (End-clamp); Lipid + mtAO, n = 10. Representative blots ( n = 2 biological replicates from each muscle biopsy sample for each patient). ( F ) Purity of the isolated plasma membrane protein fractions (used to determine GLUT4 translocation), as determined by immunoblot analysis of actin (cytosolic protein marker) and Na + /K + -ATPase subunit α1 (plasma membrane protein marker) in plasma membrane homogenates (PM) as compared with the corresponding whole-muscle homogenates (WM). PM and WM samples were obtained by pooling a given volume of each individual sample. ( G ) Pearson’s correlation between mtAO-induced changes in plasma membrane GLUT4 and leg glucose uptake under insulin stimulation. n = 9. Data [(B) to (E)] presented as observed individual values with estimated means ±95% confidence limits. Linear mixed models were used to estimate within- and between-treatment differences at End-clamp [(B) to (E)]. *Different from Pre-clamp ( P < 0.05). n = 10, unless otherwise stated. Illustrations in (A) were created with BioRender.com .

Article Snippet: Primary human skeletal muscle (HSKM) myoblasts obtained from m. rectus abdominis of a 17-year-old, nondiabetic male (BMI: 27 kg/m 2 ) (SK-1111, Donor: P01052-17M, Cook Myosite) were grown to confluency in Myotonic Basal Media (MB-2222) supplemented with 10% Myotonic Growth Supplement (MS-3333) and 1% antibiotic-antimycotic.

Techniques: Phospho-proteomics, Translocation Assay, Quantitative Proteomics, Clinical Proteomics, Membrane, Staining, Control, Isolation, Western Blot, Marker

Journal: Science Advances

Article Title: Reducing the mitochondrial oxidative burden alleviates lipid-induced muscle insulin resistance in humans

doi: 10.1126/sciadv.adq4461

Figure Lengend Snippet: ( A ) Workflow to quantitatively analyze redox-sensitive proteins and lipid peroxidation in skeletal muscle biopsy samples. ( B ) Cytosolic oxidative burden as determined by protein abundance of peroxiredoxin 2 (PRDX2) dimers relative to monomers in whole-muscle homogenates. ( C ) Mitochondrial oxidative burden as determined by protein abundance of peroxiredoxin 3 (PRDX3) dimers relative to monomers in whole-muscle homogenates. ( D ) Overall peroxiredoxin oxidation as determined by protein abundance of oxidized/hyperoxidized peroxiredoxin (PRDX-SO 2/3 ) dimers in whole-muscle homogenates. ( E ) Whole-muscle lipid peroxidation as determined by protein abundance of 4-hydroxynonenal (4-HNE) adducts in whole-muscle homogenates. ( F ) Representative blots related to the experiments in human skeletal muscle biopsy samples ( n = 2 biological replicates from each muscle biopsy sample for each patient). Coomassie blue staining was used as a loading control for PRDX-SO 2/3 and 4-HNE. ( G and H ) Cytosolic and mitochondrial oxidative burden in human myotubes treated with either vehicle BSA (control; n = 5), 250 μM palmitate ( n = 5), or 500 μM palmitate. Relative PRDX2 and PRDX3 dimer abundance (dimer-to-monomer ratio) was normalized to cell plate–specific mean dimerization to adjust for N -ethylmaleimide treatment efficiency. Human data are presented as observed individual values with estimated means ±95% confidence limits. Linear mixed models were used to estimate within- and between-treatment differences (B to E). *Different from Baseline ( P < 0.05). n = 10 for all measurements. Human muscle cell data are presented as observed values with means ±95% confidence limits. A one-way ANOVA was used to estimate between-treatment differences (H). Illustrations in (A) and (G) were created with BioRender.com .

Article Snippet: Primary human skeletal muscle (HSKM) myoblasts obtained from m. rectus abdominis of a 17-year-old, nondiabetic male (BMI: 27 kg/m 2 ) (SK-1111, Donor: P01052-17M, Cook Myosite) were grown to confluency in Myotonic Basal Media (MB-2222) supplemented with 10% Myotonic Growth Supplement (MS-3333) and 1% antibiotic-antimycotic.

Techniques: Quantitative Proteomics, Staining, Control

Journal: Science Advances

Article Title: Reducing the mitochondrial oxidative burden alleviates lipid-induced muscle insulin resistance in humans

doi: 10.1126/sciadv.adq4461

Figure Lengend Snippet: ( A ) Workflow to determine ex vivo mitochondrial bioenergetics in human skeletal muscle, i.e., in permeabilized fiber bundles (PmFBs), using a substrate-inhibitor titration (SUIT) protocol reflecting normal (20 μM P-CoA) versus high (60 μM P-CoA) intramyocellular lipid conditions. PmFBs were preincubated in either decylTPP (control compound) or mitoquinone to evaluate the effects of mtAO on lipid-induced mitochondrial stress. ( B ) SUIT protocol used to simultaneously measure mitochondrial O 2 consumption ( J O 2 ) and H 2 O 2 emission ( J H 2 O 2 ) rates. Succinate (Succ); pyruvate (Pyr); malate (Mal); adenosine diphosphate (ADP); glutamate (Glut); cytochrome C (Cyt C); oligomycin (Omy). Data are means ± SEM. ( C ) Muscle mitochondrial content, as determined by citrate synthase (CS) activity in muscle homogenates obtained from separate portions of the biopsy specimen used for assessments of mitochondrial bioenergetics. Data are presented as individual values with estimated mean ± 95% confidence limits. ( D ) Maximal mitochondrial oxidative phosphorylation capacity (OXPHOS), oligomycin-induced leak respiration (LEAK Omy ), and OXPHOS efficiency [calculated as 1 − RCR = 1 − LEAK Omy /OXPHOS ]. Data are presented as individual values with estimated means ±95% confidence limits. ( E ) Sensitivity of mitochondrial J O 2 to ADP. The apparent half-maximal effective concentration (EC 50 ) for ADP was determined using [agonist] versus response (three parameters) analysis in GraphPad Prism. Data are means ± SEM. ( F ) Maximal and submaximal mitochondrial H 2 O 2 emission rates. Data are presented as individual values with estimated means ±95% confidence limits. ( G ) Sensitivity of mitochondrial J H 2 O 2 to ADP. The apparent half-maximal inhibitory concentration (IC 50 ) for ADP was determined using [inhibitor] versus response (three parameters) analysis in GraphPad Prism. Data are means ± SEM. A linear mixed model [(D) and (F)] or one-way ANOVA [(E) and (G)] was used to estimate between-treatment differences. n = 10 for all measurements. Illustrations in (A) were created with BioRender.com .

Article Snippet: Primary human skeletal muscle (HSKM) myoblasts obtained from m. rectus abdominis of a 17-year-old, nondiabetic male (BMI: 27 kg/m 2 ) (SK-1111, Donor: P01052-17M, Cook Myosite) were grown to confluency in Myotonic Basal Media (MB-2222) supplemented with 10% Myotonic Growth Supplement (MS-3333) and 1% antibiotic-antimycotic.

Techniques: Ex Vivo, Titration, Control, Activity Assay, Phospho-proteomics, Concentration Assay