Fig. 6.

Ethanol impairs and HMB restores mitochondrial respiration in differentiated C2C12 myotubes and ATP content in muscle from mice model of ALD. (A). Representative tracings of high-resolution respirometry to quantify intact cell respiration of differentiated C2C12 myotubes. After initial stabilization, ATP synthetase inhibitor, oligomycin (O) was added, and oxygen consumption quantified to determine the oligomycin-sensitive and -insensitive respiration. Uncoupler of oxidative phosphorylation, FCCP (U) at 0.5 μM increments was then added to quantify maximum respiratory capacity. This was followed by rotenone (R) 375 nM final concentration, to inhibit complex I of the ETC, and then 2.5 μM antimycin A (Aa), which inhibits complex III, was added to determine non-mitochondrial respiration. (B) Intact, non-permeabilized C2C12 myotubes in basal DMEM medium were either untreated or treated with 100 mM ethanol for 6h with and without 50μM HMB for 6h. Basal cell respiration, proton leak, ATP-linked respiration, maximum respiratory capacity (Max. Resp.) from the response to FCCP and reserve respiratory capacity (Reserve Resp.) were measured. (C) Representative tracings of high-resolution respirometry to quantify respiration of permeabilized differentiated C2C12 myotubes. After initial stabilization, 2 mM malate (M) and 2.5 mM pyruvate (P) were added. This was followed by 4.1 mM digitonin (Dig) to permeabilize the cell membrane without losing the integrity of cells or mitochondria for permitting entry of mitochondrial substrates inside the cells; 2.5 mM ADP (D); 10 mM glutamate (G); 10 mM succinate (S); 2 mM increments of FCCP (U) for measuring maximum respiration; 375 nM rotenone (R); 125 nM antimycin A (Aa); 2 mM ascorbate and 2 mM TMPD (tetramethyl p-phenylene diamine) (AT) to test complex IV activity; 50 mM sodium azide (Az) to inhibit complex IV activity. (D) Oxygen consumption was measured in intact non-permeabilized C2C12 myotubes treated with 100 mM ethanol with and without HMB in mitochondrial respiration buffer followed by digitonin permeabilization and ETC complex specific substrates and inhibitors sequentially in the concentrations as stated above. Intact cell respiration, oxidative phosphorylation (OXPHOS) in response to M, P, D, G and S, and Max. R and RR capacity (response to U) were quantified. Rotenone-sensitive and -insensitive respiration and complex II and IV function were measured. (E) Blue native gel electrophoresis of isolated mitochondria to evaluate mitochondrial supercomplex assembly. (F)Total ATP content in C2C12 myotubes either untreated or treated with 100mM ethanol for 6hours with and without 50 μM HMB. (G) ATP content in gastrocnemius muscle from mALD mice with and without HMB compared to that from PF mice with and without HMB. * p<0.05; ** p<0.01, *** p<0.001. EtOH: ethanol; HMB β-hydroxy-β-methyl butyrate; UnT: untreated controls; PF: pair-fed mice; mALD: mouse model of ALD.