The Effect of Ex Vivo Human Serum from Liver Disease Patients on Cellular Protein Synthesis and Growth

Abstract

Sarcopenia is a common complication affecting liver disease patients, yet the underlying mechanisms remain unclear. We aimed to elucidate the cellular mechanisms that drive sarcopenia progression using an in vitro model of liver disease. C2C12 myotubes were serum and amino acid starved for 1-h and subsequently conditioned with fasted ex vivo serum from four non-cirrhotic non-alcoholic fatty liver disease patients (NAFLD), four decompensated end-stage liver disease patients (ESLD) and four age-matched healthy controls (CON) for 4- or 24-h. After 4-h C2C12 myotubes were treated with an anabolic stimulus (5 mM leucine) for 30-min. Myotube diameter was reduced following treatment with serum from ESLD compared with CON (−45%) and NAFLD (−35%; p < 0.001 for both). A reduction in maximal mitochondrial respiration (24% and 29%, respectively), coupling efficiency (~12%) and mitophagy (~13%) was identified in myotubes conditioned with NAFLD and ESLD serum compared with CON (p < 0.05 for both). Myostatin (43%, p = 0.04) and MuRF-1 (41%, p = 0.03) protein content was elevated in myotubes treated with ESLD serum compared with CON. Here we highlight a novel, experimental platform to further probe changes in circulating markers associated with liver disease that may drive sarcopenia and develop targeted therapeutic interventions.

Keywords: chronic liver disease; leucine; mitochondria; protein breakdown; sarcopenia.

Figure 1

Serum from NAFLD and ESLD patients induces myotube atrophy. (a) Representative images illustrating atrophy in myotubes treated with ex vivo serum from non-alcoholic fatty liver disease (NAFLD) and end stage liver disease (ESLD) patients, in comparison to myotubes treated with serum from age-matched control participants (CON), (b) mean myotube diameter, (c) mean nuclear fusion index (NFI). Data are expressed as the mean (cross), median (central horizontal line), 25th and 75th percentiles (box) and the minimum and maximum values (vertical lines), with n = 4 per group and each data point corresponding to the average of 3 technical repeats. **** p < 0.0001, *** p < 0.001.

Figure 2

Measures of MPS and anabolic signaling in response to treatment with CON, NAFLD and ESLD serum. Myotubes were treated for 4 h with ex vivo serum from age-matched control (CON), non-alcoholic fatty liver disease (NAFLD) and end stage liver disease (ESLD) patients. (a) Representative western blot for puromycin and total protein, (b) representative western blots for anabolic signaling targets and loading control, (c) puromycin incorporation, (d) phospho-mTOR (Ser2448)/total-mTOR, (e) phospho-eEF2 (Thr56)/total-eEF2, (f) phospho-p70S6K (Thr389)/total-p70S6K, (g) phospho-AktSer473/total-Akt, (h) phospho-RPS6 (Ser240/244)/total-RPS6, (i) phospho-4EBP-1 (Thr37/46)/total-4EBP-1. Data are expressed as the mean (cross), median (central horizontal line), 25th and 75th percentiles (box) and the minimum and maximum values (vertical lines), with n = 4 per group and each data point corresponding to the average of 3 technical repeats.

Figure 3

Markers of catabolic signaling are elevated within myotubes treated with serum from ESLD patients for 4 h and a 30 min treatment with leucine. Myotubes were treated with ex vivo human serum from age-matched control (CON), non-alcoholic fatty liver disease (NAFLD) and end stage liver disease (ESLD) patients for 4-h, with and without a 30-min treatment with 5 mM leucine. (a) Representative western blot images of catabolic signaling markers, (b) myostatin, (c) MAFbx, (d) MuRF-1, (e) caspase-3, (f) LC3A/B. Data are expressed as fold change in comparison to the CON. Data are expressed as the mean (cross), median (central horizontal line), 25th and 75th percentiles (box) and the minimum and maximum values (vertical lines), with n = 4 per group and each data point corresponding to the average of 3 technical repeats. * p < 0.05, ** p < 0.01.

Figure 4

Serum from NAFLD and ESLD patients induces impairments in mitochondrial respiration, despite no changes in markers of mitochondrial protein content. (a) Raw trace of oxygen consumption rate (OCR) for myotubes treated with ex vivo serum from non-alcoholic fatty liver disease (NAFLD), end stage liver disease (ESLD) and age-matched controls (CON), (b) basal mitochondrial respiration was assessed prior to the addition of oligomycin (OM), an ATP synthase inhibitor and represents the energetic demand of the cell in baseline conditions, (c) maximal mitochondrial respiration was calculated after the addition of the uncoupler BAM-15, (d) spare respiratory capacity indicates the ability of a cell to respond to an increase in energetic demand (e) proton leak respiration represents the portion of basal respiration not associated with ATP production (f) ATP coupled respiration was calculated upon the addition of OM, (g) coupling efficiency was calculated as the percentage of respiration accounted for by ATP production, (h) quantification of OXPHOS/total protein, (i) representative western blot of OXPHOS. Respirometry data were normalized to protein content. Western blot data are expressed as fold change in comparison to CON. Data are expressed as the mean (cross), median (central horizontal line), 25th and 75th percentiles (box) and the minimum and maximum values (vertical lines), with n = 4 per group and each data point corresponding to the average of 3 technical repeats. *** p < 0.001, * p < 0.05.

Figure 5

Serum from NAFLD and ESLD patients induces a reduction in mitophagy. (a) Representative images illustrating that treatment of mitoQC cells with ex vivo serum from patients with non-alcoholic fatty liver disease (NAFLD) and end stage liver disease (ESLD) in comparison to age-matched control (CON) patients in C2C12 myoblasts stably expressing mCherry-GFP-FIS1101-152, (b) quantification of mitophagy, expressed as mCherry/GFP. Increases in the relative mCherry/GFP ratio assessed by fluorescence intensity indicate mitophagy. Data are represented a fold change from CON. Data are expressed as the mean (cross), median (central horizontal line), 25th and 75th percentiles (box) and the minimum and maximum values (vertical lines), with each data point corresponding to the mean of 25 measurements per condition. An n = 4 was utilized within each group. ** p < 0.01.