Metabolic reprogramming during hyperammonemia targets mitochondrial function and postmitotic senescence
Avinash Kumar, Nicole Welch, Saurabh Mishra, Annette Bellar, Rafaella Nasciemento Silva, Ling Li, Shashi Shekhar Singh, Mary Sharkoff, Alexis Kerr, Aruna Kumar Chelluboyina, Jinendiran Sekar, Amy H. Attaway, Charles Hoppel, Belinda Willard, Gangarao Davuluri, Srinivasan Dasarathy
Avinash Kumar, Nicole Welch, Saurabh Mishra, Annette Bellar, Rafaella Nasciemento Silva, Ling Li, Shashi Shekhar Singh, Mary Sharkoff, Alexis Kerr, Aruna Kumar Chelluboyina, Jinendiran Sekar, Amy H. Attaway, Charles Hoppel, Belinda Willard, Gangarao Davuluri, Srinivasan Dasarathy
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Research Article Cell biology Hepatology

Metabolic reprogramming during hyperammonemia targets mitochondrial function and postmitotic senescence

Abstract

Ammonia is a cytotoxic metabolite with pleiotropic molecular and metabolic effects, including senescence induction. During dysregulated ammonia metabolism, which occurs in chronic diseases, skeletal muscle becomes a major organ for nonhepatocyte ammonia uptake. Muscle ammonia disposal occurs in mitochondria via cataplerosis of critical intermediary metabolite α-ketoglutarate, a senescence-ameliorating molecule. Untargeted and mitochondrially targeted data were analyzed by multiomics approaches. These analyses were validated experimentally to dissect the specific mitochondrial oxidative defects and functional consequences, including senescence. Responses to ammonia lowering in myotubes and in hyperammonemic portacaval anastomosis rat muscle were studied. Whole-cell transcriptomics integrated with whole-cell, mitochondrial, and tissue proteomics showed distinct temporal clusters of responses with enrichment of oxidative dysfunction and senescence-related pathways/proteins during hyperammonemia and after ammonia withdrawal. Functional and metabolic studies showed defects in electron transport chain complexes I, III, and IV; loss of supercomplex assembly; decreased ATP synthesis; increased free radical generation with oxidative modification of proteins/lipids; and senescence-associated molecular phenotype–increased β-galactosidase activity and expression of p16INK, p21, and p53. These perturbations were partially reversed by ammonia lowering. Dysregulated ammonia metabolism caused reversible mitochondrial dysfunction by transcriptional and translational perturbations in multiple pathways with a distinct skeletal muscle senescence-associated molecular phenotype.

Authors

Avinash Kumar, Nicole Welch, Saurabh Mishra, Annette Bellar, Rafaella Nasciemento Silva, Ling Li, Shashi Shekhar Singh, Mary Sharkoff, Alexis Kerr, Aruna Kumar Chelluboyina, Jinendiran Sekar, Amy H. Attaway, Charles Hoppel, Belinda Willard, Gangarao Davuluri, Srinivasan Dasarathy

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Figure 6

Hyperammonemia reversibly impairs mitochondrial respiration in differentiated C2C12 myotubes.

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Hyperammonemia reversibly impairs mitochondrial respiration in different...
(A) Representative oxygraph tracings from myotubes that were untreated (UnT), 24 hours ammonium acetate treated (AmAc), or following ammonia withdrawal for 24 hours (WD). Oxygen consumption was measured in intact nonpermeabilized myotubes in DMEM in response to ATP synthase inhibitor (oligomycin), uncoupler of oxidative phosphorylation trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP) (U), rotenone, and a complex I inhibitor (R) and complex III inhibitor, antimycin A (Aa). (B) High-resolution respirometry to quantify oxygen consumption of permeabilized, differentiated C2C12 myotubes in mitochondrial respiration buffer by digitonin permeabilization and electron transport chain (ETC) complex–specific substrates and inhibitors sequentially in the concentrations as stated below. Proton leak, oxidative phosphorylation (OXPHOS) in response to malate (M), pyruvate (P), ADP (D), glutamate (G), and succinate (S), and maximum respiration (Max. R) and reserve respiratory capacity (R.R.) i.e., the response to FCCP (U), were quantified. Rotenone-sensitive and -insensitive respiration followed by complex IV function were measured. After initial stabilization, 2 mM malate and 2.5 mM pyruvate were added. This was followed by 4.1 μM digitonin 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, 10 mM glutamate, 10 mM succinate, 2 μM increments of FCCP for measuring maximum respiration, 375 nM rotenone (R), 125 nM Aa, 2 mM ascorbate (AT) and 2 mM tetramethyl phenylene diamine (TMPD) to test complex IV activity, and 50 mM sodium azide (Az) to inhibit complex IV activity. All data expressed as mean ± SD from at least 6 biological replicates for oxygraph studies in myotubes. *P < 0.05; **P < 0.01; ***P < 0.001 compared with respective controls by 1-way ANOVA followed by Bonferroni’s post hoc comparison tests.