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 5

Skeletal muscle proteome from hyperammonemic or control rats with and without ammonia-lowering therapy.

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Skeletal muscle proteome from hyperammonemic or control rats with and wi...
(A) Heatmap of the hierarchically arranged differentially expressed proteins (DEPs) of the proteome from gastrocnemius muscle from rats that underwent either a sham surgery (Sham) or a portacaval anastomosis (PCA) surgery with/without the ammonia-lowering agents L-ornithine-L-aspartate and rifaximin (LOLA). Each row represents 1 protein and each column represents 1 biological replicate (red = increased expression, blue = decreased expression). (B) Venn diagram of unique and shared DEPs in the tissue proteome from skeletal muscle from sham- and PCA-operated rats with/without LOLA. (C) Functional enrichment analyses of canonical pathways specifically curated for critical protein homeostasis and mitochondrial function in the skeletal muscle proteome from PCA rats treated with LOLA compared with Sham rats (green = decreased expression, red = increased expression). (D) Functional enrichment analyses of canonical pathways specifically curated for critical protein homeostasis and mitochondrial function in the skeletal muscle proteome from PCA rats treated with/without LOLA (green = decreased expression, red = increased expression). (E) Venn diagram of unique and shared DEPs in the muscle proteome data set overlaid with the genes listed in MitoCarta3.0 (shaded in yellow) and the DEPs known to be expressed in skeletal muscle within MitoCarta3.0 (shaded in purple). Verified mitochondrial DEPs in the PCA+LOLA versus Sham comparison (enclosed by a red line), PCA+LOLA versus PCA (green line), and PCA versus Sham (blue line). Mitochondrial DEPs refer to those matched with MitoCarta3.0. P value cutoff for all DEPs was set at P < 0.10 using a 2-tailed Student’s t test. All studies were performed in n = 5 biological replicates. Significance cutoff for all canonical pathways was set at –log(P value) ≥ 1.3 by the right-sided Fisher’s exact test.