Metabolic reprogramming during hyperammonemia targets mitochondrial function and postmitotic senescence.

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

doi:10.1172/jci.insight.154089

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.

Highlights

Ammonia is a cytotoxic metabolite generated during the biological processes of amino acid catabolism, purine breakdown, and gut microbial metabolism [1, 2]
Whole-cell transcriptomics were integrated with proteomics from mitochondria isolated from hyperammonemic myotubes and from gastrocnemius muscle from the portacaval anastomosis (PCA)/sham rats and myotubes in response to ammonia withdrawal/lowering
Temporal cluster analyses have been reported by us earlier and are described in the Methods section. These clusters were grouped based on the similarity of the direction of responses during hyperammonemia and following ammonia withdrawal compared with UnT myotubes (Figure 1A)

Summary

Introduction

Ammonia is a cytotoxic metabolite generated during the biological processes of amino acid catabolism, purine breakdown, and gut microbial metabolism [1, 2]. In nonureagenic cells, increased uptake and metabolism of ammonia to glutamate and glutamine occurs via cataplerosis or loss of critical tricarboxylic acid (TCA) cycle intermediate, α-ketoglutarate (αKG) [4]. Insufficient anaplerotic response with continued cataplerosis results in mitochondrial oxidative dysfunction, decreased ATP synthesis, and increased generation of free radicals with oxidative modifications of proteins and senescence [4,5,6]. Dysregulated cellular protein homeostasis with decreased protein synthesis, one of the most energy-consuming processes within a cell, and increased autophagy, to support critical cellular substrate demands, occur during cellular ATP deficiency and an impaired response to anabolic stimuli that are observed in senescence [7,8,9]

Methods

Results

Conclusion