In liver the mitochondrial sirtuin, SIRT5, controls ammonia detoxification by regulating CPS1, the first enzyme of the urea cycle. However, while SIRT5 is ubiquitously expressed, urea cycle and CPS1 are only present in the liver and, to a minor extent, in the kidney. To address the possibility that SIRT5 is involved in ammonia production also in nonliver cells, clones of human breast cancer cell lines MDA-MB-231 and mouse myoblast C2C12, overexpressing or silenced for SIRT5 were produced. Our results show that ammonia production increased in SIRT5-silenced and decreased in SIRT5-overexpressing cells. We also obtained the same ammonia increase when using a new specific inhibitor of SIRT5 called MC3482. SIRT5 regulates ammonia production by controlling glutamine metabolism. In fact, in the mitochondria, glutamine is transformed in glutamate by the enzyme glutaminase, a reaction producing ammonia. We found that SIRT5 and glutaminase coimmunoprecipitated and that SIRT5 inhibition resulted in an increased succinylation of glutaminase. We next determined that autophagy and mitophagy were increased by ammonia by measuring autophagic proteolysis of long-lived proteins, increase of autophagy markers MAP1LC3B, GABARAP, and GABARAPL2, mitophagy markers BNIP3 and the PINK1-PARK2 system as well as mitochondrial morphology and dynamics. We observed that autophagy and mitophagy increased in SIRT5-silenced cells and in WT cells treated with MC3482 and decreased in SIRT5-overexpressing cells. Moreover, glutaminase inhibition or glutamine withdrawal completely prevented autophagy. In conclusion we propose that the role of SIRT5 in nonliver cells is to regulate ammonia production and ammonia-induced autophagy by regulating glutamine metabolism.
Keywords: ACTB, actin, β; ATG, autophagy-related; BNIP3, BCL2/adenovirus E1B 19kDa interacting protein 3; BPTES, bis-2-(5-phenylacetamido-1, 3, 4-thiadiazol-2-yl)ethyl sulfide; COX4I1, cytochrome c oxidase subunit IV isoform 1; CPS1, carbamoyl-phosphate synthase 1, mitochondrial; GABARAP, GABA(A) receptor-associated protein; GABARAPL2, GABA(A) receptor-associated protein-like 2; GLS, glutaminase; GLUD1, glutamate dehydrogenase 1; GLUL, glutamate-ammonia ligase; MAP1LC3B, microtubule-associated protein 1 light chain 3 β; MFN2, mitofusin 2; OPA1, optic atrophy 1 (autosomal dominant); PARK2, parkin RBR E3 ubiquitin protein ligase; PEG, polyethylene glycol; PINK1, PTEN induced putative kinase 1; SIRT5, sirtuin 5; SQSTM1, sequestosome 1; TCA, tricarboxylic acid cycle; TEM, transmission electron microscopy; ammonia; autophagy; glutaminase; glutamine; hexachlorophene, 2, 2′-methylenebis(3, 4, 6-trichlorophenol); mitochondrial dynamics; mitophagy; molecular rehabilitation; sirtuin 5; succinylation.