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α-Ketoglutarate inhibits autophagy

α-ketoglutarate autophagy

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#1 Engadin

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Posted 28 June 2019 - 12:11 PM


F U L L   T E X T :   AgING

 

 

 

Abstract

The metabolite α-ketoglutarate is membrane-impermeable, meaning that it is usually added to cells in the form of esters such as dimethyl −ketoglutarate (DMKG), trifluoromethylbenzyl α-ketoglutarate (TFMKG) and octyl α-ketoglutarate (O-KG). Once these compounds cross the plasma membrane, they are hydrolyzed by esterases to generate α-ketoglutarate, which remains trapped within cells. Here, we systematically compared DMKG, TFMKG and O-KG for their metabolic and functional effects. All three compounds similarly increased the intracellular levels of α−ketoglutarate, yet each of them had multiple effects on other metabolites that were not shared among the three agents, as determined by mass spectrometric metabolomics. While all three compounds reduced autophagy induced by culture in nutrient-free conditions, TFMKG and O-KG (but not DMKG) caused an increase in baseline autophagy in cells cultured in complete medium. O-KG (but neither DMKG nor TFMK) inhibited oxidative phosphorylation and exhibited cellular toxicity. Altogether, these results support the idea that intracellular α-ketoglutarate inhibits starvation-induced autophagy and that it has no direct respiration-inhibitory effect.

 

 

Introduction

α-Ketoglutarate, also known as 2-oxoglutaric acid (IUPAC name: 2-oxopentanedioic acid) is an intermediate of the Krebs cycle, as well as the keto acid produced by deamination of glutamate. In the Krebs cycle, α-ketoglutarate is generated by oxidative decarboxylation of isocitrate (catalyzed by isocitrate dehydrogenase) and then used to generate succinyl coenzyme A (CoA) (catalyzed by α-ketoglutarate dehydrogenase) [1]. During glutaminolysis, glutamine is converted into glutamate and then α-ketoglutarate, which can be introduced into the Krebs cycle as an anaplerotic substrate [2] and is often used in cancer cells (which heavily rely on glutaminolysis) for reductive carboxylation to generate succinate [3].

 

Macroautophagy (here referred to as ‘autophagy’) is a phylogenetically conserved degradation pathway in which portion of the cytoplasm are wrapped into double-membraned organelles, the autophagosomes, which then fuse with lysosomes for the enzymatic hydrolysis of macromolecules contained in the autophagic cargo into micromolecules that can be used for anabolic reactions or bioenergetic purposes [4,5] Macroautophagy has prominent cytoprotective functions, meaning that it increases the resistance of cells to a variety of external stress signals [6]. In vivo, chronic or cyclic induction of autophagy can cause the extension of lifespan in model organisms including yeast, nematodes, flies and mice [79]. Thus, genetic manipulations designed to increase autophagy can increase the health span and longevity of mice [7,10]. Moreover, a few universally effective anti-aging interventions such as caloric restriction [8,1113], inhibition of mechanistic target of rapamycin complex 1 (MTORC1) [1416] or supplementation of spermidine [1720] rely on autophagy, meaning that knockout of essential autophagy genes abolishes their benefits.

 

Recently, several papers have been published that claim contradictory effects of α-ketoglutarate on autophagy. Several works indicate that addition of the α-ketoglutarate precursor dimethyl α-ketoglutarate (DMKG) to human cell cultures or its intraperitoneal injection into mice effectively inhibits autophagy through an anaplerosis-dependent increase in acetyl-CoA (AcCoA) levels, thus, increasing the acetylation of cytoplasmic proteins [2125]. In contrast, another paper claims that addition of another α-ketoglutarate precursor, octyl α-ketoglutarate (O-KG) induces autophagy both in human cells and in Caenorhabditis elegans, because it inhibits the mitochondrial ATP synthase as well as MTORC1 [26,27].

 

We hence decided to systematically compare the effects of distinct α-ketoglutarate precursors on cellular metabolism and functions in vitro. Here, we report that three distinct α-ketoglutarate precursors have rather distinct effects and that respiratory chain inhibition and autophagy induction are not universally found in conditions in which intracellular α-ketoglutarate are successfully elevated. Rather, at least in starvation conditions, autophagy inhibition appears to be the universal result of intracellular α-ketoglutarate increases.

 

 

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