Spontaneous hydrolysis and spurious metabolic properties of α-ketoglutarate esters

Abstract

Abstractα-ketoglutarate (KG), also referred to as 2-oxoglutarate, is a key intermediate of cellular metabolism with pleiotropic functions. Cell-permeable esterified analogs are widely used to study how KG fuels bioenergetic and amino acid metabolism and DNA, RNA, and protein hydroxylation reactions, as cellular membranes are thought to be impermeable to KG. Here we show that esterified KG analogs rapidly hydrolyze in aqueous media, yielding KG that, in contrast to prevailing assumptions, imports into many cell lines. Esterified KG analogs exhibit spurious KG-independent effects on cellular metabolism, including extracellular acidification, arising from rapid hydrolysis and de-protonation of α-ketoesters, and significant analog-specific inhibitory effects on glycolysis or mitochondrial respiration. We observe that imported KG decarboxylates to succinate in the cytosol and contributes minimally to mitochondrial metabolism in many cell lines cultured in normal conditions. These findings demonstrate that nuclear and cytosolic KG-dependent reactions may derive KG from functionally distinct subcellular pools and sources.

Metabolic requirement for GOT2 in pancreatic cancer depends on environmental context

“…However, we quantified Asp at 15 µM and αKG at 50 µM in CAF CM ( Figure 5—figure supplement 2A ), well below the reported values from mouse serum ( Sullivan et al, 2019 ). Furthermore, millimolar levels of Asp are required to impact intracellular levels, as PDA cell lines do not express an Asp transporter ( Birsoy et al, 2015 ), and dimethyl-αKG must be used as αKG has limited membrane permeablility ( Parker et al, 2021 ). Nevertheless, we tested the rescue activity of reported serum levels of Asp and αKG (50 µM and 500 µM, respectively; Sullivan et al, 2019 ), again utilizing the dimethyl-αKG and compared this to serum levels of pyruvate (250 µM).…”

Disrupted Alpha-Ketoglutarate Homeostasis: Understanding Kidney Diseases from the View of Metabolism and Beyond

“…Consequently, alpha-ketoglutaric acid and alcohol are released, which may result in off-target effect, considering the acidified microenvironments and unappreciated contributions to other cellular responses. 142 The off-target effect also leads to another question, namely, among the oral versus intravenous and bolus versus continuous administration, as well as the mode of release (controlled versus rapid), which is a more effective route in humans? Further studies are yet to be conducted.…”

Separation of α‐ketoglutaric acid by salting‐out extraction coupled with solar‐driven distillation

“…Alpha‐ketoglutaric acid, an organic acid containing both carboxyl and ketone groups, has a carbonyl group at the alpha site. It is a crucial intermediate of the tricarboxylic acid cycle and a node connecting carbon‐nitrogen metabolism, 1 participating in various metabolic processes in the body 2 . In recent years, α‐KG has attracted attention as a promising anti‐aging drug to extend life span 3 .…”