Citric acid cycle intermediates as ligands for orphan G-protein-coupled receptors

Abstract

The citric acid cycle is central to the regulation of energy homeostasis and cell metabolism 1 . Mutations in enzymes that catalyse steps in the citric acid cycle result in human diseases with various clinical presentations 2 . The intermediates of the citric acid cycle are present at micromolar concentration in blood and are regulated by respiration, metabolism and renal reabsorption/ extrusion. Here we show that GPR91 (ref.3), a previously orphan G-protein-coupled receptor (GPCR), functions as a receptor for the citric acid cycle intermediate succinate. We also report that GPR99 (ref. 4), a close relative of GPR91, responds to a-ketoglutarate, another intermediate in the citric acid cycle. Thus by acting as ligands for GPCRs, succinate and a-ketoglutarate are found to have unexpected signalling functions beyond their traditional roles. Furthermore, we show that succinate increases blood pressure in animals. The succinate-induced hypertensive effect involves the renin-angiotensin system and is abolished in GPR91-deficient mice. Our results indicate a possible role for GPR91 in renovascular hypertension, a disease closely linked to atherosclerosis, diabetes and renal failure 5,6 .In a search for natural ligands for orphan GPCRs, we tested extracts from various animal tissues for their ability to evoke an increase in intracellular Ca 2þ concentration ([Ca 2þ ] i ) using the aequorin assay 7 . We found that fractions from pig kidney extracts specifically activated cells expressing GPR91 (Fig. 1a). GPR91 is an orphan GPCR highly expressed in the kidney and shares 33% amino acid identity with GPR99/GPR80 (refs 4, 8). On the basis of their homology with the purinergic receptor P2Y1, nucleotide ligands were predicted for GPR91 and GPR99 (ref. 4). However, the GPR91 ligand activity in pig kidney extracts was resistant to various stringent treatments including alkaline phosphatase, peptidase, and hydrolysis in 6 M HCl at 100 8C. Accordingly, the supposition that GPR91 might be activated by a nucleotide or peptide ligand was unlikely. We purified the natural ligand for GPR91 by ion-exchange, size-exclusion and reversed-phase fast performance liquid chromatography/high-performance liquid chromatography (Fig. 1a).A major molecular ion [M þ H] þ at m/z 119.2 was observed by mass spectrometry (Fig. 1b). 1 H NMR analysis revealed a single type of proton in the highly purified GPR91 ligand (Fig. 1c). 13 C NMR analysis further suggested the presence of -CH 2 -(methylene) and ¼C¼O (carbonyl) groups (Fig. 1d). Combined with mass spectrometry results and the biochemical properties of the ligand, the purified GPR91 ligand was predicted and confirmed to be succinic acid (Fig. 1c, d).Commercially obtained succinate (the physiological form of succinic acid) increased [Ca 2þ ] i dose-dependently in the aequorin assay (Fig. 2a). Succinate also activated mouse and rat orthologues of GPR91 (Fig. 2a). The succinate-induced increase in [Ca 2þ ] i was further confirmed with a fluorimetric imaging plate reader (FLIPR) system in the 293-hGP...

Succinate: a metabolic signal in inflammation

“…PKA is known to phosphorylate and activate the p65 subunit of nuclear factor kB (NF-kB) [46] and cAMP response element-binding protein (CREB since renamed SUCNR1 (Figure 3). This indicates that succinate has a signaling role beyond HIF-1a stabilization [27,28]. SUCNR1 is highly expressed in mouse kidney, liver, spleen, small intestine [27], and on DCs [8].…”

“…This indicates that succinate has a signaling role beyond HIF-1a stabilization [27,28]. SUCNR1 is highly expressed in mouse kidney, liver, spleen, small intestine [27], and on DCs [8]. The signaling cascade activated by SUCNR1 has only recently emerged [29] (Figure 3).…”

“…The signaling cascade activated by SUCNR1 has only recently emerged [29] (Figure 3). Human embryonic kidney (HEK) 293 cells stably expressing SUCNR1 were stimulated with succinate, resulting in inositol trisphosphate (IP3) accumulation, calcium mobilization, and extracellular signalregulated kinase (ERK) phosphorylation [27] similar to Gi and Gq signaling cascades. Indeed, the use of the respective inhibitors of Gq and Gi signaling, YM254890 and pertussis toxin, impaired succinate-induced responses [30].…”

“…Mice overexpressing renin developed glucose intolerance, and angiotensin II generated in response to renin impaired insulin receptor signaling and glucose transport [48]. In vivo succinate administration induced hypertension, but this did not occur in SUCNR1-deficient mice, which have diminished renin accumulation [27]. Finally, circulating succinate was increased in spontaneously hypertensive rats, as well as in obese and diabetic rats [49] and this may contribute to disease by stimulation of the RAS.…”

L’offre et la demande : l’influence du métabolisme énergétique sur l’angiogenèse

“…Grâce à la découverte de son récepteur, le GPR91 [6], le ligand succinate semble bien être la solution au chaînon manquant unissant le compromis énergétique induit par l'ischémie rétinienne et le processus de réparation vasculaire. Le GPR91 est un récepteur couplé aux protéines G ; il est fortement exprimé dans l'oeil par les neurones ganglionnaires de la rétine interne.…”