A dynamic anchor domain in slc13 transporters controls metabolite transport.

Metabolite transport across cellular membranes is required for bioenergetic processes and metabolic signaling. The solute carrier family 13 (slc13) transporters mediate transport of the metabolites succinate and citrate and hence are of paramount physiological importance. Nevertheless, the mechanisms of slc13 transport and regulation are poorly understood. Here, a dynamic structural slc13 model suggested that an interfacial helix, H4c, which is common to all slc13s, stabilizes the stationary scaffold domain by anchoring it to the membrane, thereby facilitating movement of the SLC13 catalytic domain. Moreover, we found that intracellular determinants interact with the H4c anchor domain to modulate transport. This dual function is achieved by basic residues that alternately face either the membrane phospholipids or the intracellular milieu. This mechanism was supported by several experimental findings obtained using biochemical methods, electrophysiological measurements in Xenopus oocytes, and fluorescent microscopy of mammalian cells. First, a positively charged and highly conserved H4c residue, Arg108, was indispensable and crucial for metabolite transport. Furthermore, neutralization of other H4c basic residues inhibited slc13 transport function, thus mimicking the inhibitory effect of the slc13 inhibitor, slc26a6. Our findings suggest that the positive charge distribution across H4c domain controls slc13 transporter function and is utilized by slc13-interacting proteins in the regulation of metabolite transport.

Systemic Succinate Homeostasis and Local Succinate Signaling Affect Blood Pressure and Modify Risks for Calcium Oxalate Lithogenesis.

BACKGROUND: In the kidney, low urinary citrate increases the risk for developing kidney stones, and elevation of luminal succinate in the juxtaglomerular apparatus increases renin secretion, causing hypertension. Although the association between stone formation and hypertension is well established, the molecular mechanism linking these pathophysiologies has been elusive. METHODS: To investigate the relationship between succinate and citrate/oxalate levels, we assessed blood and urine levels of metabolites, renal protein expression, and BP (using 24-hour telemetric monitoring) in male mice lacking slc26a6 (a transporter that inhibits the succinate transporter NaDC-1 to control citrate absorption from the urinary lumen). We also explored the mechanism underlying this metabolic association, using coimmunoprecipitation, electrophysiologic measurements, and flux assays to study protein interaction and transport activity. RESULTS: Compared with control mice, slc26a6-/- mice (previously shown to have low urinary citrate and to develop calcium oxalate stones) had a 40% decrease in urinary excretion of succinate, a 35% increase in serum succinate, and elevated plasma renin. Slc26a6-/- mice also showed activity-dependent hypertension that was unaffected by dietary salt intake. Structural modeling, confirmed by mutational analysis, identified slc26a6 and NaDC-1 residues that interact and mediate slc26a6's inhibition of NaDC-1. This interaction is regulated by the scaffolding protein IRBIT, which is released by stimulation of the succinate receptor SUCNR1 and interacts with the NaDC-1/slc26a6 complex to inhibit succinate transport by NaDC-1. CONCLUSIONS: These findings reveal a succinate/citrate homeostatic pathway regulated by IRBIT that affects BP and biochemical risk of calcium oxalate stone formation, thus providing a potential molecular link between hypertension and lithogenesis.

A transepithelial pathway delivers succinate to macrophages, thus perpetuating their pro-inflammatory metabolic state.

The gut metabolite composition determined by the microbiota has paramount impact on gastrointestinal physiology. However, the role that bacterial metabolites play in communicating with host cells during inflammatory diseases is poorly understood. Here, we aim to identify the microbiota-determined output of the pro-inflammatory metabolite, succinate, and to elucidate the pathways that control transepithelial succinate absorption and subsequent succinate delivery to macrophages. We show a significant increase of succinate uptake into pro-inflammatory macrophages, which is controlled by Na+-dependent succinate transporters in macrophages and epithelial cells. Furthermore, we find that fecal and serum succinate concentrations were markedly augmented in inflammatory bowel diseases (IBDs) and corresponded to changes in succinate-metabolizing gut bacteria. Together, our results describe a succinate production and transport pathway that controls the absorption of succinate generated by distinct gut bacteria and its delivery into macrophages. In IBD, this mechanism fails to protect against the succinate surge, which may result in chronic inflammation.

Systemic inflammatory response syndrome-related lymphopenia is associated with adenosine A1 receptor dysfunction.

SIRS is associated with lymphopenia, and prolonged lymphopenia of septic patients has been associated with increased mortality risk. We hypothesize that elevated adenosine during SIRS down-regulates Gi-coupled A1R, which signals an effect that sensitizes a cAMP-dependent lymphotoxic response. In this study, we evaluate the role of adenosine in SIRS-mediated lymphopenia and impaired IL-15 production. Cecal ligation and puncture was used to induce sepsis-associated SIRS in mice. BMDCs were cultured and used to measure the effect of adenosine on IL-15. We found that A1R mRNA levels were significantly down-regulated and A1R-dependent Gi activity was abolished in T cells of septic mice. In accordance, cAMP was elevated in isolated T cells from cecal ligation and puncture compared with sham-treated mice. Similar to septic mice, leukopenia was evident in sham A1R-KO mice, after treatment with the A1R antagonist (8-cyclopentyl-1,3-dipropylxanthine), or after A1R desensitization. In contrast, A2AR-KO mice were protected from leukopenia. In addition, we observed that septic A1R-KO mice exhibited low IL-15 levels. Cultured BMDC agonists of A2AR and A2BR inhibited IL-15 production and adenosine blocked IL-15-dependent proliferation of cytotoxic T cells that were cocultured with stimulated BMDCs. To conclude, we suggest that SIRS-associated lymphopenia is initiated by A1R desensitization and adenosine-mediated inhibition of IL-15 production is part of the mechanism that accounts for the delay in leukopenia recovery in patients with severe sepsis. Interference with adenosine signaling may thus be potentially beneficial for septic patients with leukopenia.