Y-27632 mg Others demonstrated that Paneth cells

Others demonstrated that Paneth cells maintain stem cell function and crypt homeostasis by providing metabolic lactate to sustain the enhanced mitochondrial oxidative phosphorylation in the Lgr5+ ISCs (Rodriguez-Colman et al., 2017), which substantially agrees with our findings in this study. Furthermore, deletion of lactate dehydrogenase in Lgr5+ cells prevented the activation of hair follicle stem cells (Flores et al., 2017). We suggest that lactate directly stimulates CD24+ Paneth cells and αSMA+ intestinal stromal cells through Gpr81 and activates the Wnt3-β-catenin pathway, which plays an important role in maintaining stemness of Lgr5+ ISCs. By taking advantage of the essential roles of LAB-derived lactate and Gpr81 interactions in Wnt3 secretion, we propose prophylactic clinical use of LAB-type symbionts or lactate to protect gut injury in response to therapy such as radiation and chemotherapy.
STAR★Methods
Acknowledgments This work was supported by the National Research Foundation of Korea (NRF-2017R1A2B3002132), the Asan Institute for Life Sciences, Asan Medical Center (2018-678), and the Korean Healthcare Technology R&D Project, Ministry of Health, Welfare and Family Affairs, Republic of Korea (HI13C0016).
GPR81 is an orphan G-protein-coupled receptor of unknown function . Expression of GPR81 is highly restricted in the adipose tissues , suggesting that this orphan GPCR may play a role in energy metabolism. GPR81 is most homologous (52% amino Y-27632 mg sequence identity in humans) to GPR109A, the receptor for the lipid lowering agent nicotinic acid , , . A well-studied pharmacologic effect of nicotinic acid, a small carboxylic acid, is its ability to inhibit triglyceride (TG) lipolysis in adipose tissue, which results in reduced levels of plasma FFA , . Another small carboxylic acid that can reduce FFA is the ketone body β-hydroxybutyrate , , and we have recently shown that this metabolite is an endogenous ligand for GPR109A . β-Hydroxybutyrate is significantly increased during starvation , and we postulate that it acts in a negative feed-back loop to control lipolysis in adipose tissue via activation of GPR109A, thereby enhancing the efficient use of adipose lipid stores during starvation . Lactic acid (lactate) differs from β-hydroxybutyrate by being one methylene group smaller. Like β-hydroxybutyrate , , infusion of lactate reduces lipolysis , , as does treatment of adipose tissue , . The levels of lactate in plasma increase significantly following physical exercise or as a consequence of oxygen deficit (may reach up to 30mM) , , , and this lactate elevations often links with a reduction of plasma FFA , , . We thus investigated whether lactate might be an endogenous ligand for GPR81 and whether GPR81 plays a role in lactate-induced inhibition of lipolysis. Experimental procedures GPR81-, GPR109A- and GPR109B stable cell lines. For the generation of stable cell lines, 5×106 Chinese Hamster Ovary (CHO)-K1 cells were transfected with 12μg plasmid DNA pHM6 (Invitrogen, Carlsbad, CA) containing GPR81 or plasmid DNA pCDNA3.1 (Invitrogen) containing GPR109A or GPR109B expressed from the cytomegalovirus promoter. Two days after transfection, the growth medium was supplemented with 400μg/ml G418 to select for antibiotic resistant cells. Clonal CHO-K1 cell lines that stably express GPR81 were selected based on immunostaining with anti-HA antibodies. Clonal CHO-K1 cell lines that stably express GPR109A and GPR109B were selected based on the ability of nicotinic acid (GPR109A-specific agonist) or 1-isopropylbenzotriazole-5-carboxylic acid (GPR109B-specific agonist, [26]) to inhibit forskolin-induced cAMP production, as previously described [16]. 35S-GTPγS binding assay: Membranes prepared from CHO-K1 cells stably expressing GPR109A, GPR109B, GPR81 or vector control (7μg/assay) were diluted in assay buffer (100mM HEPES, 100mM NaCl and 10mM MgCl2, pH 7.4) in Wallac Scintistrip plates and pre-incubated with test compounds diluted in assay buffer containing 40μM GDP (final [GDP] was 10μM) for ∼10min before addition of 35S-GTPγS to 0.3nM. Binding was allowed to proceed for one hour before centrifuging the plates at 2500rpm for 20min at room temperature and subsequent counting in a TopCount scintillation counter. Non-linear regression analysis of the binding curves was performed in GraphPad Prism.