Bacterial metabolite indole modulates incretin secretion from intestinal enteroendocrine L cells.
Bottom Line: These effects were attributed to the ability of indole to affect two key molecular mechanisms in L cells.On the one hand, indole inhibited voltage-gated K(+) channels, increased the temporal width of action potentials fired by L cells, and led to enhanced Ca(2+) entry, thereby acutely stimulating GLP-1 secretion.On the other hand, indole slowed ATP production by blocking NADH dehydrogenase, thus leading to a prolonged reduction of GLP-1 secretion.
Affiliation: Institute of Metabolic Science and MRC Metabolic Diseases Unit, University of Cambridge, Addenbrooke's Hospital, Hills Road, Cambridge CB2 0QQ, UK; Cavendish Laboratory, Department of Physics, University of Cambridge, JJ Thomson Avenue, Cambridge CB3 0HE, UK. Electronic address: email@example.com.Show MeSH
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Mentions: As indole was found to reduce the rate of GLP-1 secretion during long-term exposures (30-240 min; Figure 1B), we investigated its effects on cellular metabolism. Indole is known to affect mitochondrial ATP production by blocking NADH dehydrogenase and by facilitating proton permeation (“uncoupling”) through the mitochondrial membrane (Chimerel et al., 2013). To monitor NAD(P)H levels in GLUTag cells, we excited the intrinsic fluorescence of NAD(P)H molecules at 360 nm (±15 nm). In the presence of indole, NAD(P)H autofluorescence was observed to increase (Figure 3A), as would be expected when the oxidation of NADH to NAD+ is blocked. As a positive control, we used 1 μM rotenone, which is an established blocker of NADH dehydrogenase. Rotenone, like indole, resulted in an increase in NAD(P)H autofluorescence. We also assessed the ability of indole to block oxidative phosphorylation, by monitoring the intracellular ATP/ADP ratio using the genetically encoded sensor, Perceval, which was transiently transfected into GLUTag cells (Berg et al., 2009; Tarasov et al., 2012). Addition of indole to the extracellular medium resulted in a decrease in the Perceval fluorescence intensity, indicative of a fall in the intracellular ATP/ADP ratio (Figure 3B). Perceval fluorescence decreased with a rate of 1.5% ± 0.3%/min in the presence of 1 mM indole and with a rate of 7.5 ± 1.5%/min in 1 μM rotenone. These data suggest that indole affects intracellular ATP generation. In light of this result, we hypothesize that the observed inhibitory effect of indole on GLP-1 secretion over longer time periods is a consequence of the lowered intracellular ATP concentration in GLUTag cells.
Affiliation: Institute of Metabolic Science and MRC Metabolic Diseases Unit, University of Cambridge, Addenbrooke's Hospital, Hills Road, Cambridge CB2 0QQ, UK; Cavendish Laboratory, Department of Physics, University of Cambridge, JJ Thomson Avenue, Cambridge CB3 0HE, UK. Electronic address: firstname.lastname@example.org.