Ubated in media containing significantly less than three mM glucose to recruit aUbated in media

Ubated in media containing significantly less than three mM glucose to recruit a
Ubated in media containing significantly less than 3 mM glucose to recruit a adequate quantity of KATP channels for the plasma membrane (six). We therefore hypothesized that there ought to be an endogenous p70S6K web ligand in vivo that promotes AMPK-dependent KATP channel trafficking sufficiently to stabilize pancreatic -cells at physiological fasting glucose levels. Leptin is definitely an adipocyte-derived hormone that regulates meals intake, body weight, and glucose homeostasis (eight, 9). In additionTAuthor contributions: S.-H.P., S.-H.L., P.-O.B., J.-H.J., and W.-K.H. made research; S.-H.P., S.-Y.R., W.-J.Y., Y.E.H., Y.-S.J., K.O., J.-P.J., and H.L. performed investigation; S.-H.P., S.-Y.R., Y.-S.J., K.-H.L., and W.-K.H. analyzed data; and S.-H.P., S.-Y.R., J.-W.S., A.L., P.-O.B., J.-H.J., and W.-K.H. wrote the paper. The authors declare no conflict of interest. This article is actually a PNAS Direct Submission.To whom correspondence could be addressed. E-mail: [email protected] or jhjeon2@ snu.ac.kr.This article contains supporting info on the internet at pnas.org/lookup/suppl/doi:ten. 1073/pnas.1216351110/-/DCSupplemental.pnas.org/cgi/doi/10.1073/pnas.PNAS | July 30, 2013 | vol. 110 | no. 31 | 12673CELL BIOLOGYIn our prior in vitro study utilizing the insulin-secreting cell line INS-1, glucose concentration much less than 3 mM was necessary to induce maximal AMPK activation and KATP channel trafficking (6). On the other hand, the mean blood glucose level inside the WT fed mice was 244 14 mg/dL (13.five mM, n = ten), and that in the WT fasted mice was 138 11 mg/dL (7.7 mM, n = ten), which might not be adequate to totally activate AMPK activity. Thus, we supposed the presence of an endogenous ligand in vivo that induces AMPK activation and KATP channel trafficking and tested the idea that leptin plays this function utilizing ob/ob mice lacking this hormone. In contrast to observations in WT mice, a distinct staining pattern indicating surface translocation of SUR1 and Kir6.2 was lost in islet cells of ob/ob mice obtained immediately after a 12-h fasting period (Fig. 1B and Fig. S1). Interestingly, this pattern was restored when ob/ob mice have been treated with leptin for 4 d (two g/d) (15) (Fig. 1B and Fig. S1), indicating that leptin is critical for the surface translocation of Kir6.two during fasting in vivo. Intracellular localization of KATP channels has been studied by numerous groups, but final results are controversial (4, 16). Since endosomal recycling is essential for regulation with the density of surface proteins (17), we tested the colocalization of KATP channels with early endosomal antigen 1 (EEA1), an endosomal marker. The outcomes show significant colocalization of Kir6.two with EEA1 (Fig. 1A, Decrease and Fig. S1B). Interestingly, EEA1 also is translocated toward the cell periphery and colocalized almost absolutely with Kir6.2 in -cells within the islets of WT fasted and leptin-treated fasted ob/ob mice (Fig. 1 A and B, Reduced and Fig. S1B). To confirm irrespective of whether regulation of KATP channel trafficking by feeding status has functional significance, we measured wholecell K+ currents in -cells in pancreatic slices obtained from fed and fasted mice. To mimic the distinction in glucose concentrations depending on feeding status in vivo, slices obtained from fed mice had been superfused with 17 mM glucose, and those from fasted mice have been superfused with 6 mM glucose. To maximize KATP channel open probability and to lessen channel rundown, we made use of ATP- and Mg2+-free internal solutions (6, 18). Based on the P2X7 Receptor MedChemExpress preceding report (19), we identified -cells in sl.