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    • Our data suggest that Slc a is important

    2022-03-09

    Our data suggest that Slc38a5 is important for amino-acid-induced α cell proliferation and expansion of α cell mass following GCGR inhibition, but not for formation and maintenance of α cell mass. This is supported by the finding that Slc38a5−/− mice have normal α cell mass. This is interesting since Slc38a5 has recently been proposed as a marker of early α cell commitment (Stanescu et al., 2017). The importance of Slc38a5 for cell proliferation was confirmed in αTC1-6 cells, where knockdown of this amino JNJ-10198409 transporter strongly reduced their proliferative capacity in response to glutamine or alanine. Since αTC1-6 cells express high levels of Slc38a5, we were not surprised that overexpression of the transporter only marginally increased the cell proliferation rate. Interestingly, we found that implantation of αTC1-6 cells into mice treated with GCGR antibody doubled their growth over αTC1-6 cells implanted into control mice. These data suggest that innervation or intra-islet paracrine mechanisms are unlikely to govern α cell proliferation following GCGR inhibition. GCGR is expressed at very low levels in mouse α cells, whereas no expression was detected in αTC1-6 cells. The αTC1-6 cell implantation data therefore suggest that α cell GCGR does not act as a negative regulator of cell division. This is different from AMPK, which has been shown to restrict proliferation of proglucagon-expressing intestinal cells (Sayers et al., 2016). A role for innervation in the control of α cell proliferation following GCGR inhibition has been excluded previously (Longuet et al., 2013). Our data suggest that α cell proliferation is independent of their electrical activity, Ca2+ influx, or secretory capacity. This is supported by the observations that arginine, a well-known and efficacious glucagon secretagogue, did not affect proliferation (this study; Dean et al., 2017), whereas glutamine strongly stimulated proliferation, but not glucagon release. This is supported by the findings that glutamine-induced αTC1-6 cell proliferation was not affected by diazoxide, which causes membrane hyperpolarization and inhibition of electrical activity. Furthermore, αTC1-6 cell proliferation was similarly increased in cells incubated in normal or high K+ medium, which causes cell depolarization and continuous electrical activity. These findings suggest that α cell proliferation occurs independent of electrical activity, calcium influx, and glucagon secretion. Since circulating glutamine is elevated to the highest level when glucagon action is disrupted, and glutamine shows the greatest efficacy in promoting the growth of αTC1-6 cells, both our data and those from Dean et al. (2017), suggest glutamine being the key factor responsible for α cell hyperplasia in the presence of inhibited glucagon signaling. Relatively little is known about other functions of Slc38a5, except that it is expressed in cells in the brain, eye, and liver to regulate glutamine flux (Baird et al., 2004, Umapathy et al., 2008, Zielińska et al., 2016). We found that Slc38a5−/− mice breed normally and do not have gross abnormalities. Even when exposed to high circulating amino acid levels, including glutamine, we did not observe abnormal phenotypes. This suggests that other amino acid transporters compensate for loss of Slc38a5 and that careful analysis is required to understand the precise physiological function of this amino acid transporter. It is generally believed that the Slc38a5-dependent transport process is electroneutral since it co-transports neutral amino acids with sodium but in antiport with H+ in a 1:1 ratio (Bröer, 2014). Thus, Slc38a5 transport activity would not be expected to affect α cell membrane potential and glucagon secretion. However, overexpression studies of the close family member Slc38a3 in oocytes caused significant inward currents resulting from uncoupled movements of ions during the transport cycle (Chaudhry et al., 2001, Schneider et al., 2007). In future studies, it would be interesting to explore whether inward currents are also observed in Slc38a5-positive α cells and contribute to membrane depolarization and hyperglucagonemia. Indirect evidence suggests that this might be the case since glucagon release in response to alanine stimulation was reduced in the perfused pancreas from Slc38a5−/− mice. A trend toward reduced plasma glucagon was also observed in Slc38a5−/− mice when administered an alanine bolus. However, it is important to emphasize that hyperglucagonemia is mainly mediated by Slc38a5-independent mechanisms and likely reflects amino acid uptake through one or more of the other transporters expressed in α cells. Our studies using arginine stimulation showed no difference in glucagon secretion between Slc38a5−/− and control mice. Thus, reduced glucagon release in response to alanine stimulation does not reflect reduced capacity to secrete glucagon. Consistent with this, we found that glucagon (and insulin) content in pancreas from Slc38a5−/− mice was normal.