Genetic disruption or pharmacologic inhibition of the hepati

Genetic disruption or pharmacologic inhibition of the hepatic glucagon pathway has invariably been shown to increase pancreatic α cell mass. This has been observed in glucagon receptor (GCGR) knockout (Gcgr−/−) mice (Gelling et al., 2003), glucagon knockout mice (Hayashi et al., 2009), prohormone convertase 2 knockout mice (Webb et al., 2002), liver-specific Gcgr−/− mice (Longuet et al., 2013), and liver-specific Gα knockout mice (Chen et al., 2005). Pharmacologic knockdown of hepatic GCGR using antisense oligonucleotides (Sloop et al., 2004, Liang et al., 2004) or administration of GCGR-blocking antibodies (Gu et al., 2009, Okamoto et al., 2015) also increases α cell mass in rodents. Furthermore, glucagon cell hyperplasia has been observed in patients with inactivating mutations in GCGR (Zhou et al., 2009, Larger et al., 2016, Sipos et al., 2015). Finally, a recent report showed that the GCGR inhibition-induced increase in plasma amino acids regulates α cell hyperplasia in an mTOR-dependent manner (Solloway et al., 2015). Regulation of pancreatic α and β cell proliferation (in response to glucagon and insulin blockade, respectively) has been extensively studied due to potential therapeutic implications for diabetes. Initial claims that Angptl8 was the long-sought-after “betatrophin” that induced β cell growth (Yi et al., 2013) were contradicted by an independent group (Gusarova et al., 2014) and eventually by the original authors (Yi et al., 2017). Similarly, the claim that Angptl4 mediates hyperglucagonemia and α cell proliferation following GCGR inhibition (Ben-Zvi et al., 2015) has been discounted (Okamoto et al., 2017). Here, we confirm the findings by Solloway et al. (2015) that GCGR inhibition reduces expression of hepatic amino Sulfamethazine australia transporters and metabolism genes and that the ensuing increase in plasma amino acids triggers an mTOR-dependent increase in α cell mass. To further elucidate the mechanism through which hyperaminoacidemia promotes α cell hyperplasia, we performed RNA sequencing of pancreatic islets from mice treated with a GCGR-blocking antibody or in Gcgr−/− mice. GCGR inhibition or deficiency was associated with select upregulation in the expression of the neutral amino acid transporter Slc38a5 in a subset of highly proliferative α cells. Mice deficient in Slc38a5 had normal metabolic phenotype but showed reduced expansion of α cell mass following GCGR antibody inhibition. Reduced α cell mass was also observed in mice deficient in both Slc38a5 and Gcgr. α cell growth was primarily triggered by glutamine and, to a lesser extent, alanine. The concentration of these amino acids were dramatically increased in the circulation of mice when glucagon signaling is blocked. Interestingly, α cell proliferation was independent of their membrane potential and degree of electrical activity, Ca2+ influx, and glucagon secretion. These data show that Slc38a5 is an integral component of the amino acid sensing machinery linking circulating amino acids to control of pancreatic α cell mass. This mechanism is part of a finely regulated feedback loop between glucagon signaling in the liver and the pancreatic α cells.
Results
Discussion In this study, we confirm previous findings that inhibition of glucagon signaling with a monoclonal blocking antibody to the glucagon receptor, or using Gcgr−/− mice, lowered blood glucose and reduced hepatic expression of genes involved in uptake and metabolism of amino acids and gluconeogenesis, resulting in elevated plasma amino acid levels and expanded α cell mass via an mTOR-dependent pathway. We extend the understanding of this pathway by showing that blocking the glucagon pathway increased expression of the amino acid transporter Slc38a5 in a subset of highly proliferative pancreatic α cells and that this transporter was selectively and prominently involved in mediating the α cell hyperplasia triggered by hyperaminoacidemia; most importantly, we showed that mice deficient in Slc38a5 had reduced α cell mass following treatment with the GCGR antibody or in a background of Gcgr deficiency. These data demonstrate that amino acids and the amino acid transporter Slc38a5 are key components in the feedback loop between glucagon receptor signaling in the liver and compensatory changes in circulating glucagon levels and α cell mass to ensure sufficient capacity and robustness of this circuit to maintain normal blood glucose levels.