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MTORC1 inhibition drives crinophagic degradation of glucagon.

Molecular metabolism (2021-07-06)
Sangam Rajak, Sherwin Xie, Archana Tewari, Sana Raza, Yajun Wu, Boon-Huat Bay, Paul M Yen, Rohit A Sinha
ABSTRACT

Crinophagy is a secretory granule-specific autophagic process that regulates hormone content and secretion in endocrine cells. However, despite being one of the earliest described autophagic processes, its mechanism of action and regulation in mammalian cells remains unclear. Here, we examined mammalian crinophagy and its modulation that regulate hormone secretion in a glucagon-producing mouse pancreatic α-cell line, alpha TC1 clone 9 (αTC9), and in vivo. Western blot, electron microscopy, and immunofluorescence analyses were performed to study crinophagy and glucagon secretion in αTC9 cells and C57BL/6 mice, in response to the mammalian target of rapamycin complex 1 (MTORC1) inhibitor rapamycin. Amino acid depletion and pharmacological inhibition of MTORC1 increased the shuttling of glucagon-containing secretory granules into lysosomes for crinophagic degradation to reduce glucagon secretion through a macroautophagy-independent mechanism. Furthermore, MTORC1 inhibition reduced both intracellular and secreted glucagon in rapamycin-treated mice, in response to hypoglycaemia. In summary, we have identified a novel crinophagic mechanism of intracellular glucagon turnover in pancreatic α-cells regulated by MTORC1 signalling.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Monoclonal Anti-Glucagon antibody produced in mouse, clone K79bB10, ascites fluid
Sigma-Aldrich
D-(+)-Trehalose dihydrate, ≥99% (HPLC), from Saccharomyces cerevisiae
Sigma-Aldrich
mTOR Inhibitor XI, Torin1, mTOR Inhibitor XI, Torin1, CAS 1222998-36-8, is a cell-permeable, highly potent, ATP-competitive inhibitor of mTOR and DNA-PK (IC50 = 4.32 and 6.34 nM, respectively).
Sigma-Aldrich
Tat-Beclin 1
Sigma-Aldrich
Lysosome Isolation Kit, sufficient for 25 g (tissue), sufficient for 20 mL (packed cells), enrichment of lysosomes from tissues and packed cells
Sigma-Aldrich
PP242 hydrate, ≥98% (HPLC), powder
Sigma-Aldrich
Rapamycin from Streptomyces hygroscopicus, ≥95% (HPLC), powder
Sigma-Aldrich
Triton X-100, laboratory grade
Sigma-Aldrich
CelLytic M, Cell Lysis Reagent, Suitable for Mammalian cell lysis and protein solubilization.