Fig. 5: Long-term glucagon treatment promoted lipid accumulation by activating mTORC1 signaling.

a Intracellular TG in HK2 cells treated with 100 nM glucagon for different durations. b Intracellular TG in primary PTECs from Gcgr^fl/fl and Gcgr^fl/flKsp^Cre mice treated with 100 nM glucagon for 24 h using 5 μM PA as a substrate. c–d BODIPY staining of HK2 cells under identical conditions. Scale bar: 20 μm. e–f qRT-PCR analyses of the lipogenesis and fatty acid oxidation genes in HK2 cells after 48 h glucagon treatment. g GSEA shows mTORC1 signaling enrichment in Ctrl shRNA vs. control kidneys. h–j Western blot of p-mTORC1, p-S6 in kidneys (8 wk post-STZ). k Intracellular cAMP in HK2 cells during glucagon treatment. l p-CREB western blot in HK2 cells. m ChIP-qPCR analysis using an anti-CREB antibody. n Luciferase assay of Rraga/S6k promoters in HepG2 cells. o Western blot of S6K/S6 in HK2 cells. p–s BODIPY staining and TG measurement in HK2 cells with PKA/mTORC1 inhibitors. Scale bar: 20 μm. t PTECs derived from Gcgr^fl/fl and Gcgr^fl/flKsp^Cre mice were treated with 10 μM MHY1485 (an mTORC1 activator) for 24 h, and the intracellular TG was subsequently measured. Mice were male and sampled after 4 weeks of shRNA injections (g), and mice were male and sampled after 8 weeks of STZ injections (h–j). The error bars represent the SEMs; n = 3-8 biologically independent cells (a–f, l–n, q–t); n = 3 in each group (h–k, o). PA, palmitate acid; BODIPY, boron-dipyrromethene.