Extended Data Fig. 5: ORM2 activates CaMKK2-AMPK signaling pathway.
From: Orosomucoid 2 maintains hepatic lipid homeostasis through suppression of de novo lipogenesis
Related to Fig. 6. a, b, Immunoblots of AMPK signaling pathway in MPHs (a) and HepG2 cells (b). Cells were treated with ORM2 recombinant protein (100 ng/ml) or vehicle control (PBS) for 6 hr. n = 3 independent samples per condition. c, d, MPHs were pre-incubated with palmitic acid (200 μM) for 12 hr, and then treated with ORM2 protein (100 ng/ml) or vehicle control (PBS) for 6 hr, in the presence or absence of Compound C (20 μM). c, Cellular TG contents. n = 3 independent samples per condition. d, Relative mRNA levels of genes related to de novo lipogenesis. n = 3 independent samples per condition. e, f, HepG2 Cells were pre-incubated with palmitic acid (200 μM) for 12 hr, and then treated with ORM2 protein (100 ng/ml) or vehicle control (PBS) for 6 hr, in the presence or absence of Compound C (20 μM). e, Cellular TG contents. n = 3 independent samples per condition. f, Relative mRNA levels of genes related to de novo lipogenesis. n = 3 independent samples per condition. g-i, MPHs from ORM2 WT or KO mice were administered with Ad-GFP, Ad-CA-AMPK α1 (T172D TC312) or Ad-CA-AMPK α2 (T172D TC312). g, Immunoblots of AMPK signaling. h, Cellular TG contents. n = 3 independent samples per condition. i, Relative mRNA levels of genes related to de novo lipogenesis. n = 3 independent samples per condition. j, k, MPHs were pre-incubated with palmitic acid (200 μM) for 12 hr, and then treated with ORM2 protein (100 ng/ml) or vehicle control (PBS) for 6 hr, in the presence or absence of CaMKK2 inhibitor (STO-609, 5 μM) or LKB1 inhibitor (PIM1, 10 μM). j, Cellular TG contents in MPHs. n = 3 independent samples per condition. k, Relative mRNA levels of genes related to lipogenesis in MPHs. n = 3 independent samples per condition. l, Immunoblots of CaMKK2 and AMPK signaling in MPHs. Cells were transfected with adenoviral shRNA targeting CaMKK2 or negative control for 24 hr, then treated with ORM2 protein (100 ng/ml) or vehicle control (PBS) for 6 hr. m, Immunoblots of LKB1 and AMPK signaling in MPHs. Cells were transfected with adenoviral shRNA targeting LKB1 or negative control for 24 hr, then treated with ORM2 protein (100 ng/ml) or vehicle control (PBS) for 6 hr. n, Immunoblots of AMPK in the MPHs from ORM2 WT and KO mice. Cells were treated with ORM2 recombinant protein, Ca2+ ionophore (Ionomycin, 0.5 μM) or vehicle control (DMSO) for 3 hr. o, Genomic sequencing of WT or ACC1 S80A knockin mutation (KI) HepG2 cells. p, Immunoblots of AMPK signaling in WT and KI cells treated with metformin (2 mM) or vehicle control (PBS) for 6 hr. q, r, Cells were incubated for 6 hr with 13C-acetate (1 mM) containing vehicle control (PBS), ORM2 (100 ng/ml), AMPK allosteric activators (A-769662, 10μM and AICAR, 100μM). The rate of de novo lipogenesis in WT cells (q, n = 4 independent samples per condition) and KI cells (r, n = 3 independent samples per condition) were measured. s, t, Relative mRNA levels of genes related to de novo lipogenesis in WT (s) and KI cells (t) were analyzed. n = 3 independent samples per condition. u, v, Mouse primary hepatocytes were transfected with adenoviral shRNA targeting ACC1 or negative control. Cells were then treated with ORM2 (100 ng/ml) or vehicle control (PBS) for 6 hr. ACC1 protein expression (u, n = 3 independent samples per condition) and relative mRNA levels of genes related to de novo lipogenesis (v, n = 3 independent samples per condition) were measured. Data are shown as mean ± SEM (c-f, h-k, q-t, v). P values are determined by one-way ANOVA followed by Dunnett’s multiple comparisons test (q-t) and Bonferroni’s multiple comparisons test (c-f, h-k, v).
