Fig 2: K1 and AMPK associate in membranes in the perinuclear area of the cell.(A) HEK-293 cells stably expressing empty vector (EV) or FLAG-K1 (K1) were lysed and fractionated into cytoplasmic, membrane and nuclear fractions. Immunoblots were probed for endogenous AMPK subunit and isoforms. (B) HUVEC stably expressing empty (EV) or FLAG-K1 (K1) were fixed and permeabilized. Cells were then stained with anti-FLAG directly conjugated to FITC antibody, and an AMPKβ1/2 antibody, followed by an anti-rabbit conjugated to Alexa Fluor 647. Nuclei were stained with DAPI. Stained HUVEC were evaluated with a Zeiss 700 confocal microscope using a 63X oil objective. Mander’s overlap coefficient (R = 0.83) was generated using ImageJ. (C) HEK-293 cells were transiently transfected with EV (pcDNA3), K1WT, K1ΔCT and K1ΔNT. Cell lysates were separated into membrane, cytoplasmic and nuclear fractions. We evaluated the membrane fraction for expression of the K1 domain deleted mutants by immunoblot. We also immunoblotted for AIF (membrane), MEK1/2 (cytoplasm) and Histone H3 (nucleus). (D) HEK-293 cells were transiently transfected with the constructs described in (C) with the addition of AMPKγ1. Cell lysates were separated into membrane, cytoplasmic and nuclear fractions. We immunoprecipitated FLAG K1 and immunoblotted for AMPKγ1 or FLAG using only the membrane fraction.

Fig 3: Characterofization of circulating iron, bone marrow erythropoiesis, and erythrocyte half-life of Prkab1-deficient mice. (A) Plasma ferritin concentration. (B) Plasma iron concentration. (C) Representative H&E stained bone marrow sections from Prkab1+/+ and Prkab1tm1b/tm1b mice (400× magnification). (D) Erythroid (Ter119)/Leukocyte (CD45) ratio of bone marrow from Prkab1+/+ and Prkab1tm1b/tm1b mice. (E) Characterization of erythropoiesis in the bone marrow of Prkab1+/+ and Prkab1tm1b/tm1b mice. In vivo half-life of erythrocytes transferred into Prkab1+/+ (F) or Prkab1tm1b/tm1b (G) mice. For all, *p < 0.05, ***p < 0.001, and ****p < 0.0001, unpaired two-tailed Student t test, except for Ter119/CD45 ratio, which was analyzed with a Mann–Whitney test. Data are representative of two independent experiments or four mice for histology analysis. Each symbol represents an individual mouse with the line at the mean, except for (F) and (G), where n = 5 with mean ± standard error of the mean.

Fig 4: Molecular and phenotypic characterization of Prkab1-deficient mice. (A) Mean gene expression of AMPK subunits in Prkab1+/+ and Prkab1tm1b/tm1b spleen RNA. n = 5 per genotype, with error bars representing standard errors of the mean. (B) Immunoblot analysis of AMPK subunits in Prkab1+/+ and Prkab1tm1b/tm1b spleen protein lysates. *IgG heavy chain. (C) White blood cell count. (D) Mean platelet volume. (E) Platelet count of 16-week-old Prkab1+/+ and Prkab1tm1b/tm1b mice. *p < 0.05, **p < 0.01, *** p < 0.001, and ****p < 0.0001. Student’s t test, hematology data are representative of three independent experiments. Each symbol represents an individual mouse with the line at the mean.

Fig 5: ETC-1002 mediates effects on lipid metabolism independently of AMPK β1.Apoe−/− and Apoe−/−/Ampkβ1−/− (DKO) mice were fed a HFHC diet for 12 weeks with or without ETC-1002 targeted to achieve a 30 mg kg−1 per day dose and (a) total and phosphorylated (T172) AMPKα and total and phosphorylated (S79) ACC measured in livers from Apoe−/− (n=4) and DKO (n=3). (b) Slc27a2 (ACSVL1) expression in liver and tibialis anterior from untreated Apoe−/− mice (n=6). (c) Representative fast performance liquid chromatography tracings from chow- and HFHC diet-fed±ETC-1002 Apoe−/− and DKO mice. Plasma (d) very-low-density lipoprotein (VLDL), (e) LDL, (f) HDL (n=4 for chow-fed mice and 6 for remaining treatment groups) and (g) total cholesterol (n=10), liver (h) cholesterol and (i) triglycerides (n=4 for chow-fed and 6 for remaining treatment groups) determined at the end of study. (j) RER was measured over 24 h following 10 weeks on diet and mean RER during (k) dark (19:00–07:00) and (l) light (07:00–19:00) cycles calculated. (m) Total lipid synthesis in primary hepatocytes isolated from WT and AMPK β1 KO mice treated for 4 h with the indicated concentration of ETC-1002, A-769622 or vehicle (CON) in the presence of 3H-acetate (n=3–8 independent experiments performed in duplicate). (n) Srebf2, Ldlr, Pcsk9, Hmgr, Acly, Srebf1c and Slc27a2 gene expression (n=4 chow-fed and 5 for remaining treatment groups) determined at the end of study. (o) Representative images for immunohistochemistry staining of LDLR in frozen liver sections from ETC-1002-treated Apoe−/− and DKO mice (20 × ); scale bar, 100 μM. Data are expressed as mean±s.e.m. Multiple comparisons were made using an *one-way (within Apoe−/− treatment groups) or #two-way ANOVA (between Apoe−/− and DKO treatment groups); Bonferroni's multiple comparisons test; *and #P<0.05.