Figure 1.
Enhanced Nrf2 activity prevents fasting effects in Keap1-KD mice.
Serum content of (A) glucose, (B) insulin, (C) β-hydroxybutyrate, (D) TG, (E) FFA, and (F) cholesterol were determined in fed and fasted C57BL/6 (WT) and Keap1-KD (KD) mice (n=4-9 per group). *, P<0.05, WT-Fasted compared with WT-Fed mice; #, P<0.05, KD-Fasted compared with KD-Fed mice; §, P<0.05, KD-Fasted compared with WT-Fasted mice.
Figure 2.
Enhanced Nrf2 activity prevents fasting-induced fatty liver in Keap1-KD mice.
(A) Representative images of Oil Red O staining of livers sections in fed and fasted C57BL/6 (WT) and Keap1-KD (KD) mice. Magnification: 200×. Scale bar=50 μm (n=3 per group). Hepatic (B) TG, (C) FFA, and (D) cholesterol from fed and fasted C57BL/6 (WT) and Keap1-KD (KD) mice (n=4-6 per group). *, P<0.05, WT-Fasted compared with WT-Fed mice; #, P<0.05, KD-Fasted compared with KD-Fed mice; §, P<0.05, KD-Fasted compared with WT-Fasted mice.
Figure 3.
Enhanced Nrf2 activity attenuates Pparα signaling induction and decreases lipogenic gene expression in liver by fasting.
(A) The induction of Cyp4a14 and CPT 1a by fasting was attenuated in liver of Keap1-KD (KD) mice. (B, C) Immunoblot analysis of Pparα in liver from fed or fasted C57BL/6 (WT) and Keap1-KD (KD) mice. (D)The expression of lipogenic genes, such as Fas, Acc1, and Scd1, was decreased in liver of Keap1-KD (KD) mice compared to C57BL/6 (WT) mice by fasting (n=4-6 per group). *, P<0.05, WT-Fasted compared with WT-Fed mice; #, P<0.05, KD-Fasted compared with KD-Fed mice; §, P<0.05, KD-Fasted compared with WT-Fasted mice.
Figure 4.
Enhanced Nrf2 activity increases AMPKα phosphorylation in liver of Keap1-KD mice.
Immunoblot analysis of (A) Acc-1, Scd-1, and (B) total and p-AMPKα, total-AMPKβ1/2, and p-AMPKβ1 in livers of C57BL/6 (WT) and Keap1-KD (KD) mice. *, P<0.05, WT-Fasted compared with WT-Fed mice; #, P<0.05, KD-Fasted compared with KD-Fed mice; §, P<0.05, KD-Fasted compared with WT-Fasted mice. (C) p-AMPKα levels were increased in primary hepatocytes isolated from C57BL/6 (WT) and Keap1-KD (KD) mice. &, P<0.05, KD compared with WT mice hepatocytes.
Figure 5.
Enhanced Nrf2 activity decreases lipid clearance rate and VLDL secretion in Keap1-KD mice.
(A) Enhanced Nrf2 activity impairs lipid clearance. Ten-week-old C57BL/6 (WT) and Keap1-KD (KD) mice were fasted overnight (more than 16 hrs), and then administered olive oil (15ml/kg) by oral gavage, serum TG content was measured 0, 1, 3, and 6 hrs after oil administration (n=5 to 8 per group) (&, P<0.05, Keap1-KD compared with WT mice). (B) The expression of fatty acid transporters of CD36 and FATP2 was deceased in liver from Keap1-KD mice compared with WT mice by fasting (n=4 to 6 per group). VLDL secretion was impaired in Keap1-KD mice (C and D). Ten-week-old C57BL/6 (WT) and Keap1-KD (KD) fasted for 5 hrs were injected with Tyloxapol (0.5 mg/g) and serum TG content was measured at 0, 1, and 2 hrs after injection (n=5 to 8 per group) (&, P<0.05, Keap1-KD compared with WT mice). The induction of (E) ApoB and (F) MTTP expression was attenuated in liver of Keap1-KD mice by fasting. The relative mRNA levels were quantified by quantitative real-time PCR and normalized with 18S as loading control (n=4-6 per group). *, P<0.05, WT-Fasted compared with WT-Fed mice; #, P<0.05, KD-Fasted compared with KD-Fed mice; §, P<0.05, KD-Fasted compared with WT-Fasted mice.
Figure 6.
Enhanced Nrf2 activity decreases fatty acid transport and results in increased fatty acids content in white adipose tissue.
(A) TG and (B) FFA content were measured in white adipose tissue of fed and fasted C57BL/6 (WT) and Keap1-KD (KD) mice (n=5-6 per group). (C) Nrf2 increased serum glycerol contents in Keap1-KD mice. (D, E, F) The induction of Pparα signaling and fatty acid transporter expression by fasting was attenuated in white adipose tissue of Keap1-KD mice. Total RNA was isolated from fed or fasted C57BL/6 (WT) and Keap1-KD (KD) mice and the relative mRNA levels were quantified by quantitative real-time PCR and normalized with β-2 microglobulin as loading control (n=4-6 per group). (G, H, I) Immunoblot analysis of Pparα and FATP1 in white adipose tissue from fed or fasted C57BL/6 (WT) and Keap1-KD (KD) mice. *, P<0.05, WT-Fasted compared with WT-Fed mice; $, P<0.05, KD-Fed compared with WT-Fed mice; #, P<0.05, KD-Fasted compared with KD-Fed mice; §, P<0.05, KD-Fasted compared with WT-Fasted mice.
Figure 7.
Enhanced Nrf2 activity increases glucose tolerance and Akt phosphorylation upon insulin challenge.
(A and B) Keap1-KD mice have a lower glucose load. Ten-week-old C57BL/6 (WT) and Keap1-KD (KD) mice fasted overnight were challenge with glucose (2g/kg) and blood glucose levels were determined at 0, 30, 60, 90, 120, 150 mins after glucose administration (n=6 per group) (*, P<0.05, Keap1-KD compared with WT mice). (C) Gene expression in skeletal muscle from C57BL/6 (WT) and Keap1-KD (KD) at 16-week-old age. The relative mRNA levels were quantified by quantitative real-time PCR and normalized with 18S as loading control (n=5 to 6 per group) (*, P<0.05, Keap1-KD compared with WT mice). (D) Immunoblot analysis of Glut4 in skeletal muscle of C57BL/6 (WT) and Keap1-KD (KD) mice at 16-week-old age. GAPDH was used as loading control. (E) Enhanced Nrf2 activity increased p-Akt levels upon insulin administration in skeletal muscle. Fifteen-week-old C57BL/6 (WT) and Keap1-KD (KD) mice fasted for 6 hrs were injected with a maximal bolus of insulin (5 U/kg) via portal vein. Five minutes later, gastrocnemius muscles were removed and snap frozen in lipid nitrogen. &, P<0.05 as indicated.