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. 2016 Feb 1;591:57-65.
doi: 10.1016/j.abb.2015.11.040. Epub 2015 Dec 14.

Keap1/Nrf2 Pathway Activation Leads to a Repressed Hepatic Gluconeogenic and Lipogenic Program in Mice on a High-Fat Diet

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Free PMC article

Keap1/Nrf2 Pathway Activation Leads to a Repressed Hepatic Gluconeogenic and Lipogenic Program in Mice on a High-Fat Diet

Stephen L Slocum et al. Arch Biochem Biophys. .
Free PMC article

Abstract

The Keap1/Nrf2 pathway, known to regulate the expression of a series of cytoprotective and antioxidant genes, has been studied in the context of obesity and type 2 diabetes; diseases that are characterized by chronic oxidative stress. There is increasing evidence, however, that the transcription factor Nrf2 can crosstalk with pathways not directly related to cytoprotection. Our present work focuses on the effect of Nrf2 on hepatic gluconeogenesis and lipogenesis, two metabolic processes which are dysregulated in the obese/diabetic state. To this end, a genetic mouse model of Nrf2 pathway activation was used (Keap1-hypo; both Keap1 alleles are hypomorphic) and was exposed to a 3-month high-fat diet along with the relevant control wild-type mice. The Keap1-hypo mice were partially protected from obesity, had lower fasting glucose and insulin levels and developed less liver steatosis compared to the wild-type. Key gluconeogenic and lipogenic enzymes were repressed in the Keap1-hypo livers with concomitant activated Ampk signaling. Primary Keap1-hypo hepatocyte cultures also show increased Ampk signaling and repressed glucose production. In conclusion, increased Keap1/Nrf2 signaling in the liver is accompanied by repressed gluconeogenesis and lipogenesis that can, at least partially, explain the ameliorated diabetic phenotype in the Keap1-hypo mice.

Keywords: Ampk; Diabetes; Gluconeogenesis; Keap1; Lipogenesis; Nrf2.

Figures

Figure 1
Figure 1. Keap1-hypo mice gained less weight, had ameliorated glucose tolerance and developed less hepatic steatosis than the WT after 3 months on HFD
A. Body weights of WT or Keap1-hypo mice fed either a HFD or a StD are plotted against time. Data show mean ± SEM. n = 9–10 per genotype per diet type. *P<0.05 (WT-HFD vs. Keap1-hypo-HFD). B. Representative H&E stained liver sections from WT or Keap1-hypo mice fed a StD or a HFD. C. Hepatic triglycerides (TG) levels normalized to total protein amount. Data show mean ± SEM. n = 9–10 per genotype per diet type. *P<0.05 (WT vs Keap1-hypo).
Figure 2
Figure 2. Keap1-hypo mice show repressed hepatic expression of gluconeogenic and lipogenic enzymes
A. mRNA levels of Pepck, G6pase and Fasn as assessed by qRT-PCR. Data show mean ± SEM. n = 9–10 per genotype. *P<0.05. B. Pepck and Fasn expression as assessed by immunoblotting. Coomassie blue staining and Gapdh expression is shown for loading control purposes. C. Relative quantification of Pepck and Fasn protein levels. Coomassie blue staining intensity was used as loading control. Data show mean ± SEM. n = 5 per genotype. *P<0.05
Figure 3
Figure 3. Keap1-hypo primary hepatocytes show repressed glucose production and increased Ampk signaling
A. In vitro gluconeogenesis is repressed in Keap1-hypo primary hepatocytes compared to WT. Glucose production induced by incubation with 1 μM Dexamethasone and 250 μM 8-Br-cAMP is attenuated in the Keap1-hypo primary hepatocytes. Data show mean ± SEM from 4 individual experiments with 6–11 technical replicates in each experiment. *P<0.05 (induced glucose production of WT vs Keap1-hypo primary hepatocytes). B. Immunoblots of p-Ampkα-Thr172, p-Ampkβ1-Ser108, p-Torc2-Ser171 show that AMPK signaling is activated in primary hepatocytes derived from Keap1-hypo mice compared to WT ones. A representative immunoblot per protein is shown from a total number of n=4 independent experiments.
Figure 4
Figure 4. Ampk signaling is activated in the Keap1-hypo livers after HFD
A. Immunoblotting analysis of p-Ampkα-Thr172, total Ampkα, p-Ampkβ1-Ser108, total Ampkβ1 show that Ampk is activated in the Keap1-hypo mice after 90 days on HFD. Downstream targets of Ampk, Torc2 and Acc1 show increased phosphorylation at Ser171 and Ser79 correspondingly. Total Acc1 levels are lower in the Keap1-hypo mice. B. Relative quantification of p-Ampkα-Thr172, p-Ampkβ1-Ser108, p-Torc2-Ser171, p-Acc1-Ser79 and Acc1. Data show mean ± SEM. n = 5 per genotype. *P<0.05 (WT-HFD vs. Keap1-hypo-HFD). C. Immunoblotting analysis of p-Ampkα-Thr172 and total Ampkα show that Ampk is activated in the Keap1-hypo mice on StD.
Figure 5
Figure 5. In vivo liver insulin signaling and indirect calorimetry in Keap1-hypo mice
A. Insulin signaling study. Mice that have been on high-fat diet for 90 days were fasted for 16h overnight and then injected intraportally with 10 units/kg insulin. Liver biopsies were obtained before and five minutes after the insulin injection. Immunoblots show phosphorylation of Akt at Ser473, Thr308 and total Akt in the livers of WT and Keap1-hypo mice before (−) and after (+) intraportal insulin administration. The diagrams show relative protein amount of p-Akt Ser473 and p-Akt Thr308 after normalization to total Akt levels. n = 5 per genotype. *P<0.05 compared to WT-HFD. B. Raw energy expenditure (EE) values plotted against body weights (BW) or lean body mass (LBM) (as calculated by Echo-MRI) for both WT and Keap1-hypo mice after 50 days on HFD. The diagrams show body weight-normalized and lean body mass-normalized energy expenditure. Data show mean ± SEM. N=5 per genotype. *P<0.05 (WT-HFD vs. Keap1-hypo-HFD). C. Respiratory exchange ratio (RER) and energy intake (EI) of WT and Keap1-hypo mice after 50 days on HFD.

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