Nrf2 deficiency improves glucose tolerance in mice fed a high-fat diet

Abstract

Nrf2, a master regulator of intracellular redox homeostasis, is indicated to participate in fatty acid metabolism in liver. However, its role in diet-induced obesity remains controversial. In the current study, genetically engineered Nrf2-null, wild-type (WT), and Nrf2-activated, Keap1-knockdown (K1-KD) mice were fed either a control or a high-fat Western diet (HFD) for 12 weeks. The results indicate that the absence or enhancement of Nrf2 activity did not prevent diet-induced obesity, had limited effects on lipid metabolism, but affected blood glucose homeostasis. Whereas the Nrf2-null mice were resistant to HFD-induced glucose intolerance, the Nrf2-activated K1-KD mice exhibited prolonged elevation of circulating glucose during a glucose tolerance test even on the control diet. Feeding a HFD did not activate the Nrf2 signaling pathway in mouse livers. Fibroblast growth factor 21 (Fgf21) is a liver-derived anti-diabetic hormone that exerts glucose- and lipid-lowering effects. Fgf21 mRNA and protein were both elevated in livers of Nrf2-null mice, and Fgf21 protein was lower in K1-KD mice than WT mice. The inverse correlation between Nrf2 activity and hepatic expression of Fgf21 might explain the improved glucose tolerance in Nrf2-null mice. Furthermore, a more oxidative cellular environment in Nrf2-null mice could affect insulin signaling in liver. For example, mRNA of insulin-like growth factor binding protein 1, a gene repressed by insulin in hepatocytes, was markedly elevated in livers of Nrf2-null mice. In conclusion, genetic alteration of Nrf2 does not prevent diet-induced obesity in mice, but deficiency of Nrf2 improves glucose homeostasis, possibly through its effects on Fgf21 and/or insulin signaling.

Figure 1. Body weight and liver weight

(A) Growth curves of Nrf2-null, wild-type (WT), and Keap-1 knockdown (K1-KD) mice fed with either a high-fat Western Diet (HFD) or a control diet over a 12-week period. (B) End point body weight as well as liver/body weight ratio. Data are presented as mean ± S.E. (n=6). Two-way ANOVA was performed with genotype and treatment as main factors. Significant main effects and interactions were followed by Student-Newman-Keuls comparisons to assess the differences between groups. Asterisks (*) represent statistical differences (p < 0.05) between mice with altered Nrf2 activity and WT mice on the same diet. Pounds (#) represent statistical differences (p < 0.05) between HFD-treated and control diet-treated mice of the same genotype.

Figure 2. Glucose and insulin parameters in Nrf2-null, WT, K1-KD mice fed a control diet or HFD

(A) Glucose tolerance test (GTT) as well as area under the curves (AUCs) of serial glucose concentrations in the GTT. Asterisks (*) represent statistically significant differences (p < 0.05) compared with WT on the same diet at that time point. (B) Insulin tolerance test (ITT). (C) Blood insulin concentrations. For GTT, a bolus of intraperitoneal injection of 20% D-glucose was given after the basal blood glucose (time 0) was taken. Serial glucose levels were taken at indicated time points thereafter. The procedure of ITT resembled that of GTT except a single dose of Humulin N (0.75 U/kg) was given at time 0. The blood glucose levels at indicated time points in ITT were presented as percentages of time 0. Blood insulin concentrations were determined after euthanasia via a bolus of pentobarbital. Asterisks (*) represent statistical differences (p < 0.05) between mice with altered Nrf2 activity and WT mice on the same diet. Pounds (#) represent statistical differences (p < 0.05) between HFD-treated and control diet-treated mice of the same genotype.

Figure 3. Hepatic mRNA and protein expression of Nrf2 and Nrf2-target genes

(A) Messenger RNA of Nrf2, Nqo1, Gstm1, and Gclc were quantified by RT-qPCR, normalized to Gapdh, and presented as a ratio to WT control. The mRNA expression of Gapdh in mouse livers was not affected by either Nrf2 genotype or diet. (B) Western blots were performed with nuclear (Nrf2) or cytosol (Nqo1) fraction from control- and HFD-treated Nrf2-null, WT, and K1-KD mouse livers. Histone 3 (H3) and β-actin were used as nuclear and cytosol loading control, respectively. Density of blots was determined by Quantity One 1D Analiysis software, normalized to respective loading control, and presented as a ratio to wild-type control. Asterisks (*) represent statistical differences (p < 0.05) between mice with altered Nrf2 activity and WT mice on the same diet. Pounds (#) represent statistical differences (p < 0.05) between HFD-treated and control diet-treated mice of the same genotype.

Figure 4. Quantification of genes involved in glucose metabolism in the liver

(A) Messenger RNA of genes involved in hepatic glucose metabolism. Messenger RNA were determined by RT-qPCR, normalized to Gapdh, and presented as a ratio to WT control. (B) Nuclear accumulation of ChREBP protein. Density of ChREBP blots was normalized to the loading control TBP and presented as a ratio to wild-type control. Asterisks (*) represent statistical differences (p < 0.05) between mice with altered Nrf2 activity and WT mice on the same diet. Pounds (#) represent statistical differences (p < 0.05) between HFD-treated and control diet-treated mice of the same genotype.

Figure 5. Hepatic mRNA and protein expression of fatty acid metabolism-related genes

(A) Messenger RNA of genes involved in fatty acid biosynthesis. (B) Messenger RNA expression of genes involved in fatty acid catabolism. (C) Immunoblots of PPARα protein using nuclear fraction prepared from livers of mice with various Nrf2 activity. Density of PPARα protein blots was normalized to the loading control H3 and presented as a ratio to wild-type control. Asterisks (*) represent statistical differences (p < 0.05) between mice with altered Nrf2 activity and WT mice on the same diet. Pounds (#) represent statistical differences (p < 0.05) between HFD-treated and control diet-treated mice of the same genotype.

Figure 6. Hepatic mRNA expression of liver-derived endocrine hormones

(A) Messenger RNA expression of insulin-like growth factor-1 (IGF-1), IGF-1 binding protein-1 (Igfbp-1), and Fgf21. (B) Immunoblots of Fgf21 in mouse livers with graded activation of Nrf2. Density of Fgf21 protein blots was normalized to the loading control β-actin and presented as a ratio to wild-type control. Asterisks (*) represent statistical differences (p < 0.05) between mice with altered Nrf2 activity and WT mice on the same diet. Pounds (#) represent statistical differences (p < 0.05) between HFD-treated and control diet-treated mice of the same genotype.