Keap1 knockdown increases markers of metabolic syndrome after long-term high fat diet feeding

Abstract

The nuclear factor E2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway upregulates antioxidant and biotransformation enzyme expression to counter cellular oxidative stress. The contributions of Nrf2 to other cellular functions, such as lipid homeostasis, are emerging. This study was conducted to determine how enhanced Nrf2 activity influences the progression of metabolic syndrome with long-term high-fat diet (HFD) feeding. C57BL/6 and Keap1-knockdown (Keap1-KD) mice, which exhibit enhanced Nrf2 activity, were fed a HFD for 24 weeks. Keap1-KD mice had higher body weight and white adipose tissue mass compared to C57BL/6 mice on HFD, along with increased inflammation and lipogenic gene expression. HFD feeding increased hepatic steatosis and inflammation to a greater extent in Keap1-KD mice compared to C57BL/6 mice, which was associated with increased liver Cd36, fatty acid-binding protein 4, and monocyte chemoattractant protein 1 mRNA expression, as well as increased acetyl-CoA carboxylase 1 and stearoyl-CoA desaturase-1 protein expression. The HFD altered short-term glucose homeostasis to a greater degree in Keap-KD mice compared to C57BL/6 mice, which was accompanied by downregulation of insulin receptor substrate 1 mRNA expression in skeletal muscle. Together, the results indicate that Keap1 knockdown, on treatment with HFD, increases certain markers of metabolic syndrome.

Keywords: Free radicals; Keap1; Metabolic syndrome; Nrf2; Steatosis.

Figure 1. Body, liver and WAT weight and adipocyte size of C57BL/6 and Keap1-KD mice fed a 10% kCal low fat diet (LFD) or 60% kCal high fat diet (HFD)

A) Body weights (starting from age 6 weeks) and B) food consumption of C57BL/6 (C57) and Keap1-KD mice fed a LFD or HFD from weaning age to 27 weeks (starting at age 6 weeks). C) Abdominal adipose tissue weight and liver to body weight ratio. Differences between the groups were analyzed by a one-way ANOVA followed by a Duncan’s post hoc test. Different letters indicate statistically significant difference between the groups (p<0.05). For example, letter “a” is significantly different from “b”, but not different from “a”. Also, “a” is significantly different from “b,c” but not different from “a,b”.

Figure 2. Hepatic lipid accumulation and triglyceride (TG) content in C57BL/6 (C57) and Keap1-KD mice fed a 10% kCal low fat diet (LFD) or 60% kCal high fat diet (HFD)

A) Hematoxylin and eosin staining of formaldehyde fixed paraffin embedded liver tissues (200X magnification). B) Oil red O staining of liver sections. Frozen liver tissues were sectioned in 5 m sections, stained with Oil red O, and counter stained with hematoxylin (200X magnification). C) Hepatic TG content. Lipids were extracted using a methanol-chloroform based protocol and the resulting TG content was assayed using kit from Pointe Scientific (Canton, MI). Differences between the groups were analyzed by a one-way ANOVA followed by a Duncan’s post hoc test. Different letters indicate statistically significant difference between the groups (p<0.05).

Figure 3. Lipogenic, Nrf2, and Nrf2 target gene expression in livers and white adipose tissue (WAT) of C57BL/6 (C57) and Keap1-KD mice fed a 10% kCal low fat diet (LFD) or 60% kCal high fat diet (HFD)

Total RNA was extracted from the livers by phenol-chloroform extraction and mRNA was quantified using Quantigene Plex 2.0 or Branched DNA Signal Amplification assay (Affymetrix, Santa Clara, CA). A) Peroxisome proliferator activated receptor (Ppar-γ), Steroyl CoA desaturase (Scd1), Fatty acid binding protein 4 (Fabp4), Lipoprotein lipase (Lpl), and Cluster of differentiation (Cd36) mRNA expression. B) Nrf2, NADPH:quinone oxidoreductase (Nqo1), glutamate cysteine ligase (Gclc) mRNA expression in liver. C) Nrf2 and Nqo1 mRNA expression in WAT. Differences between the groups were analyzed by a one-way ANOVA followed by a Duncan’s post hoc test. Different letters indicate statistically significant difference between the groups (p<0.05). For example, letter “a” is significantly different from “b”, but not different from “a”. Also, “a” is significantly different from “b,c” but not different from “a,b”.

Figure 4. Protein expression of lipogenic enzymes in livers of C57BL/6 (C57) and Keap1-KD mice fed a 10% kCal low fat diet (LFD) or 60% kCal high fat diet (HFD)

A) Western blots for lipogenic enzymes in liver. Total protein extracts were separated on polyacrylamide gel, immunoblotted, and chemiluminescence was captured on X-ray films. B) Western blot quantification. The resulting blots obtained were evaluated using Quantity One® software (Biorad, Hercules, CA) and band density is plotted as percent of C57BL/6 fed LFD. Differences between the groups were analyzed by a one-way ANOVA followed by a Duncan’s post hoc test. Different letters indicate statistically significant difference between the groups (p<0.05).

Figure 5. Inflammatory markers in liver and serum of C57BL/6 (C57) and Keap1-KD mice fed a 10% kCal low fat diet (LFD) or 60% kCal high fat diet (HFD)

A) Neutrophil (NIMP R14) staining of paraffin embedded liver sections. Images displayed in 200X magnification. B) Serum cytokine Monocyte chemoattractant protein 1 (Mcp1) levels C) mRNA expression of inflammatory cytokine markers Mcp1, Tumor necrosis factor (Tnf) and Cd11c in liver. Total RNA was extracted from liver tissue by phenol-chloroform extraction and mRNA was quantified using Quantigene Plex 2.0 assay (Affymetrix, Santa Clara, CA). by a Duncan’s post hoc test. Different letters indicate statistically significant difference between the groups (p<0.05).

Figure 6. White adipose tissue (WAT) cellularity and inflammation in C57BL/6 (C57) and Keap1-KD mice fed a 10% kCal low fat diet (LFD) or 60% kCal high fat diet (HFD)

A) Hematoxylin and eosin stained sections of paraffin embedded WAT. Images displayed in 200X magnification. B) Messenger RNA expression of proinflammatory macrophage markers monocyte chemoattractant protein 1 (Mcp1), tumor necrosis factor (Tnf) and Cd11c in WAT. Differences between the groups were analyzed by a one-way ANOVA followed by a Duncan’s post hoc test. Different letters indicate statistically significant difference between the groups (p<0.05).

Figure 7. Glucose tolerance test (GTT) and expression of insulin signaling molecules in skeletal muscle (SKM) of C57BL/6 (C57) and Keap1-KD mice fed a 10% kCal low fat diet (LFD) or 60% kCal high fat diet (HFD)

A) GTT after 23 weeks of LFD or HFD treatment. Mice were fasted overnight and challenged with an oral glucose bolus (1g/kg). Blood glucose levels were recorded at 0, 15, 30, 60, and 120-minute time points using Contour® glucose meter (Bayer HealthCare LLC, Tarrytown, NY). *Represents statistically significant difference of glucose levels between C57BL/6 and Keap1-KD mice fed same diet. B) Area under the curve (AUC) plotted for the GTT. C) Messenger RNA expression of insulin receptor substrate 1 (Irs1) and glucose transporter Glut4 in SKM. Total RNA was extracted from liver tissue by phenol-chloroform extraction and mRNA was quantified using Quantigene Plex 2.0 assay (Affymetrix, Santa Clara, CA). For parts Differences between the groups were analyzed by a one-way ANOVA followed by a Duncan’s post hoc test. Different letters indicate statistically significant difference between the groups (p<0.05). For example, letter “a” is significantly different from “b”, but not different from “a”. Also, “a” is significantly different from “b,c” but not different from “a,b”. D) Protein expression of Glut4 from skeletal muscle of C57BL/6 and Keap1-KD mice fed LFD and HFD by western blot (n=2 per group).