Deficiency in the nuclear factor E2-related factor-2 transcription factor results in impaired adipogenesis and protects against diet-induced obesity

Abstract

Nuclear factor E2-related factor 2 (Nrf2) is a cap-n-collar basic leucine zipper (CNC-bZIP) transcription factor that is well established as a master regulator of phase II detoxification and antioxidant gene expression and is strongly expressed in tissues involved in xenobiotic metabolism including liver and kidney. Nrf2 is also abundantly expressed in adipose tissue; however, the exact function of Nrf2 in adipocyte biology is unclear. In the current study we show that targeted knock-out of Nrf2 in mice decreases adipose tissue mass, promotes formation of small adipocytes, and protects against weight gain and obesity otherwise induced by a high fat diet. In mouse embryonic fibroblasts, 3T3-L1 cells, and human subcutaneous preadipocytes, selective deficiency of Nrf2 impairs adipocyte differentiation. Deficiency of Nrf2 also leads to decreased expression of peroxisome proliferator-activated receptor gamma (PPARgamma), CCAAT enhancer-binding protein alpha (C/EBPalpha), and their downstream targets during adipocyte differentiation. Conversely, activation of Nrf2 in 3T3-L1 cells by stable knockdown of its negative regulator Keap1 enhances and accelerates hormone-induced adipocyte differentiation. Transfection of Nrf2 stimulates Ppargamma promoter activity, and stable knockdown of Keap1 enhances PPARgamma expression in 3T3-L1 cells. In addition, chromatin immunoprecipitation studies show that Nrf2 associates with consensus binding sites for Nrf2 in the Ppargamma promoter. These findings demonstrate a novel biologic role for Nrf2 beyond its participation in detoxification and antioxidant pathways and place Nrf2 within the limited network of transcription factors that control adipocyte differentiation by regulating expression of PPARgamma.

FIGURE 1.

Nrf2 knock-out mice have lower amounts of adipose tissue. A, shown is body weight in adult (age 12 weeks) male wild type (WT; n = 8) and Nrf2 KO (n = 10) mice fed a regular chow diet. B, shown is organ weight in wild type and Nrf2 KO mice. BW, body weight. C, the top panel shows the white adipose tissue (WAT) in wild type and Nrf2 KO mice. The bottom panel shows the total fat pad weight (epididymal, retro-peritoneal, and inguinal) in wild type and Nrf2 KO mice. All the measurements were normalized to body weights of individual mice, n = 8 per group. *, p < 0.05. D, representative histological sections of white adipose tissues stained with hematoxylin and eosin are shown. E, shown are cell numbers per mm² in the sections of adipose tissues shown in panel D. *, p < 0.05.

FIGURE 2.

Nrf2 knock-out animals on HFD gain less weight over time. A, shown are body weights of 4-week-old wild type (WT) and Nrf2 KO mice before high fat feeding and weights after 12 weeks on a high fat diet. All mice in this experiment were males (n = 8 per group). *, p < 0.05. B, shown are total fat pad weights (epididymal, retro-peritoneal, and inguinal) of wild type and Nrf2 KO mice on a high fat diet for 12 weeks. n = 8; *, p < 0.05. WAT, white adipose tissue; BW, body weight. C, shown is the measurement of daily food intake of wild type and Nrf2 KO mice on HFD. D, fecal triglyceride (TG) levels are shown of wild type and Nrf2 KO mice. n = 8; *, p < 0.05. E, horizontal activity of wild type and Nrf2 KO mice is shown. AU, arbitrary units. n = 4; *, p < 0.05.

FIGURE 3.

Adipogenesis and induction of adipogenic genes are impaired in Nrf2-deficient MEFs. A, MEFs derived from E13.5 wild type (WT) and Nrf2 KO embryos were cultured with DMI to induce differentiation into adipocytes. Cells were then stained after 7 days with ORO to visualize lipid accumulation. A representative of three independent experiments is shown. B, shown is reverse transcription-PCR analysis of key lipogenic genes in wild type and Nrf2 KO MEFs differentiated for 5 days. Differentiated 3T3-L1 cells were used as positive control for adipogenic induction. C, protein levels of PPARγ and C/EBPγ in wild type and Nrf2 KO white adipose tissue (epididymal, retro-peritoneal, and inguinal) extracts measured by Western blotting. β-Actin levels are shown as loading controls.

FIGURE 4.

Stable knockdown of Nrf2 by shRNA lentivirus suppresses adipogenesis in 3T3-L1 cells and primary human preadipocytes. A, shown is expression of Nrf2 mRNA and protein (inset: S, sh-Scr; N, sh-Nrf2-I) in 3T3-L1 cells transduced with shRNA lentivirus targeted against mouse Nrf2. n = 3. B, ORO staining of scramble (Scr) and knockdown cells treated with a hormonal mixture (detailed under “Experimental Procedures”) for 3 days followed by 3 days of culture in DMEM supplemented with 10% FBS. The number in parentheses after each plate name is the relative volume (density × area) of ORO staining. A representative of 15 independent experiments is shown. C, expression is shown of Nrf2 in subcutaneous human preadipocytes infected with shRNA lentivirus targeting human Nrf2. n = 3. D, shown is ORO staining of differentiated human adipocytes (ORO staining). Confluent cells were kept in adipocyte differentiation medium (Zen-Bio #DM-2) for 7 days followed by 5 days of culture in adipocyte medium (Zen-Bio #AM-1). A representative of three independent experiments is shown. E, expression of adipogenic genes is shown. Levels of mRNA are expressed as -fold of sh-Scr at day 0, n = 3. F, protein levels of adipogenic markers are shown. A representative experiment of 3–5 independent experiments is shown. C, control.

FIGURE 5.

Stable knockdown of Keap1 by shRNA lentivirus enhances hormonal mixture-induced adipogenesis in 3T3-L1 cells. A, expression is shown of Keap1 mRNA and protein (inset) in 3T3-L1 cells transduced with shRNA lentivirus targeting mouse Keap1. Sh-K-I∼V represent five different shRNAs targeting Keap1. n = 3. B, ORO staining of differentiated cells is shown. Cells were treated with differentiation medium for 3 days followed by 3 days of culture in DMEM supplemented with 10% FBS. The number in parentheses after each plate name is the relative volume of ORO staining. A representative of five independent experiments is shown. C, expression of adipogenic genes is shown. Levels of mRNA are expressed as -fold sh-Scr at day 0. n = 3. D, shown are protein levels of adipogenic markers. A representative of 3–5 independent experiments is shown.

FIGURE 6.

Nrf2 regulates PPARγ expression. A, shown is transactivation of the mouse Pparγ promoter by Nrf2. MEF cells were transfected with the luciferase gene under the mouse Pparγ promoter along with either vector or Nrf2 cDNA. Cells were harvested 48 h later, and luciferase activity was determined. Transfection efficiency was normalized to Renilla luciferase under control of the cytomegalovirus promoter. Bars represent the mean of three independent experiments ± S.E. (*, p < 0.05). B, the luciferase gene under the mouse Ppar γ promoter was transfected into wild type (WT), Nrf2 KO, or Nrf2 KO fibroblasts along with Nrf2 cDNA. Luciferase activity was measured 48 h after. Bars represent the mean ± S.E., n = 3 independent experiments; *, p < 0.05. C, expression of Pparγ in 3T3-L1 cells with Nrf2- or Keap1 knockdown is shown. *, p < 0.05 versus sh-Scr. D, Western blotting of PPARγ in 3T3-L1 cells with Nrf2- or Keap1 knockdown is shown.

FIGURE 7.

Chromatin immunoprecipitation assays indicate a physical association between Nrf2 and the Pparγ promoter region. Chromatin immunoprecipitation assays were performed on 3T3-L1 chromatin using either Nrf2 or preimmune antisera as the negative control. A primer walk was performed across a 3-kb region encompassing the Pparγ2 promoter. The diagram indicates the location of the primer sets. Open boxes indicate the locations of the Nrf2 consensus binding site. PCR amplification of the Nqo1 promoter was used as a positive control. Non-immunoprecipitated chromatin (1%) was used as an input control.