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. 2006 Oct;26(20):7372-87.
doi: 10.1128/MCB.00580-06. Epub 2006 Aug 14.

Role of transcription factor NFAT in glucose and insulin homeostasis

Teddy T C Yang  1 , Hee Yun Suk, Xiaoyong Yang, Opeyemi Olabisi, Raymond Y L Yu, Jorge Durand, Linda A Jelicks, Ja-Young Kim, Philipp E Scherer, Yuhua Wang, Yun Feng, Luciano Rossetti, Isabella A Graef, Gerald R Crabtree, Chi-Wing Chow
Affiliations

Role of transcription factor NFAT in glucose and insulin homeostasis

Teddy T C Yang et al. Mol Cell Biol. 2006 Oct.

Abstract

Compromised immunoregulation contributes to obesity and complications in metabolic pathogenesis. Here, we demonstrate that the nuclear factor of activated T cell (NFAT) group of transcription factors contributes to glucose and insulin homeostasis. Expression of two members of the NFAT family (NFATc2 and NFATc4) is induced upon adipogenesis and in obese mice. Mice with the Nfatc2-/- Nfatc4-/- compound disruption exhibit defects in fat accumulation and are lean. Nfatc2-/- Nfatc4-/- mice are also protected from diet-induced obesity. Ablation of NFATc2 and NFATc4 increases insulin sensitivity, in part, by sustained activation of the insulin signaling pathway. Nfatc2-/- Nfatc4-/- mice also exhibit an altered adipokine profile, with reduced resistin and leptin levels. Mechanistically, NFAT is recruited to the transcription loci and regulates resistin gene expression upon insulin stimulation. Together, these results establish a role for NFAT in glucose/insulin homeostasis and expand the repertoire of NFAT function to metabolic pathogenesis and adipokine gene transcription.

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Figures

FIG. 1.
FIG. 1.
Expression of NFAT upon adipocyte differentiation, in obesity, and in Nfatc2−/− Nfatc4−/− mice. Expression of NFATc2 and NFATc4 were determined upon adipocyte differentiation using 3T3/L1 cells (A) and with a genetic model of obesity using ob/ob mice (B). Cell extracts from different stages of adipocyte differentiation were examined by immunoblotting analysis to assess the expression of NFAT and C/EBP members (A). The expression of PPARγ and β-actin is also indicated. Semiquantitative RT-PCR was performed to assess the expression level of NFAT messages in epididymal fat depots of 8-week-old control and ob/ob mice (B). Relative intensity of NFAT messages was normalized to the expression of GAPDH and presented (C). The level of NFAT members in epididymal fat depots of Nfatc2−/− Nfatc4−/− mice (DKO) and control Nfatc2−/+ Nfatc4−/+ mice was determined by RT-PCR (D) and immunoblotting analysis (E). The expression level of adipocyte markers (PPARγ, C/EBPα, and fatty acid binding protein aP2) in Nfatc2−/− Nfatc4−/− mice (DKO) or control Nfatc2−/+ Nfatc4−/+ mice was also shown (D and E).
FIG. 2.
FIG. 2.
Nfatc2−/− Nfatc4−/− mice are resistant to high-fat-diet-induced obesity. Four-week-old Nfatc2−/− Nfatc4−/− mice (DKO) and control Nfatc2−/+ Nfatc4−/+ mice (n ≥ 15 mice per group) were fed ad libitum with regular chow (10% fat content) or a high-fat diet (59% fat content). Body weight was measured once a week for 20 weeks (panel A). Food intake (panel B) was measured for 12-week-old mice, and weights of various adipose depots (panel C) and tissue organs (panel D) harvested from 24-week-old mice were also shown. Epi, epididymal fat; SC, subcutaneous fat; PR, perirenal fat; BAT, brown adipose tissue.
FIG. 3.
FIG. 3.
Nfatc2−/− Nfatc4−/− mice exhibit reduced adiposity. Cross-sectional image of 12-week-old Nfatc2−/− Nfatc4−/− mice (DKO) and control Nfatc2−/+ Nfatc4−/+ mice by magnetic resonance imaging (A). The measured in vivo adiposity was also shown (B). Pathohistological analysis of white (WAT) (C) or brown (BAT) (D) adipose tissues of DKO and control mice was also shown. Effect of high-fat-diet-elicited obesity was indicated. Asterisks illustrate enlarged adipocytes.
FIG. 4.
FIG. 4.
Altered fasting insulin and glucose levels in Nfatc2−/− Nfatc4−/− mice. Glucose and insulin levels of 16-h-fasted Nfatc2−/− Nfatc4−/− (DKO) or control mice were measured (A and B). Levels of cholesterol, triglycerol, and FFA in Nfatc2−/− Nfatc4−/− (DKO) and control mice were also shown (C, D, and E).
FIG. 5.
FIG. 5.
Altered metabolic rate in Nfatc2−/− Nfatc4−/− mice. Oxygen consumption (VO2) (A and B), carbon dioxide production (VCO2) (C and D), heat production (E and F), and locomotor activity (G and H) of Nfatc2−/− Nfatc4−/− (DKO) and control mice (n = 8) were measured using an indirect open-circuit calorimeter system.
FIG. 6.
FIG. 6.
Increased insulin sensitivity and heightened glucose handling in Nfatc2−/− Nfatc4−/− mice. Nfatc2−/− Nfatc4−/− mice (DKO) and control Nfatc2−/+ Nfatc4−/+ mice fed ad libitum with regular chow or a high-fat diet for 20 weeks (n ≥ 15 mice per group) were subjected to an insulin tolerance test (ITT) (A) and glucose tolerance test (GTT) (C and E). Glucose (A and C) and insulin (E) levels were measured at indicated times and are presented. The area under the curve was also determined (B, D, and F).
FIG. 7.
FIG. 7.
Increased glucose uptake in Nfatc2−/− Nfatc4−/− mice. In vivo glucose uptake was determined by using 2-DOG during the glucose tolerance test. The amounts of [3H]glucose in various tissues were determined by scintillation counter and normalized to protein content and are presented.
FIG. 8.
FIG. 8.
Sustained activation of insulin signaling in Nfatc2−/− Nfatc4−/− mice. Activation of insulin receptor (IR) in adipose tissue (A), skeletal muscle (B), and liver (C) of 8-week-old Nfatc2−/− Nfatc4−/− (DKO) and control mice fed ad libitum with regular chow was determined at times indicated after insulin challenge (0.75 U/kg). Activation of insulin receptor downstream effectors, such as Akt protein kinase and ribosomal S6 kinase (S6K), was also determined by using phospho-Akt (P-Akt) and phospho-S6K (P-S6K) antibodies. Activation of IR was assessed by immunoprecipitation (IP) and subsequent immunoblotting (IB) analysis using phospho-Tyr antibody (P-Tyr). Time course analysis of insulin receptor activation and quantitation of insulin receptor phosphorylation were also illustrated (D).
FIG. 9.
FIG. 9.
Sustained activation in insulin signaling in Nfatc2−/− Nfatc4−/− mice is not cell autonomous. Primary hepatocytes isolated from Nfatc2−/− Nfatc4−/− (DKO) and control mice were challenged with insulin in vitro for times indicated (A). Cell extracts prepared were used to determine activation of insulin downstream effectors, including Akt and S6K protein kinases, by immunoblotting analysis using phospho-Akt (P-Akt) and phospho-S6K (P-S6K) antibodies. Expression of total Akt and S6K was used as the control. Activation of insulin signaling in primary macrophages isolated from peritoneal cavities of Nfatc2−/− Nfatc4−/− (DKO) or control mice was also determined (B).
FIG. 10.
FIG. 10.
Dysregulation in AMPK signaling in Nfatc2−/− Nfatc4−/− mice is also not cell autonomous. Tissue extracts prepared from adipose depots of overnight-fasted Nfatc2−/− Nfatc4−/− (DKO) and control mice were examined by immunoblotting analysis using phospho-AMPK (P-AMPK) and AMPK antibodies (A). Normalization of P-AMPK/AMPK was also presented (B). In vitro activation of AMPK in primary fibroblasts of Nfatc2−/− Nfatc4−/− (DKO) and control mice using AICAR (500 μM) and sorbitol (500 mM) is also shown (C and D).
FIG. 11.
FIG. 11.
Altered adipokine profile in Nfatc2−/− Nfatc4−/− mice. Blood from Nfatc2−/− Nfatc4−/− mice (DKO) and control Nfatc2−/+ Nfatc4−/+ mice fed ad libitum with regular chow or a high-fat diet for 20 weeks was harvested by cardiac puncture. Adipokine levels in serum of Nfatc2−/− Nfatc4−/− (DKO) and control mice (n ≥ 8 mice per group) were determined by Lincoplex analysis.
FIG. 12.
FIG. 12.
NFAT regulates resistin adipokine gene expression. Chromatin immunoprecipitation assays were performed to determine binding of NFAT to resistin transcription loci in the epididymal fat depot upon insulin challenge (A; see hatched box in panel D for region investigated). Recruitment of NFAT to the resistin transcription loci upon adipocyte differentiation was also shown (B). Isotype-matching IgG and/or amplification of the GAPDH loci were used as controls. Regulation of resistin gene transcription by NFAT upon adipocyte differentiation was also assessed by RT-PCR analysis (C). Effect of NFAT activation, using constitutive nuclear NFATc4 (cnNFATc4; shaded bars) or constitutive active calcineurin (ΔCn; filled bars), on the resistin promoter (−1 to −2000) and promoterless pGL3 plasmid was determined (D). Luciferase activity was normalized to β-galactosidase activity and presented. Resistin loci investigated by chromatin immunoprecipitation were illustrated as a hatched box, and filled triangles represent NFAT binding elements on the resistin promoter (D). Formation of NFAT-DNA complex at the −700-bp and −2,000-bp NFAT binding elements of the resistin promoter was assessed by gel mobility shift assays (E). Specificity of the NFAT:DNA complex was assessed by supershift analysis using antibody against NFAT (see asterisk for supershifted complex) and competition using excess amounts of unlabeled NFAT oligonucleotides.
FIG. 13.
FIG. 13.
Serum of Nfatc2−/− Nfatc4−/− mice elicits increased activation in insulin signaling. HepG2 cells were challenged with serum isolated from Nfatc2−/− Nfatc4−/− mice (DKO) and control Nfatc2−/+ Nfatc4−/+ mice for the time indicated (A). Activation of Akt and S6K was determined by immunoblotting analysis using phospho-Akt (P-Akt) and phospho-S6K (P-S6K) antibodies. The extent of ERK phosphorylation was also shown. Similar amounts of Akt, S6K, ERK, and β-actin were used as controls. The effect of recombinant resistin on Akt and S6K phosphorylation (B) and AMPK activation (C) elicited by the serum of Nfatc2−/− Nfatc4−/− mice (DKO) and control mice was also shown.
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