TET proteins play an important role in transcriptional regulation by serving as transcriptional activators or repressors. Nevertheless, the role of TET proteins in adipocyte biology and energy metabolism in vivo remains to be fully determined. In this article I aimed to identify the function of TET proteins in adipocyte biology and metabolism.
Fat-specific ablation of all TET proteins in mice prevents body weight gain and metabolic disturbances on a high-fat diet via enhanced fat browning, lipolysis, and thermogenesis. Second, to clarify the role of TET proteins in brown fat activation and white fat browning by thermogenic stimuli, I demonstrated that TET proteins are important physiological suppressors of mitochondrial oxidative capacity in brown and beige adipocytes via the reduction of mitophagy. Genetic ablation of TET proteins increases mitochondrial mass and function via increased mitochondrial biogenesis and decreased mitophagy.
Overall, modulation of adipocyte TET proteins may provide a new therapeutic avenue for the treatment of obesity and related metabolic disorders. TET proteins are critical for high-fat diet-induced transcriptomic changes in visceral adipose tissue.
Introduction
- TET proteins facilitate DNA demethylation in mammals
- Obesity and adipose tissues
- β-adrenergic signaling in brown and beige adipocytes
- β-adrenergic receptors in adipose thermogenesis
- Mitophagy in brown and beige adipose tissue
Adipose tissue (AT) is a highly plastic organ that possesses a remarkable capacity to change its size and function in response to environmental inputs, such as nutritional status and temperature (9). White adipose tissue (WAT) mainly contains white adipocytes that store excess calories in the form of triglycerides (TG) in unilocular lipid droplets (LDs). Strikingly, brown adipose tissue (BAT) from β-less mice display features similar to white adipose tissue (WAT), populated mainly by markedly enlarged adipocytes with unilocular lipid droplets (LDs) that lack UCP1 expression .
In rodents, selective β3-AR agonists exert excellent antiobesity and antidiabetic effects by increasing energy expenditure due to their ability to induce lipolysis, mitochondrial biogenesis, lipid oxidation, and the thermogenic program in adipose tissue ( 47 – 51 ). Together, these results strongly suggest that strategies to enhance β3-AR expression and catecholamine sensitivity with minimal side effects in adipose tissue may hold promise in obesity treatments. Activation of brown/beige adipose tissue is associated with enhanced lipolysis and inhibition of mitophagic processes.
On the other hand, inactivation of brown/beige adipose tissue means loss of mitochondria and disappearance of beige adipocytes (bleaching). Inactivation of brown/beige adipose tissue is associated with lipid accumulation and the induction of autophagy and mitophagy, which clear cellular components responsible for thermogenesis (Figure 6).
Results
Loss of adipose TET proteins enhances β-adrenergic responses and protects against obesity by
- TET deficiency increases β-adrenergic responsiveness by upregulating β3-AR expression in
- TET proteins suppress 3-AR expression and signaling, which is mainly independent of
- Adipocyte-specific TET ablation increases the responsiveness to 3-AR signals in vivo. …26
- Adipose TET deficiency protects against the detrimental metabolic effects of obesity
- Adipocyte-specific Tet TKO mice resist 3-AR reduction and catecholamine resistance during
- Adipose TET deficiency leads to the maintenance of higher capacity for thermogenesis and
- TET deficiency prevents HFD-induced transcriptional alterations in visceral adipose
- TET proteins cooperate with HDACs to directly repress 3-AR transcription in adipocytes
In vitro lipolysis in WT and Tet-TKO adipocytes stimulated with ISO (5 μM), forskolin (20 μM), or isobutylmethylxanthine (IBMX) (0.2 mM). H and I) Hematoxylin and eosin (H&E) staining (H, n = 10) and frequency distribution of adipocyte cell size (I) in iWAT and eWAT from WT and Tet TKO mice fed CD or HFD. Daily food intake of female WT and Tet TKO mice fed CD or HFD.
B and C) Oral glucose tolerance (OGTT) (B) and insulin tolerance (IPITT) (C) tests in WT and Tet TKO mice fed a CD. Plasma concentrations of high-density lipoprotein (HDL) and triglycerides (TG) in CD- or HFD-fed WT and Tet TKO mice. H and I) mRNA levels of Gapdh-related thermogenesis-related genes in BAT (H) or iWAT (I) from HFD-fed WT and Tet TKO mice.
B and C) Relative mitochondrial DNA content (B) and ex vivo fatty acid oxidation (C) in the indicated adipose depots from WT and Tet TKO mice fed a HFD.
Adipose TET depletion induces white fat browning and BAT activation in cold-induced
- Adipose TET expression is repressed by thermogenic stimuli
- TET ablation in adipocyte promotes fat browning and thermogenesis
- Adipose TET deficiency leads to the maintenance of higher capacity for thermogenesis and
- Ablation of TET reduces mitophagy and increases mitochondrial oxidative respiration
To determine whether TET proteins affect fat browning in vivo, WT and TKO mice were acclimated to 4 °C for 7 days. Compared to WT mice, TKO mice lost more weight primarily due to a decrease in WAT mass (Figure 33A-D), whereas the weight of mice housed at room temperature (RT, 22 °C) remained comparable. iWAT of TKO mice appeared browner in color (Figure 34A), had more abundant Ucp1+ multilocular beige adipocytes (Figure 34B and C), and showed higher mRNA levels of genes associated with beiging (Cd137, Tbx1 and Tmem26), thermogenesis (Ucp1), Ppargcla, Ppara, etc.), lipolysis (Adrb3, Atgl, etc.), and fatty acid oxidation (Acadm, Acadvl, Acox1, etc.) (Figure 34, D-G).
A and B) Representative photographs of dissected mice (A) and iWAT, eWAT and BAT (B) of WT and TKO mice maintained at 4 °C for 1 week. -G) mRNA levels of beige marker (D), thermogenesis (E), lipolysis (F), and fatty acid oxidation (G) genes relative to Gapdh in iWAT from cold-exposed CD-fed WT and TKO mice. E and F) mRNA levels of lipolysis (E) and fatty acid oxidation genes (F) relative to Gapdh in BAT from CD-fed WT and TKO mice housed at 4 °C for 1 week.
Cold-exposed TKO mice showed higher serum glycerol and free fatty acid levels than WT mice, indicating increased lipolysis to provide fuel for adaptive thermogenesis (Figure 36A). Primary iWAT and BAT explants from cold-exposed TKO mice had an increased capacity to oxidize fatty acids (Figure 36B). Notably, assessment of metabolic rate by indirect calorimetry showed that TKO mice showed significantly higher rates of oxygen consumption (VO2), carbon dioxide production (VCO2), and heat production at 4 °C, but not at 30 °C or 22 °C, than their WT counterparts, despite comparable levels of motor activity (Figure 36C-F).
Consistent with these observations, TKO mice maintained higher surface and core (rectal) temperatures than WT littermates under basal conditions and after cold exposure (Figure 36G and H). Ex vivo fatty acid oxidation in iWAT and BAT from CD-fed WT and TKO mice, kept at RT or 4°C for 1 week. TET deletion in immortalized brown preadipocytes and TET deficiency increase thermogenic gene expression in vitro.
Immunoblot analysis showed that the levels of LC3II relative to LC3I were lower in TKO adipocytes (Figure 38B). To further investigate the functional function of TET in adipocytes, I monitored WT and TKO adipocytes using guinea pig respirometry. Flow cytometry analysis of mitophagy in WT and TKO immortalized brown adipocytes expressing mt-Keima.
Discussion
Wu et al., Dual functions of Tet1 in transcriptional regulation in mouse embryonic stem cells. Lee et al., Increased O2 consumption in adipocytes triggers HIF-1alpha, resulting in inflammation and insulin resistance in obesity. Chondronikola et al., Brown adipose tissue improves whole-body glucose homeostasis and insulin sensitivity in humans.
Orava et al., Different metabolic responses of human brown adipose tissue to cold and insulin activation. Widen et al., Association of a polymorphism in the beta 3-adrenergic receptor gene with features of the insulin resistance syndrome in fins. Collins et al., Impaired expression and functional activity of beta 3 and beta 1 adrenergic receptors in adipose tissue of congenitally obese (C57BL/6J ob/ob) mice.
Shenoy et al., beta-arrestin-dependent, G protein-independent activation of ERK1/2 by the beta2 adrenergic receptor. Arch et al., The atypical beta-adrenoceptor on brown adipocytes as a target for antiobesity drugs. Finlin et al., The beta3-adrenergic receptor agonist mirabegron improves glucose homeostasis in obese humans.
O'Mara et al., Chronic mirabegron treatment increases human brown fat, HDL cholesterol, and insulin sensitivity. et al., Acute loss of TET function results in aggressive myeloid cancer in mice. Ferrari et al., HDAC3 is a molecular brake of the metabolic switch that supports browning of white adipose tissue.
Li et al., Histone Deacetylase 1 (HDAC1) Negatively Regulates the Thermogenic Program in Brown Adipocytes via Coordinated Regulation of Histone H3 Lysine 27 (H3K27). Tahiliani et al., Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Lee et al., TonEBP/NFAT5 promotes obesity and insulin resistance through epigenetic suppression of white adipose tissue formation.
Materials & Methods
Animals
To delete all Tet-floxed alleles in adipose tissue, Tet1fl/fl Tet2 fl/fl Tet3fl/fl mice were crossed with Adiponectin-Cre mice. For inducible excision of Tet-floxed alleles, Tet1fl/fl Tet2fl/fl Tet3fl/fl mice were crossed with Cre-ERT2 transgenic mice. Mice were bred and housed in the In Vivo Research Center (IVRC) at Ulsan National Institute of Science and Technology (UNIST, Ulsan, Korea) under specific pathogen-free conditions.
All animal experiments were approved by the Institutional Animal Care and Use Committee of UNIST (UNISTACUC) and were performed in accordance with institutional guidelines.
Cell Lines
Establishment of Immortalized Preadipocytes
Adipogenic Differentiation in vitro
Lentivirus transduction
Diet-Induced Obesity, Cold Exposure, and CL-316,243-Induced Browning
Glucose Tolerance Test (GTT) and Insulin Tolerance Test (ITT)
Metabolic Analyses
ELISA and Lipid Analyses
Histology
Lipolysis Assays
Fatty Acid Oxidation
Assessment of oxygen consumption rate (OCR)
Mitochondrial DNA quantification
Analysis of RNA Sequencing Data
Flow Cytometry
Cloning of TET Expression Vectors and Co-Immunoprecipitation
Chromatin Immunoprecipitation Coupled with qPCR
Dot Blot Analysis
RNA Purification, Reverse Transcription, and Real-Time RT-PCR
Immunoblot Analysis
Luciferase Reporter Assay
Mitophagy Analysis
Statistical Analysis
List of oligonucleotides used in this study
Zhang et al., Tet2 is required to resolve inflammation by recruiting Hdac2 to specifically suppress IL-6. Wu et al., Beige adipocytes are a distinct type of thermogenic adipose cells in mice and humans. Yuan et al., Vitamin C inhibits the metabolic changes induced by Tet1 insufficiency during high-fat stress.
Montagner et al., TET2 Regulates Mast Cell Differentiation and Proliferation Through Catalytic and Non-Catalytic Activities. Wu et al., Glucose-regulated phosphorylation of TET2 by AMPK reveals a pathway linking diabetes to cancer. Ko et al., Ten-Eleven-Translocation 2 (TET2) negatively regulates hematopoietic stem cell homeostasis and differentiation in mice.
Kang et al., Simultaneous deletion of the methylcytosine oxidases Tet1 and Tet3 increases transcriptome variability in early embryogenesis.
