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Adipocytes fail to maintain cellular identity during obesity due to reduced PPAR? activity and elevated TGF?-SMAD signaling.

ABSTRACT: OBJECTIVE:Obesity due to overnutrition causes adipose tissue dysfunction, which is a critical pathological step on the road to type 2 diabetes (T2D) and other metabolic disorders. In this study, we conducted an unbiased investigation into the fundamental molecular mechanisms by which adipocytes transition to an unhealthy state during obesity. METHODS:We used nuclear tagging and translating ribosome affinity purification (NuTRAP) reporter mice crossed with Adipoq-Cre mice to determine adipocyte-specific 1) transcriptional profiles (RNA-seq), 2) promoter and enhancer activity (H3K27ac ChIP-seq), 3) and PPAR? cistrome (ChIP-seq) profiles in mice fed chow or a high-fat diet (HFD) for 10 weeks. We also assessed the impact of the PPAR? agonist rosiglitazone (Rosi) on gene expression and cellular state of adipocytes from the HFD-fed mice. We integrated these data to determine the transcription factors underlying adipocyte responses to HFD and conducted functional studies using shRNA-mediated loss-of-function approaches in 3T3-L1 adipocytes. RESULTS:Adipocytes from the HFD-fed mice exhibited reduced expression of adipocyte markers and metabolic genes and enhanced expression of myofibroblast marker genes involved in cytoskeletal organization, accompanied by the formation of actin filament structures within the cell. PPAR? binding was globally reduced in adipocytes after HFD feeding, and Rosi restored the molecular and cellular phenotypes of adipocytes associated with HFD feeding. We identified the TGF?1 effector protein SMAD to be enriched at HFD-induced promoters and enhancers and associated with myofibroblast signature genes. TGF?1 treatment of mature 3T3-L1 adipocytes induced gene expression and cellular changes similar to those seen after HFD in vivo, and knockdown of Smad3 blunted the effects of TGF?1. CONCLUSIONS:Our data demonstrate that adipocytes fail to maintain cellular identity after HFD feeding, acquiring characteristics of a myofibroblast-like cell type through reduced PPAR? activity and elevated TGF?-SMAD signaling. This cellular identity crisis may be a fundamental mechanism that drives functional decline of adipose tissues during obesity.

SUBMITTER: Roh HC

PROVIDER: S-EPMC7559520 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Adipocytes fail to maintain cellular identity during obesity due to reduced PPARγ activity and elevated TGFβ-SMAD signaling.

Roh Hyun Cheol HC Kumari Manju M Taleb Solaema S Tenen Danielle D Jacobs Christopher C Lyubetskaya Anna A Tsai Linus T-Y LT Rosen Evan D ED

Molecular metabolism 20200928

<h4>Objective</h4>Obesity due to overnutrition causes adipose tissue dysfunction, which is a critical pathological step on the road to type 2 diabetes (T2D) and other metabolic disorders. In this study, we conducted an unbiased investigation into the fundamental molecular mechanisms by which adipocytes transition to an unhealthy state during obesity.<h4>Methods</h4>We used nuclear tagging and translating ribosome affinity purification (NuTRAP) reporter mice crossed with Adipoq-Cre mice to determ ...[more]

PMID: 32992037

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Project description:Obesity due to overnutrition causes adipose tissue dysfunction, serving as a critical pathological step on the road to Type 2 diabetes (T2D) and other metabolic disorders. Here, we performed an unbiased investigation into the fundamental molecular mechanisms by which adipocytes transition to an unhealthy state during obesity. We fed NuTRAP (Nuclear tagging and Translating Ribosome Affinity Purification) mice crossed with Adipoq-Cre with chow or high fat diet (HFD) for 10 weeks and determined adipocyte-specific transcriptomic profiles by RNA-seq, active promoter and enhancer activities by H3K27ac ChIP-seq, and the PPARg cistrome by ChIP-seq. We also assessed the impact of the PPARg agonist rosiglitazone (Rosi) on gene expression and cellular state of adipocytes from HFD-fed mice. We used bioinformatic approaches to find transcriptional pathways and performed functional studies using shRNA-mediated loss-of-function approaches in 3T3-L1 adipocytes. Adipocytes from HFD-fed mice exhibited reduced expression of adipocyte markers and metabolic genes while showing enhanced expression of myofibroblast marker genes involved in cytoskeletal organization, accompanied by the formation of actin filament structures within the cell. PPARg binding was globally reduced in adipocytes after HFD feeding, and Rosi restored the molecular and cellular phenotypes of adipocytes associated with HFD feeding. SMAD binding motifs were over-represented in HFD-induced promoters and enhancers, while TGFb1 expression was elevated in adipose tissues after HFD feeding. TGFb1 treatment of mature 3T3-L1 adipocytes induced gene expression and cellular changes similar to those seen after HFD in vivo, and knockdown of Smad3 blunted the effects of TGFb1. Our data demonstrate that adipocytes fail to maintain cellular identity after HFD, acquiring characteristics of a myofibroblast-like cell type through reduced PPARg activity and elevated TGFb-SMAD signaling. This cellular identity crisis may serve as a fundamental mechanism that drives functional decline of adipose tissues during obesity.

Project description:The effect of endogenous progesterone and/or exogenous pre- or postnatal progesterone application on lung function of preterm infants is poorly defined. While prenatal progesterone substitution may prevent preterm birth, in vitro and in vivo data suggest a benefit of postnatal progesterone replacement on the incidence and severity of bronchopulmonary dysplasia (BPD). However, the molecular mechanisms responsible for progesterone's effects are undefined. Numerous factors are involved in lung development, airway inflammation, and airway remodeling: the transforming growth factor beta (TGF-?)/mothers against decapentaplegic homolog (Smad) signaling pathway and TGF-?-regulated genes, such as connective tissue growth factor (CTGF), transgelin (TAGLN), and plasminogen activator inhibitor-1 (PAI-1). These processes contribute to the development of BPD. The aim of the present study was to clarify whether progesterone could affect TGF-?1-activated Smad signaling and CTGF/transgelin/PAI-1 expression in lung epithelial cells. The pharmacological effect of progesterone on Smad signaling was investigated using a TGF-?1-inducible luciferase reporter and western blotting analysis of phosphorylated Smad2/3 in A549 lung epithelial cells. The regulation of CTGF, transgelin, and PAI-1 expression by progesterone was studied using a promoter-based luciferase reporter, quantitative real-time PCR, and western blotting in the same cell line. While progesterone alone had no direct effect on Smad signaling in lung epithelial cells, it dose-dependently inhibited TGF-?1-induced Smad3 phosphorylation, as shown by luciferase assays and western blotting analysis. Progesterone also antagonized the TGF-?1/Smad-induced upregulation of CTGF, transgelin, and PAI-1 at the promoter, mRNA, and/or protein levels. The present study highlights possible new molecular mechanisms involving progesterone, including inhibition of TGF-?1-activated Smad signaling and TGF-?1-regulated genes involved in BPD pathogenesis, which are likely to attenuate the development of BPD by inhibiting TGF-?1-mediated airway remodeling. Understanding these mechanisms might help to explain the effects of pre- or postnatal application of progesterone on lung diseases of preterm infants.

Project description:BackgroundGlioblastoma multiforme (GBM) is characterized by lethal aggressiveness and patients with GBM are in urgent need for new therapeutic avenues to improve quality of life. Current studies on tumor invasion focused on roles of cytokines in tumor microenvironment and numerous evidence suggests that TGF-β2 is abundant in glioma microenvironment and vital for glioma invasion. Autopagy is also emerging as a critical factor in aggressive behaviors of cancer cells; however, the relationship between TGF-β2 and autophagy in glioma has been poorly understood.MethodsU251, T98 and U87 GBM cell lines as well as GBM cells from a primary human specimen were used in vitro and in vivo to evaluate the effect of TGF-β2 on autophagy. Western blot, qPCR, immunofluorescence and transmission-electron microscope were used to detect target molecular expression. Lentivirus and siRNA vehicle were introduced to establish cell lines, as well as mitotracker and seahorse experiment to study the metabolic process in glioma. Preclinical therapeutic efficacy was evaluated in orthotopic xenograft mouse models.ResultsHere we demonstrated that TGF-β2 activated autophagy in human glioma cell lines and knockdown of Smad2 or inhibition of c-Jun NH2-terminal kinase, attenuated TGF-β2-induced autophagy. TGF-β2-induced autophagy is important for glioma invasion due to the alteration of epithelial-mesenchymal transition and metabolism conversion, particularly influencing mitochondria trafficking and membrane potential (△Ψm). Autopaghy also initiated a feedback on TGF-β2 in glioma by keeping its autocrine loop and affecting Smad2/3/7 expression. A xenograft model provided additional confirmation on combination of TGF-β inhibitor (Galunisertib) and autophagy inhibitor (CQ) to better "turn off" tumor growth.ConclusionOur findings elucidated a potential mechanism of autophagy-associated glioma invasion that TGF-β2 could initiate autophagy via Smad and non-Smad pathway to promote glioma cells' invasion.

Dataset Information

Adipocytes fail to maintain cellular identity during obesity due to reduced PPAR? activity and elevated TGF?-SMAD signaling.

Publications

Adipocytes fail to maintain cellular identity during obesity due to reduced PPARγ activity and elevated TGFβ-SMAD signaling.

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