Adipose tissue, which is primarily composed of adipocytes, is crucial for maintaining energy and metabolic homeostasis. Adipogenesis is thought to occur in two stages: commitment of mesenchymal stem cells to a preadipocyte fate and terminal differentiation. Cell shape and extracellular matrix remodelling have recently been found to regulate preadipocyte commitment and competency by modulating WNT and RHO-family GTPase signalling cascades. Adipogenic stimuli induce terminal differentiation in committed preadipocytes through the epigenomic activation of peroxisome proliferator-activated receptor-γ (PPARγ). The coordination of PPARγ with CCAAT/enhancer-binding protein (C/EBP) transcription factors maintains adipocyte gene expression. Improving our understanding of these mechanisms may allow us to identify therapeutic targets against metabolic diseases that are rapidly becoming epidemic globally.

Peroxisome proliferator-activated receptor ␥(PPAR␥), a nuclear receptor and the target of anti-diabetic thiazolinedione drugs, is known as the master regulator of adipocyte biology. Although it regulates hundreds of adipocyte genes, PPAR␥ binding to endogenous genes has rarely been demonstrated. Here, utilizing chromatin immunoprecipitation (ChIP) coupled with whole genome tiling arrays, we identified 5299 genomic regions of PPAR␥ binding in mouse 3T3-L1 adipocytes. The consensus PPAR␥/RXR␣ "DR-1"-binding motif was found at most of the sites, and ChIP for RXR␣ showed colocalization at nearly all locations tested. Bioinformatics analysis also revealed CCAAT/enhancer-binding protein (C/EBP)-binding motifs in the vicinity of most PPAR␥-binding sites, and genome-wide analysis of C/EBP␣ binding demonstrated that it localized to3350 of the locations bound by PPAR␥. Importantly, most genes induced in adipogenesis were bound by both PPAR␥ and C/EBP␣, while very few were PPAR␥-specific. C/EBP␤ also plays a role at many of these genes, such that both C/EBP␣ and ␤ are required along with PPAR␥ for robust adipocyte-specific gene expression. Thus, PPAR␥ and C/EBP factors cooperatively orchestrate adipocyte biology by adjacent binding on an unanticipated scale.[Keywords: PPAR␥; C/EBP; adipocyte; genome wide; ChIP-chip] Supplemental material is available at http://www.genesdev.org.

Adipocyte differentiation is controlled by many transcription factors, but few known downstream targets of these factors are necessary for adipogenesis. Here we report that retinol saturase (RetSat), which is an enzyme implicated in the generation of dihydroretinoid metabolites, is induced during adipogenesis and is directly regulated by the transcription factor peroxisome proliferator activated receptor γ (PPARγ). Ablation of RetSat dramatically inhibited adipogenesis but, surprisingly, this block was not overcome by the putative product of RetSat enzymatic activity. On the other hand, ectopic RetSat with an intact, but not a mutated, FAD/NAD dinucleotide-binding motif increased endogenous PPARγ transcriptional activity and promoted adipogenesis. Indeed, RetSat was not required for adipogenesis when cells were provided with exogenous PPARγ ligands. In adipose tissue, RetSat is expressed in adipocytes but is unexpectedly downregulated in obesity, most likely owing to infiltration of macrophages that we demonstrate to repress RetSat expression. Thiazolidinedione treatment reversed low RetSat expression in adipose tissue of obese mice. Thus, RetSat plays an important role in the biology of adipocytes, where it favors normal differentiation, yet is reduced in the obese state. RetSat is thus a novel target for therapeutic intervention in metabolic disease.

Nuclear peroxisome proliferator-activated receptor-␥ (PPAR␥) is required for adipocyte differentiation, but its role in mature adipocytes is less clear. Here, we report that knockdown of PPAR␥ expression in 3T3-L1 adipocytes returned the expression of most adipocyte genes to preadipocyte levels. Consistently, down-regulated but not up-regulated genes showed strong enrichment of PPAR␥ binding. Surprisingly, not all adipocyte genes were reversed, and the adipocyte morphology was maintained for an extended period after PPAR␥ depletion. To explain this, we focused on transcriptional regulators whose adipogenic regulation was not reversed upon PPAR␥ depletion. We identified GATA2, a transcription factor whose down-regulation early in adipogenesis is required for preadipocyte differentiation and whose levels remain low after PPAR␥ knockdown. Forced expression of GATA2 in mature adipocytes complemented PPAR␥ depletion and impaired adipocyte functionality with a more preadipocyte-like gene expression profile. Ectopic expression of GATA2 in adipose tissue in vivo had a similar effect on adipogenic gene expression. These results suggest that PPAR␥-independent down-regulation of GATA2 prevents reversion of mature adipocytes after PPAR␥ depletion.Peroxisome proliferator-activated receptors (PPARs) 2 are ligand-activated transcription factors that bind to PPAR response elements as heterodimers with the retinoid X receptor ␣ (1). Among all PPARs, the expression of PPAR␥ exhibits the greatest specificity for adipose tissue (2, 3), and the antidiabetic thiazolidinedione drugs are high affinity PPAR␥ ligands that promote adipogenesis (4). One of the most common models used to study adipocyte differentiation is the mouse 3T3-L1 cell line. After hormonal stimulation of growth-arrested preadipocytes, cells undergo a clonal expansion phase before they permanently exit the cell cycle for the final adipocyte commitment (5). During this process, PPAR␥ is induced and stimulates the expression of many adipocyte-specific genes. PPAR␥ is both necessary (6) and sufficient (7) for the differentiation of murine fibroblasts into adipocytes, and no factor is known to stimulate adipocyte differentiation in the absence of PPAR␥.PPAR␥ itself is activated mainly by hormonally induced changes in the expression of transcriptional activators and repressors. Upstream of the induction of PPAR␥ are, for instance, EGR2 (early growth response 2) and members of the Krüppel-like factors and C/EBP (CCAAT/enhancer binding protein) families. C/EBP␤ has been shown to directly or indirectly induce PPAR␥ expression (5). On the other hand, several inhibiting factors upstream of PPAR␥ are regulators of alternative cell fates, including some members of the WNT family (8) as well as factors highly expressed in preadipocytes and downregulated during differentiation, including PREF-1 and GATA2/3 (9 -11).In contrast to the thoroughly studied role of PPAR␥ during adipocyte differentiation, our knowledge about the role of PPAR␥ in maintaining the adipocyte phenotype in mat...

This study provides a comprehensive overview of IQSEC2-related encephalopathy in males and females, and suggests that an accurate dosage of IQSEC2 at the synapse is crucial during normal brain development.

Mouse RetSat catalyzes the saturation of the C13-C14 double bond of all-trans-retinol to produce all-trans-13,14-dihydroretinol 8 1 (Figure 1). A related enzyme in zebrafish catalyzes the saturation of the C7-C8 double bond in addition to the C13-C14 double bond of all-transretinol to produce both 8 and all-trans-7,8-dihydroretinol. 2 Further oxidation of 8 and of alltrans-7,8-dihydroretinol by retinol dehydrogenases and then by retinaldehyde dehydrogenase enzymes leads to formation of all-trans-13,14-dihydroretinoic acid 9 and all-trans-7,8-dihydroretinoic acid, compounds whose levels are exquisitely controlled in vivo by the enzymes that catalyze their synthesis and breakdown. 2,3 Both 9 and all-trans-7,8-dihydroretinoic acid are highly selective agonists in activating the retinoic acid receptor (RAR) but not the retinoid X receptor (RXR). 3,4 Because 13,14-dihydroretinoids are chiral compounds it is important to determine their absolute configuration and evaluate how the different enantiomers interact with binding proteins, receptors, and enzymes. In this study we investigated the absolute configuration of biologically derived 8 and consequently of 9 and evaluated the activation of RAR by the enantiomers of 9.To establish the absolute configuration of 8 we examined the products of mouse and zebrafish RetSat by chiral HPLC. We also examined the endogenous form of compound 8 purified from livers of mice gavaged with all-trans-retinyl palmitate. For our analyses by chiral HPLC we established authentic standards by stereospecific syntheses of the two enantiomers of 8 (Scheme 1 and Supporting Information Scheme S-2). The synthetic scheme leading to the biological material (R)-all-trans-13,14-dihydroretinol (R)-8 is depicted in Scheme 1. The chirality was transferred from that of the γ-alkoxy group in (4S)-4,5-(O-isopropylidene)pent-2-enoate enantiomer (4S)-1, 5 and Suzuki coupling was selected as the connective method to construct the polyene skeleton. 6 An analogous sequence produced (S)-all-trans-13,14-dihydroretinol (S)-8 from (4R)-4,5-(O-isopropylidene)pent-2-enoate (4R)-1. Determination of the enantiomeric excess of the target compounds was based on HPLC separation by a Chiracel OD-H 0.46 cm × 15 cm column that afforded an enantiomeric excess (ee) value of >96% for each enantiomer of 8.We analyzed compound 8 purified from previously described human embryonic kidney cells (HEK) 293 cells that express mouse or zebrafish RetSat (HEK-mRetSat and HEK-zRetSat, E-mail: ram50@case.edu. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript respectively). 1,2 This analysis showed that both mouse and zebrafish RetSat produce the (R)-8 enantiomer as it had chromatographic properties on chiral HPLC identical to synthetic (R)-8 but distinct from (S)-8 ( Figure 1 and Figure S-1). Therefore, both mouse and zebrafish RetSat have the same stereospecificity at the C13-C14 double bond. We also examined the endogenous form of compound 8 purified from the livers of mice gavaged with all-trans...

The identification of factors that define adipocyte precursor potential has important implications for obesity. Preadipocytes are fibroblastoid cells committed to becoming round lipid-laden adipocytes. In vitro, this differentiation process is facilitated by confluency, followed by adipogenic stimuli. During adipogenesis, a large number of cytostructural genes are repressed before adipocyte gene induction. Here we report that the transcriptional repressor transcription factor 7-like 1 (TCF7L1) binds and directly regulates the expression of cell structure genes. Depletion of TCF7L1 inhibits differentiation, because TCF7L1 indirectly induces the adipogenic transcription factor peroxisome proliferator-activated receptor γ in a manner that can be replaced by inhibition of myosin II activity. TCF7L1 is induced by cell contact in adipogenic cell lines, and ectopic expression of TCF7L1 alleviates the confluency requirement for adipocytic differentiation of precursor cells. In contrast, TCF7L1 is not induced during confluency of non-adipogenic fibroblasts, and, remarkably, forced expression of TCF7L1 is sufficient to commit nonadipogenic fibroblasts to an adipogenic fate. These results establish TCF7L1 as a transcriptional hub coordinating cell-cell contact with the transcriptional repression required for adipogenic competency.A dipose tissue is a highly specialized compartment of cells actively involved in maintaining global metabolic homeostasis through lipid synthesis and storage, adipokine secretion, and insulin responsiveness (1). Adipocytes compose the majority of cells in adipose tissue and play a critical role in normal physiology, but their dysfunction is also at the center of a diverse range of diseases, including obesity, diabetes, and lipodystrophies (2). Furthermore, primary preadipocytes and adipose-derived stem cells have shown promise in treating multiple conditions (3-5). Therefore, it is critical to understand the process by which spindly fibroblastic precursor cells undergo conversion into round lipid-laden fat cells.In vitro models of adipogenesis, such as the extensively studied committed preadipocyte cell line 3T3-L1 cells, have elucidated two major phases of adipogenesis: commitment and terminal differentiation (6, 7). Terminal differentiation is characterized by the induction of metabolic genes, many of which are the direct targets of the transcription factors peroxisome proliferator-activated receptor γ (PPARγ) and C/CAAT-binding protein (C/ EBP) α and β (8-14). Recent efforts have focused on identifying committed preadipocyte populations in vivo (15, 16), as well as on determining molecular factors that define the committed preadipocytes phenotype. Zinc finger protein 423 (Zfp423) is a critical preadipocyte factor upstream of PPARγ that is not present in non-adipogenic fibroblasts (17). However, Zfp423 also has been identified as a regulator of neurologic development (18), suggesting that other factors also may be involved in specifying adipogenic competency and commitment of precursor cells up...

Autism spectrum disorders (ASDs) are characterized by a deficit in social communication, pathologic repetitive behaviors, restricted interests, and electroencephalogram (EEG) aberrations. While exhaustive analysis of nuclear DNA (nDNA) variation has revealed hundreds of copy number variants (CNVs) and loss-of-function (LOF) mutations, no unifying hypothesis as to the pathophysiology of ASD has yet emerged. Based on biochemical and physiological analyses, it has been hypothesized that ASD may be the result of a systemic mitochondrial deficiency with brain-specific manifestations. This proposal has been supported by recent mitochondrial DNA (mtDNA) analyses identifying both germline and somatic mtDNA variants in ASD. If mitochondrial defects do predispose to ASD, then mice with certain mtDNA mutations should present with autism endophenotypes. To test this prediction, we examined a mouse strain harboring an mtDNA ND6 gene missense mutation (P25L). This mouse manifests impaired social interactions, increased repetitive behaviors and anxiety, EEG alterations, and a decreased seizure threshold, in the absence of reduced hippocampal interneuron numbers. EEG aberrations were most pronounced in the cortex followed by the hippocampus. Aberrations in mitochondrial respiratory function and reactive oxygen species (ROS) levels were also most pronounced in the cortex followed by the hippocampus, but absent in the olfactory bulb. These data demonstrate that mild systemic mitochondrial defects can result in ASD without apparent neuroanatomical defects and that systemic mitochondrial mutations can cause tissue-specific brain defects accompanied by regional neurophysiological alterations.