Palmoplantar keratoderma along with neuromuscular and metabolic phenotypes in Slurp1-deficient mice.

Mutations in SLURP1 cause mal de Meleda, a rare palmoplantar keratoderma (PPK). SLURP1 is a secreted protein that is expressed highly in keratinocytes but has also been identified elsewhere (e.g., spinal cord neurons). Here, we examined Slurp1-deficient mice (Slurp1(-/-)) created by replacing exon 2 with β-gal and neo cassettes. Slurp1(-/-) mice developed severe PPK characterized by increased keratinocyte proliferation, an accumulation of lipid droplets in the stratum corneum, and a water barrier defect. In addition, Slurp1(-/-) mice exhibited reduced adiposity, protection from obesity on a high-fat diet, low plasma lipid levels, and a neuromuscular abnormality (hind-limb clasping). Initially, it was unclear whether the metabolic and neuromuscular phenotypes were due to Slurp1 deficiency, because we found that the targeted Slurp1 mutation reduced the expression of several neighboring genes (e.g., Slurp2, Lypd2). We therefore created a new line of knockout mice (Slurp1X(-/-) mice) with a simple nonsense mutation in exon 2. The Slurp1X mutation did not reduce the expression of adjacent genes, but Slurp1X(-/-) mice exhibited all of the phenotypes observed in the original line of knockout mice. Thus, Slurp1 deficiency in mice elicits metabolic and neuromuscular abnormalities in addition to PPK

Impaired Preadipocyte Differentiation in Human Abdominal Obesity: Role of Wnt, Tumor Necrosis Factor-α, and Inflammation

OBJECTIVE—We examined preadipocyte differentiation in obese and nonobese individuals and the effect of cytokines and wingless-type MMTV (mouse mammary tumor virus) integration site family, member 3A (Wnt3a) protein on preadipocyte differ-entiation and phenotype. RESEARCH DESIGN AND METHODS—Abdominal subcuta-neous adipose tissue biopsies were obtained from a total of 51 donors with varying BMI. After isolation of the adipose and stromalvascular cells, inflammatory cells (CD14- and CD45-positive cells) were removed by immune magnetic separation. CD133-positive cells, containing early progenitor cells, were also isolated and quantified. The CD14- and CD45-negative preadipo-cytes were cultured with tumor necrosis factor (TNF)-, inter-leukin (IL)-6, resistin, or Wnt3a with or without a differentiation cocktail

Activation of Human Stearoyl-Coenzyme A Desaturase 1 Contributes to the Lipogenic Effect of PXR in HepG2 Cells

The pregnane X receptor (PXR) was previously known as a xenobiotic receptor. Several recent studies suggested that PXR also played an important role in lipid homeostasis but the underlying mechanism remains to be clearly defined. In this study, we found that rifampicin, an agonist of human PXR, induced lipid accumulation in HepG2 cells. Lipid analysis showed the total cholesterol level increased. However, the free cholesterol and triglyceride levels were not changed. Treatment of HepG2 cells with rifampicin induced the expression of the free fatty acid transporter CD36 and ABCG1, as well as several lipogenic enzymes, including stearoyl-CoA desaturase-1 (SCD1), long chain free fatty acid elongase (FAE), and lecithin-cholesterol acyltransferase (LCAT), while the expression of acyl:cholesterol acetyltransferase(ACAT1) was not affected. Moreover, in PXR over-expressing HepG2 cells (HepG2-PXR), the SCD1 expression was significantly higher than in HepG2-Vector cells, even in the absence of rifampicin. Down-regulation of PXR by shRNA abolished the rifampicin-induced SCD1 gene expression in HepG2 cells. Promoter analysis showed that the human SCD1 gene promoter is activated by PXR and a novel DR-7 type PXR response element (PXRE) response element was located at -338 bp of the SCD1 gene promoter. Taken together, these results indicated that PXR activation promoted lipid synthesis in HepG2 cells and SCD1 is a novel PXR target gene. © 2013 Zhang et al

De-Novo Identification of PPARγ/RXR Binding Sites and Direct Targets during Adipogenesis

BACKGROUND: The pathophysiology of obesity and type 2 diabetes mellitus is associated with abnormalities in endocrine signaling in adipose tissue and one of the key signaling affectors operative in these disorders is the nuclear hormone transcription factor peroxisome proliferator-activated receptor-gamma (PPARgamma). PPARgamma has pleiotropic functions affecting a wide range of fundamental biological processes including the regulation of genes that modulate insulin sensitivity, adipocyte differentiation, inflammation and atherosclerosis. To date, only a limited number of direct targets for PPARgamma have been identified through research using the well established pre-adipogenic cell line, 3T3-L1. In order to obtain a genome-wide view of PPARgamma binding sites, we applied the pair end-tagging technology (ChIP-PET) to map PPARgamma binding sites in 3T3-L1 preadipocyte cells. METHODOLOGY/PRINCIPAL FINDINGS: Coupling gene expression profile analysis with ChIP-PET, we identified in a genome-wide manner over 7700 DNA binding sites of the transcription factor PPARgamma and its heterodimeric partner RXR during the course of adipocyte differentiation. Our validation studies prove that the identified sites are bona fide binding sites for both PPARgamma and RXR and that they are functionally capable of driving PPARgamma specific transcription. Our results strongly indicate that PPARgamma is the predominant heterodimerization partner for RXR during late stages of adipocyte differentiation. Additionally, we find that PPARgamma/RXR association is enriched within the proximity of the 5' region of the transcription start site and this association is significantly associated with transcriptional up-regulation of genes involved in fatty acid and lipid metabolism confirming the role of PPARgamma as the master transcriptional regulator of adipogenesis. Evolutionary conservation analysis of these binding sites is greater when adjacent to up-regulated genes than down-regulated genes, suggesting the primordial function of PPARgamma/RXR is in the induction of genes. Our functional validations resulted in identifying novel PPARgamma direct targets that have not been previously reported to promote adipogenic differentiation. CONCLUSIONS/SIGNIFICANCE: We have identified in a genome-wide manner the binding sites of PPARgamma and RXR during the course of adipogenic differentiation in 3T3L1 cells, and provide an important resource for the study of PPARgamma function in the context of adipocyte differentiation

Sphingosine kinases regulate ER contacts with late endocytic organelles and cholesterol trafficking

Membrane contact sites (MCS), close membrane apposition between organelles, are platforms for interorganellar transfer of lipids including cholesterol, regulation of lipid homeostasis, and co-ordination of endocytic trafficking. Sphingosine kinases (SphKs), two isoenzymes that phosphorylate sphingosine to the bioactive sphingosine-1-phosphate (S1P), have been implicated in endocytic trafficking. However, the physiological functions of SphKs in regulation of membrane dynamics, lipid trafficking and MCS are not known. Here, we report that deletion of SphKs decreased S1P with concomitant increases in its precursors sphingosine and ceramide, and markedly reduced endoplasmic reticulum (ER) contacts with late endocytic organelles. Expression of enzymatically active SphK1, but not catalytically inactive, rescued the deficit of these MCS. Although free cholesterol accumulated in late endocytic organelles in SphK null cells, surprisingly however, cholesterol transport to the ER was not reduced. Importantly, deletion of SphKs promoted recruitment of the ER-resident cholesterol transfer protein Aster-B (also called GRAMD1B) to the plasma membrane (PM), consistent with higher accessible cholesterol and ceramide at the PM, to facilitate cholesterol transfer from the PM to the ER. In addition, ceramide enhanced in vitro binding of the Aster-B GRAM domain to phosphatidylserine and cholesterol liposomes. Our study revealed a previously unknown role for SphKs and sphingolipid metabolites in governing diverse MCS between the ER network and late endocytic organelles versus the PM to control the movement of cholesterol between distinct cell membranes

High Efficiency Lipid-Based siRNA Transfection of Adipocytes in Suspension

BACKGROUND:Fully differentiated adipocytes are considered to be refractory to introduction of siRNA via lipid-based transfection. However, large scale siRNA-based loss-of-function screening of adipocytes using either electroporation or virally-mediated transfection approaches can be prohibitively complex and expensive. METHODOLOGY/PRINCIPAL FINDINGS:We present a method for introducing small interfering RNA (siRNA) into differentiated 3T3-L1 adipocytes and primary human adipocytes using an approach based on forming the siRNA/cell complex with the adipocytes in suspension rather than as an adherent monolayer, a variation of "reverse transfection". CONCLUSIONS/SIGNIFICANCE:Transfection of adipocytes with siRNA by this method is economical, highly efficient, has a simple workflow, and allows standardization of the ratio of siRNA/cell number, making this approach well-suited for high-throughput screening of fully differentiated adipocytes

PPAR? Downregulation by TGF in Fibroblast and Impaired Expression and Function in Systemic Sclerosis: A Novel Mechanism for Progressive Fibrogenesis

The nuclear orphan receptor peroxisome proliferator-activated receptor-gamma (PPAR-γ) is expressed in multiple cell types in addition to adipocytes. Upon its activation by natural ligands such as fatty acids and eicosanoids, or by synthetic agonists such as rosiglitazone, PPAR-γ regulates adipogenesis, glucose uptake and inflammatory responses. Recent studies establish a novel role for PPAR-γ signaling as an endogenous mechanism for regulating transforming growth factor-ß (TGF-ß)- dependent fibrogenesis. Here, we sought to characterize PPAR-γ function in the prototypic fibrosing disorder systemic sclerosis (SSc), and delineate the factors governing PPAR-γ expression. We report that PPAR-γ levels were markedly diminished in skin and lung biopsies from patients with SSc, and in fibroblasts explanted from the lesional skin. In normal fibroblasts, treatment with TGF-ß resulted in a time- and dose-dependent down-regulation of PPAR-γ expression. Inhibition occurred at the transcriptional level and was mediated via canonical Smad signal transduction. Genome-wide expression profiling of SSc skin biopsies revealed a marked attenuation of PPAR-γ levels and transcriptional activity in a subset of patients with diffuse cutaneous SSc, which was correlated with the presence of a ''TGF-ß responsive gene signature'' in these biopsies. Together, these results demonstrate that the expression and function of PPAR-γ are impaired in SSc, and reveal the existence of a reciprocal inhibitory cross-talk between TGF-ß activation and PPAR-γ signaling in the context of fibrogenesis. In light of the potent anti-fibrotic effects attributed to PPAR-γ, these observations lead us to propose that excessive TGF-ß activity in SSc accounts for impaired PPAR-γ function, which in turn contributes to unchecked fibroblast activation and progressive fibrosis. © 2010 Wei et al