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Protein kinase B/Akt protein kinases control an array diverse functions, including cell growth, survival, proliferation, and metabolism. We report here the identification of pleckstrin homology-like domain family B member 1 (PHLDB1) as an insulin-responsive protein that enhances Akt activation. PHLDB1 contains a pleckstrin homology domain, which we show phosphatidylinositol PI(3,4)P2, PI(3,5)P2, and PI(3,4,5)P3, well as a Forkhead-associated domain and coiled coil regions. PHLDB1 expression is increased during adipocyte differentiation, and it is abundant in many mouse tissues. Both endogenous and HA- or GFP-tagged PHLDB1 displayed a cytoplasmic disposition in unstimulated cultured adipocytes but translocated to the plasma membrane in response to insulin. Depletion of PHLDB1 by siRNA inhibited insulin stimulation of phosphorylation but not tyrosine phosphorylation of IRS-RNAi-based silencing of PHLDB1 in cultured adipocytes attenuated insulin-stimulated deoxyglucose transport Myc-GLUT4-EGFP translocation to the plasma membrane, whereas knockdown of the PHLDB1 isoform PHLDB2 failed attenuate insulin-stimulated deoxyglucose transport. Furthermore, adenovirus-mediated expression of PHLDB1 in adipocytes enhanced insulin-stimulated Akt and p70 S6 kinase phosphorylation, as well as GLUT4 translocation. These results indicate that PHLDB1 is a novel modulator of Akt protein kinase activation by insulin. [ABSTRACT FROM AUTHOR]

The receptor peroxisome proliferator-activated receptor γ (PPARγ) is considered a master regulator of adipocyte differentiation and promotes glucose and lipid metabolism in mature adipocytes. We recently identified the yeast Sterile 20 (Ste20) protein kinase ortholog, Map4k4, in an RNA interference-based screen as an inhibitor of PPARγ expression in cultured adipocytes. Here, we show that RNA interference-mediated silencing of Map4k4 elevates the levels of both PPAR'γ1 and PPARγ2 proteins in 3T3-L1 adipocytes without affecting PPARγ mRNA levels, suggesting that Map4k4 regulates PPARγ at a post-transcriptional step. PPARγ degradation rates are remarkably rapid as measured in the presence of cycloheximide (t½ = 2 h), but silencing Map4k4 had no effect on PPARγ degradation. However, depletion of Map4k4 significantly enhances [35S]methionine/cysteine incorporation into proteins, suggesting that Map4k4 signaling decreases protein translation. We show a function of Map4k4 is to inhibit rapamycin-sensitive mamma- Jian target of rapamycin (mTOR) activity, decreasing AE-BP1 phosphorylation. In addition, our results show mTOR and 4E-BP1 are required for the increased PPARγ protein expression upon Map4k4 knockdown. Consistent with this concept, adenovirus-mediated expression of Map4k4 decreased PPARγ protein levels and mTOR phosphorylation. These data show that Map4k4 negatively regulates PPARγ post-transcriptionally, by attenuating mTOR signaling and a 4E-BP 1-dependent mechanism. [ABSTRACT FROM AUTHOR]

Fat-specific protein (FSP)27/Cidec is most highly expressed in white and brown adipose tissues and increases in abundance by over 50-fold during adipogenesis. However, its function in adipocytes has remained elusive since its discovery over 15 years ago. Here we demonstrate that FSP27/Cidec localizes to lipid droplets in cultured adipocytes and functions to promote lipid accumulation. Ectopically expressed FSP27-GFP surrounds lipid droplets in 3T3-L1 adipocytes and colocalizes with the known lipid droplet protein perilipin. Immunostaining of endogenous FSP27 in 3T3-L1 adipocytes also confirmed its presence on lipid droplets. FSP27-GFP expression also mark- edly increases lipid droplet size and enhances accumulation of total neutral lipids in 3T3-L1 preadipocytes as well as other cell types such as COS cells. Conversely, RNA interference-based FSP27/Cidec depletion in mature adipocytes significantly stimulates lipolysis and reduces the size of lipid droplets. These data reveal FSP27/Cidec as a novel adipocyte lipid droplet protein that negatively regulates lipolysis and promotes triglyceride accumulation. [ABSTRACT FROM AUTHOR]

Insulin stimulates glucose uptake in muscle and adipose cells by mobilizing intracellular membrane vesicles containing GLUT4 glucose transporter proteins to the plasma membrane. Here we show in live cultured adipocytes that intracellular membranes co [ABSTRACT FROM AUTHOR]