adipocyte ͉ Cide ͉ diabetes ͉ fat droplet ͉ fat metabolism

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 markedly 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.Adipose tissue is a major determinant of whole body glucose homeostasis and insulin sensitivity, as evidenced by its ability to secrete bioactive peptides and control lipid storage (1-6). The nuclear receptor peroxisome proliferator-activated receptor-␥ promotes adipogenesis and enhances these functions, acting in mice and humans to increase insulin signaling and glucose tolerance (7-12). Several proteins that are highly and selectively expressed in adipocytes, such as the secreted proteins adiponectin and leptin as well as adipsin, are under the control of peroxisome proliferator-activated receptor-␥ (3-5, 13, 14). These and other adipocyte proteins are highly up-regulated during adipogenesis and confer unique characteristics to these cells, including high capacity to store triglyceride and release fatty acids.Fat-specific protein (FSP)27, also denoted as CIDEC for the human homolog (15), was discovered over 15 years ago to be strikingly up-regulated during adipogenesis and is highly expressed in both white and brown adipose tissues (15-18). However, the question of FSP27/Cidec function has remained unsolved. The results we present here demonstrate FSP27/Cidec to be a novel lipid droplet protein that shares many features characteristic of the lipid droplet protein perilipin (19,20), including the ability to enhance neutral lipid accumulation when expressed in 3T3-L1 preadipocytes or even COS cells. Our data also reveal that a mechanism whereby FSP27/Cidec functions to promote triglyceride deposition in adiocytes is by inhibiting lipolysis. FSP27 is thus a major new modulator of lipid droplet function that is required for optimal storage of triglycerides by adipocytes. EXPERIMENTAL PROCEDURESMaterials-C57BL/6J (male, 10 weeks old) mice were obtained from The Jackson Laboratory. Human insulin was obtained from Lilly. Fetal bovine serum was purchased from Atlanta Biologicals, Inc. (Lawrenceville, GA). Other reagents ...

The insulin-regulated glucose transporter GLUT4 is a key modulator of whole body glucose homeostasis, and its selective loss in adipose tissue or skeletal muscle causes insulin resistance and diabetes. Here we report an RNA interference-based screen of protein kinases expressed in adipocytes and identify four negative regulators of insulin-responsive glucose transport: the protein kinases PCTAIRE-1 (PCTK1), PFTAIRE-1 (PFTK1), I B kinase ␣, and MAP4K4͞NIK. Integrin-linked protein kinase was identified as a positive regulator of this process. We characterized one of these hits, MAP4K4͞NIK, and found that it is unique among mitogenactivated protein (MAP) kinases expressed in cultured adipocytes in attenuating hexose transport. Remarkably, MAP4K4͞NIK suppresses expression of the adipogenic transcription factors C͞EBP␣, C͞EBP␤, and PPAR␥ and of GLUT4 itself in these cells. RNA interference-mediated depletion of MAP4K4͞NIK early in differentiation enhances adipogenesis and triglyceride deposition, and even in fully differentiated adipocytes its loss up-regulates GLUT4. Conversely, conditions that inhibit adipogenesis such as TNF-␣ treatment or depletion of PPAR␥ markedly up-regulate MAP4K4͞ NIK expression in cultured adipocytes. Furthermore, TNF-␣ signaling to down-regulate GLUT4 is impaired in the absence of MAP4K4͞NIK, indicating that MAP4K4 expression is required for optimal TNF-␣ action. These results reveal a MAP4K4͞NIK-dependent signaling pathway that potently inhibits PPAR␥-responsive gene expression, adipogenesis, and insulin-stimulated glucose transport.GLUT4 function ͉ adipocyte differentiation ͉ protein kinase screening

Calcium signaling is important for differentiation-dependent gene expression, but is also involved in other cellular functions. Therefore mechanisms must exist to distinguish calcium signaling relevant to differentiation. Calcineurin is a calcium-regulated phosphatase that is required for myogenic gene expression and skeletal muscle differentiation. Here, we demonstrate that inhibition of calcineurin blocks chromatin remodeling and that the Brg1 ATPase of the SWI/SNF chromatin remodeling enzyme, which is required for the activation of myogenic gene expression, is a calcineurin substrate. Furthermore, we identify the calcium-regulated classical protein kinase C beta (PKCβ) as a repressor of myogenesis and as the enzyme that opposes calcineurin function. Replacement of endogenous Brg1 with a phosphomimetic mutant in primary myoblasts inhibits myogenesis, while replacement with a non-phosphorylatable mutant allows myogenesis despite inhibition of calcineurin signaling, demonstrating the functionality of calcineurin/PKC modified residues. Thus the Brg1 chromatin remodeling enzyme integrates two antagonistic calcium-dependent signaling pathways that control myogenic differentiation.

Regulation of adipose tissue formation by adipogenic-regulatory proteins has long been a topic of interest given the ever-increasing health concerns of obesity and type 2 diabetes in the general population. Differentiation of precursor cells into adipocytes involves a complex network of cofactors that facilitate the functions of transcriptional regulators from the CCATT/enhancer binding protein, and the peroxisome proliferator-activated receptor (PPAR) families. Many of these cofactors are enzymes that modulate the structure of chromatin by altering histone-DNA contacts in an ATP-dependent manner or by posttranslationally modifying the histone proteins. Here we report that inhibition of protein arginine methyltransferase 5 (Prmt5) expression in multiple cell culture models for adipogenesis prevented the activation of adipogenic genes. In contrast, overexpression of Prmt5 enhanced adipogenic gene expression and differentiation. Chromatin immunoprecipitation experiments indicated that Prmt5 binds to and dimethylates histones at adipogenic promoters. Furthermore, the presence of Prmt5 promoted the binding of ATP-dependent chromatin-remodeling enzymes and was required for the binding of PPARγ2 at PPARγ2-regulated promoters. The data indicate that Prmt5 acts as a coactivator for the activation of adipogenic gene expression and promotes adipogenic differentiation.