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The potential function of syndecans in triglyceride metabolism are explored and it is found that syndecan-1 and syndecan-4 are expressed by adipocytes in adipose tissue and by 3T3-L1 adipocytes and are shed as soluble ectodomains in the extracellular space by insulin-stimulated 3T3-L1 adipocytes. The studies have shown that syndecan extodomains, shed by adipocytes in response to insulin, are physiological extracellular chaperons for lipoprotein lipase as it translocates from its site of synthesis to its site of action.

Lipid rafts and insulin signaling. Am J Physiol Endocrinol Metab 282: E1-E10, 2002.--Lipid rafts are domains within the plasma membrane that are enriched in cholesterol and lipids with saturated acyl chains. Specific proteins, including many signaling proteins, segregate into lipid rafts, and this process is important for certain signal transduction events in a variety of cell types. Within the past decade, data have emerged from many laboratories that implicate lipid rafts as critical for proper compartmentalization of insulin signaling in adipocytes. A subset of lipid rafts, caveolae, are coated with membrane proteins of the caveolin family. Direct interactions between resident raft proteins (caveolins and flotillin-1) and insulin-signaling molecules may organize these molecules in space and time to ensure faithful transduction of the insulin signal, at least with respect to the glucose-dependent actions of insulin in adipocytes. The in vivo relevance of this model remains to be determined. adipocyte; caveolin; flotillin; glucose transport; Cbl-associated protein

Author(s): Christian A. Baumann [1]; Vered Ribon [1]; Makoto Kanzaki [2]; Debbie C. Thurmond [2]; Silvia Mora [2]; Satoshi Shigematsu [2]; Perry E. Bickel [3]; Jeffrey E. Pessin [2]; Alan [...]

The identification of robust alpha oxidation of unbranched long chain fatty acids in a mammalian cell line and the obligatory sequential ordered alpha oxidation of fatty acids prior to their delta9 desaturation is studied. The results show that proximal peroxisomal fatty acids are an important metabolic determinant of the lipid content in the differentiating adipocyte.

The identification of robust alpha oxidation of unbranched long chain fatty acids in a mammalian cell line and the obligatory sequential ordered alpha oxidation of fatty acids prior to their delta9 desaturation is studied. The results show that proximal peroxisomal fatty acids are an important metabolic determinant of the lipid content in the differentiating adipocyte.

In this paper, we studied the fate of endocytosed glycosyiphosphatidyl inositol anchored proteins (GPI-APs) in mammalian cells, using aerolysin, a bacterial toxin that binds to the GPI anchor, as a probe. We find that GPI-APs are transported down the endocytic pathway to reducing late endosomes in BHK cells, using biochemical, morphological and functional approaches. We also find that this transport correlates with the association to raft-like membranes and thus that lipid rafts are present in late endosomes (in addition to the Golgi and the plasma membrane). In marked contrast, endocytosed GPI-APs reach the recycling endosome in CHO cells and this transport correlates with a decreased raft association. GPI-APs are, however, diverted from the recycling endosome and routed to late endosomes in CHO cells, when their raft association is increased by clustering seven or less GPI-APs with an aerolysin mutant. We conclude that the different endocytic routes followed by GPI-APs in different cell types depend on the residence time of GPI-APs in lipid rafts, and hence that raft partitioning regulates GPI-APs sorting in the endocytic pathway. [ABSTRACT FROM AUTHOR]

Alterations in lipid secondary messenger generation and lipid metabolic flux are essential in promoting the differentiation of adipocytes. To determine whether specific subtypes of intracellular phospholipases A2 (PLA2s) facilitate hormone-induced differentiation of 3T3-L1 cells into adipocytes, we examined alterations in the mRNA level, protein mass, and activity of three previously characterized mammalian intracellular PLA2s. Hormone-induced differentiation of 3T3-L1 cells resuited in 7.3 ± 0.5- and 7.4 ± 1.4-fold increases of mRNA encoding the calcium-independent phospholipases, iPLA2β and iPLA2γ, respectively. In contrast, the temporally coordinated loss of at least 90% of cPLA2α mRNA was manifest. Western analysis demonstrated the near absence of both iPLA2β and iPLA2γ protein mass in resting 3T3-L1 cells that increased dramatically during differentiation. In vitro measurement of PLA2 activities demonstrated an increase in both iPLA2β and iPLA2γ activities that were discriminated using the chiral mechanism based inhibitors (S)- and (R)-BEL, respectively. Remarkably, treatment of 3T3-L1 cells with small interfering RNA directed against either iPLA2β or iPLA2γ prevented hormone-induced differentiation. Moreover, analysis of the temporally programmed expression of transcription factors demonstrated that the small interfering RNA knockdown of iPLA2β or iPLA2γ resulted in down-regulation of the expression of peroxisome proliferator-activated receptor γ and the CCAAT enhances-binding protein α (C/EBPα). No alterations in the expression of the early stage transcription factors C/EBPβ and C/EBPδ were observed. Collectively, these results demonstrate prominent alterations in intracellular PLA2s during 3T3-L1 cell differentiation into adipocytes and identify the requirement of iPLA2β and iPLA2γ for the adipogenic program that drives resting 3T3-L1 cells into adipocytes after hormone stimulation. [ABSTRACT FROM AUTHOR]

Organisms store energy for later use during times of nutrient scarcity. Excess energy is stored as triacylglycerol in lipid droplets during lipogenesis. When energy is required, the stored triacylglycerol is hydrolyzed via activation of lipolytic pathways. The coordination of lipid storage and utilization is regulated by the perilipin family of lipid droplet coat proteins [perilipin, adipophilin/adipocyte differentiation-related protein (ADRP), S3-12, tail-interacting protein of 47 kilodaltons (TIP47), and myocardial lipid droplet protein (MLDP)/oxidative tissues-enriched PAT protein (OXPAT)/lipid storage droplet protein 5 (LSDP5)]. Lipid droplets are dynamic and heterogeneous in size, location, and protein content. The proteins that coat lipid droplets change during lipid droplet biogenesis and are dependent upon multiple factors, including tissue-specific expression and metabolic state (basal vs. lipogenic vs. lipolytic). New data suggest that proteins previously implicated in vesicle trafficking, including Rabs, soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs), and motor and cytoskeletal proteins, likely orchestrate the movement and fusion of lipid droplets. Thus, rather than inert cytoplasmic inclusions, lipid droplets are now appreciated as dynamic organelles that are critical for management of cellular lipid stores. That much remains to be discovered is suggested by the recent identification of a novel lipase [adipocyte triglyceride lipase (ATGL)] and lipase regulator [Comparative Gene Identification-58 (CGI-58)], which has led to reconsideration of the decades-old model of lipolysis. Future discovery likely will be driven by the exploitation of model organisms and by human genetic studies.

Alterations in lipid secondary messenger generation and lipid metabolic flux are essential in promoting the differentiation of adipocytes. To determine whether specific subtypes of intracellular phospholipases A(2) (PLA(2)s) facilitate hormone-induced differentiation of 3T3-L1 cells into adipocytes, we examined alterations in the mRNA level, protein mass, and activity of three previously characterized mammalian intracellular PLA(2)s. Hormone-induced differentiation of 3T3-L1 cells resulted in 7.3 +/- 0.5- and 7.4 +/- 1.4-fold increases of mRNA encoding the calcium-independent phospholipases, iPLA(2)beta and iPLA(2)gamma, respectively. In contrast, the temporally coordinated loss of at least 90% of cPLA(2)alpha mRNA was manifest. Western analysis demonstrated the near absence of both iPLA(2)beta and iPLA(2)gamma protein mass in resting 3T3-L1 cells that increased dramatically during differentiation. In vitro measurement of PLA(2) activities demonstrated an increase in both iPLA(2)beta and iPLA(2)gamma activities that were discriminated using the chiral mechanism based inhibitors (S)- and (R)-BEL, respectively. Remarkably, treatment of 3T3-L1 cells with small interfering RNA directed against either iPLA(2)beta or iPLA(2)gamma prevented hormone-induced differentiation. Moreover, analysis of the temporally programmed expression of transcription factors demonstrated that the small interfering RNA knockdown of iPLA(2)beta or iPLA(2)gamma resulted in down-regulation of the expression of peroxisome proliferator-activated receptor gamma and the CCAAT enhancer-binding protein alpha (C/EBPalpha). No alterations in the expression of the early stage transcription factors C/EBPbeta and C/EBPdelta were observed. Collectively, these results demonstrate prominent alterations in intracellular PLA(2)s during 3T3-L1 cell differentiation into adipocytes and identify the requirement of iPLA(2)beta and iPLA(2)gamma for the adipogenic program that drives resting 3T3-L1 cells into adipocytes after hormone stimulation.

Lipid rafts are detergent-insoluble membrane domains that play a key role in signal transduction by the T-cell antigen receptor. Proteome analysis revealed the presence of amidosulfobetaine-soluble signal transducing, integral membrane, cytoskeletal, heat shock, and GTP-binding proteins in rafts prepared from Jurkat cells. Several of these proteins were recruited to rafts by CD3/CD28 costimulation. Of particular interest is the inducible association of activated IkappaB kinase complexes with raft vesicles that could be captured with anti-flotillin-1 antibodies. Following amidosulfobetaine solubilization, flotillin-beta and IKKbeta underwent reciprocal co-immunoprecipitation. Treatment of Jurkat cells with methyl-beta-cyclodextrin disrupted the assembly and activation of this raft complex and also interfered in CD3/ CD28-induced activation of a NF-kappaB response element in the IL-2 promoter.