Gangliosides are known as modulators of transmembrane signaling by regulating various receptor functions. We have found that insulin resistance induced by tumor necrosis factor-␣ (TNF-␣) in 3T3-L1 adipocytes was accompanied by increased GM3 ganglioside expression caused by elevating GM3 synthase activity and its mRNA. We also demonstrated that TNF-␣ simultaneously produced insulin resistance by uncoupling insulin receptor activity toward insulin receptor substrate-1 (IRS-1) and suppressing insulin-sensitive glucose transport. Pharmacological depletion of GM3 in adipocytes by an inhibitor of glucosylceramide synthase prevented the TNF-␣-induced defect in insulin-dependent tyrosine phosphorylation of IRS-1 and also counteracted the TNF-␣-induced serine phosphorylation of IRS-1. Moreover, when the adipocytes were incubated with exogenous GM3, suppression of tyrosine phosphorylation of insulin receptor and IRS-1 and glucose uptake in response to insulin stimulation was observed, demonstrating that GM3 itself is able to mimic the effects of TNF on insulin signaling. We used the obese Zucker fa/fa rat and ob/ob mouse, which are known to overproduce TNF-␣ mRNA in adipose tissues, as typical models of insulin resistance. We found that the levels of GM3 synthase mRNA in adipose tissues of these animals were significantly higher than in their lean counterparts. Taken together, the increased synthesis of cellular GM3 by TNF may participate in the pathological conditions of insulin resistance in type 2 diabetes.
Sphingosine 1-phosphate (S1P) is a bioactive lipid molecule that acts as both an extracellular signaling mediator and an intracellular second messenger. S1P is synthesized from sphingosine by sphingosine kinase and is degraded either by S1P lyase or by S1P phosphohydrolase. Recently, mammalian S1P phosphohydrolase (SPP1) was identified and shown to constitute a novel lipid phosphohydrolase family, the SPP family. In this study we have identified a second human S1P phosphohydrolase, SPP2, based on sequence homology to human SPP1. SPP2 exhibited high phosphohydrolase activity against S1P and dihydrosphingosine 1-phosphate. The dihydrosphingosine-1-phosphate phosphohydrolase activity was efficiently inhibited by excess S1P but not by lysophosphatidic acid, phosphatidic acid, or glycerol 3-phosphate, indicating that SPP2 is highly specific to sphingoid base 1-phosphates. Immunofluorescence microscopic analysis demonstrated that SPP2 is localized to the endoplasmic reticulum. Although the enzymatic properties and localization of SPP2 were similar to those of SPP1, the tissue-specific expression pattern of SPP2 was different from that of SPP1. Thus, SPP2 is another member of the SPP family that may play a role in attenuating intracellular S1P signaling.Sphingosine 1-phosphate (S1P), 1 a sphingolipid metabolite, regulates diverse biological processes including mitogenesis, differentiation, migration, and apoptosis both as an extracellular mediator and as an intracellular second messenger (1-3). Extracellular effects of S1P are known to be mediated via the endothelial differentiation gene (Edg) family of plasma membrane G-protein-coupled receptors, whereas its intracellular targets have yet to be determined (2, 3). S1P is synthesized by the phosphorylation of sphingosine and catalyzed by sphingosine kinase. Once formed, S1P is rapidly degraded by S1P lyase to hexadecenal and phosphoethanolamine or dephosphorylated by S1P phosphohydrolase.The existence of a S1P-specific phosphohydrolase had been suggested by biochemical analyses using cultured skin fibroblasts and rat liver (4, 5). In 2000, murine S1P phosphohydrolase (mSPP1) was cloned as a S1P phosphohydrolase based on sequence homology to the yeast sphingoid base 1-phosphate phosphatase, Lcb3p/Lbp1p/Ysr2p (6). Recently, a human homolog of mSPP1, hSPP1, which exhibits 76% identity and 81% similarity to mSPP1, was identified (7). These mammalian SPP1s and their two yeast homologs, Lcb3p and Ysr3p, constitute the SPP family, which is distinct from another lipid phosphohydrolase family, the type 2 lipid phosphate phosphohydrolases (LPP), both in sequence and in biochemical properties. Accordingly, the SPP family members are highly specific to sphingoid base 1-phosphates, including S1P, dihydrosphingosine 1-phosphate (dihydro-S1P), and phytosphingosine 1-phosphate (6 -9); yet the LPP family members have broad substrate specificities including S1P, phosphatidate (PA), lysophosphatidate (LPA), ceramide 1-phosphate, and diacylglycerol pyrophosphate (10 -14). Proteins from bot...
Peroxisome proliferator-activated receptor ␥ coactivator (PGC)-1 is a critical transcriptional regulator of energy metabolism. Here we found that PGC-1␣ is a short lived and aggregation-prone protein. PGC-1␣ localized throughout the nucleoplasm and was rapidly destroyed via the ubiquitin-proteasome pathway. Upon proteasome inhibition, PGC-1␣ formed insoluble polyubiquitinated aggregates. Ubiquitination of PGC-1␣ depended on the integrity of the C terminus-containing arginine-serine-rich domains and an RNA recognition motif. Interestingly, ectopically expressed C-terminal fragment of PGC-1␣ was autonomously ubiquitinated and aggregated with promyelocytic leukemia protein. Cooperation of the N-terminal region containing two PEST-like motifs was required for prevention of aggregation and targeting of the polyubiquitinated PGC-1␣ for degradation. This region thereby negatively controlled the aggregation properties of the C-terminal region to regulate protein turnover and intranuclear compartmentalization of PGC-1␣. Exogenous expression of the PGC-1␣ C-terminal fragment interfered with degradation of full-length PGC-1␣ and enhanced its coactivation properties. We concluded that PGC-1␣ function is critically regulated at multiple steps via intramolecular cooperation among several distinct structural domains of the protein.
Methylation is an important event in the biotransformation pathway for many drugs and xenobiotic compounds. We screened DNA from 48 Japanese individuals for single-nucleotide polymorphisms (SNPs) in six methyltransferase (MT) genes (catechol-O-MT, COMT; guanidinoacetate N-MT, GAMT; histamine N-MT, HNMT; nicotinamide N-MT, NNMT; phosphatidylethanolamine N-MT, PEMT; and phenylethanolamine N-MT, PNMT) by direct sequencing of their entire genomic regions except for repetitive elements. This approach identified 190 SNPs and seven insertion/deletion polymorphisms among the six genes. Of the 190 SNPs, 33 were identified in the COMT gene, 6 in GAMT, 41 in HNMT, 8 in NNMT, 98 in PEMT, and 4 in PNMT. Nine were located in 5' flanking regions, 156 in introns, 10 in exons, and 15 in 3' flanking regions. These variants may contribute to a more precise understanding of possible correlations between genotypes and disease-susceptibility phenotypes or risk for side effects from drugs.
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