-ichi Osuga, Shun Ishibashi, and Fredric B. Kraemer. Resistance to high-fat diet-induced obesity and altered expression of adipose-specific genes in HSL-deficient mice. Am J Physiol Endocrinol Metab 285: E1182-E1195, 2003. First published September 3, 2003 10.1152/ajpendo.00259.2003.-To elucidate the role of hormone-sensitive lipase (HSL) in diet-induced obesity, HSLdeficient (HSL Ϫ/Ϫ ) and wild-type mice were fed normal chow or high-fat diets. HSL Ϫ/Ϫ mice were resistant to diet-induced obesity showing higher core body temperatures. Weight and triacylglycerol contents were decreased in white adipose tissue (WAT) but increased in both brown adipose tissue (BAT) and liver of HSL Ϫ/Ϫ mice. Serum insulin levels in the fed state and tumor necrosis factor-␣ mRNA levels in adipose tissues were higher, whereas serum levels of adipocyte complement-related protein of 30 kDa (ACRP30)/adiponectin and leptin, as well as mRNA levels of ACRP30/adiponectin, leptin, resistin, and adipsin in WAT, were lower in HSL Ϫ/Ϫ mice than in controls. Expression of transcription factors associated with adipogenesis (peroxisome proliferator-activated receptor-␥, CAAT/enhancer-binding protein-␣) and lipogenesis (carbohydrate response element-binding protein, adipocyte determination-and differentiation-dependent factor-1/sterol regulatory element-binding protein-1c), as well as of adipose differentiation markers (adipocyte lipid-binding protein, perilipin, lipoprotein lipase), lipogenic enzymes (glycerol-3-phosphate acyltransferase, acyl-CoA:diacylglycerol acyltransferase-1 and -2, fatty acid synthase, ATP citrate lyase) and insulin signaling proteins (insulin receptor, insulin receptor substrate-1, GLUT4), was suppressed in WAT but not in BAT of HSL Ϫ/Ϫ mice. In contrast, expression of genes associated with cholesterol metabolism (sterol-regulatory element-binding protein-2, 3-hydroxy-3-methylglutaryl-CoA reductase, acyl-CoA:cholesterol acyltransferase-1) and thermogenesis (uncoupling protein-2) was upregulated in both WAT and BAT of HSL Ϫ/Ϫ mice. Our results suggest that impaired lipolysis in HSL deficiency affects lipid metabolism through alterations of adipose differentiation and adipose-derived hormone levels. adipocyte; differentiation; insulin; leptin; fatty liver HORMONE-SENSITIVE LIPASE (HSL) mediates the cytosolic hydrolysis of triacylglycerols (lipolysis) and cholesteryl esters (12). HSL is expressed in various tissues, including white (WAT) and brown adipose tissues (BAT), cardiomyocytes, adrenocortical cells, and gonads (11). Because HSL is responsible for the release of free fatty acids (FFA) from stored triacylglycerols in adipose tissues, the enzyme has been proposed to play an essential role in the regulation of body weight and fat mass. We previously reported, however, that the body weight of HSL-deficient (HSL Ϫ/Ϫ ) mice generated by homologous recombination fed a normal chow diet did not differ from that of wild-type (HSL ϩ/ϩ ) mice despite the presence of a markedly suppressed hydrolysis of triacylglycerols and cholester...

Glioblastoma multiforme (GBM) is a highly aggressive form of brain cancer associated with a very poor prognosis. Recently, the initiation and growth of GBM has been linked to brain tumor-initiating cells (BTICs), which are poorly differentiated and share features with neural stem cells (NSCs). Here we describe a kinome-wide RNA interference screen to identify factors that control the tumorigenicity of BTICs. We identified several genes whose silencing induces differentiation of BTICs derived from multiple GBM patients. In particular, knockdown of the adaptor protein TRRAP significantly increased differentiation of cultured BTICs, sensitized the cells to apoptotic stimuli, and negatively affected cell cycle progression. TRRAP knockdown also significantly suppressed tumor formation upon intracranial BTIC implantation into mice. Together, these findings support a critical role for TRRAP in maintaining a tumorigenic, stem cell-like state.

Hormone-sensitive lipase (HSL)1 is an intracellular neutral lipase that is highly expressed in adipose and steroidogenic tissues (1). The enzyme has broad substrate specificity, displaying hydrolytic activity against triacylglycerol, diacylglycerol, and cholesteryl ester (2). Observations from HSL-null mice have shown that HSL is responsible for ϳ50% of the neutral triglyceride lipase activity and all of the neutral cholesteryl ester hydrolase activity in white adipose tissue (3). Thus, HSL plays an important role in regulating lipolysis and the release of fatty acids from adipose tissue. The sequence of HSL is unrelated to other mammalian lipases, but it shares sequence and structural similarity with several bacterial and fungal lipases (4 -11). This structural similarity is based on the ability to model a large portion of the C-terminal ϳ450 amino acids of HSL as an ␣/␤ hydrolase (7); however, the initial ϳ320 amino acids of the protein share no sequence or structural homology with any known proteins. Within the C-terminal region of the protein lies a 150-amino acid sequence that contains a number of sites phosphorylated in response to lipolytic stimulation (7,12,13). In this regard HSL is unique among lipases for the ability of its activity to be up-regulated by phosphorylation. In addition to phosphorylation, HSL activity appears to be regulated by oligomerization, with the dimeric enzyme exhibiting markedly increased activity (14).Utilizing a yeast two-hybrid screen of a rat adipose tissue library, we previously demonstrated that HSL specifically interacts with adipocyte lipid-binding protein (ALBP or aP2) and identified the N-terminal 300 amino acids of HSL as the region responsible for this interaction (15). ALBP is highly expressed in adipose tissue and is a member of the family of intracellular fatty acid-binding proteins that bind fatty acids, retinoids and other hydrophobic ligands (16). It has been proposed that intracellular fatty acid-binding proteins function to sequester fatty acids, thus serving as an intracellular buffer or participating in facilitating the movement of fatty acids within the cell. In view of our observation that HSL and ALBP interact, we proposed that ALBP might prevent feedback inhibition of HSL by high local concentrations of free fatty acids released at the site of hydrolysis. Consistent with this view, adipocytes from ALBP-null mice exhibit markedly reduced basal and stimulated lipolysis both in situ and in vivo (17,18). In the present studies we have addressed the functional significance of the interaction of HSL with ALBP and provide evidence that the interaction of ALBP with HSL constitutes an additional mechanism whereby the hydrolytic activity of HSL is regulated. Furthermore, we have explored the identification of the sequences in HSL that mediate its interaction with ALBP.

Hormone-sensitive lipase (HSL) is a cytosolic neutral lipase that hydrolyzes intracellular stores of triacylglycerols and cholesteryl esters. HSL activity is regulated via phosphorylation-dephosphorylation, with cyclic AMP-dependent protein kinase increasing activity following phosphorylation of a single serine and Ca2+/calmodulin-dependent protein kinase II phosphorylating another serine at a basal site. The current studies used site-directed mutagenesis to show that Ser-563 of rat HSL is phosphorylated by cyclic AMP-dependent protein kinase and that Ser-565 is phosphorylated by Ca2+/calmodulin-dependent protein kinase II. Mutation of Ser-563-->Ala eliminated HSL hydrolytic activity against cholesteryl ester, triacylglycerol, and diacylglycerol substrates to the same extent as mutation of Ser-423-->Ala, the presumed catalytic site. Mutation of Ser-565-->Ala modestly decreased HSL activity. In contrast, mutation of Ser-563-->Asp preserved HSL hydrolytic activity and even increased activity 20% above the control wild-type enzyme. Molecular modeling of the catalytic pocket of HSL suggested the involvement of Val-710. Mutation of Val-710-->Ala resulted in an 85% loss of HSL hydrolytic activity. The results of these studies illustrate the importance of the presence of a hydroxyl group or negative charge at residue 563, either for proper conformation of rat HSL or for proper stabilization of substrate to allow maintenance of hydrolytic activity, as well as the importance of the involvement of additional amino acids in the catalytic pocket of the enzyme.