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Richard Lehner

Publications and source records attributed to Richard Lehner.

16 recordsLinked to original sources
PubMed2005

Mutation of F417 but not of L418 or L420 in the lipid binding domain decreases the activity of triacylglycerol hydrolase.

Human triacylglycerol hydrolase (hTGH) has been shown to play a role in hepatic lipid metabolism. Triacylglycerol hydrolase (TGH) hydrolyzes insoluble carboxylic esters at lipid/water interfaces, although the mechanism by which the enzyme adsorbs to lipid droplets is unclear. Three-dimensional modeling of hTGH predicts that catalytic residues are adjacent to an alpha-helix that may mediate TGH/lipid interaction. The helix contains a putative neutral lipid binding domain consisting of the octapeptide FLDLIADV (amino acid residues 417-424) with the consensus sequence FLXLXXXn (where n is a nonpolar residue and X is any amino acid except proline) identified in several other proteins that bind or metabolize neutral lipids. Deletion of this alpha-helix abolished the lipolytic activity of hTGH. Replacement of F417 with alanine reduced activity by 40% toward both insoluble and soluble esters, whereas replacement of L418 and L420 with alanine did not. Another potential mechanism of increasing TGH affinity for lipid is via reversible acylation. Molecular modeling predicts that C390 is available for covalent acylation. However, neither chemical modification of C390 nor mutation to alanine affected activity. Our findings indicate that F417 but not L418, L420, or C390 participates in substrate hydrolysis by hTGH.

Acylation↗
PubMed2005

Insulin, glucagon and fatty acid treatment of hepatocytes does not result in phosphorylation or changes in activity of triacylglycerol hydrolase.

It is recognized that the majority of very low density lipoprotein (VLDL) associated triacylglycerol (TG) is synthesized from fatty acids and partial acylglycerols generated by lipolysis of intra-hepatic storage rather than made de novo. Triacylglycerol hydrolase (TGH) is involved in mobilizing stored TG. Modulating the ability of TGH to hydrolyze stored lipids represents a potentially regulated and rate limiting step in VLDL assembly. Phosphorylation of lipases and carboxylesterases trigger diverse but functionally significant events. We explored the potential for regulating the mobilization of hepatic TG through phosphorylation of TGH. Insulin is known to suppress VLDL secretion from liver, and glucagon can be considered an opposing hormone. However, neither insulin nor glucagon treatment of hepatocytes led to phosphorylation of TGH or changes in its activity. Augmenting intracellular TG stores by incubations with oleic acid also did not lead to changes in TGH activity. Therefore, changes in phosphorylation state are not a mechanism for regulating TGH activity, access to TG substrate pools or for TGH-mediated contributions to VLDL assembly and secretion.

Alkaline Phosphatase↗
PubMed2005

C/EBPalpha activates the transcription of triacylglycerol hydrolase in 3T3-L1 adipocytes.

TGH (triacylglycerol hydrolase) catalyses the lipolysis of intracellular stored triacylglycerol. To explore the mechanisms that regulate TGH expression in adipose tissue, we studied the expression of TGH during the differentiation of 3T3-L1 adipocytes. TGH mRNA and protein levels increased dramatically in 3T3-L1 adipocytes compared with pre-adipocytes. Electrophoretic mobility shift assays demonstrated enhanced binding of nuclear proteins of adipocytes to the distal murine TGH promoter region (-542/-371 bp), yielding one adipocyte-specific migrating complex. Competitive and supershift assays demonstrated that the distal TGH promoter fragment bound C/EBPalpha (CCAAT/enhancer-binding protein alpha). Transient transfections of different mutant TGH promoter-luciferase constructs into 3T3-L1 adipocytes and competitive electromobility shift assays showed that the C/EBP-binding elements at positions -470/-459 bp and -404/-390 bp are important for transcriptional activation. Co-transfection with C/EBPalpha cDNA and TGH promoter constructs in 3T3-L1 pre-adipocytes demonstrated that C/EBPalpha increased TGH promoter activity. Ectopic expression of C/EBPalpha in NIH 3T3 cells activated TGH mRNA expression without causing differentiation into adipocytes. These experiments directly link increased TGH expression in adipocytes to transcriptional regulation by C/EBPalpha. This is the first evidence that C/EBPalpha participates directly in the regulation of an enzyme associated with lipolysis.

3T3-L1 Cells↗
PubMed2005

Techniques to measure lipase and esterase activity in vitro.

Lipases and esterases constitute a large category of enzymes. They are ubiquitous in nature, found in bacteria, fungi, and animals. The family members address a wide variety of structurally diverse substrates. Appropriately, a large number of assays have been developed to analyze their activity in vitro. Here, we present an overview of these enzymes, along with protocols for common assays performed in solution. An emphasis is placed on assays for enzymes that can hydrolyze triacylglycerols.

Animals↗
PubMed2004

Triacylglycerol hydrolase is localized to the endoplasmic reticulum by an unusual retrieval sequence where it participates in VLDL assembly without utilizing VLDL lipids as substrates.

The majority of hepatic intracellular triacylglycerol (TG) is mobilized by lipolysis followed by reesterification to reassemble TG before incorporation into a very-low-density lipoprotein (VLDL) particle. Triacylglycerol hydrolase (TGH) is a lipase that hydrolyzes TG within hepatocytes. Immunogold electron microscopy in transfected cells revealed a disparate distribution of this enzyme within the endoplasmic reticulum (ER), with particularly intense localization in regions surrounding mitochondria. TGH is localized to the lumen of the ER by the C-terminal tetrapeptide sequence HIEL functioning as an ER retention signal. Deletion of HIEL resulted in secretion of catalytically active TGH. Mutation of HIEL to KDEL, which is the consensus ER retrieval sequence in animal cells, also resulted in ER retention and conservation of lipolytic activity. However, KDEL-TGH was not as efficient at mobilizing lipids for VLDL secretion and exhibited an altered distribution within the ER. TGH is a glycoprotein, but glycosylation is not required for catalytic activity. TGH does not hydrolyze apolipoprotein B-associated lipids. This suggests a mechanism for vectored movement of TGs onto developing VLDL in the ER as TGH may mobilize TG for VLDL assembly, but will not access this lipid once it is associated with VLDL.

Animals↗
PubMed2004

Enhanced membrane fusion in sterol-enriched vacuoles bypasses the Vrp1p requirement.

Organization of lipids into membrane microdomains is a vital mechanism of protein processing. Here we show that overexpression of ERG6, a gene involved in ergosterol synthesis, elevates sterol levels 1.5-fold on the vacuole membrane and enhances their homotypic fusion. The mechanism of sterol-enhanced fusion is not via more efficient sorting, but instead promotes increased kinetics of fusion subreactions. We initially isolated ERG6 as a suppressor of a vrp1Delta growth defect selective for vacuole function. VRP1 encodes verprolin, an actin-binding protein that colocalizes to vacuoles. The vrp1Delta mutant has fragmented vacuoles in vivo and isolated vacuoles do not fuse in vitro, indicative of a Vrp1p requirement for membrane fusion. ERG6 overexpression rescues vrp1Delta vacuole fusion in a cytosol-dependent manner. Cytosol prepared from the vrp1Delta strain remains active; therefore, cytosol is not resupplying Vrp1p. Las17p (Vrp1p functional partner) antibodies, which inhibit wild-type vacuole fusion, do not inhibit the fusion of vacuoles from the vrp1Delta-ERG6 overexpression strain. Vacuole-associated actin turnover is decreased in the vrp1Delta strain, but recovered by ERG6 overexpression linking sterol enrichment to actin remodeling. Therefore, the Vrp1p/Las17p requirement for membrane fusion is bypassed by increased sterols, which promotes actin remodeling as part the membrane fusion mechanism.

Actins↗
PubMed2004

Carboxylesterase 3 (EC 3.1.1.1) is a major adipocyte lipase.

Hydrolysis of triglycerides is central to energy homeostasis in white adipose tissue (WAT). Hormone-sensitive lipase (HSL) was previously felt to mediate all lipolysis in WAT. Surprisingly, HSL-deficient mice show active HSL-independent lipolysis, suggesting that other lipase(s) also mediate triglyceride hydrolysis. To clarify this, we used functional proteomics to detect non-HSL lipase(s) in mouse WAT. After cell fractionation of intraabdominal WAT, most non-HSL neutral lipase activity is localized in the 100,000 x g infranatant and fat cake fractions. By oleic acid-linked agarose chromatography of infranatant followed by elution in a 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid gradient, we identified two peaks of esterase activity using p-nitrophenyl butyrate as a substrate. One of the peaks contained most of the lipase activity. In the corresponding fractions, gel permeation chromatography and SDS-PAGE, followed by tandem mass spectrometric analysis of excised Coomassie Blue-stained peptides, revealed carboxylesterase 3 (triacylglycerol hydrolase (TGH); EC 3.1.1.1). TGH is also the principle lipase of WAT fat cake extracts. Partially purified WAT TGH had lipase activity as well as lesser but detectable neutral cholesteryl ester hydrolase activity. Western blotting of subcellular fractions of WAT and confocal microscopy of fibroblasts following in vitro adipocytic differentiation are consistent with a distribution of TGH to endoplasmic reticulum, cytosol, and the lipid droplet. TGH is responsible for a major part of non-HSL lipase activity in WAT in vitro and may mediate some or all HSL-independent lipolysis in adipocytes.

Adipocytes↗
PubMed2004

Calmodulin antagonist W-7 inhibits de novo synthesis of cholesterol and suppresses secretion of de novo synthesized and preformed lipids from cultured hepatocytes.

The effects of a calmodulin antagonist W-7 were studied on the synthesis and secretion of lipids in primary rat hepatocytes and McArdle-RH7777 cells. In time course experiments, W-7 (20 microM) inhibited secretion of newly synthesized triacyl[(3)H]glycerol by 35%. When the cells were pre-treated overnight with W-7 (20 microM), followed by incubation with [(3)H]oleate, a significant decrease in the secretion of triacylglycerol (TG) and cholesteryl ester (CE) was observed. De novo synthesis of cholesterol from acetate or mevalonolactone was inhibited by W-7, but not glycerolipid synthesis from glycerol and oleic acid precursors. Concentration-response curves for the effects of overnight pre-incubation with W-7 followed labeling with [(3)H]glycerol and [(14)C]mevalonolactone revealed that: (1). the inhibitory effect of W-7 was concentration-dependent and appeared even at the lowest concentration examined (1 microM). W-7 at a concentration of 20 microM suppressed secretion of TG by 60% (P<or=0.002), phosphatidylcholine (PC) by 31% (P<or=0.05), CE by 59% (P<or=0.002) and cholesterol by 64% (P<or=0.002). (2). The incorporation of [(14)C]mevalonolactone into cellular cholesterol and CE was decreased significantly, while W-7 did not have any significant effect upon incorporation of [(3)H]glycerol into glycerolipids, except at the highest concentration examined (50 microM), where synthesis of both TG and PC was significantly suppressed. (3). While the percentage of secreted de novo synthesized glycerolipids and CE decreased proportionally with increasing concentration of W-7, the percentage of secreted newly made cholesterol remained unaffected at any concentration of W-7. In the absence of W-7, about 19% of newly formed cholesterol became esterified into CE, whereas W-7 increased cholesterol esterification in a concentration-dependent manner. (4) W-7 (20 microM) also suppressed the secretion of preformed cholesterol by 24% and CE by 55% but did not affect the recruitment of preformed cholesterol for esterification. About 6.5% of pre-labeled cholesterol and 20% of CE were directed to secretion, which was suppressed in the presence of W-7 by 17% (P<or=0.09) and 48% (P<or=0.001), respectively. These results suggest that, W-7 in the range of 1-20 microM inhibited de novo synthesis of cholesterol and the secretion of both de novo synthesized and preformed lipids.

Animals↗
PubMed2004

Regulation of the enzymes of hepatic microsomal triacylglycerol lipolysis and re-esterification by the glucocorticoid dexamethasone.

Hepatic VLDL (very-low-density lipoprotein) assembly is a complex process that is largely regulated by the provision of lipid for apolipoprotein B assembly. Intracellular stored TAG (triacylglycerol) undergoes an initial lipolysis followed by re-esterification of the lipolytic products to form TAG prior to their incorporation into a VLDL particle. TGH (TAG hydrolase) is a lipase that hydrolyses intracellular TAG within the hepatocyte. We have utilized both dexamethasone-injected mouse and primary hepatocyte models to address whether stimulation of TAG biosynthesis by the synthetic glucocorticoid, dexamethasone, altered hepatic lipolysis and re-esterification and the provision of stored TAG for lipoprotein secretion. Dexamethasone treatment resulted in decreased TGH expression, primarily due to a dexamethasone-induced decrease in TGH mRNA stability. The expression and activities of diacylglycerol acyltransferases 1 and 2 were stimulated by dexamethasone. The combination of reduced intracellular TAG lipolysis and increased TAG biosynthesis contributed to the accumulation of TAG within the livers of dexamethasone-injected mice. The rate of hepatic TAG secretion in dexamethasonetreated mice was maintained at similar levels as in control mice. Our data demonstrate that stimulation of de novo TAG synthesis by dexamethasone increased the proportion of secreted TAG that was derived from de novo sources, while the utilization of stored TAG for secretion was reduced. The results show that, during markedly increased TAG synthesis, some TAGs are diverted from the cytosolic storage pool and are utilized directly for VLDL assembly within the endoplasmic reticulum lumen.

Acyltransferases↗
PubMed2004

ABCA1-dependent lipid efflux to apolipoprotein A-I mediates HDL particle formation and decreases VLDL secretion from murine hepatocytes.

High levels of expression of the ATP binding cassette transporter A1 (ABCA1) in the liver and the need to over- or underexpress hepatic ABCA1 to impact plasma HDL levels in mice suggest a major role of the liver in HDL formation and in determining circulating HDL levels. Cultured murine hepatocytes were used to examine the role of hepatic ABCA1 in mediating the lipidation of apolipoprotein A-I (apoA-I) for HDL particle formation. Exogenous apoA-I stimulated cholesterol efflux to the medium from wild-type hepatocytes, but not from ABCA1-deficient (abca1(-/-)) hepatocytes. ApoA-I induced the formation of new HDL particles and enhanced the lipidation of endogenously secreted murine apoA-I in ABCA1-expressing but not abca1(-/-) hepatocytes. ABCA1-dependent cholesterol mobilization to apoA-I increased new cholesterol synthesis, indicating depletion of the regulatory pool of hepatocyte cholesterol during HDL formation. Secretion of triacylglycerol and apoB was decreased following apoA-I incubation with ABCA1-expressing but not abca1(-/-) hepatocytes. These results support a major role for hepatocyte ABCA1 in generating a critical pool of HDL precursor particles that enhance further HDL generation and passive cholesterol mobilization in the periphery. The results also suggest that diversion of hepatocyte cholesterol into the "reverse" cholesterol transport pathway diminishes cholesterol availability for apoB-containing lipoprotein secretion by the liver.

ATP Binding Cassette Transporter 1↗
PubMed2003

Regulation of triacylglycerol hydrolase expression by dietary fatty acids and peroxisomal proliferator-activated receptors.

Triacylglycerol hydrolase (TGH) is an enzyme that catalyzes the lipolysis of intracellular stored triacylglycerol (TG). Peroxisomal proliferator-activated receptors (PPAR) regulate a multitude of genes involved in lipid homeostasis. Polyunsaturated fatty acids (PUFA) are PPAR ligands and fatty acids are produced via TGH activity, so we studied whether dietary fats and PPAR agonists could regulate TGH expression. In 3T3-L1 adipocytes, TGH expression was increased 10-fold upon differentiation, compared to pre-adipocytes. 3T3-L1 cells incubated with a PPARgamma agonist during the differentiation process resulted in a 5-fold increase in TGH expression compared to control cells. Evidence for direct regulation of TGH expression by PPARgamma could not be demonstrated as TGH expression was not affected by a 24-h incubation of mature 3T3-L1 adipocytes with the PPARgamma agonist. Feeding mice diets enriched in fatty acids for 3 weeks did not affect hepatic TGH expression, though a 3-week diet enriched in fatty acids and cholesterol increased hepatic TGH expression 2-fold. Two weeks of clofibrate feeding did not significantly affect hepatic TGH expression or microsomal lipolytic activities in wild-type or PPARalpha-null mice, indicating that PPARalpha does not regulate hepatic TGH expression. Therefore, TGH expression does not appear to be directly regulated by PPARs or fatty acids in the liver or adipocytes.

3T3 Cells↗
PubMed2003

Inhibitors of hepatic microsomal triacylglycerol hydrolase decrease very low density lipoprotein secretion.

The presence of elevated circulating triacylglycerol (TG)-rich very low density lipoprotein (VLDL) and apolipoprotein B-100 (apoB-100) levels represents an independent risk factor for coronary artery disease. Triacylglycerol hydrolase catalyzes the mobilization of cytoplasmic TG stores. To test the hypothesis that the enzyme plays a role in the provision of core lipids for the assembly of VLDL, we inhibited the lipase activity in primary rat hepatocytes and analyzed lipid and apoB synthesis and secretion. Inhibition of lipolysis resulted in a dramatic decrease in secretion of TGs. In addition, secretion of cholesteryl ester and phosphatidylcholine was substantially decreased. Analysis of secreted apolipoproteins indicated that apoB-100 secretion was much more sensitive to lipase inhibition than was apoB-48 secretion, perhaps because of the ability of apoB-48 to be secreted as a relatively lipid-poor particle. The results agreed with those obtained with hepatoma cells transfected with triacylglycerol hydrolase cDNA, in which preferential lipidation of apoB-100 was observed. Together, our findings provide evidence that inhibition of intracellular TG hydrolysis significantly decreases apoB-100 secretion and suggest that triacylglycerol hydrolase may be a suitable pharmacological target in efforts to lower plasma lipid levels.

Animals↗
PubMed2002

Membrane topography of human phosphatidylethanolamine N-methyltransferase.

In liver, phosphatidylethanolamine is converted to phosphatidylcholine through a series of three sequential methylation reactions. Phosphatidylethanolamine N-methyltransferase (PEMT) catalyzes each transmethylation reaction, and S-adenosylmethionine is the methyl group donor. Biochemical analysis of human liver revealed that the methyltransferase activity is primarily localized to the endoplasmic reticulum and mitochondria-associated membranes. Bioinformatic analysis of the predicted amino acid sequence suggested that the enzyme adopts a polytopic conformation in those membranes. To elucidate the precise membrane topography of PEMT and thereby provide the basis for in-depth functional characterization of the enzyme, we performed endoproteinase-protection analysis of epitope-tagged, recombinant protein. Our data suggest a topographical model of PEMT in which four transmembrane regions span the membrane such that both the N and C termini of the enzyme are localized external to the ER. Two hydrophilic connecting loops protrude into the luminal space of the microsomes whereas a corresponding loop on the cytosolic side remains proximate to the membrane. Further support for this model was obtained following endoproteinase-protection analysis of mutant recombinant PEMT derivatives in which specific protease cleavage sites had been genetically engineered or ablated.

Adult↗
PubMed2002

Structure-function analysis of human triacylglycerol hydrolase by site-directed mutagenesis: identification of the catalytic triad and a glycosylation site.

Triacylglycerol hydrolase is a microsomal enzyme that hydrolyzes stored cytoplasmic triacylglycerol in the liver and participates in the lipolysis/re-esterification cycle during the assembly of very-low-density lipoproteins. The structure-activity relationship of the enzyme was investigated by site-directed mutagenesis and heterologous expression. Expression of human TGH in Escherichia coli yields a protein without enzymatic activity, which suggests that posttranslational processing is necessary for the catalytic activity. Expression in baculovirus-infected Sf-9 cells resulted in correct processing of the N-terminal signal sequence and yielded a catalytically active enzyme. A putative catalytic triad consisting of a nucleophilic serine (S221), glutamic acid (E354), and histidine (H468) was identified. Site-directed mutagenesis of the residues (S221A, E354A, and H468A) yielded a catalytically inactive enzyme. CD spectra of purified mutant proteins were very similar to that of the wild-type enzyme, which suggests that the mutations did not affect folding. Human TGH was glycosylated in the insect cells. Mutagenesis of the putative N-glycosylation site (N79A) yielded an active nonglycosylated enzyme. Deletion of the putative C-terminal endoplasmic reticulum retrieval signal (HIEL) did not result in secretion of the mutant protein. A model of human TGH structure suggested a lipase alpha/beta hydrolase fold with a buried active site and two disulfide bridges (C87-C116 and C274-C285).

Animals↗
PubMed2002

Heterologous expression, purification, and characterization of human triacylglycerol hydrolase.

Triacylglycerol hydrolase mobilizes stored triacylglycerol some of which is used for very-low-density lipoprotein assembly in the liver. A full-length cDNA coding for a human triacylglycerol hydrolase (hTGH) was isolated from a human liver cDNA library. The cDNA has an open reading frame of 576 amino acids with a cleavable 18-amino-acid signal sequence. The deduced amino acid sequence shows that the protein belongs to the carboxylesterase family. The hTGH was highly expressed in Escherichia coli as a 6xHis-tagged fusion protein, with the tag at the N-terminus in place of the signal peptide. However, the expressed protein was insoluble and inactive. Expression was confirmed by immunoblotting and N-terminal amino acid sequencing of the purified protein. Expression of hTGH with its native signal sequence and a C-terminal 6xHis-tag in Sf9 cells using the baculovirus expression system yielded active enzyme. N-terminal amino acid sequencing of the purified expressed protein showed correct processing of the signal peptide. The enzyme also undergoes glycosylation within the endoplasmic reticulum lumen. The results suggest that hTGH expressed in insect cells is properly folded. Therefore, baculovirus expression of hTGH and facile purification of the His-tagged enzyme will allow detailed characterization of the structure/activity relationship.

Amino Acid Sequence↗