Richard Lehner

Acyl-CoA:diacylglycerol acyltransferase: molecular biology, biochemistry and biotechnology.

Triacylglycerol (TG) is a storage lipid which serves as an energy reservoir and a source of signalling molecules and substrates for membrane biogenesis. TG is essential for many physiological processes and its metabolism is widely conserved in nature. Acyl-CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) catalyzes the final step in the sn-glycerol-3-phosphate pathway leading to TG. DGAT activity resides mainly in two distinct membrane bound polypeptides, known as DGAT1 and DGAT2 which have been identified in numerous organisms. In addition, a few other enzymes also hold DGAT activity, including the DGAT-related acyl-CoA:monoacylglycerol acyltransferases (MGAT). Progress on understanding structure/function in DGATs has been limited by the lack of detailed three-dimensional structural information due to the hydrophobic properties of theses enzymes and difficulties associated with purification. This review examines several aspects of DGAT and MGAT genes and enzymes, including current knowledge on their gene structure, expression pattern, biochemical properties, membrane topology, functional motifs and subcellular localization. Recent progress in probing structural and functional aspects of DGAT1 and DGAT2, using a combination of molecular and biochemical techniques, is emphasized. Biotechnological applications involving DGAT enzymes ranging from obesity therapeutics to oilseed engineering are also discussed.

Cloning and expression of a cDNA encoding a hepatic microsomal lipase that mobilizes stored triacylglycerol

A microsomal triacylglycerol hydrolase (TGH) was recently purified from porcine liver [Lehner and Verger (1997) Biochemistry 36, 1861-1868]. To gain further insight into the function of TGH, we have cloned a cDNA encoding TGH from a rat liver cDNA library and generated McArdle RH7777 rat hepatoma cell lines that stably express the rat TGH. The putative protein derived from the cDNA sequence contains a cleavable signal sequence and a catalytic site serine residue present within a pentapeptide motif (GXSXG) that is conserved in all known lipases. TGH-transfected cells showed a 2-fold increase, compared with control cells, in the rate of depletion of prelabelled triacylglycerol stores. Thus, TGH is capable of hydrolysis of stored triacylglycerol. In contrast, the rate of turnover of labelled phosphatidylcholine was similar in both the vector- and TGH-transfected cells. Studies in TGH-transfected cells demonstrated that utilization of intracellular triacylglycerol pools for secretion was approx. 30% higher than in vector-transfected cells. Whereas phosphatidylcholine secretion was essentially the same in control and TGH-transfected cells, TGH-transfected cells also secreted an approx. 25% greater mass of triacylglycerol into the medium and had increased levels of apolipoprotein B100 in the very-low-density lipoprotein density range compared with control cells. The results suggest that the microsomal TGH actively participates in the mobilization of cytoplasmic triacylglycerol stores, some of which can be used for lipoprotein assembly.

Triacylglycerol hydrolase: role in intracellular lipid metabolism

Recent scientific advances have revealed the identity of several enzymes involved in the synthesis, storage and catabolism of intracellular neutral lipid storage droplets. An enzyme that hydrolyzes stored triacylglycerol (TG), triacylglycerol hydrolase (TGH), was purified from porcine, human and murine liver microsomes. In rodents, TGH is highly expressed in liver as well as heart, kidney, small intestine and adipose tissues, while in humans TGH is mainly expressed in the liver, adipose and small intestine. TGH localizes to the endoplasmic reticulum and lipid droplets. The TGH genes are located within a cluster of carboxylesterase genes on human and mouse chromosomes 16 and 8, respectively. TGH hydrolyzes stored TG, and in the liver, the lipolytic products are made available for VLDL-TG synthesis. Inhibition of TGH activity also inhibits TG and apolipoprotein B secretion by primary hepatocytes. A role for TGH in basal TG lipolysis in adipocytes has been proposed. TGH expression and activity is both developmentally and hormonally regulated. A model for the function of TGH is presented and discussed with respect to tissue specific functions.

Loss of TGH/Ces3 in Mice Decreases Blood Lipids, Improves Glucose Tolerance, and Increases Energy Expenditure

Excessive accumulation of triacylglycerol in peripheral tissues is tightly associated with obesity and has been identified as an independent risk factor for insulin resistance, type 2 diabetes, and cardiovascular complications. Here we show that ablation of carboxylesterase 3 (Ces3)/triacylglycerol hydrolase (TGH) expression in mice (Tgh(-/-)) results in decreased plasma triacylglycerol, apolipoprotein B, and fatty acid levels in both fasted and fed states. Despite the attenuation of very low-density lipoprotein secretion, TGH deficiency does not increase hepatic triacylglycerol levels. Tgh(-/-) mice exhibit increased food intake, respiratory quotient, and energy expenditure without change in body weight. These metabolic changes are accompanied by improved insulin sensitivity and glucose tolerance. Tgh(-/-) mice have smaller sized pancreatic islets but maintain normal glucose-stimulated insulin secretion. These studies demonstrate the potential of TGH as a therapeutic target for lowering blood lipid levels.

Recommended nomenclature for five mammalian carboxylesterase gene families: human, mouse, and rat genes and proteins

Mammalian carboxylesterase (CES or Ces) genes encode enzymes that participate in xenobiotic, drug, and lipid metabolism in the body and are members of at least five gene families. Tandem duplications have added more genes for some families, particularly for mouse and rat genomes, which has caused confusion in naming rodent Ces genes. This article describes a new nomenclature system for human, mouse, and rat carboxylesterase genes that identifies homolog gene families and allocates a unique name for each gene. The guidelines of human, mouse, and rat gene nomenclature committees were followed and “CES” (human) and “Ces” (mouse and rat) root symbols were used followed by the family number (e.g., human CES1). Where multiple genes were identified for a family or where a clash occurred with an existing gene name, a letter was added (e.g., human CES4A; mouse and rat Ces1a) that reflected gene relatedness among rodent species (e.g., mouse and rat Ces1a). Pseudogenes were named by adding “P” and a number to the human gene name (e.g., human CES1P1) or by using a new letter followed by ps for mouse and rat Ces pseudogenes (e.g., Ces2d-ps). Gene transcript isoforms were named by adding the GenBank accession ID to the gene symbol (e.g., human CES1_AB119995 or mouse Ces1e_BC019208). This nomenclature improves our understanding of human, mouse, and rat CES/Ces gene families and facilitates research into the structure, function, and evolution of these gene families. It also serves as a model for naming CES genes from other mammalian species.

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.

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.

Shedding light on the enigma of myocardial lipotoxicity: the involvement of known and putative regulators of fatty acid storage and mobilization

Excessive fatty acid (FA) uptake by cardiac myocytes is often associated with adverse changes in cardiac function. This is especially evident in diabetic individuals, where increased intramyocardial triacylglycerol (TG) resulting from the exposure to high levels of circulating FA has been proposed to be a major contributor to diabetic cardiomyopathy. At present, our knowledge of how the heart regulates FA storage in TG and the hydrolysis of this TG is limited. This review concentrates on what is known about TG turnover within the heart and how this is likely to be regulated by extrapolating results from other tissues. We also assess the evidence as to whether increased TG accumulation protects against FA-induced lipotoxicity through limiting the accumulations of ceramides and diacylglycerols versus whether it is a maladaptive response that contributes to cardiac dysfunction.

The cloning and expression of a murine triacylglycerol hydrolase cDNA and the structure of its corresponding gene

A novel murine cDNA for triacylglycerol hydrolase (TGH), an enzyme that is involved in mobilization of triacylglycerol from storage pools in hepatocytes, has been cloned and expressed. The cDNA consists of 1962 bp with an open reading frame of 1695 bp that encodes a protein of 565 amino acids. Murine TGH is a member of the CES1A class of carboxylesterases and shows a significant degree of identity to other carboxylesterases from rat, monkey and human. Expression of the cDNA in McArdle RH7777 hepatoma cells showed a 3-fold increase in the hydrolysis of p-nitrophenyl laurate compared to vector-transfected cells. The highest expression of TGH was observed in the livers of mice, with lower expression in kidney, heart, adipose and intestinal (duodenum/jejunum) tissues. The murine gene that encodes TGH was cloned and exon-intron boundaries were determined. The gene spans approx. 35 kb and contains 14 exons. The results will permit future studies on the function of this gene via gene-targeting experiments and analysis of transcriptional regulation of the TGH gene.

Obesity-induced lysine acetylation increases cardiac fatty acid oxidation and impairs insulin signalling

AIMS Lysine acetylation is a novel post-translational pathway that regulates the activities of enzymes involved in both fatty acid and glucose metabolism. We examined whether lysine acetylation controls heart glucose and fatty acid oxidation in high-fat diet (HFD) obese and SIRT3 knockout (KO) mice. METHODS AND RESULTS C57BL/6 mice were placed on either a HFD (60% fat) or a low-fat diet (LFD; 4% fat) for 16 or 18 weeks. Cardiac fatty acid oxidation rates were significantly increased in HFD vs. LFD mice (845 ± 76 vs. 551 ± 87 nmol/g dry wt min, P < 0.05). Activities of the fatty acid oxidation enzymes, long-chain acyl-CoA dehydrogenase (LCAD), and β-hydroxyacyl-CoA dehydrogenase (β-HAD) were increased in hearts from HFD vs. LFD mice, and were associated with LCAD and β-HAD hyperacetylation. Cardiac protein hyperacetylation in HFD-fed mice was associated with a decrease in SIRT3 expression, while expression of the mitochondrial acetylase, general control of amino acid synthesis 5 (GCN5)-like 1 (GCN5L1), did not change. Interestingly, SIRT3 deletion in mice also led to an increase in cardiac fatty acid oxidation compared with wild-type (WT) mice (422 ± 29 vs. 291 ± 17 nmol/g dry wt min, P < 0.05). Cardiac lysine acetylation was increased in SIRT3 KO mice compared with WT mice, including increased acetylation and activity of LCAD and β-HAD. Although the HFD and SIRT3 deletion decreased glucose oxidation, pyruvate dehydrogenase acetylation was unaltered. However, the HFD did increase Akt acetylation, while decreasing its phosphorylation and activity. CONCLUSION We conclude that increased cardiac fatty acid oxidation in response to high-fat feeding is controlled, in part, via the down-regulation of SIRT3 and concomitant increased acetylation of mitochondrial β-oxidation enzymes.