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The Role of Diacylglycerol Acyltransferase (DGAT) 1 and 2 in Cardiac Metabolism and Function.

ABSTRACT: It is increasingly recognized that synthesis and turnover of cardiac triglyceride (TG) play a pivotal role in the regulation of lipid metabolism and function of the heart. The last step in TG synthesis is catalyzed by diacylglycerol:acyltransferase (DGAT) which esterifies the diacylglycerol with a fatty acid. Mammalian heart has two DGAT isoforms, DGAT1 and DGAT2, yet their roles in cardiac metabolism and function remain poorly defined. Here, we show that inactivation of DGAT1 or DGAT2 in adult mouse heart results in a moderate suppression of TG synthesis and turnover. Partial inhibition of DGAT activity increases cardiac fatty acid oxidation without affecting PPAR? signaling, myocardial energetics or contractile function. Moreover, coinhibition of DGAT1/2 in the heart abrogates TG turnover and protects the heart against high fat diet-induced lipid accumulation with no adverse effects on basal or dobutamine-stimulated cardiac function. Thus, the two DGAT isoforms in the heart have partially redundant function, and pharmacological inhibition of one DGAT isoform is well tolerated in adult hearts.

SUBMITTER: Roe ND

PROVIDER: S-EPMC5862879 | biostudies-other | 2018 Mar

REPOSITORIES: biostudies-other

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The Role of Diacylglycerol Acyltransferase (DGAT) 1 and 2 in Cardiac Metabolism and Function.

Roe Nathan D ND Handzlik Michal K MK Li Tao T Tian Rong R

Scientific reports 20180321 1

It is increasingly recognized that synthesis and turnover of cardiac triglyceride (TG) play a pivotal role in the regulation of lipid metabolism and function of the heart. The last step in TG synthesis is catalyzed by diacylglycerol:acyltransferase (DGAT) which esterifies the diacylglycerol with a fatty acid. Mammalian heart has two DGAT isoforms, DGAT1 and DGAT2, yet their roles in cardiac metabolism and function remain poorly defined. Here, we show that inactivation of DGAT1 or DGAT2 in adult ...[more]

PMID: 29563512

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Project description:BackgroundDiacylglycerol acyltransferase families (DGATs) catalyze the final and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. Understanding the roles of DGATs will help to create transgenic plants with value-added properties and provide clues for therapeutic intervention for obesity and related diseases. The objective of this analysis was to identify conserved sequence motifs and amino acid residues for better understanding of the structure-function relationship of these important enzymes.Results117 DGAT sequences from 70 organisms including plants, animals, fungi and human are obtained from database search using tung tree DGATs. Phylogenetic analysis separates these proteins into DGAT1 and DGAT2 subfamilies. These DGATs are integral membrane proteins with more than 40% of the total amino acid residues being hydrophobic. They have similar properties and amino acid composition except that DGAT1s are approximately 20 kDa larger than DGAT2s. DGAT1s and DGAT2s have 41 and 16 completely conserved amino acid residues, respectively, although only two of them are shared by all DGATs. These residues are distributed in 7 and 6 sequence blocks for DGAT1s and DGAT2s, respectively, and located at the carboxyl termini, suggesting the location of the catalytic domains. These conserved sequence blocks do not contain the putative neutral lipid-binding domain, mitochondrial targeting signal, or ER retrieval motif. The importance of conserved residues has been demonstrated by site-directed and natural mutants.ConclusionsThis study has identified conserved sequence motifs and amino acid residues in all 117 DGATs and the two subfamilies. None of the completely conserved residues in DGAT1s and DGAT2s is present in recently reported isoforms in the multiple sequences alignment, raising an important question how proteins with completely different amino acid sequences could perform the same biochemical reaction. The sequence analysis should facilitate studying the structure-function relationship of DGATs with the ultimate goal to identify critical amino acid residues for engineering superb enzymes in metabolic engineering and selecting enzyme inhibitors in therapeutic application for obesity and related diseases.

Project description:Recently, we isolated a novel Streptomyces strain which can accumulate extraordinarily large amounts of triacylglycerol (TAG) and consists of 64% fatty acids (dry weight) when cultivated with glucose and 50% fatty acids (dry weight) when cultivated with cellobiose. To identify putative gene products responsible for lipid storage and cellobiose utilization, we analyzed its draft genome sequence. A single gene encoding a wax ester synthase/acyl coenzyme A (CoA):diacylglycerol acyltransferase (WS/DGAT) was identified and heterologously expressed in Escherichia coli The purified enzyme AtfG25 showed acyltransferase activity with C12- or C16-acyl-CoA, C12 to C18 alcohols, or dipalmitoyl glycerol. This acyltransferase exhibits 24% amino acid identity to the model enzyme AtfA from Acinetobacter baylyi but has high sequence similarities to WS/DGATs from other Streptomyces species. To investigate the impact of AtfG25 on lipid accumulation, the respective gene, atfG25, was inactivated in Streptomyces sp. strain G25. However, cells of the insertion mutant still exhibited DGAT activity and were able to store TAG, albeit in lower quantities and at lower rates than the wild-type strain. These findings clearly indicate that AtfG25 has an important, but not exclusive, role in TAG biosynthesis in the novel Streptomyces isolate and suggest the presence of alternative metabolic pathways for lipid accumulation which are discussed in the present study.A novel Streptomyces strain was isolated from desert soil, which represents an extreme environment with high temperatures, frequent drought, and nutrient scarcity. We believe that these harsh conditions promoted the development of the capacity for this strain to accumulate extraordinarily large amounts of lipids. In this study, we present the analysis of its draft genome sequence with a special focus on enzymes potentially involved in its lipid storage. Furthermore, the activity and importance of the detected acyltransferase were studied. As discussed in this paper, and in contrast to many other bacteria, streptomycetes seem to possess a complex metabolic network to synthesize lipids, whereof crucial steps are still largely unknown. This paper therefore provides insights into a range of topics, including extremophile bacteria, the physiology of lipid accumulation, and the biotechnological production of bacterial lipids.

Project description:Diacylglycerol acyltransferase (DGAT) is the rate-limiting enzyme in triacylglycerol biosynthesis in eukaryotic organisms. Triacylglycerols are important energy-storage oils in plants such as peanuts, soybeans and rape. In this study, Arachis hypogaea type 2 DGAT (AhDGAT2) genes were cloned from the peanut cultivar 'Luhua 14' using a homologous gene sequence method and rapid amplification of cDNA ends. To understand the role of AhDGAT2 in triacylglycerol biosynthesis, two AhDGAT2 nucleotide sequences that differed by three amino acids were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli Rosetta (DE3). Following IPTG induction, the isozymes (AhDGAT2a and AhDGAT2b) were expressed as 64.5 kDa GST fusion proteins. Both AhDGAT2a and AhDGAT2b occurred in the host cell cytoplasm and inclusion bodies, with larger amounts in the inclusion bodies. Overexpression of AhDGATs depressed the host cell growth rates relative to non-transformed cells, but cells harboring empty-vector, AhDGAT2a-GST, or AhDGAT2b-GST exhibited no obvious growth rate differences. Interestingly, induction of AhDGAT2a-GST and AhDGAT2b-GST proteins increased the sizes of the host cells by 2.4-2.5 times that of the controls (post-IPTG induction). The total fatty acid (FA) levels of the AhDGAT2a-GST and AhDGAT2a-GST transformants, as well as levels of C12:0, C14:0, C16:0, C16:1, C18:1n9c and C18:3n3 FAs, increased markedly, whereas C15:0 and C21:0 levels were lower than in non-transformed cells or those containing empty-vectors. In addition, the levels of some FAs differed between the two transformant strains, indicating that the two isozymes might have different functions in peanuts. This is the first time that a full-length recombinant peanut DGAT2 has been produced in a bacterial expression system and the first analysis of its effects on the content and composition of fatty acids in E. coli. Our results indicate that AhDGAT2 is a strong candidate gene for efficient FA production in E. coli.

Dataset Information

The Role of Diacylglycerol Acyltransferase (DGAT) 1 and 2 in Cardiac Metabolism and Function.

Publications

The Role of Diacylglycerol Acyltransferase (DGAT) 1 and 2 in Cardiac Metabolism and Function.

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