Studies on the substrate and stereo/regioselectivity of adipose triglyceride lipase, hormone-sensitive lipase, and diacylglycerol-O-acyltransferases - PubMed (original) (raw)

Studies on the substrate and stereo/regioselectivity of adipose triglyceride lipase, hormone-sensitive lipase, and diacylglycerol-O-acyltransferases

Thomas O Eichmann et al. J Biol Chem. 2012.

Abstract

Adipose triglyceride lipase (ATGL) is rate-limiting for the initial step of triacylglycerol (TAG) hydrolysis, generating diacylglycerol (DAG) and fatty acids. DAG exists in three stereochemical isoforms. Here we show that ATGL exhibits a strong preference for the hydrolysis of long-chain fatty acid esters at the sn-2 position of the glycerol backbone. The selectivity of ATGL broadens to the sn-1 position upon stimulation of the enzyme by its co-activator CGI-58. sn-1,3 DAG is the preferred substrate for the consecutive hydrolysis by hormone-sensitive lipase. Interestingly, diacylglycerol-O-acyltransferase 2, present at the endoplasmic reticulum and on lipid droplets, preferentially esterifies sn-1,3 DAG. This suggests that ATGL and diacylglycerol-O-acyltransferase 2 act coordinately in the hydrolysis/re-esterification cycle of TAGs on lipid droplets. Because ATGL preferentially generates sn-1,3 and sn-2,3, it suggests that TAG-derived DAG cannot directly enter phospholipid synthesis or activate protein kinase C without prior isomerization.

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Figures

FIGURE 1.

Schematic depiction of enzymatic TAG hydrolysis and formation of potential DAG products. Hydrolysis of the prochiral TAG may lead to formation of the regioisomer _sn_-1,3 DAG upon cleavage of the _sn_-2 position or to the enantiomers _sn_-1,2 or _sn_-2,3 DAG by hydrolysis of the ester bond at the _sn_-3 or _sn_-1 position, respectively. The numbers denoted on the carbon atoms of the glycerol backbone indicate the stereospecific numbering (sn). The asterisk indicates the chiral center of the DAG at the _sn_-2 position.

FIGURE 2.

ATGL hydrolyzes TAGs with FAs of different chain length and saturation in vitro. A, recombinant proteins were expressed in Cos-7 cells, and expression of His-tagged proteins was assessed by immunoblotting. B and C, homogenates of Cos-7 cells overexpressing ATGL were incubated in the absence or presence of CGI-58 with different, homogeneously (B) or heterogeneously esterified TAG species (C) as substrates for 1 h at 37 °C. Released FAs were measured using a NEFA-C kit. Data are normalized to LacZ and presented as the means ± S.D. and are representative of two independent experiments (***, p < 0.001; **, p < 0.01). O = oleic acid, C18:1; P = palmitic acid, C16:0.

FIGURE 3.

ATGL exhibits a preference for unsaturated FAs in vivo. A, WAT lipids of fed wt and ATGLko mice were extracted according to Folch et al. (21) and separated by TLC. TAGs were isolated and transesterified. FAMEs were separated and analyzed using GC/ flame ionization detector. B, plasma lipids of overnight-fasted wt and ATGLko mice were extracted and analyzed as in A. C, numeric changes in FA species of WAT TAGs and plasma FAs from wt and ATGLko mice are shown. D, plasma lipids of fed and overnight-fasted wt mice were extracted and analyzed as in A. E, numeric changes in plasma FA species of fed and fasted wt mice are shown. Data are presented as the means ± S.D. and are representative of two independent experiments (***, p < 0.001; **, p < 0.01; *, p < 0.05). n = 4 (each genotype).

FIGURE 4.

ATGL specifically cleaves the _sn_-2 ester bond and expands its selectivity to the _sn_-1 position upon co-activation by CGI-58. A and B, cytosolic fractions of Cos-7 cells overexpressing ATGL were incubated in the absence (A) or presence (B) of GST-tagged CGI-58 with 3H-labeled triolein substrate in the presence of an HSL-specific inhibitor (76-0079) at 37 °C. Reactions were stopped at different time points, DAG regioisomers were separated by TLC, and radioactivity in the corresponding bands was measured by liquid scintillation counting. n.s., not significant. C, to assess transesterification of DAG isomers, _sn_-1,3 and _rac_-1,2/2,3 DAG were incubated with lysates of Cos-7 cells in the presence of an HSL-specific inhibitor (76-0079) for 120 min at 37 °C. DAG species were analyzed by TLC before and after incubation. D--F, chiral-phase HPLC resolution of different DAG species is shown. DAGs were analyzed as their corresponding 3,5-dinitrophenylurethanes by chiral-phase HPLC. Analysis of a racemic diolein reference mix is shown in D. Analysis of the reaction products of “egg yolk lecithin” hydrolyzed by purified PLC (B. cereus) is shown. The three peaks within the retention time range 11.5–13 min display the different FA composition of the separated DAGs; I, 16:0–18:1 + 18:1–18:1; II, 16:0–18:2 + 18:1–18:2; III, 18:2–18:2 (E). Analysis of the reaction products of triolein substrate hydrolyzed by CGI-58 co-activated ATGL contained in the cytosolic fraction of Cos-7 cells and in the presence HSL-specific inhibitor (76-0079, F). Data are normalized to LacZ and are presented as the means ± S.D. and are representative of two independent experiments (***, p < 0.001). X = unknown compound. mAU, milliabsorbance units.

FIGURE 5.

Distinct accumulation of _sn_-1,3 DAG in WAT of HSL-deficient mice. A, lipids were extracted according to Folch et al. (21), and total acylglycerol levels in WAT of wt and HSLko mice were determined using an Infinity-TAG kit (Thermo Scientific Fisher). C, neutral lipids of wt and HSL-deficient WAT were separated by TLC using chloroform/acetone/acetic acid (90/8/1; v/v/v). B and D, bands corresponding to TAG and DAG were scraped off and extracted, the acylglycerol content was determined using an Infinity-TAG kit (B), and DAG isomers were analyzed by chiral-phase HPLC (D). Data are presented as the means ± S.D. (***, p < 0.001), n = 4 (each genotype). FChol, free cholesterol.

FIGURE 6.

HSL hydrolyzes all isoforms of DAG. A, immunoblot analysis of lysates from Cos-7 cells expressing HSL or, as a control, LacZ using anti-His antibody is shown. B, lysate of Cos-7 cells expressing HSL were incubated with triolein or stereochemically different diolein substrates emulsified with phospholipids at 37 °C for 1 h. Generated FAs were measured using a NEFA-C kit (Wako Chemicals). Data are normalized to LacZ and presented as the means ± S.D. and are representative of two independent experiments (***, p < 0.001).

FIGURE 7.

DGAT1 and DGAT2 exhibit different preferences for DAG regioisomers. A, expression of FLAG-tagged murine DGAT1 and DGAT2 in Cos-7 cells was assessed by immunoblotting. B, lysates of Cos-7 cells expressing FLAG-tagged DGAT1 or DGAT2 were incubated with either _sn_-1,2, _rac_-1,2/2,3, or _sn_-1,3 diolein substrate emulsified with phospholipids and 14C-labeled oleoyl-CoA at 37 °C for 10 min. Lipids were extracted and separated by TLC, and radioactivity in the TAG bands was determined by scintillation counting. C, regioselectivity of recombinant DGAT enzymes against _sn_-1,2 or _sn_-1,3 diolein is expressed as a percentage of total acyltransferase activity. D, endogenous expression of DGAT1 and DGAT2 in WAT was determined by immunoblotting using anti-DGAT1 and anti-DGAT2 antibody. E, microsomal fractions of WAT from fed C57Bl/6-mice were assayed for DGAT activity in the presence and absence of a specific DGAT1 inhibitor (D1 inh., 2-((1s,4s)-4-(4-(4-amino-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]oxazin-6-yl)phenyl)cyclohexyl)acetic acid) using _sn_-1,2 or _sn_-1,3 diolein as substrate as described in A. F, regioselectivity of endogenous DGATs in WAT expressed as a percentage of total acyltransferase activity is shown. Data are normalized to LacZ and presented as means ± S.D. and are representative of two independent experiments (***, p < 0.001).

FIGURE 8.

Stereo/regioselectivity of lipolytic enzymes. TAG is hydrolyzed to _sn_-1,3 DAG by ATGL or to _sn_-1,3 and _sn_-2,3 DAGs by ATGL co-activated by CGI-58. HSL preferentially hydrolyzes FAs at the _sn_-3 position of DAGs, yielding a mixture of MAGs (_sn_-2 and _sn_-1). MGL exhibits no selectivity in degradation of MAGs. G, glycerol.

FIGURE 9.

Three-pool model of intracellular DAG distribution. Pool I consists of _sn_-1,2 DAGs generated during de novo lipogenesis at the ER. DGAT1 and DGAT2 as well as CDP-choline:1,2-diacylglycerol choline phosphotransferase can metabolize newly generated _sn_-1,2 DAG. Pool II consists of _sn_-1,3 ± _sn_-2,3 DAGs, which are generated on cytosolic LDs by ATGL ± CGI-58-dependent TAG hydrolysis. Both DAG species display targets for further hydrolysis catalyzed by HSL or re-esterification catalyzed by DGAT2. _sn_-1,2 DAGs of pool III are generated by PLC at the plasma membrane and function as activators of various PKCs. Furthermore, these DAGs are substrates for DAG kinases and DAG lipases. CPT, choline:1,2-diacylglycerol cholinephosphotransferase; DAGL, diacylglycerol lipase; DAGK, diacylglycerol kinase; PA, phosphatidic acid; PL, phospholipid.

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Studies on the substrate and stereo/regioselectivity of adipose triglyceride lipase, hormone-sensitive lipase, and diacylglycerol-O-acyltransferases - PubMed (original) (raw)