(HSL) is also a retinyl ester hydrolase: evidence from mice lacking ...

More documents

Recommendations

Info

genic process (11). Low doses of RA, however, have been shown to promote preadipocyte differentiation (12). RA is believed to exert its effects on adipogenesis mainly through its role as a ligand for the retinoid X receptor (RXR), which heterodimerizes with peroxisome proliferator-activated receptor � (PPAR�), the crucial transcription factor for adipogenesis and survival of mature adipocytes (13, 14). RALD, which up to now was believed to play a role only in the eye, was recently shown to be a potent inhibitor of adipogenesis, operating through both RXR-dependent and RXRindependent mechanisms (15). HSL is best known as a lipase hydrolyzing acylglycerides. Its role in catecholamine-stimulated hydrolysis of stored triacylglycerols and, in particular, diacylglycerols has been demonstrated in studies of HSL-null mice (16, 17). Despite its prominent role in lipolysis, HSL-null mice are not obese and exhibit a remarkable resistance to development of obesity following challenge with a long-term high fat diet (HFD) (18, 19). The resistance to diet-induced obesity is accompanied by impaired adipogenesis (18, 19) and attainment of brown adipocyte features of WAT (18), suggesting that HSL plays additional, yet unexplored, roles in adipose tissue. HSL exhibits broad substrate specificity and besides acylglycerides it hydrolyzes cholesteryl esters, REs, lipoidal esters, and water-soluble esters. The biological significance of these other activities, however, is poorly understood. Results from studies using partially purified preparations of HSL as well as transfected CHO cells, have indicated that the REH activity of HSL is approximately one tenth of the activity against triacylglycerol (7). Furthermore, following stimulation of cultured adipocytes with dibuturyl cAMP, ROH was released to the medium in parallel with decreased RE stores of the cell, suggesting that a cAMP-regulated enzyme, such as HSL, is responsible for this mobilization. The biological significance of HSL as an REH in adipose tissue has, however, never been directly addressed. We therefore utilized purified preparations of recombinant HSL, as well as HSL-null mice, to investigate the REH activity of HSL and the consequences of HSL deficiency for ROH metabolism in adipose tissue. MATERIALS AND METHODS Animal experiments The study was reviewed and approved by the Ethical Committee in Malmö/Lund, Lund, Sweden (license no. M162-05) and is in accordance with the Council of Europe Convention (ETS 123). HSL-null mice were generated by targeted disruption of the HSL gene in 129SV-derived embryonic stem cells as described elsewhere (16). Animals used were from the same embryonic stem cell colony. Animals in the different groups were littermates and had a mixed genetic background from the inbred strains C57BL/6J and SV129 (16). The animals were maintained in a temperature-controlled room (22 � C) on a 12-h light-dark cycle. Mice, 16–20 wk of age, were fed a chow diet ad libitum (control) (11% energy from fat) or a high-fat diet (HFD) (58% energy from fat) (Research Diets; products D12310 and D12309, respectively; Research Diets, New Brunswick, NJ, USA). After sacrificing the mice, tissues were rapidly dissected, snap frozen, and stored in liquid nitrogen before in vitro analyses. For the diet intervention studies, regular HFD (see above), containing 0.8 g/kg diet of retinyl palmitate, and retinyl ester-free HFD, supplemented with 10 mg all-trans retinoic acid (atRA)/kg diet (product D05081101), were obtained from Research Diets. The studies were initiated by feeding mating mice the two respective diets. Thus, diets were provided from pregnancy to weaning of the pups and maintained until the age of 5 mo, after which the mice were killed and tissues were dissected. Mice fed the atRA-supplemented diet were divided into three groups; one group was fed the higher concentration throughout the entire study, and two groups were initially fed the higher concentration, but after 2.5 mo were switched to a diet containing 1 and 0.5 mg atRA/kg diet, respectively, by mixing the original atRA-containing diet (10 mg/kg diet) with retinyl ester-free HFD. Body weight and food intake were measured throughout the study. Blood samples were drawn by retro-orbital puncture from female animals in the fed state. Recombinant HSL and enzyme activity measurements Recombinant rat and human HSL, purified to apparent homogeneity (�95% protein purity) (20–22), were assayed against trioloein (TO), 1-mono-oleoyl-2-O-mono-oleylglycerol (MOME, a diolein analog), cholesteryl oleate (CO), and retinyl palmitate (RP) substrates, as described previously (20, 23, 24). Briefly, unlabeled and labeled substrates were emulsified with phospholipids in 100 mM KH 2PO 4 (pH 7.0) and 5% defatted BSA (fatty acid acceptor) using sonication, to yield final substrate concentrations of 5 mM (TO and MOME), 0.45 mM (CO), and 0.5 mM (RP). The purified enzyme preparations were in 5 mM NaH 2PO 4 (pH 7.4), 1 mM dithioerythritol, 50% glycerol, and 0.2% C 13E 12 (a detergent from the polyoxyethylene series) and were diluted with 20 mM KH 2PO 4 (pH 7.0), 1 mM EDTA, 1 mM dithioerythritol, and 0.02% defatted BSA to noninhibitory detergent concentrations before assay (23). Unit (U) is defined as micromoles of fatty acids released per minute at 37 � C. Determination of RALD and RA in WAT with LC-MS/MS To extract RALD and RA from WAT, 3 vol of 2-propanol containing a 13 C-labeled stable isotope of atRA as internal standard was added to WAT pieces (200 mg), followed by homogenization with a motorized homogenizer (Pro Scientific, Oxford, CT, USA), shaking for 15 min, and centrifugation (10 min, 10 � C, 2700 g). The whole procedure was performed under red light. An aliquot of 100 �l was analyzed on a 4000 Q TRAP LC-MS/MS, triple quadruple mass spectrometer with APCI ionization (Applied Biosystems, Foster City, CA, USA), as described previously (25) except that the separating column was a Supelco ABZ Plus, 70 mm � 3mm ID, 3-�m particles (Supelco/Sigma-Aldrich, St. Louis, MO, USA). The additional MRM transitions monitored for retinal were 285.2-161 (quantifier) and 385.2-133 (qualifier). Determination of ROH in plasma and WAT, and RE in WAT using LC-UV One hundred microliters of plasma or �200 mg of WAT was diluted with 450 �l 2-propanol containing retinyl propionate as an internal standard and butylated hydroxytoluene as an antioxidant. After thorough mixing (15 min) and centrifugation (10 min, 4000 g at 10 � C), an aliquot of 20 �l was injected 2308 Vol. 23 July 2009 The FASEB Journal STRÖM ET AL.
from the supernatant into the HPLC system. HPLC was performed with an HP 1100 liquid chromatograph (Agilent Technologies, Santa Clara, CA, USA) with an HP1100 diode array detector. Retinoids were separated at 40 � C on a chromolith speedrod RP-18e 4.6- � 50-mm reverse-phase column. A linear gradient was generated from Mobile Phase A (1:9 v/v H 2O:MeOH) and Mobile Phase B (65:35 v/v 2-propanol- MeOH). A 3-point calibration curve was made from analysis of an MeOH solution enriched with known ROH and RP concentrations. For the plasma samples, recovery was �97%; the method was linear at least from 0.1 to 10 �M, and the detection limit was 0.22 pmol. RSD was 4.8%. For the WAT samples, recovery was 92–95%; the method was linear at least from 50 to 1000 �g/g, and the detection limit for ROH was 195 ng on column. RSD was 3.2%. The detection limit for RE was 98 ng on column, and RSD was 5.1%. Tissue homogenization Tissues were homogenized in 0.25 M sucrose, 1 mM EDTA (pH 7.0), 1 mM dithiothreitol, 20 �g/ml leupeptin, 10 �g/ml antipain, and 1 �g/ml pepstatin A using a glass/Teflon homogenizer (10–20 strokes), followed by a centrifugation at 10 000 g for 25 min at 4 � C. The infranatant fraction was used for activity measurements against RP (24), and the pellet fraction was used for Western blot analysis. The pellet fraction was solubilized in homogenization buffer containing 5% SDS for 30 min at 50 � C prior to determination of protein concentration. Protein was determined using the BCA assay (Pierce, Rockford, IL, USA). DNA was isolated from WAT using a DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany), and the concentration was determined using spectrophotometry (NanoDrop; NanoDrop Technologies, Wilmington, DE, USA). Western blot analysis Periovarial WAT was homogenized, and 50 �g of total protein was resolved by SDS-PAGE and electroblotted onto nitrocellulose membranes (HyBond-c extra, Amersham Pharmacia Biotech, Sunnyvale, CA, USA). Primary antibodies used were retinoic acid receptor alpha (RAR�; 1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and �-Actin (1:5000; Sigma). Actin was used as loading control. Secondary antibodies were horseradish peroxidase-conjugated donkey anti-rabbit IgG (RAR�), and sheep anti-mouse IgG (�- Actin; Amersham Biosciences, Piscataway, NJ, USA). Western blot analysis was performed using a chemiluminescence system (Luminol), and detection was made using a CCD camera (LAS 1000, Fujifilm Corporation, Tokyo, Japan). Relative protein levels were calculated after normalization to the loading control. RNA preparation and real-time quantitative PCR Total RNA was isolated using RNeasy Lipid Tissue Mini Kit (Qiagen), according to the manufacturer’s recommendations. Total RNA (1 �g) was treated with DNase I (DNase I amplification grade; Invitrogen, Carlsbad, CA, USA) and then reversely transcribed using random hexamers (Amersham) and SuperScriptII RNaseH reverse transcriptase (Invitrogen), according to the manufacturer’s recommendations. The cDNA was used in quantitative PCR reactions using Taqman chemistry [RAR�, RAR�, RXR�, RXR�, alcohol dehydrogenase 1 and 3 (Adh1, Adh3), retinaldehyde dehydrogenase 1 and 2 (Raldh1, Raldh2), RBP4, S14, TIF2, receptor interacting protein 140 (RIP140), and retinoblastoma protein (pRb)] or SYBR green chemistry [PPAR�, PPAR�, sterol regulatory IMPAIRED RETINOL METABOLISM IN HSL-NULL MICE element binding protein 1c (SREBP1c; also known as ADD1), fatty acid binding protein 4 (Fabp4; also known as aP2) and uncoupling protein 1 (UCP1)] with an ABI 7900 system (Applied Biosystems). Primers were designed using the software Primer Express 1.5 (Applied Biosystems); the sequences or assay IDs are presented in Supplemental Data. Relative abundance of mRNA was calculated using the ��Ct method with normalization to the ribosomal protein 29S (whole tissue) or the TATA box binding protein (TBP; isolated adipocytes). Preparation of isolated adipocytes and BAT Adipocytes were isolated after incubation in Krebs-Ringers solution (pH 7.4) supplemented with 3.5% BSA, 2 mM glucose, 200 nM adenosine and collagenase (1 mg/ml; Sigma) in a shaking incubator at 37 � C for �60 min according to a modification (26) of the Rodbell method (27). The digested tissue was filtered, and the isolated cells were washed twice in Krebs-Ringer buffer (pH 7.4) with 1% BSA, 2 mM glucose, and 200 nM adenosine by allowing the isolated adipocytes to float to the surface and then aspirate the underlying buffer. Excised BAT pads from the interscapular depot were incubated in Krebs-Ringer phosphate buffer (pH 7.4) with 4% crude BSA and 0.83 mg/ml collagenase (Sigma) for 5 min in a shaking water bath at 37 � C and then filtered to eliminate the remaining white adipocytes. Statistics Data are expressed as means � se unless otherwise stated. Statistical analyses were made using either a nonparametric Mann-Whitney U test or by 1-way analysis of variance (ANOVA) followed by a Bonferroni post hoc test (GraphPad Prism 4 software; GraphPad, San Diego, CA, USA). Data were considered significant if P � 0.05. RESULTS HSL displays prominent REH activity It has previously been demonstrated that HSL exhibits REH activity (7). However, its activity against this substrate has never been compared to the activity against other known substrates using homogenous preparations of HSL. Thus, preparations of recombinant rat and human HSL that had been purified to homogeneity (�95%) were assayed against TO, MOME (a diolein analog), CO, and RP, and the activity against these different lipid substrates was compared under V max conditions and noninhibitory detergent concentrations. We have previously observed that the inhibitory action of detergents in HSL assays based on lipid substrates varies according to the nature of the lipid substrate (23). In the comparisons made in the present study, the inhibitory action of the detergent used was found to be most pronounced for the RP assay. As shown in Fig. 1A, the activity of rat HSL against RP was the highest activity, followed by the activities against MOME, CO, and TO. Compared to rat, human HSL showed a lower activity for all tested substrates (Fig. 1B). Similar to rat HSL, human HSL exhibited high 2309
Page 1: The FASEB Journal Research Communic
Page 5 and 6: phenotype of HSL-null mice challeng
Page 7 and 8: tion with RA caused a decrease in B
Page 9 and 10: emerging view of a multifunctional

(HSL) is also a retinyl ester hydrolase: evidence from mice lacking ...

Create successful ePaper yourself

Delete template?

Save as template?