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

The FASEB Journal Research Communication 

Hormone-sensitive lipase (HSL) is also a retinyl ester 

hydrolase: evidence from mice lacking HSL 

Kristoffer Ström,* ,1 Thomas E. Gundersen, † Ola Hansson,* Stéphanie Lucas,* 

Céline Fernandez,* Rune Blomhoff, † and Cecilia Holm* 

*Department of Experimental Medical Science, Lund University, Lund, Sweden; and † Department of 

Nutrition, Faculty of Medicine, University of Oslo, Oslo, Norway 

ABSTRACT Here, we investigated the importance of 

hormone-sensitive lipase (HSL) as a retinyl ester hydrolase 

(REH). REH activity was measured in vitro using 

recombinant HSL and retinyl palmitate. The expression 

of retinoic acid (RA)-regulated genes and retinoid 

metabolites were measured in high-fat diet fed HSLnull 

mice using real-time quantitative PCR and triplestage 

liquid chromatography/tandem mass spectrometry, 

respectively. Age- and gender-matched wild-type 

littermates were used as controls. The REH activity of 

rat HSL was found to be higher than that against the 

hitherto best known HSL substrate, i.e., diacylglycerols. 

REH activity in white adipose tissue (WAT) of HSL-null 

mice was completely blunted and accompanied by 

increased levels of retinyl esters and decreased levels of 

retinol, retinaldehyde and all-trans RA. Accordingly, 

genes known to be positively regulated by RA were 

down-regulated in HSL-null mice, including pRb and 

RIP140, key factors promoting differentiation into the 

white over the brown adipocyte lineage. Dietary RA 

supplementation partly restored WAT mass and the 

expression of RA-regulated genes in WAT of HSL-null 

mice. These findings demonstrate the importance of 

HSL as an REH of adipose tissue and suggest that HSL 

via this action provides RA and other retinoids for 

signaling events that are crucial for adipocyte differentiation 

and lineage commitment.—Ström, K., Gundersen, 

T. E., Hansson, O., Lucas, S., Fernandez, C., 

Blomhoff, R., Holm, C. Hormone-sensitive lipase 

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

mice lacking HSL. FASEB J. 23, 2307–2316 (2009) 

Key Words: adipocyte differentiation � brown adipocyte � cAMP 

� retinoic acid 

Retinoids originate from the diet, in which they 

are present either as vitamin A or as provitamin A, i.e., 

carotenoids. Dietary retinoids are carried as retinyl 

esters (REs) in chylomicrons and are taken up as 

chylomicron remnants by the hepatocytes, for subsequent 

storage in the stellate cells, which are the primary 

retinoid-storing cells (1). In addition, a significant 

fraction, i.e., 15–20%, of the retinoids is stored in 

adipose tissue (2). Uptake of RE into adipose tissue is 

facilitated by lipoprotein lipase through two distinct 

0892-6638/09/0023-2307 © FASEB 

mechanisms, i.e., internalization of RE carried in lipoproteins 

and extracellular catalysis of RE to form 

retinol (ROH), which is taken up by the adipocyte (3). 

Inside the adipocyte, ROH is esterified to RE or bound 

to cellular retinol-binding proteins, shown to be expressed 

at low levels in adipocytes (2). In contrast to the 

liver, in which the esterification of retinol is catalyzed 

by lecithin:retinol acyltransferase (4), the enzyme 

responsible for this process in the adipose tissue is 

not known. Although diacylglycerol acyltransferase 1 

(DGAT1), an enzyme highly expressed in adipose 

tissue, has been shown to possess the ability to 

esterify retinol to RE in vitro (5), a recent study has 

shown that DGAT1 does not catalyze RE synthesis in 

adipose tissue in vivo (6). In the adipocyte, �50–70% 

of total retinoids are in the form of RE (3, 4), 

presumably contained within the lipid droplet. ROH 

can be released to plasma from RE stores in adipose 

tissue, and the importance of these stores is underscored 

by the observation that they decline before 

the hepatic REs in times of dietary retinoid insufficiency 

(4). The enzyme responsible for the mobilization 

of ROH from RE in adipose tissue has not been 

identified. Of the two major triglyceride lipases in 

adipocytes, hormone-sensitive lipase (HSL) and adipose 

triglyceride lipase (ATGL), only HSL has been 

shown to possess RE hydrolase (REH) activity (7, 8). 

The importance of adipose tissue in the mobilization of 

ROH to plasma is further emphasized by the observation 

that adipocytes synthesize and secrete retinolbinding 

protein (RBP), later known as RBP4 (2, 9). 

RBP4 is involved in circulating retinol transport and 

has recently been shown to impair insulin sensitivity via 

mechanisms that are not yet fully understood (10). 

Adipose tissue, as well as most other tissues, oxidizes 

ROH to retinaldehyde (RALD) and retinoic acid (RA), 

through the action of alcohol dehydrogenases and 

retinaldehyde dehydrogenases, respectively. RA has 

been recognized as an inhibitor of adipogenesis, at 

least if applied at high doses early during the adipo- 

1 Correspondence: Division of Diabetes, Metabolism and 

Endocrinology, Department of Experimental Medical Science, 

Lund University, BMC C11, SE-221 84 Lund, Sweden. 

E-mail: kristoffer.strom@med.lu.se 

doi: 10.1096/fj.08-120923 

2307

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

Figure 1. HSL exhibits high REH activity. Homogenous 

preparations of recombinant rat (A) and human (B) HSL 

were assayed against TO, MOME (a diolein analog), CO, and 

RP, and the activity against these substrates was compared 

under V max conditions (n�3–5). Values are means � se. 

activity against RP, although diacylglycerols were the 

preferred substrate for the human enzyme. 

REH activity is reduced in WAT and BAT of HSL-null 

mice 

Recently, it was reported that REH activity is dramatically 

reduced in periovarial WAT of HSL-null mice 

compared to wild-type (WT) littermates fed either 

control or HFD (24). Here, these analyses were extended 

to include both visceral and subcutaneous 

WAT, as well as BAT. As shown in Fig. 2, REH activity 

was dramatically decreased in all these depots of HSLnull 

mice, suggesting that HSL is the major REH in 

adipose tissue. The largest decrease in activity was seen 

in visceral WAT from HSL-null mice, exhibiting only 

1% of remaining activity against RP compared to WT 

littermates. 

ROH metabolism in WAT is perturbed in HSL-null 

mice 

To investigate ROH metabolism in HSL-null mice, 

retinoid metabolites were measured. Regarding RA 

metabolites, the methodology employed allowed confident 

quantification of atRA and 13-cis RA in WAT, 

whereas the levels of 9-cis RA were too low to allow 

confident determination in all samples (Supplemental 

Fig. 1). In line with the reduced REH activity (ref. 24 

and Fig. 2), a marked increase in RE content and a 

decrease in ROH, RALD, atRA, and 13-cis RA levels 

were observed in perigonadal WAT from HSL-null mice 

fed an HFD (Fig. 3A–D). No differences in plasma RA 

or ROH concentration were observed between HSLnull 

mice and WT littermates in either feeding category 

(data not shown). To investigate the enzymes involved 

in the conversion of ROH to RA, we measured the 

expression of two members of the family of enzymes 

catalyzing the oxidation of ROH to RALD, i.e., Adh1 

and Adh3, as well as the expression of Raldh1 and 

Raldh2, which catalyze the terminal step of RA biosynthesis. 

The mRNA levels of Adh1 and Adh3 were 

down-regulated by 40 and 44%, respectively (Fig. 4A). 

Also, the mRNA level of Raldh1 was decreased by 62%, 

whereas Raldh2 was increased 2.6-fold in HSL-null mice 

compared to WT littermates (Fig. 4B). Next, we investigated 

the mRNA levels of genes known to be transcriptionally 

regulated by RA (28). In agreement with 

the lowered RA level in WAT from HSL-null mice, 

decreased mRNA levels of RIP140, RBP4, S14, and 

SREPB1c (Fig. 4C), as well as RAR� (Fig. 4D), were 

found in HSL-null mice compared to WT littermates. A 

reduction of RAR� protein by 60% in WAT of HSL-null 

mice was confirmed using Western blot analysis (Fig. 

4E). Also, the mRNA level of RXR� was decreased in 

WAT of HSL-null mice compared to WT littermates 

(Fig. 4D). 

Dietary administration of RA rescues the WAT and 

BAT phenotype of HSL-null mice 

HSL-null mice are resistant to diet-induced obesity, 

which is accompanied by impaired adipogenesis (18, 

19) and attainment of brown adipocyte features of 

WAT (18). Having established that retinoid metabolism 

is perturbed in WAT of HSL-null mice, we investigated 

to what extent this contributes to the adipose tissue 

Figure 2. REH activity is reduced in WAT and BAT of 

HSL-null mice. REH activity was determined in visceral (epididymal; 

eWAT) and subcutaneous (inguinal; iWAT) WAT as 

well as BAT (interscapular; iBAT) from 6-mo-old male mice 

fed a normal chow diet (n�4). Values are means � se; *P � 

0.05; 1-way ANOVA with by Bonferroni’s multiple comparison 

test. 


STRÖM ET AL.

phenotype of HSL-null mice challenged with an HFD. 

To this end, diet-intervention studies were performed. 

Animals were fed either the ordinary HFD or an HFD 

supplemented with atRA. A marked decrease in body 

weight was observed in animals fed the HFD supplemented 

with 10 mg/kg RA compared to control mice 

(Fig. 5A). Although this was seen in both genotypes, the 

largest decrease (28%, P�0.001) occurred in WT mice, 

whereas a considerably smaller decrease (11%, nonsignificant) 

occurred in HSL-null mice. After the 5-mo 

diet period, no difference in body weight was found 

between the two genotypes in mice fed an HFD with 

addition of RA. RA supplementation caused a dosedependent 

increase in WAT weight of HSL-null mice 

compared to HSL-null mice fed a regular HFD, most 

pronounced at the highest RA concentration used (Fig. 

Figure 3. ROH metabolism in WAT is perturbed in HSL-null mice. 

A, B) Measurements of RE (A) and ROH (B) in WAT from 10-mo-old male 

and female WT and HSL-null mice fed either control diet (n�8–11) or 

HFD (n�9–17) for 6 mo. C) Measurement of RALD in WAT from 9-mo-old 

female WT and HSL-null mice (n�5) fed HFD for 7 mo. D) Measurements 

of atRA and 13-cis RA in WAT from 3-mo-old male and female WT and 

HSL-null mice (n�12–16) fed HFD for 2 wk. Values are means � se. **P � 

0.01, ***P � 0.001; Mann-Whitney U test. 

5B). The opposite effect was observed in WT mice, 

showing a 50% decrease in WAT weight when the 

highest concentration of RA was administered, compared 

to control mice. At the highest concentration of 

RA, the difference in WAT weight between WT and 

HSL-null mice found on an HFD was reduced to a 

nonsignificant level. 

As shown by real-time quantitative PCR analysis of 

isolated adipocytes from periovarial WAT, PPAR� was 

found to be decreased in HSL-null mice compared to 

WT littermates (Fig. 6A), as has previously been demonstrated 

both in our HSL-null line and in another 

independent study (18, 19). The addition of RA to the 

diet decreased the expression of PPAR� in WT mice, 

whereas the levels were unchanged in the HSL-null 

mice. Decreased expression was also observed for the 

Figure 4. Altered expression of RA-regulated 

genes in WAT of HSL-null mice. 

A–C) mRNA levels, analyzed with realtime 

quantitative PCR, of Adh1 and Adh3 

(n�4–7) (A), Raldh1 and Raldh2 (n�5–7) 

(B), and RBP4, S14, RIP140 and SREBP1c 

(n�4–7) (C) in isolated adipocytes from periovarial WAT from 5-mo-old WT and HSL-null mice fed HFD for 5 mo. 

D) mRNA levels of RAR� and RXR� in periovarial WAT from 10- to 12-mo-old WT and HSL-null mice fed HFD for 6 mo 

(n�5). E) Protein level of RAR� analyzed with Western blot in periovarial WAT from 10- to 12-mo-old WT and HSL-null 

mice fed HFD for 6–7 mo (n�6). Bottom band is loading control (�-actin). Values are means � se. *P � 0.05, **P � 0.01; 

Mann-Whitney U test. 


2311

Figure 5. RA partly restores WAT mass of HSL-null mice. 

Diet-intervention studies showing body weight (A) and weight 

of periovarial WAT (B) from 5-mo-old WT and HSL-null 

female mice fed either a regular HFD (n�10–12) or an HFD 

supplemented with either 10 (n�7–9), 1.0 (n�3–4), or 0.5 

(n�4–6) mg atRA/kg diet (RA). Values are means � se. **P � 

0.01, ***P � 0.001; �� P � 0.01, HSL-null HFD vs. HSL-null RA 

(10 mg/kg); 000 P � 0.001, WT HFD vs. WT RA (10 mg/kg); 

1-way ANOVA with Bonferroni’s multiple comparison test. 

terminal differentiation marker aP2 in HSL-null mice 

compared to WT littermates (Fig. 6B). The addition of 

RA caused an increase in aP2 by 1.7-fold in HSL-null 

mice compared to HSL-null mice on a regular HFD, 

whereas no significant effect was observed in WT mice. 

pRb was decreased by 54% in HSL-null mice compared 

to WT littermates (Fig. 6C). In WT mice, RA caused a 

decrease in mRNA expression of pRb compared to WT 

mice fed a regular HFD, whereas in HSL-null mice, RA 

caused a 1.7-fold increase in pRb compared to HSL-null 

mice on a regular HFD. RAR� is suggested to be the 

retinoid receptor that is the most tightly regulated by 

retinol status, being decreased in vitamin A-deficient 

states and being rapidly increased when RA is administered 

(29). On the regular HFD, the mRNA level of 

RAR� was decreased in WAT from HSL-null mice (Fig. 

6D), as were the mRNA levels of both Raldh1 (Fig. 6E) 

and RXR� (Fig. 6F). The decrease in RAR�, Raldh1, 

and RXR� in HSL-null mice was reversed by RA treatment 

(Fig. 6D–F). An investigation of cofactors to 

nuclear receptors revealed that the mRNA levels of 

TIF2 and RIP140 were decreased by 30 and 60%, 

respectively, in WAT from HSL-null mice compared to 

WT littermates (Fig. 6G, H). Supplementation with RA 

caused a 1.5- and 1.9-fold increase in TIF2 and RIP140 

expression, respectively, in HSL-null mice compared to 

HSL-null mice on a regular HFD. Although mRNA 

levels of RIP140 were still significantly higher in WT 

mice fed a RA-supplemented HFD compared to HSLnull 

littermates, the positive effect of RA on stimulating 

mRNA expression of RIP140 was considerably higher in 

HSL-null mice (Fig. 6H). 

HSL-null mice fed regular HFD displayed an almost 

3-fold increase in the weight of BAT compared to WT 

littermates (Supplemental Fig. 2A). Diet supplementa- 

Figure 6. RA normalizes mRNA levels of 

RA-regulated genes in WAT of HSL-null 

mice. mRNA levels of PPAR� (A), aP2 

(B), pRb (C), RAR� (D), Raldh1 (E), 

RXR� (F), TIF2 (G), and RIP140 (H) 

analyzed with real-time quantitative 

PCR in isolated adipocytes from 

periovarial WAT of 5-mo-old WT and 

HSL-null mice fed either a regular 

HFD (n�6–7) or an HFD supplemented with 10 mg/kg atRA (RA) (n�5–6). Values are means � se. *P � 0.05, **P � 

0.01, ***P � 0.001; Mann-Whitney U test. 


STRÖM ET AL.

tion with RA caused a decrease in BAT weight in both 

genotypes. The decrease was more pronounced in 

HSL-null mice, showing an almost 40% lowering of 

BAT weight compared to HSL-null mice fed a regular 

HFD, whereas the decrease in WT mice did not attain 

statistical significance. A screening for genes known to 

be highly expressed in differentiated BAT was performed. 

The mRNA levels of UCP1, PPAR�, and glycerol 

kinase (GlyK) were decreased by 40, 50, and 42%, 

respectively, in BAT from HSL-null mice compared to 

WT littermates (Supplemental Fig. 2B–D). A significant 

increase in the mRNA level for UCP1, PPAR�, and GlyK 

by 1.9-, 1.8-, and 2.8-fold, respectively, was found in 

HSL-null mice fed a diet supplemented with RA, compared 

to HSL-null mice on a regular HFD. Besides a 

vital role in WAT differentiation, PPAR� has also been 

shown to control the differentiation of brown adipocytes 

(30). In BAT from HSL-null mice, a 50% decrease 

in the mRNA level of PPAR� was shown compared to 

WT littermates (Supplemental Fig. 2E). RA supplementation 

caused a 1.9-fold increase in the PPAR� mRNA 

level in HSL-null mice compared to HSL-null mice fed 

a regular HFD. 

DISCUSSION 

We demonstrate here that retinoid metabolism is perturbed 

in WAT of HSL-null mice and that supplementation 

of the HFD with RA partly restores WAT mass 

and most other aspects of the phenotype, thus establishing 

a role for HSL as an REH. Previous work has 

demonstrated that HSL exhibits REH activity, although 

the activity was described to be quite low, i.e., only 

about one-tenth of the activity against triacylglycerols 

(7). In contrast, we show here that the activity against 

RP is in the same order of magnitude as the activity 

against diacylglycerols and thus at least two orders of 

magnitude higher than previously described. The reason 

for this discrepancy is not clear. The use of homogenous 

enzyme preparations in this study, compared to 

partially purified HSL preparations and transfected 

CHO cells previously (7), may be a contributing factor. 

Furthermore, we found that the REH activity of HSL is 

very sensitive to detergent inhibition, more so than its 

activity against other lipid substrates. This property may 

account for previous underestimations of the REH activity 

of HSL. In line with its high in vitro activity against RP, 

HSL was found to be the dominating REH of both WAT 

and BAT, as judged from the very low residual activity in 

adipose tissues of HSL-null mice. 

The dramatic reduction in REH activity in adipose 

tissue of HSL-null mice is reflected in increased levels 

of RE and decreased levels of ROH, RALD, atRA, and 

13-cis RA. Although the lack of HSL is likely to be the 

major reason for this change in the pattern of retinoid 

metabolites, failure to generate these metabolites may 

secondarily lead to gene expression changes that further 

contribute to alterations in the levels of retinol, 

RALD, and RA. Adh3, catalyzing the oxidation of ROH 


to RALD, and Raldh1, catalyzing the oxidation of 

RALD to RA, are both positively regulated by RA (28, 

31) and were found to be down-regulated in HSL-null 

mice. Raldh2, on the other hand, has been shown to be 

negatively regulated by RA (32), and its expression was 

elevated in HSL-null mice. Raldh2 has been shown to 

be functionally more important and also more efficient 

and selective for RALD than Raldh1 (32–34). Recently, 

it was reported that RALD represses adipogenesis and 

diet-induced obesity (15). On the basis of this report, 

decreased levels of RALD in WAT of HSL-null mice 

would be expected to positively affect adipogenesis, 

leading to the expansion of WAT, which is opposite to 

what is observed here. It is possible that in the HSL-null 

mouse model, RALD and RA exert antagonizing effects 

on adipogenesis, but the impairment of adipogenesis 

imposed by the decreased RA levels, surpasses the 

stimulation of adipogenesis expected to occur as a 

result of the decreased RALD levels, leading to a net 

decrease in adipocyte differentiation. Thus, a picture is 

emerging indicating that the regulation of adipogenesis 

by retinoids is very complex and depends on absolute, 

as well as relative levels of several retinoid metabolites 

and their respective binding proteins. 

The observation that the plasma levels of ROH did 

not differ between HSL-null mice and WT littermates 

has several implications. First, WAT appears to play a 

minor role in maintenance of normal plasma levels of 

ROH, i.e., ROH generated within WAT is mainly for 

local use. Second, plasma ROH cannot compensate for 

failure to generate RA within WAT. This is in agreement 

with the observation that chylomicron RE is an 

important source of adipocyte retinoids (3) and furthermore 

suggests that ROH taken up by adipocytes is 

rapidly reesterified to RE. Thus, the adipocyte appears 

to be critically dependent on a functional cytosolic 

REH and our studies strongly suggest that HSL is 

serving this role. 

The severely reduced capacity to mobilize ROH, and 

thus RA, in WAT of HSL-null mice, together with the 

inability of plasma ROH to compensate for this defect, 

was the rationale for using RA in the dietary intervention 

studies. Following supplementation of the HFD 

with RA, the mass of WAT in HSL-null mice increased 

in a dose-dependent manner, with a maximal increase 

of 1.6-fold at the highest RA dose administered, 

whereas in WT mice, RA supplementation caused a 

dose-dependent reduction in the size of the periovarial 

WAT compared to WT mice fed regular HFD. These 

opposite effects of RA supplementation indicate that 

while added RA in the HSL-null mice compensates 

for the inability to generate RA within WAT, added 

RA in the WT mice may exert other effects. For 

instance, a dual role of RA in adipogenesis has been 

described. Whereas low doses of RA have been shown 

to promote preadipocyte differentiation (12, 35), other 

studies have recognized RA as a potent inhibitor of 

adipogenesis (11, 36). Thus, whereas dietary RA administration 

in HSL-null mice serves to restore RA levels 

back to normal or near-normal levels and thereby exert 

2313

adipogenic effects, the same treatment in WT mice 

exerts antiadipogenic effects. RA supplementation partially 

or fully restored the expression of RA-regulated 

genes in WAT of HSL-null mice. Among these were the 

genes encoding pRb and RIP140, which both are key 

factors in the determination of differentiation into the 

white vs. the brown lineage (37, 38). The down-regulation 

of the expression of these factors is believed to 

account for the described attainment of brown adipocyte 

features of WAT of HSL-null mice (18). Consequently, 

normalized expression of these factors following 

RA administration presumably accounts for the 

expansion of WAT mass in HSL-null mice as normally 

expected during an HFD feeding. Diet intervention 

with RA to a large extent reversed the phenotypic 

changes also of BAT of HSL-null mice, i.e., BAT mass 

was significantly reduced and the mRNA levels of 

UCP1, PPAR�, and GlyK, all markers of differentiated 

brown adipocytes, were restored to normal levels. 

Although it cannot be excluded, we find it unlikely 

that the effects observed in adipose tissue in response 

to dietary RA administration are secondary to effects 

exerted elsewhere in the body. The main reason for this 

is that we have found no evidence that retinoid metabolism 

is perturbed in nonadipose tissues of HSL-null 

mice. In particular, in the liver, the main retinoidstoring 

organ, HSL appears to account for a very minor 

fraction of the REH activity (unpublished results). In 

agreement with this, no differences in the hepatic levels 

of RE, ROH, and atRA were found between WT and 

HSL-null mice. Also, on analyses of the liver transcriptome, 

no concerted alterations in RA-regulated genes 

have been observed (39). 

The exact mechanisms whereby perturbed retinoid 

metabolism in HSL-null mice causes the observed disturbances 

in the differentiation program of adipocytes 

remain to be elucidated. We propose that HSL generates 

ROH from RE stores, which, following conversion 

to atRA, 9-cis-RA, and possibly other RA species, act as 

ligands for RAR and RXR (Fig. 7). This ensures a 

proper expression of RA-regulated genes, such as the 

genes encoding pRb and RIP140 (40, 41), with crucial 

roles in the determination of adipocyte cell fate. Provision 

of RA by HSL would also allow proper activation of 

the PPAR�:RXR heterodimer, the crucial transcription 

factor for adipogenesis and survival of mature adipocytes 

(13, 14). RA signaling events not relying on the 

retinoid receptors, such as RA activation of the ERK 

pathway, shown to be important for the differentiation 

of mesenchymal stem cells to preadipocytes (42), may 

also be involved, although it should be pointed out that 

HSL is induced late in the adipogenic program (43). 

The late induction of HSL in adipogenesis could suggest 

that the main role of HSL is to supply RA crucial 

for the survival of mature adipocytes. Besides RA, the 

REH action of HSL will give rise to the generation of 

RALD, which, as discussed above, recently was found to 

inhibit adipogenesis, presumably through both RXRdependent 

and RXR-independent mechanisms (15). 

Finally, it should be mentioned that ROH, without 

Figure 7. Working hypothesis illustrating how HSL, through 

its action as an REH in WAT, is involved in the regulation of 

adipogenesis/adipocyte survival and adipocyte cell fate. 

further conversion to RA, could act as a signaling 

molecule on its own (44), which may, in part, explain 

why a complete phenotypic rescue was not obtained on 

RA administration. Thus, HSL, together with the other 

enzymes in the pathway, i.e., Adh and Raldh, determines 

the levels and the ratios of the different retinoid 

metabolites and this, in turn, regulates adipogenesis 

and adipocyte function. 

Although the present study strongly suggests that the 

perturbations in retinoid metabolism account for the 

adipose tissue phenotype of HSL-null mice, other contributing 

factors cannot be excluded. In view of the 

broad substrate specificity of HSL, it is not unlikely that 

HSL also generates other lipidic signaling molecules 

and that failure to do so in the HSL-null mice contributes 

to the phenotype. For instance, diglycerides have 

been shown to accumulate in WAT of HSL-null mice 

(ref. 17, and unpublished observations). Another possibility 

is that the fatty acid profile in adipose tissue 

changes in the absence of HSL. Preliminary results 

from experiments employing the same GC-MS approach 

recently used to analyze the fatty acid profile of 

liver of HSL-null mice (39), however, give no indications 

of that any specific fatty acid is lacking, or 

accumulated, in HSL-null mice (unpublished results). 

In conclusion, our data show that HSL is the major 

REH of white and brown adipocytes. In its absence 

retinoid metabolism is perturbed, potentially leading to 

failure to provide RA for signaling events that are 

crucial for adipogenesis and determination of adipocyte 

fate. The data also underscore the importance of 

the internal RE stores for WAT function and, furthermore, 

that plasma ROH cannot compensate for failure 

to generate RA within WAT. The novel role of HSL 

described here should be added to the growing list of 

actions of HSL in and outside WAT. It is clear that the 

traditional view of HSL as a fatty acid-mobilizing enzyme 

in WAT has to be abandoned in favor of an 


STRÖM ET AL.

emerging view of a multifunctional enzyme, which has 

the capacity to hydrolyze a wide variety of lipid substrates. 

Thus, in addition to its key role in energy 

homeostasis, HSL appears to play an important role in 

the generation of lipids for transcriptional regulation 

and various lipid-signaling events. Detailed knowledge 

about these processes seem highly warranted in order 

to be able to explore HSL as a therapeutic target in the 

area of obesity and diabetes. 

We thank Birgitta Danielsson and Ann-Helen Thorén- 

Fischer for excellent technical assistance. Financial support 

was provided by the Swedish Research Council (project no. 

11284 to C.H); a Center of Excellence grant from the Juvenile 

Diabetes Foundation, USA; the Knut and Alice Wallenberg 

Foundation, Sweden; and the Swedish Diabetes Association; 

as well as the following foundations: Novo Nordisk, A. Påhlsson, 

Salubrin/Druvan, and Torsten and Ragnar Söderberg. 

O.H. was supported by the Cell Factory for Functional 

Genomics, a program funded by the Swedish Foundation for 

Strategic Research, S.L was supported by the Foundation of 

Tage Blücher for Medical Research, and C.F. was supported 

by the Swedish Research School of Genomics and Bioinformatics. 

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Received for publication September 12, 2008. 

Accepted for publication February 5, 2009. 


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