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

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.
2310 Vol. 23 July 2009 The FASEB Journal
STRÖM ET AL.