International Journal of Obesity (2002) 26, 1277–1280
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SHORT COMMUNICATION
The chlorophyll-derived metabolite phytanic acid
induces white adipocyte differentiation
A Schlüter1, P Yubero1, R Iglesias1, M Giralt1 and F Villarroya1*
1
Department de Bioquı́mica i Biologia Molecular, Universitat de Barcelona, Barcelona, Spain
Phytanic acid is a derivative of the phytol side-chain of chlorophyll. It appears in humans following the ingestion of fatcontaining foods and is present in human blood at a low micromolar concentration. It may activate retinoid X receptors (RXR) or
peroxisome proliferator-activated receptor (PPAR) a in vitro. Phytanic acid induced the adipocyte differentiation of 3T3-L1 cells
in culture as assessed by accumulation of lipid droplets and induction of the aP2 mRNA marker. This effect was mimicked by a
synthetic activator of RXR but not by a PPARa agonist or by palmitic acid. In human pre-adipocytes in primary culture, phytanic
acid also induced adipocyte differentiation. These findings indicate that phytanic acid may act as a natural rexinoid in adipose
cells and suggest a potential use in the treatment of human type 2 diabetes and obesity.
International Journal of Obesity (2002) 26, 1277 – 1280. doi:10.1038=sj.ijo.0802068
Keywords: phytanic; phytol; adipocyte; retinoid-X receptor
Introduction
Phytanic (3,7,11,15-tetramethylhexadecanoic) acid is a derivative of the phytol side chain of chlorophyll. In humans it
appears either following ingestion of fat-containing foods of
animal origin or oxidation of the phytol from animal or
vegetable foods. It is metabolized in peroxisomes which
requires a specific enzyme, phytanoyl-CoA hydroxylase.
Lack of this enzyme causes Refsum disease.1 In the search
for natural products that could activate nuclear hormone
receptors, phytanic acid was found to be capable of binding
and activating retinoid X receptor (RXR)2 and peroxisome
proliferator-activated receptor (PPAR) a.3 Although the affinity of phytanic acid for RXR and PPARa was lower than 9cis-retinoic acid or fibrates, respectively, phytanic acid levels
in human blood are in the micromolar range,4 which is
compatible with a physiological role for this molecule.
Drugs that activate RXR or PPARa have a marked impact
on whole body metabolism and act as insulin-sensitizers or
hypolipidemic agents, respectively.5,6 Thus, phytanic acid
appears as a natural product with potential effects on meta-
*Correspondence: F Villarroya, Department de Bioquimica i Biologia
Molecular, Universitat de Barcelona, Facultat de Biologia, Avda Diagonal
645, 08028-Barcelona, Spain.
E-mail: [email protected]
Received 29 January 2002; revised 4 April 2002;
accepted 19 April 2002
bolic disorders such as diabetes or obesity. However, to date
few studies have assessed the influence of phytanic acid on
gene expression or physiology. We have reported that phytanic acid promotes the expression of the uncoupling protein-1 gene and activates brown adipocyte differentiation.7
However, phytanic acid not only activated gene markers
specific to brown adipocyte such as uncoupling protein-1
but also gene markers common to brown and white adipocytes. Considering the differential roles of brown and white
adipose tissue (energy expenditure by thermogenesis and
energy storage, respectively) and the minor metabolic relevance of brown fat in adult humans, we attempted to
establish whether white adipocyte is a target of phytanic
acid and to examine the effects of this fatty acid on white
adipocyte differentiation.
Methods
Phytanic acid, dexamethasone (DEX), 3-isobutyl-l-methylxantine (IBMX), insulin, palmitic acid, Wy 14,643, D-biotine
and Na-panthotenate were from Sigma. AGN19420 and
BRL49653 were gifts from Dr Chandraratna (Allergan,
Irvine, USA) and Dr L Casteilla, (University of Toulouse,
France), respectively. 3T3-L1 preadipocytes were maintained
in Dulbecco’s modified Eagle’s medium (DMEM) with 10%
fetal calf serum (FCS). At confluence cells were induced to
differentiate by a 2 day treatment with DMEM supplemented
Chlorophyll-derived metabolite phytanic acid
A Schlüter et al
1278
with 10% FCS, 10 mg=ml insulin, 0.5 mM IBMX and 1 mM
DEX (differentiating medium). Thereafter, cells were maintained in DMEM supplemented with 10% FCS only. When
indicated, exposure to the differentiation medium was suppressed and cells were maintained after confluence in the
medium containing DMEM plus 10% FCS. Cells were studied
1 week or 2 weeks after treatment with differentiation
medium or non-differentiation medium to which phytanic
acid had been added or not. Phytanic acid was dissolved in
dimethylsulfoxide and controls included the equivalent
amount of solvent, always less than 1 : 1000 the volume of
culture medium. The appearance of cytoplasmatic lipid
droplets was monitored by bright-field microscopy after
staining with Oil-Red O8 and the percentage of cells showing
lipid droplets was quantified. Total RNA was extracted using
an affinity column-based method (Qiagen, Germany). Samples (20 mg) of RNA were analyzed by Northern blot and
probed with the aP2 cDNA labeled by [a-32P]dCTP (Amersham). Ribosomal RNA stained with ethidium bromide was
used to confirm equal loading of samples. Human preadipocytes were obtained from Zen-Bio (Research Triangle Park,
USA). They were cultured in DMEM=Ham’s F12 medium
(1 : 1, vol=vol) supplemented with 10% FCS and, when confluent, differentiation was induced by 3 day culture with
DMEM=Ham’s F12 medium supplemented with 3% FCS,
10 mg=ml insulin, 33 mM D-biotine, 17 mM Na-panthotenate,
1 mM DEX, 0.5 mM IBMX and 1 mM BRL49653. Cells were
incubated thereafter in medium without IBMX and
BRL49653 for 2 weeks. When indicated BRL49653 was withdrawn from the differentiation – induction medium and
replaced or not by 80 mM phytanic acid. The extent of
adipocyte differentiation was estimated as for 3T3-L1 cell
cultures.
Results and discussion
When 3T3-L1 cells were treated with the differentiating
medium more than 85% of cells accumulated lipids and
acquired the adipose cell morphology 1 week after induction
(Figure 1A). When confluent 3T3-L1 cells were cultured
without the treatment with the differentiation-inducing
medium, cells did not differentiate. The addition of phytanic
acid to this non-differentiating medium resulted in around
50% (40 mM) or 65% (80 mM) of cells becoming adipocytes.
When cells were cultured for a further week, around 70% of
cells treated with 40 mM phytanic became adipocytes and
more than 85% of cells treated with 80 mM phytanic acid
acquired adipocyte morphology. The effect observed was not
common to any fatty acid: exposure of 3T3-L1 cells to 80 mM
palmitic acid did not promote differentiation. The effects of
phytanic acid were also assessed using aP2 mRNA expression
as a gene marker of adipocyte differentiation (Figure 1B). aP2
mRNA levels were high in differentiated 3T3-L1 adipocytes
whereas, in cells that were not allowed to differentiate into
adipocytes, aP2 mRNA expression was undetectable. Exposure to 80 mM phytanic acid induced aP2 mRNA expression
International Journal of Obesity
Figure 1 Effects of phytanic acid on 3T3-L1 adipocyte differentiation.
(A) Microphotographs of 3T3-L1 cells after induction of differentiation or
alternative treatment with non-differentiating medium alone or supplemented with 80 mM phytanic acid. The scale bar corresponds to 250 mm.
(B) Northern blot analysis of 20 mg of RNA from 3T3-L1 cells cultured for
1 or 2 weeks after induction of differentiation or alternative treatments
(see Methods) and probed for aP2 mRNA expression (top) and ethidium
bromide staining of the gel showing equal loading of RNA samples
(bottom). Palmitic acid was used at 80 mM.
in parallel with the acquisition of the adipocyte morphology.
A positive effect was also induced by 1 mM AGN19420, a
specific RXR activator (Figure 1B). Wy 14,643, a PPARa
activator, had no effect (not shown). Moreover, as for morphology, no major effects were induced by treatment with
palmitic acid, which led to a slight increase in aP2 mRNA
expression.
The identification of a novel natural agent capable of
promoting white adipose differentiation could be relevant
for the treatment of human disorders. Therefore, the effects
of phytanic acid were studied using primary cultures of
human white adipose precursor cells. Human white adipose
Chlorophyll-derived metabolite phytanic acid
A Schlüter et al
1279
pre-adipocytes were cultured in the optimal conditions for
differentiation (see Methods) which required supplementation of the culture medium with BRL49653, a PPARg activator. In this cell model, the absence of BRL49653 suppressed
the capacity of pre-adipocytes to differentiate into adipocytes (Figure 2). When phytanic acid was added to the
medium instead of BRL49653, cells again acquired adipocyte
morphology, although to a lesser extent (65%) respect to
differentiation obtained with the optimal conditions.
In conclusion, phytanic acid has a positive action on
white adipocyte differentiation both in an established
murine cell line, 3T3-L1, and in primary cultures of human
white preadipocytes. The doses used here to show the effects
of phytanic acid are in an upper range of the physiological
levels in humans.4 However, the amount of phytanic acid
added to cultures that enters the pre-adipose cell may be
much lower due to the binding of the fatty acid to proteins
in the culture medium and to the potential requirement of
saturable fatty acid transport systems. Moreover, the effects
observed may involve the action of compounds derived from
phytanic acid metabolism inside the cell. Finally, the extent
of the exposure of white adipose tissue to phytanic acid in
vivo may depend not only on circulating levels but also on
local production. Most of the body’s phytanic acid is stored
in white adipose tissue as triacylglycerol esters of phytanic
acid. As a consequence of lipolysis, free phytanic acid could
appear and a transient high local exposure to phytanic acid
in white adipose tissue could be expected.
Phytanic acid is likely to activate white adipocyte differentiation by acting as a natural rexinoid. The known positive
effects of rexinoids on adipogenesis are caused by the activation of the RXR moiety of the RXR=PPARg heterodimer, a
master transcription factor for adipocyte differentiation.9
Phytanic acid does not appear to play its adipogenic role
by its capacity to activate PPARa.2 White adipocytes or preadipocytes express very low levels of PPARa and, in any case,
even high-affinity synthetic PPARa activators are poorly
adipogenic.10 Synthetic rexinoids have insulin sensitizing
properties in vivo similarly to thiazolidinediones.5 However,
rexinoids also show anti-obesity effects that are not shared
by PPARg agonists.11 In agreement with recent hypotheses,12
the effects of phytanic acid promoting white adipocyte
differentiation by a mechanism similar to synthetic rexinoids should lead to further research to explore the potential
use of phytanic acid or precursor molecules as natural products for treatment of human type 2 diabetes and obesity.
Acknowledgements
Thanks are given to B Spiegelman (Dana-Farber Cancer
Institute, Boston, MA, USA) for the aP2 probe. This work
was supported by grant PM98-0188 from Ministerio de
Educación y Cultura (Spain) and SGR99-38 from Generalitat
de Catalunya.
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Figure 2 Effects of phytanic acid on human pre-adipocyte differentiation in primary culture. Microphotographs of cells two weeks after
induction of differentiation with differentiating medium, with medium
devoid of BRL49653 (non-differentiating medium) and with medium
devoid of BRL49653 supplemented with 80 mM phytanic acid (nondifferentiating medium þ phytanic acid). The scale bar corresponds to
100 mm.
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