A new gene regulated by energy balance in
human adipose tissue
Adiponutrin, a transmembrane protein corresponding
to a novel dietary- and obesity-linked mRNA specifically expressed
in the adipose lineage
We have used a mRNA differential display technique
to identify new genes involved in the reprogramming of gene expression
during the adipose conversion of mouse 3T3 preadipocyte cell lines.
We report here on the identification and cloning of a novel adipose-specific
cDNA encoding a predicted membrane protein of 413 amino acids.
The level of the corresponding 3.2-kilobase mRNA is tremendously
increased during 3T3-L1 and 3T3-F442A differentiation into adipocytes.
A single, very abundant 3.2-kilobase transcript is also found
in inguinal and epididymal white adipose tissues and in interscapular
brown adipose tissue but not in any other tissues examined. Its
expression in adipose tissue is under tight nutritional regulation.
The level of this novel 3.2-kilobase transcript becomes virtually
nondetectable during fasting but is dramatically increased when
fasted mice are refed a high carbohydrate diet. Based on its adipose
specificity and dietary regulation, the novel gene product has
been designated adiponutrin. The expression of adiponutrin mRNA
is also 50-fold elevated in genetically obese fa/fa rats, indicating
a link between adiponutrin and obesity. Western blot and confocal
imagery analyses with epitope-tagged protein transiently expressed
in 3T3-L1 adipocytes, and COS cells show that adiponutrin strictly
localizes to membranes and is absent from the cytosol. Sequence
analysis reveals homologies with several other members of related
eukaryotic proteins, including a human paralog, which has been
recently described in vesicular transport mechanisms. This leads
us to suggest that adiponutrin could be involved in vesicular
targeting and protein transport restricted to the adipocyte function.
Baulande S, et al. J Biol Chem. 2001 Sep
7;276(36):33336-44. Epub 2001 Jun 28.
Adiponutrin expression in mouse adipose tissue is dependent
upon changes in nutritional status. 8-week-old Swiss
mice were divided into three experimental groups and were either
allowed free access to food (Fed) or subjected to 19.5 h
of fast (Fast) or 12 h of fast followed by 7.5 h
of refeeding (Fast/Refed). The animals were
sacrificed at the end of 19.5 h, and 10 µg of
total RNA extracted from epididymal fat pads was used for Northern
blot analysis for adiponutrin, ADD1, and FAS mRNAs. Mean values
of insulinemia and glycemia corresponding to each nutritional
status are indicated at the top. alpha-Enolase mRNA,
probed with the 3'-untranslated region of the murine mRNA (accession
number ACX52379) was used as invariant control. 18 S ribosomal
RNA from ethydium bromide-stained gels is shown. Baulande
S. , et al. J. Biol. Chem., Vol. 276, Issue 36, 33336-33344, September
Desnutrin mRNA levels in various adult mouse tissues and cells.
A, 10 µg of total RNA from various mouse tissues was analyzed
by Northern blotting and hybridized with radiolabeled desnutrin
and adiponutrin cDNA probes. Sk, skeletal; Ing, inguinal; Gon,
gonadal; Ren, renal. B, shown is desnutrin mRNA expression in
gonadal WAT from 12-h fasted wild-type (WT), db/db, and ob/ob
mice. C, 5 µg of total RNA from cells of the stromal vascular
fraction (SVF) or adipocytes isolated from mouse inguinal adipose
tissue (left panels) or 10 µg of total RNA from 3T3-L1 cells
at the indicated days of differentiation (right panels) was examined
by Northern blot analysis for the expression of desnutrin and
various adipocyte markers. Prol. Pre., proliferating preadipocytes;
Conf. pre., confluent preadipocytes; FAS, fatty-acid synthase;
aFABP, adipocyte fatty acid-binding protein. Villena
JA, et al. J Biol Chem. 2004 Nov 5;279(45):47066-75. Epub 2004
Desnutrin mRNA levels during adipocyte differentiation of 3T3-L1
cells. Two-day post-confluent 3T3-L1 preadipocytes (day 0) were
induced to differentiate by treatment with 1 µM dexamethasone
and 0.5 mM MIX for 2 days and then maintained in differentiation
medium for an additional 5 days. Ten µg of total RNA prepared
from cells collected at the indicated time points was examined
for the expression of desnutrin and other adipocyte markers by
Northern blot analysis. aFABP, adipocyte fatty acid-binding protein;
FAS, fatty-acid synthase; PPAR, peroxisome proliferator-activated
receptor-gamma. Villena JA, et al.
J Biol Chem. 2004 Nov 5;279(45):47066-75. Epub 2004 Aug 27.
Nutritional regulation of desnutrin mRNA levels.
A, expression of desnutrin mRNA in WAT from mice fasted for 24 h
(F) or fasted and then refed for 12 h (R) as assessed by Northern
blot analysis; B, time course analysis of desnutrin and adiponutrin
mRNA expression levels in WAT during fasting. Mice were fasted for
12, 24, or 48 h, and total RNA was extracted for examination of
desnutrin mRNA levels by Northern blot analysis. Desnutrin mRNA
levels in WAT were compared with those in mice that were fasted
for 48 h and subsequently fed for 12 h. FAS, fatty-acid synthase.
Subcellular localization of
desnutrin-EGFP fusion protein. A, COS-7 cells were transfected with
the desnutrin-EGFP expression vector, and localization of the fusion
protein was assessed by confocal microscopy. B, COS-7 cells were
transfected with an HA-tagged desnutrin expression vector, and nuclear
(Nuc.), mitochondrial (Mit.), microsomal (Mic.), and cytosolic (Cyt.)
fractions were prepared as described under "Experimental Procedures."
Five µg of protein from each fraction was subjected to SDS-PAGE,
transferred to a polyvinylidene difluoride membrane, and analyzed
for the presence of HA-desnutrin using anti-HA antibody. As a positive
control for the immunodetection, 10 µg of whole cell lysate
Wiesner G., et al. Food restriction regulates
adipose-specific cytokines in pituitary gland but not in hypothalamus.
Journal of Endocrinology (2004) 180, R1–R6