(Received for publication, August 22, 1995; and in revised form, February 22, 1996)
From the
Adipose differentiation is accompanied by changes in cellular
morphology, a dramatic accumulation of intracellular lipid and
activation of a specific program of gene expression. Using an mRNA
differential display technique, we have isolated a novel adipose cDNA,
termed adipoQ. The adipoQ cDNA encodes a polypeptide of 247 amino acids
with a secretory signal sequence at the amino terminus, a collagenous
region (Gly-X-Y repeats), and a globular domain. The globular domain of
adipoQ shares significant homology with subunits of complement factor
C1q, collagen 1(X), and the brain-specific factor cerebellin. The
expression of adipoQ is highly specific to adipose tissue in both mouse
and rat. Expression of adipoQ is observed exclusively in mature fat
cells as the stromal-vascular fraction of fat tissue does not contain
adipoQ mRNA. In cultured 3T3-F442A and 3T3-L1 preadipocytes,
hormone-induced differentiation dramatically increases the level of
expression for adipoQ. Furthermore, the expression of adipoQ mRNA is
significantly reduced in the adipose tissues from obese mice and
humans. Whereas the biological function of this polypeptide is
presently unknown, the tissue-specific expression of a putative
secreted protein suggests that this factor may function as a novel
signaling molecule for adipose tissue.
Adipose tissue is highly specialized to play important roles in energy storage, fatty acid metabolism, and glucose homeostasis(1, 2) . Adipocytes synthesize and store triglyceride in periods of nutritional abundance and mobilize the lipids in response to fasting(2, 3) . Fat tissue is also involved in regulating blood glucose levels through the expression of the insulin responsive glucose transporter, Glu4(4, 5) . Fat and muscle, in fact, constitute the two major sites for insulin-regulated glucose uptake.
At a molecular
level, many genes involved in lipid metabolism and glucose homeostasis
are prominently expressed in fat(1) . These include fatty acid
synthase(6) , the fatty acid binding protein
aP2(7, 8) , lipoprotein lipase(9) ,
phosphoenolpyruvate carboxykinase(10) , malic
enzyme(11) , glyceraldehyde-3-phosphate
dehydrogenase(12) , and Glut4 (4) . Receptors for
lipogenic or lipolytic hormones such as
insulin(13, 14) , insulin-like growth factor
1(15) , and adrenergic compounds (16, 17) are
also expressed in adipocytes. In addition to these genes that clearly
participate in the metabolic functions of adipose tissue, a group of
genes that function in extracellular signaling have also been
identified in fat. A prototype of these molecules is insulin-like
growth factor 1, which is expressed in many tissues during development
and plays an important role in cell proliferation(18) . In
adipocytes, however, insulin-like growth factor 1 is found to stimulate
cell differentiation(19) . More interestingly, insulin-like
growth factor 1 is synthesized by preadipocytes in response to growth
hormone stimulation(20) , thus potentially functioning in an
autocrine or paracrine fashion to promote adipogenesis during
development. Another signaling molecule from adipose tissue is
TNF-. (
)TNF-
is secreted from fat, especially in
obesity, and acts in an autocrine or paracrine manner to interfere with
insulin action in fat and muscle(21, 22) . The recent
cloning and characterization of the ob gene product has further
illustrated that adipose tissue secretes signaling molecules that
function in an endocrine fashion(23) . The ob gene
product (leptin) is secreted from fat into the circulation and acts to
regulate body weight, perhaps via a putative receptor in the
cerebroventricular region of the
brain(15, 23, 24) . Hence, molecules secreted
from adipose tissue are capable of modulating diverse functions in fat
and other tissues, thus representing a new facet of adipose tissue
physiology.
In this study, we have used mRNA differential display to
clone a novel adipose gene termed adipoQ. Sequence analysis suggests
that adipoQ is a secreted protein that shares significant homology to
subunits of complement factor C1q and contains a collagenous structure
at the NH terminus and a globular domain at the COOH
terminus. The expression of this novel gene is highly regulated during
the adipose differentiation process and is expressed predominantly in
adipose tissue in vivo. Moreover, a significant
down-regulation in adipoQ mRNA was observed in fat tissues from obese
mice and humans. Our results provide a potentially valuable new
molecular tool to explore the physiology of adipose tissue in normal
and pathological states.
Figure 1:
Identification of a novel
adipocyte-specific mRNA. A, mRNA differential display
reactions were performed as described under ``Experimental
Procedures.'' S-Labeled PCR products were visualized
by autoradiography. Lanes 1, 2, and 3 represent samples from 3T3-C2 (C), 3T3-F442A
preadipocytes (P), and differentiated 3T3-F442A adipocytes (A). DD1 indicates a candidate PCR product that is
differentially regulated. B, Northern analysis using DD1 as a
probe. 10 µg of RNA from 3T3-C2 fibroblasts (lane 1, C2), 3T3-F442A preadipocytes (lane 2, P) and
adipocytes (lane 3, A), and 3T3-L1 preadipocytes (lane 4, P) and adipocytes (lane 5, A) was analyzed. Ethidium bromide (EtBr) staining and
hybridization to a
-actin probe were used to normalize RNA
loading.
Figure 2:
Nucleotide and deduced amino acid
sequences of adipoQ: homology to components of C1q, collagen 1(X),
and cerebellin. A, nucleotides are numbered from the 5` end of
the sequence. The amino acid sequence is derived from the longest open
reading frame. The bold region indicates the collagen-like
domain. Putative polyadenylation signals are underlined. The
accession number for adipoQ in GenBank is U49915. B, homology
of adipoQ with murine C1q A, B, and C chains. The bold region indicates identical amino acids in all four sequences. Two
interruptions in collagenous region of C1q-B and C1q-C chains are
indicated by an asterisk (see text). Symbols # and mark the
conserved cysteines (see text) that are altered in adipoQ. C,
comparisons of the globular region between C1q B chain, collagen
1(X), and cerebellin. Identical or conserved residues in all four
sequences are indicated by bold letters. The two regions that
are most homologous are underlined. Pair-wise comparisons
between adipoQ and C1q-B chain, collagen
1(X), and cerebellin give
31, 38, and 25% identity, respectively. D, in vitro transcription and translation of adipoQ cDNA. TNT in vitro translation system (Promega) was used for translation (see
``Experimental Procedures''). Varying amounts of purified
cDNA plasmid (pBluescript) were added directly to the
transcription-translation mixture with no RNA polymerase (lane
1), T3 RNA polymerase (0.5 µg DNA) (lane 2), T3 RNA
polymerase (2 µg of DNA) (lane 3), T7 RNA polymerase (0.5
µg DNA) (lane 4) and T7 RNA polymerase (2 µg of DNA) (lane 5). [
S]Methionine-labeled
products were separated on 10% SDS gel and visualized after
fluorography. Molecular mass is indicated by kDa at the left side of the gel. E, Western blot of proteins
from conditional medium and cell lysates of transiently transfected
NIH-3T3 cells (see ``Experimental Procedures''). Lane 1 and 2 were 1 µl of in vitro translated
Flag-adipoQ and empty vector. Lanes 3 and 4 were 50
µl (10 µg of protein) of culture medium from Flag-adipoQ
transfected (lane 3) and empty vector (lane 4). Lanes 5 and 6 were 10 µl (50 µg of protein)
of cell lysates from Flag-adipoQ transfected (lane 5) and
empty vector (lane 6). The proteins were separated on 12%
SDS-polyacrylamide gel electrophoresis and immunoblotted with M2-Flag
antibody from Kodak, Inc.
Analysis of the
putative protein sequence identified a hydrophobic leader from amino
acid residues 2 to 17, presumably representing a signal peptide. A
region of collagenous repeats (Gly-X-Y) was present from amino acids 45
to 110, with 22 individual Gly-X-Y repeats. Comparisons with genes in
GenBank identified several regions of homology to the subunits (A, B,
and C chains) of complement factor C1q (36, 37) , a
tissue-specific collagen 1(X)(38) , and a brain-specific
protein cerebellin(39) . The identity with the C1q chains is
approximately 31% in the globular COOH-terminal region (Fig. 2B), with the homology localized primarily in two
segments of uncharged, hydrophobic regions (Fig. 2C).
In addition, adipoQ and C1q A, B, and C chains have a similar size of
240-250 amino acids (Fig. 2B). The number of
Gly-X-Y repeats is similar as well, with 22 such repeats for adipoQ and
26-29 for the C1q chains. The similarity of this protein to
collagen
1(X) and cerebellin is found mainly at the COOH-terminal
globular domain (Fig. 2C) with 38 and 25% identity over
a 130-amino acid region. Collagen
1(X), however, encodes a much
larger protein (680 amino acids) with a long collagenous segment (154
Gly-X-Y repeats). Cerebellin, on the other hand, is a smaller
polypeptide with 193 amino acid residues and does not contain a
collagenous domain(39) . Because of the similarity between this
novel protein and all three components of C1q molecules in size, domain
structure and overall homology, we termed this novel protein adipoQ.
AdipoQ is clearly a distinct member of a proteins family
characterized by a collagenous helical structure at the NH terminus, and a globular domain at the COOH
terminus(40) . In addition to C1q A, B, and C chains (36, 37) and collagen
1(X)(38) , this
protein family includes lung surfactant proteins SP-A and
SP-D(41) , mannan binding protein(42) , and the
scavenger receptor and its homolog(43, 44) . These
proteins often homo- or hetero-oligomerize via the collagenous
structures. The presence of a collagenous domain in adipoQ suggests
that this protein is likely to form oligomeric structures by itself or
with other proteins.
Although the COOH-terminal region of adipoQ shares significant similarity with the C1q chains, it also has some notable differences. For example, a cysteine (marked in Fig. 2B) in the globular region of C1q B (residue 194) and C (residue 198) chains is known to form disulfide bonds with activator molecules, e.g. IgG(45) . In adipoQ sequence, this cysteine is not conserved and is replaced by an aspartate residue. Another conserved cysteine (residue 180 for C1q C chain, marked # in Fig. 2B) that is important for the formation of disulfide bonds and the stabilization of triplex strands in the collagenous domain (37, 46) is also altered in adipoQ. In addition, an interruption (marked * in Fig. 2B) in a collagenous motif found in C1q A (residue 61) and C (residue 65) chains is absent in adipoQ. These interruptions have been shown to be conserved between human and mice and result in a bend in the collagen triplex formation that can be observed under the electron microscope (47, 48) . These differences suggest that adipoQ may have structural and functional properties distinct from those of C1q.
In an in vitro transcription and translation system, the adipoQ cDNA generates a protein of approximately 30 kDa in size (Fig. 2E). This is in agreement with the molecular mass predicted from the cDNA sequence.
To test whether adipoQ is secreted, we constructed a flag-epitope tagged adipoQ and transiently transfected the DNA construct into NIH-3T3 cells. Western blot analysis (Fig. 2E) demonstrated that NIH-3T3 cells synthesized the adipoQ protein, and the protein is secreted into the medium.
Figure 3:
Expression of adipoQ mRNA during adipocyte
differentiation. Total RNA at different times during adipose
differentiation was analyzed for both 3T3-F442A (A) and 3T3-L1 (B) cells. 10 µg of total RNA was loaded for each sample.
Northern blots were sequentially hybridized to labeled cDNA probes
corresponding to adipoQ, aP2, lipoprotein lipase, and PPAR-. RNA
loading was normalized by hybridizing to a probe for
ribosomal-associated protein
(36B4(68) ).
Figure 4: Expression of adipoQ mRNA in various tissues from mice, rats, and humans. A, 10 µg of total RNA from different mouse tissues was analyzed by Northern blot. Tissues are designated as follows: B, brain; Fa, fat; H, heart; I, intestine; K, kidney; L, liver; M, muscle; P, pancreas; S, spleen. The blot was sequentially hybridized to the adipoQ and the aP2 cDNA probes. B, expression of adipoQ mRNA in rat tissues. 10 µg of total RNA from different rat tissues was analyzed by Northern blot. Tissue designation was identical to A, with two additional samples: mature fat cell from rat fat pads (floaters, FL), and the stromal-vascular fraction of rat fat pads (SV). C, comparison of adipoQ mRNA expression in mice, rats, and human fat. 10 µg of total fat tissue RNAs were analyzed by Northern blot. M, mouse; R, rat; H, human fat. Ethidium bromide (EtBr) stainings were used to normalize RNA loading.
Figure 5: Expression of adipoQ mRNA in lean and obese fat samples from mice and humans. A, 10 µg of total RNA from lean (ob/+) (lane 2) and obese (ob/ob) mice fat pads (lane 1) was analyzed by Northern blot. The same blot was hybridized with probes for adipoQ, adipsin, and aP2. B, 10 µg of total fat RNA from three lean human individuals (lanes 5-7) and four obese individuals (lanes 1-4) was analyzed for adipoQ expression. Expression of the TNF receptor type 1 (TNFR1) mRNA was not altered in these conditions and was used as a control for RNA loading(22) .
Adipose tissue was traditionally thought to be a relatively
passive depot for lipid storage and mobilization and was viewed to be
solely at the receiving end of hormonal and neuronal signals.
Consistent with this, receptors for hormones such as insulin,
adrenocorticotropic hormone, and epinephrine are abundantly expressed
in adipose cells in vivo and in vitro(1) .
However, recent investigations suggest that fat tissue is much more
actively involved in the energy balance systems by secreting molecules
that signal to and perhaps regulate the functions of other tissues and
organs(3, 15, 23) . One clear example of this
is the production of TNF- by adipose tissue. This cytokine is
produced by fat cells mainly in the context of animal and human
obesity(21) . It interferes with insulin action in both muscle
and fat and plays a major role in systemic insulin resistance, at least
in part through a reduction in the tyrosine kinase activity of the
insulin receptor(50) . Another example is the recently cloned
obese (ob) gene product. The ob protein is
synthesized mainly by adipocytes and is secreted into the circulation.
Injection of this protein indicates that it influences (directly or
indirectly) food intake and thermogenesis (15, 24, 51) . Another secreted molecule from
adipose tissue with signaling potential is adipsin. Originally
identified as an adipocyte-specific serine protease(52) ,
adipsin has been shown to encode a critical component of the
alternative complement pathway (factor D)(53) . Moreover, the
proximal part of this complement pathway is shown to be activated in
adipose tissue(54) , generating small bioactive molecules such
as the anaphylatoxin C3a that could affect systemic functions. Most
recently, C3a has been shown to regulate triglyceride synthesis in
fibroblasts and adipocytes(55) . These data suggest that many
important physiological functions may be controlled through secreted
proteins from adipose tissue.
The adipoQ molecule identified in this
study has several features that suggest that it could function as a
signaling molecule from adipocytes. First, adipoQ contains a
hydrophobic signal peptide sequence and is homologous to several
secreted proteins such as C1q A, B, and C chains, collagen 1(X),
and cerebellin. Consistent with this, adipoQ is secreted from
fibroblasts after transfection of a expression vector. Second, the
expression of adipoQ is highly regulated during differentiation and is
restricted to adipose tissue in vivo. Finally, the expression
of adipoQ is affected by obesity in rodents and humans, suggesting a
dysregulation in this pathological state. These properties closely
parallel those of other important signaling molecules secreted from
adipose tissue including the ob gene product and TNF-
.
Given these unique sequence features and expression patterns, it is
tempting to speculate on the possible functions of adipoQ. The sequence
homology with C1q provide a possible clue. C1q is the first component
of the classical complement activation pathway(46) . It is
composed of three homologous subunits: the A, B, and C chains. Each
chain has a NH-terminal collagenous segment (Gly-X-Y
repeats) of 78-84 amino acids and a globular carboxyl region of
approximately 130 amino acids. A functional C1q molecule contains six
subunits of each chain heteroligomerized along the collagenous helix.
C1q interacts with the aggregated IgGs and initiates the complement
cascade by proteolytically activating factors C2 and C4(56) .
However, recent evidence suggests that C1q can also regulate other
functions such as cell-mediated cytotoxicity(57) ,
phagocytosis(58) , chemotaxis(59) , and interleukin-1
production (60) via a receptor-mediated mechanism. A putative
receptor for C1q has been isolated and characterized in several human
and murine cells including macrophages, lymphocytes, and
fibroblasts(61) . The collagenous region of C1q has been shown
to be important for ligand-receptor
interaction(62, 63) . AdipoQ and C1q share significant
similarities in the structure of this domain. Thus, it is possible that
adipoQ could bind to the same or a similar receptor, thereby eliciting
a biological response.
It is also possible that adipoQ may participate in the complement activation processes. Surrogate complement activation has been previously shown to be achieved by mannan-binding protein, a carbohydrate binding protein with a domain structure similar to that of C1q(64, 65, 66) . However, because adipoQ lacks several key cysteines (see ``Results'') in the regions that are important for C1q function, it is not clear whether adipoQ functions in complement system. Further experiments will be needed to address this issue.
The identification of adipo-Q, a novel adipose tissue-specific protein, poses many questions regarding its molecular and biochemical properties that are yet to be examined. More importantly, its biological role in adipose tissue and in the overall energy balance systems remain to be defined. The production and purification of this novel protein should open a new avenue to studying adipose tissue physiology in normal and pathological states.
While this manuscript was under review, Scherer et al.(67) identified a adipocyte-specific protein named Acrp30 using a random cDNA sequencing approach. AdipoQ is identical to Acrp30 in protein sequence.