(Received for publication, July 25, 1995; and in revised form, September 12, 1995)
From the
We describe a novel 30-kDa secretory protein, Acrp30 (adipocyte complement-related protein of 30 kDa), that is made exclusively in adipocytes and whose mRNA is induced over 100-fold during adipocyte differentiation. Acrp30 is structurally similar to complement factor C1q and to a hibernation-specific protein isolated from the plasma of Siberian chipmunks; it forms large homo-oligomers that undergo a series of post-translational modifications. Like adipsin, secretion of Acrp30 is enhanced by insulin, and Acrp30 is an abundant serum protein. Acrp30 may be a factor that participates in the delicately balanced system of energy homeostasis involving food intake and carbohydrate and lipid catabolism. Our experiments also further corroborate the existence of an insulin-regulated secretory pathway in adipocytes.
Insulin-induced glucose transport occurs in heart, striated muscle, and fat tissue. In adipocytes, glucose uptake increases 20- to 30-fold in the presence of insulin. Glucose transport is mediated by the sodium-independent facilitative glucose transporters GLUT1 and GLUT4, which, in response to insulin, translocate from an intracellular compartment to the plasma membrane(1, 2) . GLUT4, which is expressed only in fat and skeletal and cardiac muscle, is the primary transporter involved in this process and is the predominant transporter expressed in these tissues(3, 4) . Insulin also causes translocation of several receptor proteins from intracellular membranes to the plasma membrane(5, 6, 7) . Adipocytes are a principal storage depot for triglycerides and express a specific transport protein allowing them to import free fatty acids(8) .
Adipocytes also secrete several proteins potentially important in
homeostatic control of glucose and lipid metabolism. Adipsin,
equivalent to Factor D of the alternative complement
pathway(9) , is synthesized exclusively in adipocytes, and its
secretion is enhanced severalfold by insulin(10) . The function
of adipsin in fat cell biology is not known, nor are the roles of
complement factors C3 and B that are also secreted by
adipocytes(11) . Tumor necrosis factor , also secreted by
adipocytes, is a key mediator of insulin resistance in animal models of
non-insulin-dependent diabetes mellitus. Tumor necrosis factor
directly interferes with the signaling of insulin through its receptor
and consequently blocks biological actions of insulin including
insulin-stimulated glucose uptake (reviewed in (12) ).
Adipocytes are the only cell type known to secrete the ob gene
product(13) . In the absence of ob (ob
/ob
mice) or
its presumed receptor, the db gene product (db
/db
mice) the
mice overeat and become obese and diabetic(14) . Here we
describe another novel protein, Acrp30, (
)that is
exclusively synthesized in adipose tissue and secreted into serum. Like
adipsin, secretion of Acrp30 is enhanced severalfold by insulin. While
we do not know the function of this protein, its sequence and
structural resemblance to complement factor C1q is intriguing.
Importantly, our experiments confirm the existence of an
insulin-regulated secretory pathway in adipocytes.
In order to identify novel adipocyte-specific proteins, we
have randomly sequenced portions of 1000 clones from a subtractive cDNA
library enriched in mRNAs induced during adipocyte differentiation of
3T3-L1 fibroblasts(15) . Northern blot analysis using one
250-bp clone showed a marked induction during adipocyte
differentiation, and thus a full-length cDNA was isolated and
sequenced. The encoded protein, Acrp30, was novel; it contained 247
amino acids with a predicted molecular mass of 28 kDa. Acrp30 consists
of a predicted amino-terminal signal sequence, followed by a stretch of
27 amino acids that does not show significant homology to any protein
in the data base and then by 22 perfect Gly-X-Pro or
Gly-X-X repeats (Fig. 1, A and B). The carboxyl-terminal globular domain exhibits striking
homology to a number of proteins, such as the globular domains of type
VIII and type X collagens(17) , the subunits of complement
factor C1q (18) and a protein found in the serum of hibernating
animals during the summer months(19) . Structurally, albeit not
at the primary sequence level, the protein resembles the lung
surfactant protein (20) and the hepatocyte mannan-binding
protein(21) , both of which have collagen-like domains and
globular domains of similar size.
Figure 1:
A, predicted structure of Acrp30. The
protein consists of an amino-terminal signal sequence (SS)
followed by a sequence of 27 amino acids lacking significant homologies
to any entries in the GenBank data base. This region is
followed by a stretch of 22 collagen repeats with 7
``perfect'' Gly-X-Pro repeats (dark hatched
boxes) clustered at the beginning and end of the domain
interspersed with 15 ``imperfect'' Gly-X-Y repeats (light hatched boxes). The C-terminal 137 amino
acids probably form a globular domain. B, alignment of the
amino acid sequences of Acrp30. Hib27, a member of the
hibernation-specific protein family; C1q-C, one of
the subunits of complement C1q; Coll type X, the globular
domain of the type X collagen. Conserved residues are shaded.
For simplicity, the other members of each family are not shown, but
shaded conserved residues are in most instances conserved within each
protein family. Only the globular domain for the type X collagen was
used for the alignment (residues 562-693). C, Northern
blot analysis of Acrp30 expression. The left panel shows
poly(A) RNA isolated from various tissues probed with the full-length
Acrp30 cDNA. The predominant Acrp30 mRNA is 1.4 kb and is expressed
only in adipose tissue and cultured 3T3-L1 adipocytes. Overexposure of
the autoradiogram does not reveal expression in any other tissue. The right panel shows induction of the Acrp30 message during
differentiation of 3T3-L1 fibroblasts to adipocytes. Induction of
Acrp30 occurs primarily between days 2 and 4 of differentiation, the
same time as induction of the insulin receptor and the
insulin-responsive glucose transporter GLUT4. Numbers on the left indicate molecular mass standards (in kb). Equal loading
of RNA was documented by probing the stripped filter with a cDNA
encoding the cytosolic hsp70 protein (lower
panel).
Northern blot analysis shows that Acrp30 is expressed exclusively in adipocytes. It is not expressed in 3T3-L1 fibroblasts and is induced over 100-fold during adipocyte differentiation. Induction occurs between days 2 and 4, at the same time as other adipocyte- specific proteins such as GLUT4 (22) and Rab3D (15) (Fig. 1C). These results were confirmed by Western blot analysis (data not shown). The amount of Acrp30 mRNA may decline somewhat from Day 6 to Day 8 (Fig. 1C), but this drop is not reproducible in other experiments. In any case, we have not studied the accumulation or stability of Acrp30 mRNA after Day 8.
An antibody raised against a
peptide corresponding to the unique amino-terminal domain of Acrp30
recognized a 3T3-L1 adipocyte protein of approximately 28 kDa (not
shown). Acrp30 contains one potential N-glycosylation site,
within the collagen domain, but apparently is not glycosylated; Endo H
treatment did not cause a shift in molecular mass of Acrp30 at any time
during a metabolic pulse-chase experiment (not shown). Acrp30 does
become modified post-translationally, since, after 20 min of chase,
there was a small but reproducible reduction in gel mobility. This most
likely represents hydroxylation of collagen domain proline residues in
the endoplasmic reticulum or Golgi compartments, by analogy to a
similar modification in the structurally related mannan-binding
protein(23) . In 3T3-L1 adipocytes unstimulated by insulin, 50%
of newly made Acrp30 is secreted into the medium at 2.5 to 3 h of chase
as judged by densitometric scanning of the immunoprecipitates of
intracellular and extracellular S-labeled Acrp30. Indeed,
Acrp30 can be detected by Western blotting in normal mouse serum. A
protein of the identical molecular weight can be detected by Western
blot analysis of 3T3-L1 adipocytes (not shown). The anti-peptide
antibody is specific for the mouse homologue, as it does not
cross-react with bovine, human, or rabbit serum (Fig. 2).
Figure 2:
Acrp30 is a secretory protein found in
blood. Acrp30 can be detected by Western blotting in serum from mice;
the antibody does not cross-react with calf, human, or rabbit serum.
One microliter of fetal calf, rabbit, mouse, and human serum was boiled
for 5 min in 2 sample buffer and analyzed by SDS-PAGE and
Western blotting with the anti-Acrp30 antibody according to standard
protocols. Antibody was visualized with an anti-rabbit IgG antibody
coupled to horseradish peroxidase using a chemiluminesence kit from
DuPont NEN.
To
examine effects of insulin on Acrp30 secretion, we monitored the
discrete population of newly made protein generated in a short pulse
with labeled amino acids followed by inhibition of further protein
synthesis by cycloheximide. This offers increased sensitivity compared
to examining secretion of the entire cellular complement of Acrp30,
particularly in light of the very long t for
secretion of Acrp30. Fig. 3shows that, during the first 60 min
of chase, insulin causes a 4-fold increase in secretion of newly made
Acrp30. After 60 min, the rates of Acrp30 secretion are the same in
unstimulated and insulin-stimulated cells. Similarly, insulin causes a
4-fold increase in adipsin secretion during the first 30 min of chase,
but, afterwards, the rate of adipsin secretion is the same in control
and insulin-treated cells ( Fig. 3and (10) ). The
ability of insulin to abolish the lag in adipsin secretion has been
seen in several separate experiments. We hypothesize that a fraction of
newly made adipsin and Acrp30 are sorted, probably in the trans-Golgi reticulum, into regulated secretory vesicles whose
exocytosis is induced (in an unknown manner) by insulin, whereas the
balance is sorted into vesicles that are constitutively exocytosed.
Partial sorting of protein hormones into regulated secretory vesicles
has been seen in other types of cultured
cells(24, 25) . We do not know how insulin causes an
increase in protein secretion; insulin could cause a more efficient
overall processing of secretory proteins in 3T3-L1 adipocytes. We are
currently isolating other adipocyte-specific secretory proteins to
study this process in detail.
Figure 3:
A, insulin stimulation of Acrp30 and
adipsin secretion by 3T3-L1 adipocytes. Two 10-cm dishes of day 8
3T3-L1 adipocytes were labeled for 10 min in medium containing
[S]methionine and cysteine as described under
``Experimental Procedures.'' The cells were then incubated in
growth medium containing cycloheximide (to prevent further protein
biosynthesis) and containing or lacking 100 nM insulin. Every
30 min, the culture medium was removed and replaced with fresh,
prewarmed medium containing or lacking 100 nM insulin. The
media were subjected to sequential immunoprecipitations with
anti-Acrp30 and anti-adipsin antibodies as described under
``Experimental Procedures'' and analyzed by electrophoresis
through a 12% polyacrylamide gel containing SDS. As Acrp30 and adipsin
contain a comparable number of cysteine and methionine residues (7 and
9, respectively) and equal exposures of the autoradiograms were used,
one can determine from the intensities of the bands that approximately
equal amounts of the two proteins are secreted. As judged by the amount
of
S-labeled proteins remaining in the cells after the 2-h
chase (not shown), all of the
S-labeled adipsin and about
40% of the
S-labeled Acrp30 has been secreted at this
time. B, quantitation of Acrp30 and adipsin secretion by
3T3-L1 adipocytes in the presence (closed circles) and absence (open circles) of insulin. The autoradiograms were scanned in
a Molecular Dynamics densitometer, and the cumulative amount secreted
at each time point was plotted. The amount of each protein secreted
after 120 min in the presence of insulin was taken as
100%.
Complement factor C1q consists of
three related polypeptides that form heterotrimeric subunits containing
a three-stranded collagen ``tail'' and three globular
``heads''; six of these subunits generate an 18-mer complex
often referred to as a ``bouquet of flowers'' (reviewed in (26) ). The experiments in Fig. 4show that Acrp30 has a
similar oligomeric structure, but is composed of a single type of
polypeptide chain. When analyzed by velocity gradient sedimentation
analysis, Acrp30 in blood serum migrates as two species of apparent
molecular masses of 90 kDa and 300 kDa (Fig. 4C).
Disregarding the presumably nonglobular shape of the complex that could
lead to a slight distortion of the molecular weight determination, the
former is probably a trimer and the latter could be a nonamer or
dodecamer. Isoelectric focusing followed by SDS-PAGE of S-Acrp30 secreted by 3T3-L1 adipocytes reveals only a
single polypeptide (not shown), suggesting that Acrp30 forms
homo-oligomeric structures. Chemical cross-linking using low
concentrations of BS
of
S medium from 3T3-L1
adipocytes, followed by specific immunoprecipitation and SDS-PAGE under
reducing conditions, shows mainly dimers and trimers (lanes 1-
5, Fig. 4A). Larger concentrations of the
BS
cross-linking agent generated Acrp30 proteins that
migrated as hexamers as well as yet larger species. As extensively
cross-linked proteins migrate aberrantly upon SDS-PAGE, it is difficult
to determine the exact size of the high molecular mass form indicated
by an asterisk. It could represent either a nonamer or a dodecameric
structure. Together, panels A and C of Fig. 4show that Acrp30 forms homotrimers that interact together
to generate nonamers or dodecamers. Nonreducing SDS-PAGE reveals that
two of the subunits in a trimer are disulfide-bonded together (Fig. 4B), similar to other proteins containing a
collagen domain, including the macrophage scavenger
receptor(27) . Besides being a homo-oligomer, Acrp30 differs
from C1q in containing an uninterrupted stretch of 22 perfect
Gly-X-X repeats; this suggests that Acrp30 has a
straight collagen stalk as opposed to the characteristic kinked
collagen domain in C1q caused by imperfect Gly-X-X repeats in two of the three subunits (26) .
Figure 4:
A,
incubation of S-labeled 3T3-L1 culture supernatant with
increasing amounts of the BS
cross-linking reagent,
followed by immunoprecipitation with Acrp30-specific antibodies,
reveals a set of cross-linked products whose molecular sizes are
multiples of 30 kDa. Predominant species are trimers, hexamers, and a
high molecular mass species (asterisk) that could correspond
to a nonamer or a dodecamer. In the lane Total, 1% of the
amount of cell medium used for the cross-linking reactions was analyzed
on the same gel; a comparison of the ``Total'' lane and lane 1 demonstrates the specificity of the antibody used for
immunoprecipitation. Immunoprecipitates were analyzed by gradient
SDS-PAGE (7-12.5% acrylamide) followed by fluorography. Rainbow
markers (Amersham) together with a phosphorylase b ladder
(Sigma) were used as molecular mass markers. B, reducing and
nonreducing SDS-PAGE of anti-Acrp30 immunocomplexes isolated from
S-labeled 3T3-L1 medium. Medium from day 8 3T3-L1
adipocytes labeled overnight with [
S]methionine
and cysteine was immunoprecipitated with anti-Acrp30 antibodies as
described under ``Experimental Procedures.'' The sample was
subjected to SDS-PAGE (7-12.5% acrylamide gradient) in the
presence (reducing) or absence (nonreducing) of 50 mM dithiothreitol. Labeled proteins were detected by fluorography. C, velocity gradient centrifugation of mouse serum displays
two discrete Acrp30-immunoreactive species. The smallest corresponds to
a trimer of Acrp30 polypeptides and the larger a nonamer or dodecamer.
One microliter of mouse serum was diluted with 50 µl of PBS and
layered on top of a 4.5-ml linear 5-20% sucrose gradient in PBS
and centrifuged for 10 h at 60,000 rpm in a SW60 rotor of a Beckman
ultracentrifuge. Thirteen 340-µl fractions were collected from the
top and analyzed by SDS-PAGE and Western blotting using anti-Acrp30
antibodies. An identical gradient was run in parallel with a set of
molecular mass standards: cytochrome c (14 kDa), carbonic
anhydrase (29 kDa), bovine serum albumin (68 kDa), alcohol
dehydrogenase (150 kDa),
-amylase (200 kDa), and apoferritin (443
kDa). The positions of these markers are indicated below the panel with arrowheads.
We do not yet know the function of Acrp30. However, its expression exclusively in adipocytes, its enhanced secretion by insulin, and its presence in normal serum, suggests that it is, like the ob protein, involved in the control of the nutritional status of the organism. Acrp30 is a relatively abundant serum protein, accounting for up to 0.05% of total serum protein as judged by quantitative Western blotting using recombinant Acrp30 as a standard (data not shown). Even though we have no evidence at this stage, we cannot exclude the possibility that Acrp30, like C3 complement released by adipocytes (28) , is converted proteolytically to a bioactive molecule.
Our experiments also corroborate the existence of a regulated secretory pathway in adipocytes. We do not yet know whether adipsin and/or Acrp30 are in the same intracellular vesicles that contain GLUT4 and that fuse with the plasma membrane in response to insulin, or whether they are in different types of vesicles. Adipocytes express two members of the Rab3 family, Rab3A and Rab3D(29) ; these are found in vesicles of different density. Rab3s are small GTP-binding proteins involved in regulated exocytic events. Except for adipocytes, Rab3A is found only in neuronal and neuroendocrine cells; in neurons, Rab3A is localized to synaptic vesicles and is important for their targeting to the plasma membrane(30) . An attractive hypothesis under test is that, in adipocytes, Rab3A is localized to vesicles containing Acrp30 and/or adipsin, and that possibly Rab3D mediates insulin-triggered exocytosis of vesicles containing GLUT4. In any case, the mechanism of signal transduction from the insulin receptor to regulated exocytosis of intracellular vesicles remains an important unsolved problem.