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INTRODUCTION |
Adipose has been under appreciated as an endocrine tissue for
decades because of the prevalent opinion that it served merely as
storage for lipids. Recently, however, the importance of adipocytes to
whole body energy homeostasis and metabolism has been underscored by
several reports focusing on secreted products of adipocytes (1-4).
There has been increased interest in adipose tissue as an endocrine
organ, and several of these secreted proteins, termed adipokines, are
currently undergoing extensive study regarding roles as divergent as
feeding behavior to cardiovascular protection. For instance, leptin,
the gene disrupted in ob/ob mice, has central roles in the
hypothalamus, as well as peripheral effects in liver, muscle, and
endothelial cells (5). Other adipose-secreted products, such as tumor
necrosis factor
and adipsin (complement factor D), have well
established functions in innate immunity (6-9). The recently
identified adipokine resistin has been implicated as a modulator of
insulin sensitivity and is also being studied for its effects on
metabolism (4, 10).
Acrp30 (also known as adiponectin, AdipoQ, and GBP28) is an adipokine
exclusively synthesized and secreted by adipocytes (11-14). Acrp30 has
recently been shown to influence glucose homeostasis and insulin
sensitivity. The mRNA expression of Acrp30 is reduced in obese and
diabetic mice (12), and plasma levels are lower in obese compared with
lean humans (15). In addition, longitudinal studies in rhesus monkeys
demonstrate that, similar to work in humans, Acrp30 levels were
negatively correlated with body weight, fat content, and resting
insulin levels and decline with progression toward the diabetic state
(16). Furthermore, the genomic locus encompassing the human Acrp30
gene, 3q27, has recently been identified as a novel susceptibility
locus for early-onset diabetes, as well as metabolic syndrome X
(17).
More recent work has described the bioactivity of Acrp30 or Acrp30
fragments on glucose and lipid homeostasis. Fruebis et al.
(18) initially demonstrated that injection of a proteolytic cleavage
product of Acrp30 (gAcrp30) ablated the rise in plasma-free fatty acids
and triglycerides accompanying a high fat/sucrose gavage (18). They
further found that chronic administration of gAcrp30 prevented
diet-induced obesity without affecting feeding behavior. Work by
Kadowaki and co-workers (3) also supported the hypothesis that Acrp30
is an important regulator of insulin sensitivity, by showing gAcrp30
and leptin synergism in improving insulin resistance in lipoatrophic
mice. Both groups suggested that gAcrp30 increases fatty acid
oxidation. Finally, work in our laboratory has focused on
administration of full-length, recombinant Acrp30 purified to
homogeneity from mammalian cells. Injection of this preparation leads
to a transient decrease in circulating glucose levels in wild-type
mice, as well as models of type I and II diabetes (2). Follow-up
studies using pancreatic clamps in vivo and cell culture
studies on primary hepatocytes indicate that the transient decrease in
serum glucose levels is because of improved hepatic insulin
sensitivity, resulting in decreased hepatic gluconeogenesis (2,
19).
Acrp30 consists of an N-terminal collagenous domain and a C-terminal
globular domain that shares homology to the globular domains of
collagens VIII and X, as well as complement factor C1q and a family of
hibernation-specific serum proteins. Other members of this family of
proteins, known as collectins, that share structural (but no sequence)
homology include surfactant proteins
(SP)1 A and D (SP-A, SP-D),
mannose-binding protein, bovine conglutinin, and cerebellin.
Experimental evidence indicates that each of these collectin family
members contains a C-terminal domain that forms either homotrimers, as
is the case for Acrp30, or heterotrimers between different isoforms of
the protein, such as C1q (20). For instance, studies have confirmed
that SP-D, a pulmonary protein secreted into the distal airways and
alveoli of the lung and recently shown to bind various pathogens and
potentiate leukocyte function, trimerizes, with a homotrimer as the
basic building block for larger oligomeric secreted complexes (21).
Further work demonstrated the necessity for higher order complex
formation of these oligomeric forms and in fact determined that the
activity of SP-D in vitro and in vivo critically
depends on the proper assembly of a higher molecular weight complex
(22). Similarly, we demonstrate here that Acrp30 exists in two discrete
complexes in serum, as a hexamer (LMW form) and a higher order complex
of between 12-18 subunits (HMW form), and these complexes are stable
both in vitro and in vivo. There is a
characteristic sexual dimorphism in circulating Acrp30 levels, with
higher levels found in females. Additionally, we demonstrate that there
is a sexual dimorphism in terms of complexes of the protein, as males
have the majority of their Acrp30 circulating as hexamers, whereas
female mice have a more balanced distribution of the two forms.
Furthermore, we show that levels of the two complexes respond
differentially to insulin and glucose treatment, with selective loss of
the HMW form. Finally, a homologous mutation to the C15S/C20S change in
SP-D that ablates higher order assembly and function of SP-D complexes
(23) was introduced into Acrp30. Biochemically, the C39S mutation in
Acrp30 prevents higher order complex formation beyond the basic
homotrimer. These homotrimers can also be visualized
electronmicroscopically by rotary shadowing. Surprisingly, and in
contrast to SP-D, this destabilized homotrimer has greater bioactivity
in vivo and in vitro than the native oligomeric complexes of Acrp30.
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EXPERIMENTAL PROCEDURES |
Materials--
Dulbecco's modified Eagle's medium (DMEM) was
prepared by the Albert Einstein Cancer Center Media Core Facility. FCS
was purchased from Invitrogen.
EXPRE35S35S protein labeling reagent was
purchased from PerkinElmer Life Sciences. DMEM lacking
methionine, cysteine, and glutamate was purchased from ICN (Costa Mesa,
CA). Dithiothreitol (DTT), glucose Trinder assays, and trypsin was
purchased from Sigma. All other chemicals were purchased from Fisher.
Antibodies--
Antibodies to the N-terminal domain of Acrp30
were described previously (2). Another antibody was raised by injecting
full-length recombinant Acrp30 produced in and purified from 293-T
cells into rabbits (Covance Research, Denver, PA).
Animals--
FVB or C57Bl/6J mice were bred in house and
used as indicated. All mice were 2 to 5 months of age and fed ad
libitum using regular mouse chow. All protocols were approved by
the Albert Einstein Animal Committee.
Velocity Sedimentation/Gel Filtration Chromatography for
Separation of Acrp30 Complexes--
5-20% sucrose gradients in 10 mM HEPES, pH 8, 125 mM NaCl were poured
stepwise (5, 10, 15, 20%) in 2-ml thin-walled ultracentrifuge tubes
(BD Biosciences) and allowed to equilibrate overnight at 4 °C.
Following layering of the sample on top (diluted 1:10 with 10 mM HEPES, pH 8, 125 mM NaCl in the case of
serum), the gradients were spun at 55,000 rpm for 4 h at 4 °C
in a TLS55 rotor in a Sorvall TL-100 table-top ultracentrifuge.
150-µl gradient fractions were sequentially retrieved from the top of
the gradient and analyzed either by quantitative Western blot analysis
or by scintillation counting in the case of iodinated protein.
Alternatively, pooled serum or recombinant protein was loaded on two
Superose 6 gel filtration columns arranged in tandem, and 0.5-ml
fractions were collected (0.2 ml/min flow rate for 4 h in
phosphate-buffered saline) and analyzed by SDS-PAGE.
Site-directed Mutagenesis to Create the C39S Mutation in
Murine Acrp30--
Complementary primers
(5'CACCCAAGGGAACTTCTGCAGGTTGGATGG) and
(5'GCCATCCAACCTGCAGAAGTTCCCTTGGGTG)
were synthesized (with the underlined sequence encoding the new
serine residue). Primers were electrophoresed on a 12% acrylamide gel
containing 8 M urea, 25 mM Tris, pH 6.8, at 20 watts for 2 h, followed by visualization by UV shadowing at
254 nM. Excised gel slices were fragmented and then
resuspended in 150 µl of phenol, pH 6.6, and 150 µl of 0.3 M sodium acetate and incubated at 37 °C. The aqueous layer was further phenol-chloroform extracted, followed by two chloroform extractions and ethanol precipitation. Pfu
polymerase (Stratagene) was used for PCR with pAB23 (bicistronic
expression vector containing the Acrp30 cDNA and green fluorescent
protein (2)) as a template. Reactions were digested by 5 units of
DpnI (New England Biolabs) to remove any remaining
double-stranded methylated template plasmid for 3 h at 37 °C
and then electroporated into DH10
bacteria. Transformants were
screened for acquisition of PstI sensitivity (new
PstI site generated by mutation italicized).
Transient Transfections in HEK 293-T Cells--
Acrp30
constructs (described below) were used for transient transfection
assays in HEK 293-T cells. Plasmids were transiently transfected into
HEK 293-T cells (10-cm dishes) by the Effectene method (Qiagen).
48 h after transfection, cells were labeled for 4 h in 3 ml
of DMEM lacking methionine and cysteine and supplemented with 0.5 mCi
(1000 Ci/mmol) of express protein labeling reagent. Cells were
thereafter washed three times with chase medium (DMEM containing
unlabeled methionine and cysteine at 1 mM and cycloheximide at 300 µM). At the end of the chase period, supernatants
were harvested, and cells were washed twice with cold
phosphate-buffered saline and then scraped into TNET-OG buffer (1%
Triton X-100, 150 mM NaCl, 2 mM EDTA, 20 mM Tris, pH 8.0, 60 mM octyl-glucoside, and
protease inhibitors).
Production of Recombinant Wild-type and C39S Mutant
Acrp30--
Wild-type and mutant Acrp30 proteins were purified as
described previously (2). Briefly, a bicistronic expression vector for
Acrp30 and green fluorescent protein was constructed and stably transfected into 293-T cells. High expressers were isolated by fluorescence-activated cell sorting and further propagated in DMEM
containing 10% FCS, penicillin/streptomycin and 0.1 g/liter ascorbic
acid. The selection process for high expressers was repeated a total of
three times, allowing sufficient recovery and growth time after each
successive cell sort. When cells reached confluence, the
serum-containing medium was removed, and cells were allowed to secrete
for 48 h into serum-free DMEM with 0.1 g/liter ascorbic acid.
Medium was collected and centrifuged for 30 min at 3000 rpm to pellet
cellular debris. Ammonium sulfate was added to 40% (w/v), and protein
was precipitated overnight at 4 °C and then centrifuged for 3 h
at 4000 rpm. Precipitated protein was resuspended in 10 mM
HEPES, pH 8, 50 mM NaCl, 1 mM CaCl2
(low salt buffer) and dialyzed overnight at 4 °C in the same buffer.
Dialyzed protein was filtered through a 0.45-µm filter before loading
onto a 5-ml EconoPac High Q anion exchange cartridge (Bio-Rad). The
column was washed extensively with low salt buffer before elution with a 50 to 500 mM NaCl salt gradient. Acrp30-containing
fractions were determined by Coomassie staining, and positive fractions were concentrated and analyzed for purity. The resulting protein was
>99% pure as judged by staining of Coomassie-stained gels.
"Headless" Recombinant Acrp30--
A purified
preparation of full-length Acrp30 lacking the globular head domain was
produced and purified from HEK 293-T cells and kindly provided by Dr.
Maximilien Murone (Apotech).
Iodination of Recombinant Protein--
50 µg of Acrp30 in 10 mM HEPES, pH 8, 125 mM NaCl was incubated with
1 mCi of Na125I and 10 µl of chloramine T (1 mg/ml) for
30 min at room temperature. The reaction was quenched with 5 mM cold NaI before loading onto a PD-10 desalting column
(Amersham Biosciences) pre-equilibrated with 20 mM BisTris,
pH 6.5, 200 mM NaCl, and 2 mg/ml bovine serum albumin to
remove unincorporated label. The column was washed with the same buffer
without the bovine serum albumin, and fractions were collected and
assayed for radioactivity and stability of protein by electrophoresis
followed by Western blot and phosphorimaging analysis.
Structural/Proteolytic Cleavage Studies of
Acrp30--
Recombinant wild-type Acrp30 was incubated in the presence
of various concentrations of DTT (Sigma) from 1 to 100 mM
final concentration, 100 mM glycine, pH 3, or 100 mM sodium carbonate, pH 11.5, for 1 h at 37 °C.
Following incubation, protein was applied to a 5-20% sucrose gradient
as described. Alternatively, recombinant wild-type Acrp30 (1 µg) was
incubated in DMEM ± 100 mM DTT for 1 h at
37 °C and then incubated in the presence of 1% serum alone, 293-T
cells alone, or 1% serum + 293-T cells for 1 h at 37 °C. Medium was collected and analyzed by SDS-PAGE followed by blotting using anti-full-length Acrp30 polyclonal antiserum. Cleavage products detected were identical to those present in 293-T cells expressing the
C39S mutant Acrp30, as verified by Edman degradation. Briefly, protein
was electrophoresed and transferred to polyvinylidene fluoride membrane
(Bio-Rad). Membranes were stained with Coomassie Brilliant Blue R250
and destained with 50% methanol, and the cleavage product band was
excised. N-terminal sequences were determined by Edman chemistry using
an Applied Biosystems Procise Sequencer by the Albert Einstein
Laboratory of Macromolecular Analysis.
In Vivo Experiments--
Blood glucose levels were measured by
Trinder assay (Sigma). Acrp30 was quantitated by Western blot analysis;
following SDS-PAGE, proteins were transferred to nitrocellulose
(Schleicher & Schuell). Rabbit polyclonal anti-Acrp30 antibody
derivatized with 125I was used to decorate the blots, and
total Acrp30 signal was determined with a PhosphorImager (Molecular
Dynamics) and analyzed with ImageQuant 1.2 software against recombinant
Acrp30 standards of known concentration. Intraperitoneal insulin
injections and glucose gavages were performed on C57Bl/6J males. Serum
was collected through tail bleeds. Purified, recombinant proteins
(wild-type Acrp30 and C39S Acrp30) or iodinated Acrp30 complexes were
injected intravenously into the tail vein of mice of the FVB background.
Generation of Transgenic Acrp30(C39S Mutant) Mice--
The
full-length untagged cDNA encoding murine Acrp30 with the C39S
mutation was put under the control of the aP2 enhancer/promoter sequence, and the rabbit
-globin 3' untranslated region poly(A) sequence was added to the 3' end. The isolated construct was purified for pronuclear injection into mouse embryos from FVB mice (Taconic Farms). Mouse embryos (fertilized one-cell zygotes) were injected and
implanted in female CD-1 mice (Charles River Breeding Laboratories) at
the Transgenic Mouse Facility at the Albert Einstein College of
Medicine. Acrp30 transgenic mice were identified by slot blot analysis using genomic DNA prepared from mouse tails and also analyzed
by Northern blot analysis. Acrp30(C39S mutant)-positive founder
transgenic mice were continuously crossed to FVB mice to maintain a
pure FVB background. The transgene was transmitted exclusively through
male mice.
Immunoblotting--
Separation of proteins by SDS-PAGE,
fluorography, and immunoblotting were performed as described previously
(24). Primary and secondary antibodies were diluted in Tris-buffered
saline with 0.1% Tween 20 and 1% bovine serum albumin. Horseradish
peroxidase-conjugated secondary antibodies were detected with enhanced
chemiluminescence according to the manufacturer's instructions (Pierce).
Measurement of Glucose Production in Primary
Hepatocytes--
Measurements were performed as described (2). In
brief, single-cell hepatocyte suspensions were isolated as described
previously and allowed to adhere to 24-well plates pre-coated with
rat-tail collagen I. Cells were cultured in RPMI 1640 medium
supplemented with 10% FCS, penicillin/streptomycin, 10 µg/ml
insulin, and 10 µM dexamethasone. Medium was changed to
RPMI with 5 mM glucose, 0.4% FCS, and cells were allowed
to equilibrate overnight in this low glucose medium. The following
morning, medium was refreshed, insulin (35 pM) and/or
Acrp30 (wild-type or Cys mutant) was added, and treatment lasted
another 24 h. After stimulation, glucose production was measured
by incubating cells for 6 h in glucose-free RPMI containing 5 mM each of alanine, valine, glycine, pyruvate, and lactate,
removing the supernatant, and assaying by Trinder assay (Sigma). Each
point represents that average of three independent measurements.
Electron Microscopy--
Samples of the various purified Acrp30
preparations were diluted in 0.2 M ammonium bicarbonate to
a final concentration of 10-20 µg/ml, mixed 1:1 (v/v) with 80%
(v/v) glycerol. Shortly after addition of the glycerol, the mixture was
sprayed onto freshly cleaved mica. The mica chips were dried at
<10
5 torr for at least 2 h. Rotary shadowing with
platinum/carbon at an angle of 9°, carbon shadowing at 90°, replica
formation, and electron microscopy followed earlier protocols
(25).
 |
RESULTS |
Sexually Dimorphic Distribution of Oligomeric Complexes in
Vivo--
Sexually dimorphic levels have been reported for many
metabolic factors, including adipocyte-specific hormones such as leptin (26). We therefore examined levels of Acrp30 in males versus female mice. We noted that there is indeed a sexual dimorphism in serum
Acrp30 levels, with a significant, ~2.5-fold increase in total
circulating levels in female mice compared with male littermates (~25
µg/ml versus ~10 µg/ml). A similar dimorphism has been
reported in humans, where the dimorphism is smaller (about a 50-100%
increase in females) (27). To further study the relevance of these
sexually dimorphic levels, we analyzed the size distribution of
oligomeric complexes of Acrp30 in mice by means of gel filtration chromatography and velocity sedimentation. Consistent with previous reports (11), Acrp30 exists in two major oligomeric forms in mouse
serum, as a hexamer of ~180 kDa and a higher order structure with the
apparent molecular mass of 400 kDa, as determined by size
standards run concurrently. Interestingly, the sexual dimorphism is not
only limited to absolute amounts of Acrp30 but is also reflected in the
distribution of oligomeric complexes in serum. The majority of Acrp30
in male mice is present in the smaller, hexameric form, whereas female
mice have a more even distribution of Acrp30 complexes, with similar
serum levels of both low and high molecular weight complexes (Fig.
1, A and B). This
suggests that male and female mice have comparable levels of the low
molecular weight form (9-12 µg/ml) but differ significantly with
respect to the levels of the high molecular form (Fig.
1C).

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Fig. 1.
Sexual dimorphism in Acrp30 complex
levels. Shown are velocity sedimentation analyses of serum Acrp30
levels of representative male and female mice, with labeled size
standards (A), and quantitated in a cohort of 10 C57Bl/6J
mice of each gender represented as %LMW (hexameric Acrp30) per total
serum Acrp30 levels (B) or as total serum concentration of
either complex (C), demonstrating similar LMW levels in
either gender.
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Acrp30 Complexes Are Stable and Do Not Interconvert in Vitro and in
Vivo--
To determine whether these oligomeric complexes are stable
or interconvert spontaneously, recombinant, HEK 293-T-produced mouse
Acrp30 protein was further separated by gel filtration chromatography into separate fractions corresponding to the hexamer and high molecular
weight forms. Following purification, the complexes were incubated at
37 °C for 1 h, either as purified proteins or in the presence
of rat serum, which contains immunologically distinct Acrp30 complexes.
Both the low and the high molecular weight form are remarkably stable
(Fig. 2A). No significant
interchange can be observed for the high molecular weight form in the
presence of rat serum; similarly, the low molecular weight form does
not assemble into a high molecular weight species, suggesting that the
two complexes do not spontaneously interchange in the presence of
serum. Because we were adding microgram amounts of recombinant Acrp30
to microliter amounts of serum, the inability of serum to catalyze an
interchange may reflect saturation of an enzymatic activity, as
recombinant Acrp30 is in large excess over endogenous Acrp30. To
address this issue, we iodinated Acrp30 and incubated nanogram amounts
of the iodinated complexes with mouse serum in vitro. This
reflects subphysiologic amounts of exogenous Acrp30 compared with
endogenous levels. Interchange between the different molecular weight
forms is assayed by monitoring the distribution of counts across a
velocity centrifugation. Even under these conditions, we found no
evidence for either a spontaneous or catalyzed interchange between the
subunits (Fig. 2B). This suggests that such an interchange may not take place in vivo or that the conditions chosen
in vitro are not effectively mimicking in vivo
conditions.

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Fig. 2.
ACRP30 complexes do not interchange in
vitro or in vivo and have different
clearance rates. Purified recombinant Acrp30 complexes were
incubated ± 20 µl of rat serum at either 4 or 37 °C for
1 h and than analyzed by velocity sedimentation and SDS-PAGE
(A). Alternatively, iodinated Acrp30 was incubated with
mouse serum for 0, 30, or 60 min, followed by velocity sedimentation
and scintillation counting; fractions containing iodinated protein
correspond to the hexamer and HMW complex peaks of endogenous Acrp30
(B). Iodinated Acrp30 was separated by gel filtration
chromatography into purified hexamer and HMW complex fractions and
injected intravenously into female FVB mice (C). Mice were
bled at 1- and 2-h time points, and the serum was subjected to velocity
sedimentation and scintillation measurements to determine interchange
of the complexes.
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Preparative amounts of iodinated complexes were isolated by velocity
sedimentation centrifugation, followed by dialysis of excess sucrose.
The purified fractions were injected intravenously into mice. The high
specific activity of the iodinated protein permitted the injection of
small amounts of protein (<1 µg) compared with the total circulating
pool of Acrp30 in female mice (~90 µg), avoiding any significant
changes to endogenous levels of the protein. Serum samples were assayed
for up to 8 h post-injection, and the complex distribution was
determined by velocity sedimentation followed by scintillation counting
to determine the size of the labeled oligomeric complexes (Fig.
2C). Even though the complexes were cleared and appeared at
reduced levels, we were unable to detect a significant redistribution
in vivo of either the iodinated hexamer or high order
structure complexes at any stage following injection. This suggests
that there is no direct precursor/mature form relationship between the
two populations of Acrp30 in serum under basal conditions.
Although no interchange was observed under basal conditions, we also
tested whether a more significant degree of interchange can be observed
during a metabolic challenge. We therefore decided to examine the
stability of the complexes in response to an insulin injection,
conditions that lead to a selective reduction of the high order complex
(see below). Mice were intravenously injected with purified, iodinated
Acrp30 complexes in the presence or absence of 1 unit insulin/kg body
weight. We failed to detect any interconversion of the complexes even
after this extreme metabolic challenge (Fig. 2C). The
possibility remains, however, that iodinated Acrp30 may not mimic
actual Acrp30 complexes in vivo; the iodinated protein may
lose the ability to either spontaneously interconvert in
vitro or interchange with unlabeled complexes in vivo.
To address this issue, we recently performed these experiments with a
FLAG-tagged version of Acrp30 and observed similar results (data not shown).
Insulin and Glucose Treatment Leads to a Selective Decrease of the
High Molecular Weight Form--
A number of studies have indicated
that Acrp30 in serum is maintained at a relatively constant level (15).
However, little attention has been given to the description of stimuli
that may lead to relatively acute changes of the protein in serum.
Because Acrp30 circulates at µg/ml (nanomolar) levels in serum with a relatively short half-life (5-6 h) under basal conditions, even relatively modest changes at the level of production and/or clearance may translate into significant changes of steady state levels in serum.
Preliminary experiments indicated that serum Acrp30 levels decreased in
female, and, to a significantly lesser degree, in male mice treated
with pharmacological amounts of insulin (not shown). Upon analysis of
the complex distribution of Acrp30 following insulin treatment, we
discovered that only the high molecular weight complex selectively
disappears in response to insulin (Fig. 3A). In both males and
females, ~30% of the high molecular weight form is lost. However,
because male mice have much lower levels of the high molecular weight
form compared with females, the selective loss of the larger complex
only has a significant impact on total circulating Acrp30 levels in
females. This selective loss of the large molecular weight form is also
observed in mice gavaged with a high glucose solution, with a delayed
onset (Fig. 3B). Both insulin injections and a glucose
gavage result in an increase in serum insulin levels, suggesting that
in both cases, the effects may be mediated by a rise in serum insulin
levels. Importantly, upon normalization of glucose levels, normal
levels of the high molecular weight form are reconstituted under both
conditions. Consistent with the data presented in Fig. 2, we do not
observe a metabolically regulated interconversion between hexamer and HMW complexes; the disappearance of the HMW complex in response to
either an insulin or glucose challenge is unaccompanied by an increase
in hexamer levels.

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Fig. 3.
Insulin/glucose treatment of mice alters the
LMW/HMW ACRP30 ratio. Male and female mice were injected
intraperitoneally with 1 unit/kg body weight recombinant human insulin
(n = 10) (A) or gavaged with 1.0 mg/g body
weight glucose (n = 8) (B). Mice were bled,
and their serum was analyzed by velocity sedimentation followed by
quantitative SDS-PAGE for levels of Acrp30 complexes. Mice of both
genders show similar profiles in response to metabolic challenges in
terms of magnitude and kinetics of response. Note similar decreases of
high order complex (HMW) Acrp30 in both treatments but with
a later onset for the glucose-treated mice (2 h peak effect
versus 0.5 h). Significant differences from
t = 0 are labeled with an asterisk
(p < 0.05).
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Trimer-Trimer but Not Higher Order Interactions Are Mediated
through Disulfide Bonds--
To analyze the quaternary structure of
Acrp30 in greater detail, a series of experiments was performed to
study the type of molecular interactions that mediate formation of
oligomeric complexes. Both serum and recombinantly produced, purified
Acrp30 form two distinct peaks as judged by velocity sedimentation
centrifugation and gel filtration chromatography, consistent with the
molecular mass of Acrp30 hexamers (~180 kDa) and a higher order
species of apparent molecular mass of greater than 400 kDa. When either serum or purified Acrp30 is incubated at low pH (pH 3), the high molecular weight species collapses to the hexamer (Fig.
4A). This is selective for low
pH, because high pH (pH 11.5) or high salt (1 M NaCl) does
not disrupt the high molecular weight form (not shown). Interestingly,
treatment of serum or recombinant protein with reducing agents (such as
dithiothreitol), in the presence (Fig. 4A) or absence (Fig.
4B) of glycine, collapses both the high molecular weight
form and the hexameric form into a trimer of ~90 kDa. We used
SDS-PAGE under non-reducing conditions as a complementary approach to
confirm that low pH disrupts exclusively HMW structures, whereas both
oligomeric forms are reduced to trimers in the presence of DTT (Fig.
4C). Under non-reducing conditions, recombinant Acrp30
migrates as a dimer at ~60 kDa, suggesting that intratrimer and
intertrimer disulfide bonds link all Acrp30 monomers in either hexamer
or HMW complexes. At low pH, no monomers are formed, consistent with
the absence of a trimer; upon addition of DTT, trimeric Acrp30 is
formed, resulting in the appearance of Acrp30 monomers in SDS-PAGE.
Similar results were obtained for endogenous serum Acrp30 (see Fig.
5E).

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Fig. 4.
High order Acrp30 complexes are sensitive to
low pH, whereas both complexes are susceptible to reduction by
DTT. Recombinant Acrp30 protein was analyzed by velocity
sedimentation and SDS-PAGE in the presence or absence of 100 mM glycine, pH 2.5 (A). High order complexes
(HMW) complexes are specifically shifted to the molecular
weight of Acrp30 hexamers, which are then further susceptible to
reduction by 20 mM DTT to the molecular weight of Acrp30
trimers (~90 kDa). B, analysis of the differential
susceptibility of Acrp30 complexes to increasing levels of reducing
agent reveals that the hexamer is slightly more sensitive to reduction
than the high order complex. C, non-reducing, denaturing
SDS-PAGE of recombinant Acrp30 in the presence of pH 2.5 glycine ± 10 mM DTT, demonstrating the presence of trimeric Acrp30
only upon incubation with reducing agent and not in glycine-treated
samples.
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Fig. 5.
Cys-39 mediates the disulfide bond that
stabilizes Acrp30 complexes. Alignment of the region surrounding
Cys-39 in Acrp30 with C1q (B subunit), hib27, and pulmonary surfactant
protein D, demonstrating the proximity of the conserved cysteine
residue (boxed) to the beginning of the collagenous domains
of these proteins (glycine residues of GXY repeats
boldface) (A). Shown is velocity sedimentation of
recombinant wild-type Acrp30, wild-type Acrp30 treated with 10 mM DTT, and Acrp30(C39S) proteins, demonstrating
co-migration of fully reduced wild-type Acrp30 and the mutant protein
(B). Increasing amounts of the homobifunctional cross-linker
BS3 (1 to 500 µM) were titrated into
solutions of purified wild-type (left panel) or Acrp30(C39S)
(right panel) and analyzed by Western blot (C).
Numbers on the left and on the right
indicate molecular weights, and numbers in between the two
panels refer to the number of subunits cross-linked. The
asterisks indicate the largest size complex obtained for
each form. Stable subunit numbers are boxed (3 subunits for
Acrp30(C39S); 3, 6, and 12-18 subunits for wild-type Acrp30). Although
purified protein was used for this experiment, fetal calf serum was
added to a final concentration of 1% prior to the addition of
cross-linking agent to prevent nonspecific cross-linking, leading to
some of the background bands seen on this blot. D, electron
micrographs following rotary shadowing of Acrp30 complexes; human and
mouse HMW forms demonstrate a rosette pattern although subunit number
is indeterminate; hexamers have a characteristic V-shape whereas
Acrp30(C39S) appears as a single trimer. In E, serum from
wild-type and Acrp30(C39S) transgenic lines is resolved by
non-reducing, denaturing SDS-PAGE as in Fig. 4C; trimeric
Acrp30 is only found in transgenic mice. F, gel filtration
chromatography of pooled serum from transgenic (top left)
and wild-type mice (top right), demonstrating a trimer peak
only in Acrp30(C39S) transgenic mice. Recombinant Acrp30(C39S) was run
under the same conditions (bottom left). Recombinant
wild-type protein was treated with DTT or purified into HMW and hexamer
forms by gel filtration. All three preparations were re-run on gel
chromatography. Chromatograms of these runs are shown (bottom
right).
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Combined, these results suggest disulfide bonds mediate trimer-trimer
interactions that lead to the formation of hexamers. Although the
formation of the high molecular weight complex is not directly mediated
by disulfide bonds and is non-covalent, the high molecular weight
complex critically depends on intact hexamers. Both oligomeric
structures therefore depend on disulfide bond formation for complex
stability. To further explore whether there is a differential
susceptibility of the two complexes to the effects of reducing agents,
we subjected recombinant Acrp30 to increasing concentrations of DTT (1 to 50 mM DTT) and incubated the samples at 37 °C for
1 h. Fig. 4C shows that at low doses of DTT, the
hexamer is more susceptible to reduction than the high molecular weight
form, underlining that the disulfide bonds are sterically more
accessible in the hexamer than in the high order complex. Because the
oligomerization of the hexamers to higher order complexes is most
likely mediated by the collagenous tail, similar to other members of
the collectin family, we decided to study the role of the only cysteine
(Cys-39) within the collagenous tail on higher order complex formation.
Cys-39 Mediates the Disulfide Bond Interaction Necessary for
Oligomeric Structures--
Several lines of evidence implicate Cys-39
as the necessary residue for disulfide bond formation. As mentioned
previously, because of the tight collagen triple-helix conformation of
Acrp30, Cys-39 would be predicted to lie in close proximity to the
corresponding residue on adjacent monomers and be readily available for
interchain disulfide bond formation. This hypothesis was confirmed by
information gained from the crystal structure solved for the trimeric
globular head of Acrp30 that demonstrated that Cys-155, the only other cysteine in Acrp30, does not appear to be properly spaced for interaction with other chains and disulfide bonding in an oligomeric structure. Additionally, analyses of structural homologues of Acrp30,
including C1q and pulmonary SP-D, suggest that many of these
proteins have conserved cysteine residues within the N-terminal region,
immediately preceding the collagenous domain critically involved in
oligomerization (Fig. 5A). We, therefore, substituted cysteine 39 in Acrp30 with a serine residue by site-directed
mutagenesis. Upon sequence confirmation of the C39S mutation, we
designed an expression construct and expressed both wild-type and the
mutant Acrp30(C39S) in HEK 293-T cells. Secreted wild-type and mutant Acrp30 were analyzed by velocity sedimentation. For the wild-type protein, we see the expected two peaks, corresponding to hexamer and
HMW structures; however, Acrp30(C39S) is unable to assemble into any
higher order structures and is secreted as a trimer similar to
DTT-treated wild-type Acrp30 (Fig. 5B). To demonstrate that the mutant Acrp30 does not form higher order structures that are perhaps destabilized during prolonged velocity sedimentation, cross-linking experiments were performed in solution. To prevent nonspecific cross-linking among the purified Acrp30 complexes, we added
fetal calf serum prior to the initiation of the cross-linking reaction.
Acrp30(C39S) can only be cross-linked to a trimeric structure with no
significant formation of higher order structures. In contrast, the
wild-type protein can be cross-linked to higher order structures, with
stable products of 6 and ~12-18 subunits, as demonstrated previously
(11) (Fig. 5C).
To further corroborate the biochemical data, we decided to visualize
individual Acrp30 molecules by rotary shadowing, a technique that was
effectively utilized to determine ultrastructural details of various
Acrp30 homologs, including C1q and SP-D. As seen in Fig. 5D,
Acrp30(C39S) is indeed secreted as a trimer only, whereas wild-type
protein complexes are found as hexamers and higher order structures.
As an initial step in determining the effect of the C39S mutation of
Acrp30 in vivo, we generated transgenic mouse lines
overexpressing the mutant Acrp30 specifically from adipose tissue under
the control of the aP2 promoter. We are still in the early stages of
the characterization of this mouse model. However, we have been able to
demonstrate that transgene expression of the mutant Acrp30 is
restricted to adipose tissue (not shown). Furthermore, the Acrp30(C39S)
forms low levels of homotrimeric mutant complexes and heterotrimeric complexes with wild-type subunits that are secreted and present in
serum as evidenced by the presence of Acrp30 monomers under non-reducing SDS-PAGE in two independent founder lines (Fig.
5E). In addition, gel filtration chromatography demonstrates
the existence of a trimeric species in serum of transgenic mice (Fig.
5F, top left) but not in wild-type mice
(top right). In parallel, purified recombinant trimeric
Acrp30(C39S) was analyzed under the same conditions and eluted in
fractions overlapping with the trimers observed in the transgenic mouse
line (Fig. 5F, bottom left). Finally, purified
wild-type recombinant protein was either treated with DTT or run as
isolated high order or hexameric complexes on the same gel filtration
column (chromatograms of these runs are shown in Fig. 5F,
bottom right). These purified complexes serve as standards
to unambiguously identify the nature serum complexes. Combined, this
underlines the notion that trimeric forms of Acrp30 usually do not
circulate at appreciable levels in wild-type mice and may only be
short-lived intermediates, generated at low levels.
Acrp30(C39S) Is Secreted More Efficiently and Is Susceptible to
Proteolytic Cleavage--
HEK 293-T cells were transiently transfected
with both wild-type and Acrp30(C39S) expression constructs. 48 h
later, the cells were pulse-labeled for 4 h with
35S-labeled Cys/Met, followed by a chase for 6 h. Intracellular and secreted material was then subjected to
immunoprecipitation with anti-Acrp30 antibodies and analyzed by
electrophoresis and autoradiography. Even though both wild-type and
mutant proteins were expressed at similar levels, Acrp30(C39S) was
secreted far more efficiently than wild-type Acrp30 (Fig.
6A). In addition, a distinct
proteolytic cleavage product of ~26 kDa was found for Acrp30(C39S).
This cleavage product was exclusively found in the extracellular
fraction, suggesting that this proteolytic processing step takes place
after export of the protein from the cell.

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Fig. 6.
ACRP30 trimers are more susceptible to
proteolysis in cell culture and in vitro compared with
higher order ACRP30 complexes. HEK 293-T cells were transiently
transfected with expression constructs for wild-type and Cys mutant
Acrp30, followed by 35S labeling and immunoprecipitation of
intracellular and secreted proteins with an antibody raised again
full-length mouse Acrp30 (A). Although similar expression
patterns exist intracellularly, secreted levels of Cys mutant Acrp30
are significantly higher and demonstrate a unique cleavage product.
Longer exposures demonstrate no cleavage product in transfected
wild-type Acrp30 constructs. Arrows demonstrate the 26-kDa
cleavage products in A and B. In B,
recombinant, purified, wild-type Acrp30 protein was incubated with or
without reducing agent in the presence of either serum or HEK 293-T
cells or both and cleavage analyzed by SDS-PAGE and Western blotting. In C and
D, recombinant wild-type and Acrp30(C39S) proteins (2 µg)
with the indicated treatments were incubated at 37 °C in the
presence of 0.02 µg of recombinant trypsin for 0, 5, 15, or 30 min
and analyzed by SDS-PAGE followed by Coomassie staining and
densitometric analysis.
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To further characterize this proteolytic step and determine whether it
was a result of altered secondary structure in Acrp30(C39S), we
performed an experiment using recombinant wild-type Acrp30. Wild-type
Acrp30 (1 µg) was left intact or pre-treated with DTT. Subsequently,
the protein was incubated in the presence or absence of serum or intact
293-T cells for 1 h at 37 °C. Cleavage was monitored by
SDS-PAGE and Western blot analysis. Only the combination of DTT
treatment followed by exposure to intact 293-T cells yielded cleavage
to the 26-kDa product (Fig. 6B). The C39S mutation or DTT
treatment likely induces destabilization of the collagenous domain.
This destabilization per se or the resulting inability to
form hexamers and higher order structures exposes the cleavage site
required for the site-specific protease. Because neither serum nor
conditioned 293-T medium contained the proteolytic activity, it is
likely that an extracellular matrix-associated protease/collagenase on
the surface of 293-T cells is responsible for the cleavage. Similar
observations were made with primary hepatocytes (not shown). Because we
see the same cleavage product in wild-type, reduced Acrp30, it is
probable that the cleavage occurs because of the absence of the
disulfide bond, resulting in trimerization, as opposed to more gross
structure abnormalities in the mutant protein. We failed to inhibit
this cleavage with various protease inhibitors (phenylmethylsulfonyl
fluoride, Pefabloc, complete protease inhibitor mixture) or metal-ion
chelating agents (EDTA, EGTA) that typically inhibit enzymes that
require either Mg2+ or Mn2+ cofactors for
activity (data not shown). N-terminal microsequencing revealed that
this cleavage product occurs roughly 30 amino acids after the signal
sequence, leading to a truncated protein with the N terminus in
position 45 (45GIPGHP).
To probe for overall structural differences between the basic trimers
and the higher order complexes in vitro, we performed limited proteolysis experiments on the various forms. Using a 1:100
ratio of protease to Acrp30 (0.02 µg trypsin/2 µg Acrp30), we
purified the different Acrp30 forms and treated them for various periods of time at 37 °C and analyzed the susceptibility to
proteolysis by SDS-PAGE, followed by Coomassie staining of the gels and
densitometry analysis (Fig. 6, C and D).
Consistent with a tightly arranged collagenous stalk in the highest
order structure, HMW Acrp30 is most resistant to proteolysis, whereas
purified hexamers are slightly more susceptible. However, both trimeric
forms of Acrp30 are highly susceptible to proteolysis, consistent with
a destabilized collagenous moiety in these forms. In all cases, the
minimal protease resistant core is the globular head domain.
Acrp30(C39S) Is Biologically More Active and Is Cleared More
Rapidly than Wild-type Acrp30--
We have shown previously (2) that
intraperitoneal injection of wild-type Acrp30 mice, resulting in a 2- to 3-fold elevation of circulating Acrp30 levels, results in a
transient reduction of glucose levels. Insulin levels were not affected
by Acrp30 treatment. To determine whether this biological activity is
dependent on the oligomeric structure of Acrp30, we required
pharmacological amounts of purified Acrp30(C39S) mutant. The protein
was produced and purified from 293-T cells in a manner similar to the
previously established protocol for wild-type Acrp30. Because 293-T
cells offer a good source for the cell surface-bound protease activity described above, the majority (>90%) of the purified protein was in
the cleaved 26-kDa form. As expected from the structure of the globular
domain that completely lacks the collagenous domain, the cleaved form
missing the N-terminal 40 amino acids with the bulk of the collagenous
domain intact maintains the basic trimeric structure (not shown). To
compare the biological activity of wild-type and mutant Acrp30, we
injected preparations intravenously into male mice at various
concentrations. Concentrations were chosen such that both wild-type
(with mostly high order structure) and wild-type glycine-treated Acrp30
(not shown) showed a similar glucose lowering effect at 1 µg Acrp30/g
body weight, with a relatively minor 20% decrease in plasma glucose at
5 h. Smaller amounts of wild-type or glycine-treated
wild-type did not significantly affect plasma glucose.
However, at these concentrations, Acrp30(C39S) had greater
glucose-lowering activity when compared with the wild-type (Fig.
7, A-D). At a 1 µg/g body
weight, plasma glucose levels are substantially reduced by 30 min, with
peak activity also reached at 5 h, but with a much more
significant glucose drop, resulting in a 60% reduction of serum
glucose levels. In addition, at doses as low as 0.03 µg/g body weight
(not shown), we still see a significant decrease in plasma glucose,
comparable with the 1 µg/g dose of wild-type Acrp30. Furthermore, the
effect of Acrp30(C39S), like that of wild-type at higher
concentrations, is saturable; injections at a 3 µg/g body weight dose
did not lead to substantial further reduction in plasma glucose
compared with the 1 µg/g body weight dose. Perhaps most
significantly, the DTT-treated wild-type Acrp30 displays a dose
response overlapping with Acrp30(C39S), indicating that reduction of
the wild-type protein results in a major activation step. Bacterially
produced globular head trimer (gAcrp30) was also tested at similar
doses and was found to have no significant bioactivity in terms of
reduction of serum glucose compared with wild-type, full-length Acrp30
purified from mammalian cells. This is consistent with previous reports
(2, 28) and may reflect the fact that that gAcrp30 may exert its
biological effects primarily through actions in muscle (18) and has
limited effects on liver gluconeogenesis. Similarly, headless
Acrp30 recombinant protein, consisting of just the collagenous domain
of Acrp30, demonstrated no significant bioactivity in our in
vivo assay (Fig. 7D).

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Fig. 7.
Acrp30(C39S) has greater glucose-lowering
potency than wild-type Acrp30 or gAcrp30 and is cleared more
efficiently than wild-type protein. Various amounts of purified,
recombinant Acrp30(C39S) (A), wild-type Acrp30 treated with
10 mM DTT (B), wild-type ACRP30 (C),
or the globular head of Acrp30 purified from bacteria (gAcrp30) or
headless Acrp30 purified from mammalian cells (D) were
intravenously injected into male FVB mice, and serum glucose levels
were measured at various time points post-injection. All significant
changes (p < 0.05) are marked by an asterisk; note that only the injections
with trimeric forms of Acrp30 result in significant changes at all
three doses tested. Wild-type Acrp30 causes a significant glucose
reduction only at the highest concentration, and the Acrp30 globular
head alone causes no significant decrease in glucose. E,
comparison of wild-type and Acrp30(C39S) ligands on in vitro
glucose production by primary hepatocytes. Dose was adjusted such that
1× is equivalent to the amount of wild-type, recombinant
Acrp30 required for half-maximal repression of glucose output,
i.e. 0.1× Cys is equivalent to 10% of that
dose. The experiment was performed in the presence of 35 pM
insulin as detailed under "Experimental Procedures." F,
five female FVB mice were injected intravenously at t = 0 with ~200 ng of 125I-labeled wild-type Acrp30 hexamers
or HMW complexes and Acrp30(C39S) protein, sampled by tail bleeds at
various intervals, and remaining serum at cpm/µl was
determined by SDS-PAGE followed by excision of the band and
scintillation counting.
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To determine whether the increased in vivo bioactivity
observed for the Acrp30(C39S) mutant compared with wild-type Acrp30 oligomeric complexes can also be observed in a in vitro
assay, we performed studies on glucose production of primary rat
hepatocytes as utilized previously by us and others (2, 28). We have demonstrated previously (29) in vivo that recombinant Acrp30 purified from mammalian cells acts directly at the level of the liver
affecting hepatic gluconeogenesis in the presence of insulin. Consistent with the increased in vivo potency of the
Acrp30(C39S) ligand, this ligand also inhibits glucose output in
isolated hepatocytes under similar conditions as reported previously.
However, Acrp30(C39S) does so at concentrations at least an order of
magnitude lower than levels required to see similar effects with
wild-type Acrp30 (Fig. 7E).
Because we demonstrate a significant increase in biological activity
with either the mutant Acrp30(C39S) or wild-type Acrp30 reduced to
trimers, we hypothesized that Acrp30 trimers might have a
significantly shortened half-life when injected exogenously into
animals. We iodinated purified wild-type Acrp30 hexamers and HMW
complexes and the mutant Acrp30(C39S) protein. These proteins were
injected intravenously into female FVB mice, and iodinated protein
remaining in serum at various time points was assayed by SDS-PAGE
followed by scintillation counting (Fig. 7F). As expected, Acrp30(C39S) has a considerably shortened serum half-life compared with
the higher order oligomeric forms of the protein. Consistent with
in vitro stability data presented above, Acrp30 HMW
structures have a significantly greater half-life than hexamers (9 versus 4.5 h), but both oligomeric structures circulate
far longer than wild-type Acrp30 reduced with DTT (not shown) or mutant
Acrp30(C39S) (45 min). This increased clearance rate is consistent with
Acrp30 trimers being short-lived intermediates in the pathway of Acrp30 activation and may indeed represent an activated moiety of the protein
generated from the circulating pool of Acrp30.
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DISCUSSION |
Acrp30, like other C1q homologues, trimerizes, and this homotrimer
is the building block for higher order complexes found circulating in
serum (11). Although the existence of these complexes has been known
for some time, regulation at the levels of these complexes has not been
studied to date. The differential regulation of these complexes is an
attractive additional level of control, particularly because overall
levels of the protein are relatively stable.
Previous studies have revealed a sexual dimorphism in Acrp30 levels in
humans, with females having significantly higher circulating levels
than males (15). In this report, we show that there is a similar sexual
dimorphism in terms of circulating Acrp30 complexes, as well; the vast
majority of Acrp30 in males is in the lower molecular weight form,
whereas in females the two complexes are more equally distributed.
Consequently, male and female mice have comparable levels of Acrp30
protein in the hexamer form, but females have a vast excess of the
higher order structure complex. It is an intriguing hypothesis that the
sexually dimorphic absolute Acrp30 levels and differential distribution
between the forms may be partly responsible for the increased insulin
sensitivity of females compared with males and the reduced
susceptibility of females to the deleterious actions of free fatty
acids (30, 31). Furthermore, it leads to the hypothesis that the
hexamer is the "necessary" form of Acrp30, required for
basal activity of this protein; despite the tremendous difference in
absolute levels of circulating Acrp30 between the genders, male and
female animals have similar levels of the hexamer. As we show in the this section, the HMW complex might be necessary for Acrp30 activity upon metabolic challenge.
To determine whether either hypoglycemia or hyperglycemia
differentially affects Acrp30 complexes, mice received either an intraperitoneal injection of insulin at 1 units/kg body weight (sufficient to decrease serum glucose by 75%) or with a gavage of a
high glucose solution (triggering a 50-100% elevation in serum
glucose). Both insulin injection and glucose gavage significantly affected the distribution of Acrp30 complexes in serum, specifically by
lowering the levels of circulating HMW complex. The fact that both
insulin and glucose provoke the same alteration and that insulin action
occurs more rapidly than glucose suggests that insulin is the common
mediator in these two experiments. In both cases, we see a significant
decrease of higher order complexes in serum. Interestingly, we do not
observe a concomitant increase in Acrp30 hexamer levels. This suggests
that the two forms are not interconverted upon stimulation; in
agreement with that, we were unable to demonstrate interconversion of
purified iodinated Acrp30 complexes in vivo, even upon
administration of insulin or glucose. These complexes are stable both
in vitro and in vivo, and we were unable to find
any evidence of a spontaneous interconversion of these two forms.
Chronic insulin treatment has been shown to suppress Acrp30 expression
in the 3T3-L1 adipocyte cell line, and ongoing experiments are testing
the hypothesis that more acute insulin action may trigger differential
production of the two complexes, producing the insulin effect of Acrp30
complexes we demonstrate here.
After demonstrating metabolic regulation of the different Acrp30
complexes, we carefully examined structural aspects of Acrp30 oligomerization. As mentioned previously, Acrp30 is a member of the C1q
family of proteins, and its complex assembly closely mirrors members of
that family of structural homologues. One of these proteins, pulmonary
SP-D has been particularly well characterized with respect to complex
assembly and bioactivity (21). In SP-D, each monomer consists of a
non-collagenous N terminus containing two conserved cysteine residues,
a collagenous domain and a C-terminal, C-type carbohydrate recognition
domain. SP-D trimerization begins with the C-terminal carbohydrate
recognition domain, and triple-helix formation of the collagenous
domain stabilizes the trimeric building block and allows accessibility
of the two N-terminal cysteines to participate in interchain disulfide
bonding to form dodecameric structures that are then secreted. In
vitro studies suggest that the degree of SP-D oligomerization
determines its functionality; trimeric subunits secreted in the
C15S/C20S mutant are comparable in potency to dodecamers in some
aspects of SP-D function (such as lectin-mediated hemagglutinination
inhibition) but are defective as viral agglutinins (22).
Although SP-D is secreted only as a dodecamer, and Acrp30 is secreted
as both hexamers and higher order structures, we wanted to determine
whether the structural aspects of the two proteins were otherwise
similar. We show that the high order species of either serum or
purified Acrp30 is susceptible to acidic (but not basic) pH treatment,
resulting in a hexameric structure. Co-treatment with reducing agent
further reduces the hexamer to a lower molecular weight species
consistent with the molecular mass of an Acrp30 trimer. These results
suggest that covalent, disulfide bonds are required for the formation
of the hexamer; higher order interactions necessary for the HMW complex
structure, although not covalent per se, also critically
depend on proper disulfide bond formation within or in between trimers.
Upon further analysis, the hexamer is more susceptible to reduction
than the high order structure, suggesting that the disulfide bonds in
the hexamer are more accessible than the bonds in the higher order
structure. The model most consistent with all of the reported
observations is that one disulfide bond is formed between two subunits
within a trimer, with the sulfhydryl contributed by the third subunit
bonding with the remaining free sulfhydryl from another trimer, leading
to the formation of a hexamer.
Because of the loss of function of the SP-D trimer with respect to
viral agglutination, we predicted that the homologous mutation in
Acrp30(C39S) would be similarly inactive when compared with the
wild-type. We produced this mutant protein and demonstrated that it was
more susceptible to specific cleavage in cell culture and more prone to
nonspecific trypsin digestion, indicating, most likely, a destabilized
conformation of the collagenous domain. Surprisingly, this mutant
protein was significantly more active than its wild-type counterpart
with respect to lowering serum glucose subsequent to intravenous
administration. Our dose-response curves indicate at least an order of
magnitude difference in bioactivity of the Acrp30(C39S) mutant when
compared with the wild-type ligand, far more so than could be accounted
for by the simple increase in molarity of the ligand compared with the
larger molecular weight complexes. It was also reassuring to see that
reduction of wild-type Acrp30 to the trimeric form also showed the same
effect, because we detected significantly higher bioactivity upon
reduction of the protein. Furthermore, we demonstrate that trimeric and
higher order forms of Acrp30 exert effects on primary hepatocytes;
treatment with both ligands leads to an inhibition of glucose output at the level of the hepatocytes. However, in this in vitro
assay, we also observe approximately ten times higher potency of the mutant protein. This confirms that the trimer is a significantly more
active protein than either the hexamer or HMW complexes.
Although the increased bioactivity of Acrp30(C39S) is clearly evident,
we do not know whether the proteolytic cleavage step that we observe
bears any in vivo relevance. Because the bulk of the
recombinant Acrp30(C39S) is in the cleaved form, we assume that the
cleaved form is mainly responsible for the activity. Importantly, even
the cleaved product still contains the bulk of the collagenous stalk.
The collagenous domain appears to be functionally important, because
equivalent amounts of the bacterially produced head domain do not
display any activity on systemic glucose levels. Nevertheless, as an
important control for these experiments, we injected a purified
preparation of Acrp30 that only has the collagenous domain and lacks
the globular head domain (headless ligand); this ligand showed no
appreciable bioactivity in our in vivo glucose-lowering
assay. There is no indication that either the globular head alone or
the collagenous tail alone have any significant positive or negative
effect in our in vivo glucose-lowering assay, suggesting
that both domains of Acrp30 are required for biological activity.
Alternatively, the globular head domain and/or the remaining
collagenous stalk in Acrp30(C39S) retains a post-translational modification essential for its action on liver that the bacterially produced head domain or the collagenous domain alone lacks. Several groups have recently provided evidence that such post-translational modifications exist and may be important for function. Wang et al. (28) have generated an Acrp30 mutant, substituting four conserved lysines in the collagenous tail with arginines. They thereby
ablated hydroxylation and further glycosylation of these residues,
leading to an inactive protein, unable to inhibit glucose output by
primary hepatocytes in the presence of insulin. However, they could not
distinguish between possible overall structural disturbances to the
collagenous protein by these mutations that may affect oligomerization
from a possible direct involvement of these modifications in Acrp30
function. Furthermore, Kitajima and co-workers (32) have provided
evidence for the presence of another glycosyl modification, an
O-linked sugar group containing an
2,8-disialic residue.
These modifications are not present on bacterially produced material.
The data presented in this report lead us to propose the following
working model for Acrp30 action: HMW Acrp30 complexes circulating in
serum represent a precursor pool that can be activated. Activation is
triggered by metabolic stimuli, such as an increase in serum insulin
levels, which may trigger the induction or activation of a serum
reductase that triggers the dissociation of the high order complex
leading to the transient appearance of the bioactive trimer. In our
studies, we have been unable to visualize trimers (either cleaved
(gAcrp30-like) or uncleaved) circulating in human and mouse serum. This
is consistent with our model of activation and a very transient
half-life of trimer in serum. However, we cannot exclude the
possibility that we are technically limited in our ability to detect
trimers because of incomplete separation from hexameric forms of
Acrp30 in serum. A recent study performed by Tsao et al.
(33) suggested that the majority of Acrp30 (>80%) in serum circulates
as the HMW form, with far less in the hexameric (<10%) and trimeric
(<10%) forms. Our studies on freshly isolated mouse and human serum
samples show a distribution more biased toward hexamers and high order
complexes. However, we do agree with Tsao et al. (33) that
trimers can be generated in vivo, albeit at much smaller
proportions. Once reduced to the basic trimer, Acrp30 may be subject to
proteolysis by membrane-bound proteases found on the cell surface of
target cells, leading to the final active ligand that is rapidly
cleared. This is consistent with the clearance data presented in this
paper, demonstrating that the trimeric Acrp30(C39S) has a significantly
shortened half-life when compared with the endogenous oligomeric
complexes. In this model, the rate-limiting step is the activation of
the serum reductase activity. We are currently trying to establish an
assay that will allow us to isolate and characterize such a serum
activity. A similar activation model, reduction followed by proteolytic
cleavage, has been proposed previously (34) for the plasmin/plasminogen system.
The past few years have revealed many roles for adipocyte-secreted
factors with a wide spectrum of effects, ranging from effects on the
central nervous system (such as the satiety response provoked by
leptin) to roles in the innate immune system (such as adipsin and
interleukin-6). More recently, additional adipokines have been
identified, such as resistin and Acrp30, which we discuss here. Both of
these proteins have been implicated in influencing systemic insulin
sensitivity. A number of studies have shown strong correlative evidence
indicating that Acrp30 influences insulin sensitivity. Matsuzawa and
colleagues (15) demonstrated that Acrp30 levels in serum are decreased
in obese patients, a finding that supported an earlier report that
Acrp30 transcript levels were lower in ob/ob mice compared
with wild-type littermates (12). Further reports showed that
circulating Acrp30 levels were lower in diabetic patients
versus non-diabetic controls (35). Other studies implicated
the genomic locus that encompasses the Acrp30 gene (3q27) as a novel
diabetes susceptibility locus (17). Finally, a study performed on a
cohort of Pima Indians (a patient population with a high propensity
toward obesity and type 2 diabetes mellitus) demonstrated that
decreased circulating Acrp30 levels correlated with the degree of
insulin resistance and hyperinsulinemia (36). These studies all
suggested a role for Acrp30 in insulin responsiveness and perhaps even
a protective effect of increased Acrp30 levels toward development of
type 2 diabetes. These reports, however, all characterized chronic
changes in Acrp30; an acute regulation of serum levels of Acrp30 had
yet to be demonstrated. The experiments described in this paper take a
first step toward the description of an acute regulation at the level
of complex formation. Furthermore, the data presented underline the
high level of complexity of the regulation of this protein, with
regulatory steps taking place at the transcriptional, translational,
and post-translational level.