Structure-Function Studies of the Adipocyte-secreted Hormone Acrp30/Adiponectin

IMPLICATIONS FOR METABOLIC REGULATION AND BIOACTIVITY*

Utpal B. PajvaniDagger , Xueliang Du§, Terry P. CombsDagger , Anders H. BergDagger , Michael W. RajalaDagger , Therese Schulthess, Jürgen Engel, Michael Brownlee§||, and Philipp E. SchererDagger ||**

From the Departments of Dagger  Cell Biology and § Medicine and Pathology and || Diabetes Research and Training Center, Albert Einstein College of Medicine, Bronx, New York 10461 and  Department of Biophysical Chemistry, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland

Received for publication, July 18, 2002, and in revised form, December 20, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

Acrp30/adiponectin is an adipocyte-specific secretory protein that has recently been implicated as a mediator of systemic insulin sensitivity with liver and muscle as target organs. Acrp30 is found as two forms in serum, as a lower molecular weight trimer-dimer and a high molecular weight complex. Little is know about the regulation and significance of these Acrp30 complexes in serum and about the events that lead to the generation of the bioactive ligand. Here, we show that there is a profound sexual dimorphism of Acrp30 levels and complex distribution in serum. Female mice display significantly higher levels of the high molecular weight complex in serum than males. In both females and males, levels of the high molecular weight complex are significantly reduced in response to a systemic increase of insulin. The ratio of the two complexes is restored upon normalization of glucose levels. Structurally, we show that oligomer formation of Acrp30 critically depends on disulfide bond formation mediated by Cys-39. Mutation of Cys-39 results in trimers that are subject to proteolytic cleavage in the collagenous domain. Surprisingly, Acrp30(C39S) or wild-type Acrp30 treated with dithiothreitol are significantly more bioactive than the higher order oligomeric forms of the protein with respect to reduction of serum glucose levels. Furthermore, treatment of primary hepatocytes with trimeric and higher order forms of Acrp30 confirms that the increased bioactivity seen in vivo is reflected in an augmented potency to reduce glucose output in the presence of gluconeogenic stimuli. Combined, these results shed new light on the regulation of this complex protein and suggest a new model for in vivo activation of the protein, implicating a serum reductase activity.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

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 alpha  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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INTRODUCTION
EXPERIMENTAL PROCEDURES
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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 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 DH10beta 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 beta -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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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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.

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.

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).

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).

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.

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 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.

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.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha 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.

    ACKNOWLEDGEMENTS

We thank members of the Scherer laboratory for valuable comments. In addition, we acknowledge the assistance of Yonathan Melman for site-directed mutagenesis and the Albert Einstein Laboratory for Macromolecular Analysis and Proteomics for Edman degradation and sequencing. We also thank Dr. Maximilien Murone (Apotech) for providing purified headless Acrp30 and helpful suggestions on various aspects of the project.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Medical Scientist Training Grant T32-GM07288 (to U. B. P. and M. W. R.), National Institutes of Health National Research Service Award DK61228 (to T. P. C.), an American Diabetes Medical Scientist Training grant (to A. H. B.), and National Institutes of Health Grant 1R01-DK55758 (to P. E. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** To whom correspondence should be addressed. Tel.: 718-430-2928; Fax: 718-430-8574; E-mail: scherer@aecom.yu.edu.

Published, JBC Papers in Press, December 20, 2002, DOI 10.1074/jbc.M207198200

    ABBREVIATIONS

The abbreviations used are: SP, surfactant protein; LMW, low molecular weight; HMW, high molecular weight; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; DTT, dithiothreitol; HEK, human embryonic kidney.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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