Hu Xiao-Bo: Cloning and Expression of Adiponectin and Its Globular Domain,and Measurement of the Biological Activity in Vivo

Short Communication

Cloning and Expression of Adiponectin and Its Globular
Domain,
and Measurement of the Biological Activity in Vivo

HU Xiao-Bo, ZHANG Yu-Jian, ZHANG
Hui-Tang, YANG Sheng-Li, GONG Yi*
( Research Center of Biotechnology, Shanghai Institutes for Biological Sciences,

the Chinese Academy of Sciences, Shanghai 200233, China )

Abstract
3T3-L1-adipocytes produce the adipocyte complement related protein of 30
kD (ACRP30), which is exclusively expressed in differentiated adipocytes.
Decreased expression of ACRP30 correlates with insulin resistance in mouse
models of altered insulin sensitivity. Adiponectin, the human homologue
of ACRP30, circulates in human plasma at high levels. Plasma adiponectin
levels have been reported to be decreased in some insulin-resistant states,
such as obesity and type II diabetes mellitus. Here, full-length adiponectin
and its C-terminal globular head domain (gAdiponectin) were expressed in
Escherichia coli and gAdiponectin was used to immunize a rabbit to obtain
polyclonal antiserum with titer of 10 000. Adiponectin was detected in human
plasma with the use of gAdiponectin anti-serum by Western blot analysis,
which was also detected by gACRP30 anti-serum. Injection in alloxan-treated
rats with purified recombinant fusion adiponectin or gAdiponectin transiently
abolished hyperglycemia. So adiponectin and gAdiponectin might have activity
as a glucose lowering agent and potentially as a therapeutic for metabolic
disease. All these results suggested that the recombinant protein had biological
activity, and provided a useful tool in further studies.

Key words
Adiponectin; gAdiponectin; cloning and expression; diabetes

Traditionally, adipose
tissue is well known to be an organ that passively stores excess energy
as fat. However, in recent years, a more active role of adipose tissue’s
function has been observed： it synthesizes and secretes a variety of biologically
active molecules, including tumor necrosis factor α (TNFα)［1－3］, PAI-1［4］,
representatives of the complement system: C1 inhibitor, C1r［5］, C3a［6, 7］,
factor D［8］ and leptin［9］.
　As an adipocyte specific protein in murine systems, ACRP30［10］, also designated
as AdipQ［11］, has been identified. ACRP30 is a protein of 247 amino acids
consisting of 　an N-terminal collagenous domain and a C-terminal globular
domain. The globular domain shares significant homology to the globular
domains of type VIII and X collagens［12］, subunits of the complement factor
C1q［13］ and the hibernation-regulated proteins hib20, 25 and 27［14］. The
mRNA expression of ACRP30 and its plasma levels are significantly decreased
in both obese and diabetic mice, indicating a possible metabolic role or
relationship to insulin resistance［11］. Consistent with this, Lodish and
colleagues have recently reported that a proteolytic cleavage product of
recombinant Acrp30 increases fatty-acid oxidation in muscles and causes
weight loss in mice［15］.
Its human homologue is adiponectin, which spans 16 kb on chromosome locus
3q27［16, 17］. Its related 4.5 kb cDNA is restricted to adipose tissue［11,
18］. It encodes a protein of 244 amino acids, consisting of a predicted
amino-terminal signal sequence(aa 1－18), followed by a specific sequence(aa
19－41), a set of 22 collagen-repeats(aa 42－107) and a globular head domain
(aa 108－244)［10, 19］. Adiponectin shares significant homologies to its murine
counterpart ACRP30(82% / 89% conservative aa). Although the expression of
adiponectin mRNA is restricted in adipose tissues, its plasma concentrations
are decreased in human obesity and insulin resistance［20］. Adiponectin is
abundantly present in the plasma of normal human in the range from 1.9 to
17 mg/L［20］, accounting for approximately 0.05% of total serum protein［10］.
Adiponectin was purified from plasma as gelatin-binding protein of 28 kD
by use of its affinity to gelatin-cellulofine［19］. However, the physiological
role of adiponectin in humans has not yet been elucidated, in our study,
we have developed a method for expressing and purification it and proved
the product is biological active.

1 Materials and
Methods
1.1 Materials
Rneasy minikit was from Qiangen; RNA PCR kit(AMV) V2.1 and DNA marker,
restriction endonucleases, T4 DNA ligase were purchased from TaKaRa; Trinder
agent from Sigma; experimental animals were from Shanghai Experiment Animal
Center, the Chinese Academy of Sciences; the cloning and expression vector,
E. coli host strain were stored in our laboratory; all other chemicals were
of analytical grade. The primers were synthesized by Sangon Inc., Shanghai.
1.2 Molecular cloning of the adiponectin coding sequence by RT-PCR
Abdominal subcutaneous fat tissue was collected from one female patient
at surgery for myoma uteri, who was of normal weight and had no history
of metabolic or endocrinological disorders, from 30 mg of adipose tissues,
total RNA was extracted using Rneasy Minikit according to the manufacture’s
protocol. About 1 μg of total RNA was reverse transcribed to cDNA using
RNA PCR kit. After that 2 μl of cDNA product were then subjected to PCR
with the following primers to obtain the coding region of adiponectin. Sense
primer: 5′-AATT GAATTC GGT CAT GAC CAG GAA AC-3′, and antisense primer:
5′-AAAA GTCGAC TCA GTT GGT GTC ATG GTA-3′, which encodes a full-length
adiponectin eliminating a leader peptide. These primers encode EcoRI and
SalI restriction sites, indicated by underlines. The PCR amplification mixture
(25 μl) contained 2 μl cDNA, 2.5 μl 10×PCR buffer, 200 μmol/L of each dNTP,
2 u pyrobest polymerase and 0.5 μmol/L of each primer respectively. Amplification
was performed under the following conditions: samples were subjected to
30 cycles of denaturation at 94 ℃ for 1 min, annealing at 55 ℃ for 1 min,
extension at 72 ℃ for 1.5 min , and a final cycle of elongation for 15 min
at 72 ℃.The PCR product was digested with EcoRI and SalI, and ligated into
EcoRI/SalI-digested pET-32a expression vector, creating pET-adiponectin.
The resulting construct, pET-Adiponectin, was confirmed by restriction mapping
and sequencing. An analogous construct encoding the globular head domain
of adiponectin (gAdiponectin) was produced only using a sense primer differently,
the sense primer is 5′-TAAA GAATTC GCC TAT GTA TAC CGC TCA-3′.
1.3 Protein expression and purification
Overnight cultures of E. coli BL21(DE3)transformed with either pET-adiponectin
or pET-gAdiponectin were stored as glycerol stocks at －80 ℃, streaked for
confluency on LB-agar plates containing 0.1 g/L of ampicillin and cultured
at 37 ℃. Bacteria, from a single plate, were collected, inoculated into
400 ml LB containing 0.1 g/L ampicillin, and shaken at 37 ℃ at 250 r/min,
to an optical absorbance of 0.4－0.6 at 600 nm, then isopropylthio-β-D-galactoside(IPTG)
was added to 0.4 mmol/L and cells were cultured for an additional 2 h. Cultures
were centrifuged at 4000 g for 10 min. The cells were suspended in 50 mmol/L
Tris・HCl (pH 8.0) for 1 h and added Triton X-100 at the final concentration
at 0.2% and sonicated. The suspended buffer was centrifuged and the pellet
was washed by the same treatment. The pellet, inclusion body was precipitated
and solubilized by 100 mmol/L Tris・HCl (pH 8.0) containing 7 mol/L guanidine
HCl, 1% β-mercato-ethanol. The solubilized protein was refolded in the presence
of 200 volumes of 2 mol/L urea, 20 mmol/L Tris・HCl (pH 8.0) for 3 days at
4 ℃. The refolded protein was concentrated by centrifugal filter, dialyzed
with 20 mmol/L Tris・HCl (pH 8.0). Purification of adiponectin and gAdiponectin
were done with Ni2+/NTA according to the manufacture’s protocol, briefly,
samples were loaded onto an equilibrated 2 ml Ni2+/NTA affinity matrix.
The column was washed with 20 ml binding buffer, then eluted contaminating
proteins from the resin at low concentrations of imidazole(0.075 mol/L)with
10 ml. The recombinant proteins were eluted with the addition of binding
buffer containing 0.25 mol/L imidazole.
1.4 Generation of specific antibody
　　Immunization of rabbit was done by injecting a rabbit four times with
a total of 400 μg gAdiponectin within 2 months according to the protocol
of reference［21］. After measuring the antiserum with titer of 10 000 by
Western blot analysis, the rabbit blood was obtained through carotid, which
were put on 4 ℃ overnight, then the antiserum were separated.
1.5 Western blot
　　1 ml of human serum was boiled for 5 min in 2×sample buffer and separated
by 12% SDS-PAGE. After transferring to a nitrocellulose membrane(1 h, 100
V), the membrane was blocked (1 h, RT) in TBS-T containing 50 g/L milk powder.
The blot was incubated in polyclonal anti-gAdiponectin antibody for 1 h
at RT. After five washes in TBS-T, the HRP-conjugated second antibody was
added (1∶1000, 1 h, RT). After washes in TBS-T (3×10 min) and TBS (1×5 min),
the blot was developed according to the manufacturer’s protocol.
1.6 Detection of the biological activity of adiponectin and gAdiponectin
in diabetic rat models
　 We obtained type I diabetic rat models by injecting normal rats with alloxan
180 μg/g BW, 2 days later these rats developed severe insulinopenia and
hyperglycemia. We measured the glucose levels with Trinder, and the rats
whose glucoses levels>2.5 g/L were used for subsequent experiments. We
divided them into five teams.

Fig.1 Identification
of the amplified adiponectin and gAdiponectincoding sequence and the plasmid
pET-adiponectin and pET-gAdiponectin
1, 5, DNA marker DL2000; 　2, the sequence of gAdiponectin (EcoRI/SalI
fragment) by RT-PCR;　3, the sequence of adiponectin (EcoRI/SalI fragment)
by RT-PCR; 4, DNA marker λ/HindIII;　6, pET-gAdiponectin vector linearized
by EcoRI/SalI enzymes;　7, pET-adiponectin vector linearized by EcoRI/SalI
enzymes.

The first team were
injected 1%NaCl to the intraperitoneal cavity, the second team were injected
purified Trx, these two teams were served as negative control; the third
were injected Metformin Hydrochloride as positive control; and the forth
were injected purified Trx-adiponectin fusion protein; the fifth were injected
purified Trx-gAdiponectin fusion protein by the same way. Importantly samples
were always injected into rats in the morning in a postabsorptive state,
and food was withdrawn after injection. Blood collection was performed through
tail bleeds. Blood glucose levels were measured by Trinder-assay. All injections
and measurements were performed in males.

2 Results
2.1 Cloning of the coding sequence of adiponectin and gAdiponectin cDNA
　 From adipocyte tissues, total RNA was isolated, and the coding region
of adiponectin and gAdiponectin were obtained by RT-PCR with the use of
the gene specific primers, which are based on the cDNA sequences reported.
The amplified full-length open reading frame of adiponectin was 678 bp and
gAdiponectin was 408 bp. pET-adiponectin and pET-gAdiponectin were confirmed
by restriction map (EcoRI/SalI) (as shown in Fig.1).
2.2 Expression and purification of recombinant fusion adiponectin and
gAdiponectin
　The E. coli host strain BL21(DE3)cells transformed with the expression
plasmid pET-Adiponectin or pET-gAdiponectin produced recom-binant fusion
protein as inclusion bodies. SDS-PAGE analysis revealed that recombinant
adiponectin and gAdiponectin accumulated up to 33% and 35% of the total
proteins of E.coli respectively, with an expected molecular weight of 42
kD and 34 kD, respectively. The fusion protein was purified by a single-step
affinity chromatography on a nickel affinity resin column. After elution,
the free recombinant protein were obtained, the yield of the protein was
about 100 mg/L of induced culture (as shown in Fig. 2).

Fig.2 SDS-PAGE
analysis of adiponectin and gAdiponectin
1, purified recombinant pET-gAdiponectin fusion protein; 2, purified
recombinant pET-adiponectin fusion protein; 3, 4, protein extracted from
BL21(DE3) transformed with pET-adiponectin and pET-gAdiponectin after IPTG
induction, respectively; 5, 6, protein extracted from BL21(DE3) transformed
with pET-adiponectin and pET-gAdiponectin but without IPTG induction, respectively;
M, protein molecular mass markers.

2.3 Adiponectin
is a secretory protein found in blood
As shown in Fig. 3, a protein in human plasma was detected by Western
blot analysis, which is accordance with the results of references［10］ and
［15］.
2.4 Effects of adiponectin and gAdiponectin in diabetic rats
Type I diabetic rats were obtained by treating them with alloxan. These
rats display hyperglycemia. To investigate the physiological role of adiponectin
and gAdiponectin, we injected type I diabetic rats Trx-adiponectin or Trx-gAdiponectin
5 μg/g body weight in the morning in a postabsorptive state, and again after
1 h. Serum glucose levels dropped significantly com-pared with negative
control at 4 h after injection

Fig.3 Western
blot analysis of the human serum using the specific antibody
(A) Adiponectin was detected by using the anti-gAdiponectin.(B) adiponectin
was detected by using the anti-gACRP30.

（as shown in Table
1）. We measured glucose levels at sixth and eighth hour, but observed no
significant differences between negative control and adiponectin /gAdiponectin
injected rats. Since the food was withdrawn after injection, serum glucose
levels dropped similarly after four hours.

Table 1 Effects
of adiponectin and gAdiponectin on plasma glucose levels in type I diabetic
rats

Five groups rats
(n=4) were treated with saline, Trx, metformin hydrochlorid, Trx-adiponectin
and Trx-gAdiponectin as described in Methods. Blood samples were taken at
0, 2, 4, 6 and 8 h separately after test, and glucose concentrations were
determined. Treatment with Trx-adiponectin and Trx-gAdiponectin significantly
decreased the glucose levels compared with negative control at 2 and 4 h
after test. Data are represented asx*P＜0.01
vs. saline group. Statistical analysis was done with the use of SPSS software,
Version 11.0.

3 Discussion

ACRP30 was first identified as a protein expressed and secreted by differentiated
murine 3T3-L1 adipocytes［10］. It was adipose-specific protein abundantly
present in the circulation［10］. Berg et al.［22］ demonstrated that elevating
the plasma level of ACRP30 two-to-three fold by intraperitoneal injection
of recombinant ACRP30 transiently decreased basal plasma glucose levels
without increasing plasma insulin concentrations. This effect was seen in
ob/ob mice, a type II diabetic model, as well as in NOD mice, a type I diabetic
model. Fruebis et al.［15］ showed that when injected into mice gACRP30 accelerated
fatty acids oxidation in muscle and decreased plasma glucoses levels. ACRP30
holds great promise as a pharmacological agent to treat metabolic disease.
Unlike potential cellular targets of pharmacological intervention, ACRP30
is a circulating hormone and its action can be achieved by injection of
recombinant forms of the protein. So ACRP30 may represent a novel approach
to control diabetes mellitus and obesity.
As a human homologue of ACRP30, adiponectin’s plasma levels in the diabetic
subjects were lower than those in nondiabetic subjects, the plasma Adiponectin
concentrations of diabetic patients with coronary artery disease were lower
than those of diabetic patients without coronary artery disease, and it
was the case both for men and women［23］. It was shown that the level of
plasma Adiponectin correlated positively with insulin-stimulated whole body
glucose disposal［24］, and it was remarkable that the most significant association
found was the negative correlation between plasma Adiponectin levels and
insulin resistance and hyperinsulinemia［24］. The gene for human adiponectin
spanning 16 kb is located at chromosome 3q27［16, 17］. The mouse ACRP30 gene
maps to the telomere region of chromosome 16, an area syntenic to human
chromosome 3q27［25］. Furthermore, Vionnet et al.［26］ mapped a diabetes-susceptibility
locus in a native French cohort to human chromosome 3q27, where the gene
encoding Adiponectin is located. All these data indicated that Adiponectin
might have potential function in curing diabetes.
Protein cleavage is a feature in the complement cascade activation, and
the adiponectin homologue precerebellin is proteolytically cleaved into
the active cerebellin peptide［27］. Furthermore, the hib family of proteins
is found in blood as a complex that indudes a homolog of α1-antitrgpsin［28］,
suggesting regulation by protease. Here we cloned gAdiponectin by RT-PCR,
and expressed it. The antibody made of it can detect a protein in human
serum which can also be detected by the antibody of mouse gACRP30 (kindly
presented by Professor Chrispher, Hug), that supports the fact that adiponectin
gene product is secreted by the adipocytes and it appears in the blood stream.
When injected adiponectin or gAdiponectin, glucose levels of rats had significant
decline in contrast with negative control. Because gAdiponectin had the
same effect as adiponectin, we presumed that adiponectin undergoes proteolysis
to generate the C-terminal fragment. But we did not detect gAdiponectin
in human serum by Western blotting. Maybe its amount is too small to be
detected, so we should do other experiments more sensitively to reveal if
gAdiponectin is present in the human plasma or not. Whether gAdiponectin
is generated from proteolytic cleavage of full-length adiponectin or from
alternative splicing of adiponectin mRNA remains to be determined. We injected
the human protein to experimental animals for the first time, and they proved
to have biological activity. That confirmed that adiponectin shares significant
homologies to its rat counterpart ACRP30［29］. Our results suggested that
adiponectin was another adipose tissue-derived hormone that affects glucose
metabolism.

References
1 Hotamisligil GS, Shargill NS, Spiegelman BM. Adipose expression of
TNF-α: Direct role in obesity-linked insulin resistance. Science, 1993,
259: 87－91
2 Hotamisligil GS, Spiegelman BM. Tumor necrosis factor-α: A key component
of the obesity diabetes link. Diabetes, 1994, 43: 1271－1278
3 Hotamisligil GS, Arner P, Caro JF, Atkinson RL, Spiegelman BM. Increased
adipose tissue expression of tumor necrosis factor-alpha in human obesity
and insulin resistance. J Clin Invest, 1995, 95: 2409－2415
4 Shimomura I, Funahashi T, Takahashi M, Maeda K, Kotani K, Nakamura T,
Yamashita S et al. Enhanced expression of PAI-1 in visceral fat: Possible
contributor to vascular disease in obesity. Nat Med, 1996, 2: 800－803
5 Maeda K, Okubo K, Shimomura I, Mizuno K, Matsuzawa Y, Matsubara K. Analysis
of an expression profile of genes in the human adipose tissue. Gene， 1997,
190: 227－235
6 Baldo A, Sniderman AD, St-Luce S, Avramoglu RK, Maslowska M, Hoang B,
Monge JC et al. The adipsin-acylation stimulating protein system and regulation
of intracellular triglyceride synthesis. J Clin Invest, 1993, 92: 1543－1547
7 Cianflone K, Maslowska M, Sniderman A. The acylation stimulating protein-adipsin
system. Int J Obes Relat Metab Disord, 1995, 19, Suppl. 1: S34－S38
8 Cook KS, Min HY, Johnson D, Chaplinsky RJ, Flier JS, Hunt CR, Spiegelman
BM. Adipsin: A circulating serine protease homolog secreted by adipose tissue
and sciatic nerve. Science, 1987, 237: 402－405
9 Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM. Positional
cloning of the mouse obese gene and its human homologue. Nature, 1994, 372:
425－432
10 Scherer PE, Williams S, Fogliano M, Baldini G, Lodish HF. A novel serum
protein similar to C1q, produced exclusively in adipocytes. J Biol chem,
1995, 270(45): 26746－26749
11 Hu E, Liang P, Spiegelman BM. AdipoQ is a novel adipose-specific gene
dysregulated in obesity. J Biol Chem, 1996, 271(18): 10697－10703
12 Reichenberger E, Beier F, Luvalle P, Olsen BR, von der Mark K, Bertling
WM. Genomic organization and full-length cDNA sequence of human collagen
Ⅹ. FEBS Lett, 1992, 311: 305－310
13 Reid KB, Gagnon J, Frampton J. Completion of the amino acid sequences
of the A and B chains of subcomponent C1q of the first component of human
complement. Biochem J, 1982, 203: 559－569
14 kondo N, Kondo J. Identification of novel blood proteins specific for
mammalian hibernation. J Biol Chem, 1992, 267: 473－478
15 Fruebis J, Tsao TS, Javorschi S, Ebbets-Reed D, Erickson MR, Yen FT,
Bihain BE et al. Proteolytic cleavage product of 30-kDa adipocyte complement-related
protein increases fatty acid oxidation in muscle and causes weight loss
in mice. Proc Natl Acad Sci USA, 2001, 98: 2005－2010
16 Saito K, Tobe T, Minoshima S, Asakawa S, Sumiya J, Yoda M, Nakano Y et
al. Organization of the gene for gelatin―binding protein (GBP28). Gene,
1999, 229: 67－73
17 Takahashi M, Arita Y, Yamagata K, Matsukawa Y, Okutomi K, Horie M, Shimomura
I et al. Genomic structure and mutations in adipose-specific gene, adiponectin.
Int J Obes Relat Metab Disord, 2000, 24(7): 861－868
18 Maeda K, Okubo K, Shimomura I, Funahashi T, Matsuzawa Y, Matsubara K.
cDNA cloning and expression of a novel adipose specific collagen-like factor,
apM1(adipose most abundant gene transcript 1). Biochem Biophys Res Commun,
1996, 221(2): 286－289
19 Nakano Y, Tobe T, Choi-Miura NH, Mazda T, Tomita M. Isolation and characterization
of GBP28, a novel gelatin-binding protein purified from human plasma. J
Biochem, 1996, 120(4): 803－812
20 Arita Y, Kihara S, Ouchi N, Takahashi M, Maeda K, Miyagawa J, Hotta K
et al. Paradoxical decrease of an adipose-specific protein, adiponectin,
in obesity. Biochem Biophys Res Commun, 1999, 257(1): 79－83
21 Sambrook J, Fritsch E F, Maniatis T. Molecular Cloning: A laboratory
Manual, 2nd ed. New York: Cold Spring Harbor Laboratory Press, 1989
22 Berg AH, Combs TP, Du Xl, Brownlee M, Scherer PE. The adipocyte-secreted
protein Acrp30 enhances hepatic insulin action. Nat Med, 2001, 7: 947－953
23 Hotta K, Funahashi T, Arita Y, Takahashi M, Matsuda M, Okamoto Y, Iwahashi
H et al. Plasma concentrations of a novel, adipose-specific protein, adiponectin,
in type 2 diabetic patients. Arterioscler Thromb Vasc Biol, 2000, 20: 1595－1599
24 Weyer C, Funahashi T, Tanaka S, Hotta K, Matsuzawa Y, Pratley RE, Tataranni
PA. Hypoadiponectinemia in obesity and type 2 diabetes: Close association
with insulin resistance and hyperinsulinemia. J Clin Endocrinol Metab, 2001,
86: 1930－1935
25 Das K, Lin Y, Widen E, Zhang Y, Scherer PE. Chromosomal localization,
expression pattern, and promoter analysis of the mouse gene encoding adipocyte-specific
secretory protein Acrp30. Biochem Biophys Res Commun, 2001, 280: 1120－1129
26 Vionnet N, Hani El-H, Dupont S, Gallina S, Francke S, Dotte S, De Matos
F et al. Genomewide search for type 2 diabetes-susceptibility genes in French
whites: Evidence for a novel susceptibility locus for early-onset diabetes
on chromosome 3q27-qter and independent replication of a type 2 diabetes
locus on chromosome 1q21-q24. Am J Hum Genet, 2000, 67: 1470－1480
27 Kishore U, Reid KB, Modular organization of proteins containing C1q-like
globular domain. Immunopharmacology, 1999, 42: 15－21
28 Takamatsu N, Kojima M, Taniyama M, Ohba K, Uematsu T, Segawa C, Tsutou
S et al. Expression of multiple α1-antitrypsin-like genes in hibernating
species of the squirrel family. Gene, 1997, 204: 127－132
29 Tsao TS, Lodish HF, Fruebis J. Acrp30, a new homone controlling fat and
glucose metabolism. Eur J Pharmacol, 2002, 404: 213－221