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Originally published online as doi:10.1189/jlb.0905521 on January 24, 2006

Published online before print January 24, 2006
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(Journal of Leukocyte Biology. 2006;79:803-808.)
© 2006 by Society for Leukocyte Biology

Different effects of adiponectin isoforms in human monocytic cells

Markus Neumeier, Johanna Weigert, Andreas Schäffler, Gabriele Wehrwein, Ulf Müller-Ladner, Jürgen Schölmerich, Christian Wrede and Christa Buechler1

Department of Internal Medicine I, University of Regensburg, Germany

1Correspondence: Department of Internal Medicine I, University of Regensburg, D-93042 Regensburg, Germany. E-mail: christa.buechler{at}klinik.uni-regensburg.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Adiponectin (APM) is an adipocyte-derived adipokine with immunosuppressive, antidiabetic, and antiatherosclerotic properties. Low molecular weight (LMW)- and higher molecular weight (HMW)-APM circulate in the serum and activate different signaling pathways. We were interested to see whether LMW-APM exerts different effects on monocytic cells compared with the HMW isoform. Therefore, the effects of recombinant LMW-APM produced in insect cells and the APM from higher eukaryotic cells containing HMW forms on monocytic cells were investigated with respect to apoptosis and inflammation. LMW- and HMW-APM induce apoptosis in nondifferentiated THP-1 cells, reduce macrophage scavenger receptor (MSR) A mRNA expression, and stimulate phosphorylation of adenosine monophosphate-activated protein kinase (AMPK). However, HMW-APM induces the secretion of interleukin (IL)-6 in human monocytes and THP-1 cells but does not suppress lipopolysaccharide (LPS)-induced IL-6 secretion. In contrast, LMW-APM reduces LPS-mediated IL-6 release and furthermore, stimulates IL-10 secretion, most likely by reducing the abundance of inhibitor of nuclear factor (NF)-{kappa}B kinase ß, leading to a diminished nuclear translocation of NF-{kappa}B p65. Our data indicate that the different APM isoforms do share common effects on monocytic cells but also induce isoform-specific responses. Although apoptosis, the activation of AMPK, and the reduction of MSR are mediated by all APM isoforms, only LMW-APM displays anti-inflammatory properties.

Key Words: endotoxin • inflammation • adipokine • diabetes


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human monocytes/macrophages accumulate in adipose tissue in obesity and in the subendothelial space in early atheroma and are a major source of inflammatory cytokines [1 , 2 ]. The adipokine adiponectin (APM) is known to exert anti-inflammatory and insulin-sensitizing effects. It is highly abundant in human serum and is secreted by adipose tissue in inverse relation to the body mass index [3 , 4 ]. APM reduces the release of the proinflammatory cytokines interleukin (IL)-6, tumor necrosis factor (TNF), and interferon-{gamma} (IFN-{gamma}) from activated monocytes and macrophages [5 6 7 ] and increases the secretion of anti-inflammatory mediators such as IL-10 and IL-1 receptor antagonist [7 ]. The nuclear factor (NF)-{kappa}B transcription factor is a major regulator of proinflammatory cytokine expression in lipopolysaccharide (LPS)-stimulated macrophages, and I{kappa}B, which is the inhibitor of NF-{kappa}B, prevents nuclear translocation of this transcription factor. I{kappa}B kinase (IKK) phosphorylates I{kappa}B in response to LPS stimulation, and I{kappa}B is subsequently degraded by the proteasome, thus liberating NF-{kappa}B [8 ]. APM reduces nuclear translocation of the NF-{kappa}B p65 subunit, and this partly explains the anti-inflammatory properties of this protein [5 ].

Besides these anti-inflammatory effects, the uptake and storage of lipids in macrophages are diminished. APM-treated macrophages have reduced phagocytosis [6 , 7 ], lowered expression of class A macrophage scavenger receptor (MSR), and reduced acyl-CoA:cholesterol acyltransferase-1 abundance [9 ]. In the apolipoprotein E-deficient mouse model, APM also reduces expression of the MSR in foam cells and lesional TNF levels [10 , 11 ]. In addition to these antiatherogenic and immunoregulatory functions, APM inhibits cell proliferation by inducing apoptosis in myelomonocytic progenitor cells [6 ].

APM circulates in blood as trimers, hexamers, and higher molecular weight (HMW) complexes [12 ]. A proteolytic cleavage product of APM, which includes its globular head group, has also been found in human plasma [13 ]. The biological activity of APM depends on its high order structure with different oligomeric complexes activating different pathways. Whereas HMW-APM correlates better with glucose tolerance than total APM in Indo-Asian males [14 ] and furthermore, was most active in reducing blood glucose in mice [15 ], only trimeric and globular APM increase phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) in myocytes [16 , 17 ]. AMPK is a fuel-sensing enzyme, and upon a decrease in the energy state of a cell, AMPK is phosphorylated. Activated AMPK phosphorylates a variety of intracellular proteins to increase adenosine 5'-triphosphate (ATP) generation and reduce ATP use [18 ].

The current literature indicates that APM isoforms may differ in their biological activity, and therefore, to better understand the effects of APM, not only the absolute amount but also the distribution of isoforms have to be considered.

Monocytes are important cells in innate immunity, inflammation, and cardiovascular disease, and the influence of APM on monocytic cells has been investigated in several studies [5 6 7 , 19 , 20 ]. However, the impact of APM isoforms on monocytes has not been analyzed in more detail yet. To understand further the molecular mechanisms of APM isoforms, the effects of low molecular weight (LMW)-APM and HMW-APM were investigated in THP-1 cells and primary monocytes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
RPMI medium was obtained from Gibco-BRL (Karlsruhe, Germany). LPS, Escherichia coli serotype 055:B5, and other laboratory reagents and chemicals were purchased from Sigma Chemical Co. (Deisenhofen, Germany). Oligonucleotides were synthesized by Metabion (Planegg-Martinsried, Germany). DuoSet enzyme-linked immunosorbent assay (ELISA) development system for human IL-6, IL-10, and APM was purchased from R&D Systems (Wiesbaden-Nordenstadt, Germany). Recombinant macrophage-colony stimulating factor (rM-CSF) and mouse cell line-expressed rAPM were from R&D Systems (purity is more than 90%; endotoxin is less than 1.0 EU). The cytotoxicity detection kit (Roche, Penzberg, Germany) was used to determine lactate dehydrogenase (LDH) activity. Antibodies to IKKß, phosphorylated (P)-AMPK, AMPK, caspase 3, 8, and 9, signal transducer and activator of transcription (STAT)1, NF-{kappa}B p65, and phospho-Akt (Ser473) were obtained from New England Biolabs (Frankfurt am Main, Germany). Phosphatidylinositol-3 kinase (PI-3K) p85 antibody was from Upstate Biotechnology (Lake Placid, NY), and the gradient gel for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was from Bio-Rad (Hercules, CA). APM antibody raised in chicken was used for immunoblot as recently described [21 ].

Expression and purification of APM using insect cells
Full-length APM cDNA (REFSEQ NM_004797.1) was amplified with the primers 5'-gggggatccttatgctgttgctgggagctgttcta-3' and 5'-gggtctagaccgttggtgtcatggtagagaagaaagcc-3' from human adipose tissue mRNA (Biocat, Heidelberg, Germany) and was cloned in-frame with the 6x HN tag in the pDNR dual donor vector (BD Biosciences, Palo Alto, CA) using the BamHI and XbaI sites (bold in the primer sequence). Following sequence verification, the DNA was transferred to the BacPak9 vector using the Cre/lox procedure (BD Biosciences). Recombinant baculovirus was generated by the BacPAKTM baculovirus expression system (BD Biosciences). Insect H5 cell (Invitrogen, Karlsruhe, Germany) supernatants were collected for purification of APM with the BD TalonTM purification kit (BD Biosciences).

Cultivation of cells
Blood leukocytes were isolated from whole blood as recently described [22 ]. Cells were cultivated in macrophage serum-free medium (Invitrogen) with M-CSF (50 ng/ml) for 1 day before stimulation with rAPM. The study protocol was approved by the local ethics committee and was carried out in accordance with the Helsinki guidelines.

THP-1 cell line was obtained from the American Type Culture Collection (Manassas, VA), and cells were cultured in RPMI-1640 medium (Sigma Chemical Co.), supplemented with 10% fetal calf serum (Biochrom, Southborough, MA), and incubated in 10% CO2 in air at 37°C. To induce phagocytic differentiation, THP-1 cells were cultured in the presence of 160 nM phorbol 12-myristate 13-acetate (PMA) for 24 h [23 ].

Monitoring of gene expression by real-time reverse transcriptase-polymerase chain reaction (RT-PCR)
Real-time RT-PCR was performed as recently described [24 ]. The primers for ß-actin were ß-actin uni: 5'-ccagggtgtgatggtgggaatg-3'; ß-actin rev: 5'-cgcacgatttccctctcagctc-3'. The primers for MSR were MSR uni: 5'-tttcttcatgtaccagatgctga-3'; MSR rev: 5'-tcacagattcggagcagcta-3'. A standard curve was used to account for different PCR efficiencies.

SDS-PAGE, silver staining, and immunoblotting
The cells were harvested, washed in phosphate-buffered saline (PBS), and solubilized in radio immunoprecipitation assay buffer. Proteins were separated by SDS-PAGE and were transferred to nitrocellulose membranes. Incubations with antibodies were performed in 5% nonfat dry milk in Tris-buffered saline, 0.1% Tween. Detection of the immune complexes was carried out with the enhanced chemiluminescence Western blot detection system (Amersham Pharmacia, Deisenhofen, Germany). Silver staining of the gels was performed as described by Heukeshoven and Dernick [25 ].

ELISA
Cells (300,000) were incubated as indicated. The supernatant (100 µl) was used for IL-6 and IL-10 determinations. The ELISAs were performed as recommended by the distributor.

Statistics
Data are represented as Box Plots, indicating the mean, the upper and lower quartile, and the highest and the lowest value in the data set (SPSS 12.0 for Windows). Variances of the data sets were compared by F-test (MS Excel). Statistical differences were analyzed by paired, two-tailed Student’s t-test, and a value of P < 0.05 was regarded as statistically significant (MS Excel).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Analysis of baculovirus- and mouse cell line-produced APM
rAPM was purified from the supernatant of H5 cells or was obtained from R&D Systems, which is recombinant protein expressed in the mouse myeloma cell line NS0. SDS-PAGE, under nonreducing and nonheat-denaturing conditions, was used to analyze various APM preparations. rAPM from the mouse myeloma cell line shows a 60-kDa protein band, and HMW forms display HMW bands starting from ~150 kDA (Fig. 1A , lanes 2 and 3). APM from insect cells had a molecular weight of 90 kDa (Fig. 1A , lane 1), most likely representing trimeric APM. Immunoblot analysis with an APM-specific antibody raised in chicken confirmed that the 90-kDa protein is APM. The baculovirus-produced APM was stable and was not converted to LMW forms by incubation at 95°C for 5 min in the absence or the presence of ßME (Fig. 1B , lanes 1 and 4). When APM from the mouse cell line was denatured at 95°C prior to nonreducing SDS-PAGE, only a 60-kDa form was detected (Fig. 1B , lanes 5 and 6). Reducing SDS-PAGE of heat-treated protein converted the 60-kDa form to a 30-kDa isoform (Fig. 1B , lanes 2 and 3). Therefore, baculovirus-expressed APM is referred to as LMW-APM and mouse cell line expressed as HMW-APM to consider that besides the 60-kDa form, which is most likely not biologically active, HMW-APM is also existent.


Figure 1
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  		Figure 1. Analysis of baculovirus-expressed APM and APM purified from a mouse cell line. (A) Mouse cell line-expressed APM (HMW-APM) was analyzed by SDS-PAGE on a 4–15% gradient gel without prior heat treatment and reduction of disulfide bonds. Dimers (60 kDa) and HMW forms are detected in two different batches of recombinant protein. (M=Molecular weight marker; RT = room temperature) (B) Baculovirus-expressed APM (LMW-APM) and APM purified from a mouse cell line (HMW-APM) were separated by SDS-PAGE on a 4–20% gradient gel. Purified protein was heated to 95°C for 10 min in Laemmli buffer with or without ß-mercaptoethanol (+ßME or –ßME, respecitvely). Baculovirus-expressed APM shows a molecular weight of 90 kDa under both conditions. Mouse cell line-expressed APM is a monomer of 30 kDa when treated with heat and reducing agent. Under nonreducing conditions, only 60 kDa forms are detected.

 
LMW-APM and HMW-APM induce apoptosis in nondifferentiated THP-1 cells
The effect of LMW-APM and HMW-APM on cell viability was investigated in THP-1 cells. Three thousand THP-1 cells were cultivated in RPMI without serum for 48 h and were treated with PBS as a control, LMW-APM (1 µg/ml), or 10 µg/ml HMW-APM. In three independent experiments, 2162 ± 142 viable cells were counted in the control wells, whereas 1548 ± 167 (P=0.03 vs. control) were viable in LMW-APM-treated wells and 1402 ± 134 (P=0.016 vs. control) in HMW-APM-incubated wells (Fig. 2A ). LDH activity as a measure of cell toxicity revealed similar results. Relative LDH activity in controls was 26.7 ± 1, 69 ± 7.5 in LMW-APM-treated cells, and 83.7 ± 6 in HMW-APM-incubated THP-1 (Fig. 2B) . Apoptotic cells were detected by flow cytometry using annexin V and propidium iodide staining, and less than 1% of the cells was found to be necrotic. Analysis of procaspase 8, 2, and 3 in THP-1 cells treated for 8 h or 24 h with rAPM revealed a reduced abundance of the procaspases in 24 h-incubated cells and an increase of active caspase 3 when compared with controls (Fig. 2C) .


Figure 2
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  		Figure 2. Effect of rAPM on apoptosis and phosphorylation of AMPK. (A) Three thousand THP-1 cells were cultivated in RPMI without serum for 48 h. Viable cells were counted, and the results of PBS-incubated controls, cells treated with baculovirus-expressed APM (LMW-APM, 1 µg/ml), or mouse cell line-expressed APM (HMW-APM, 10 µg/ml) are given. (B) LDH activity of the cells cultivated as described above is shown. (C) THP-1 cells were cultivated in RPMI without serum for 8 h and 24 h with LMW-APM or HMW-APM. An immunoblot was performed to detect procaspases 8, 2, and 3 and active caspase 3. (D) PMA-treated THP-1 cells were incubated with LMW-APM or HMW-APM for 60 min. P-AMPK and AMPK were analyzed by immunoblotting. Significance was calculated by paired, two-tailed Student’s t-test and is indicated by an asterisk for values of P < 0.05.

 
LMW-APM and HMW-APM reduce MSR mRNA and activate AMPK
The effect of LMW-APM and HMW-APM on the mRNA expression of the MSR was investigated using real-time RT-PCR. MSR mRNA in PMA-differentiated THP-1 cells treated with 2 µg/ml LMW-APM was reduced to 77 ± 7% (P=0.01), 1 µg/ml reduced MSR mRNA to 88 ± 10% (P=0.02), whereas 0.5 µg/ml did not alter MSR mRNA. HMW-APM (10 µg) suppressed MSR mRNA to 38 ± 10% (P=0.01; not shown). Thr-172 phosphorylation of the {alpha} subunit of AMPK was also analyzed. PMA-differentiated cells were treated with 1 µg/ml LMW-APM or 10 µg/ml HMW-APM for 60 min, and an enhanced phosphorylation of AMPK was detected with each of the APM isoforms (Fig. 2D) .

Anti-inflammatory effect of LMW-APM
PMA-differentiated THP-1 cells were used to study the influence of LMW-APM and HMW-APM on LPS-mediated cytokine secretion. The determination of the number of viable cells and LDH activity in the supernatants showed that there are no cytotoxic effects of rAPM. Immunoblot analysis demonstrated that caspase 3 is not activated (data not shown).

IL-6 in the supernatant of cells treated with LPS alone for 18 h was 116 ± 8.3 pg/ml and was reduced (P=0.009) significantly by LMW-APM (1 µg/ml) to 60 ± 4.3 pg/ml. HMW-APM (10 µg/ml) did not alter LPS-induced IL-6 secretion, and 119.8 ± 10.7 pg/ml IL-6 was found (Fig. 3A ). IL-10 was determined in differentiated THP-1 incubated with LPS (1 µg/ml) and rAPM in parallel for 26 h. IL-10 in control supernatants was 58 ± 9.7 pg/ml, LMW-APM induced IL-10 to 108 ± 13 pg/ml (P=0.01), whereas HMW-APM did not significantly affect IL-10 release (63±16 pg/ml; Fig. 3B ). The LMW-APM is stable (Fig. 1B) , and differentiated THP-1 cells were incubated with LPS alone or in combination with 1 µg/ml LMW-APM or 1 µg/ml LMW-APM and kept at 95°C for 5 min before being added to the cells. IL-6 level in controls was 91 ± 19.6 pg/ml, in LMW-APM-treated cells, it was 59 ± 10 pg/ml (P=0.001 vs. control), and in THP-1 incubated with heat-treated LMW-APM, it was 61 ± 14 pg/ml (P=0.006 vs. control; Fig. 3C ).


Figure 3
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  		Figure 3. Effect of rAPM on LPS-induced cytokine secretion and IL-6 release in nonactivated cells. (A) PMA-differentiated THP-1 cells (300,000) were incubated with 1 µg/ml LPS and LMW-APM (1 µg/ml) or HMW-APM (10 µg/ml) for 18 h. IL-6 was determined in the supernatants. (B) PMA-differentiated THP-1 cells (300,000) were incubated with 1 µg/ml LPS and LMW-APM (1 µg/ml) or HMW-APM (10 µg/ml) for 26 h. IL-10 was determined in the supernatants. (C) PMA-differentiated THP-1 cells were incubated with LPS and LMW-APM (1 µg/ml) or LMW-APM (1 µg/ml), which was freshly incubated at 95°C for 5 min. IL-6 was determined in the supernatants. (D) PMA-differentiated THP-1 cells were incubated with LMW-APM (1 µg/ml) or HMW-APM (10 µg/ml) for 1 h (0). LPS (1 µg/ml) was added, and cells were cultivated for an additional 20, 40, and 60 min. IKKß was determined by the immunoblot. Expression of PI-3K subunit p85 was subsequently analyzed to confirm equal loading of the membrane. (E) PMA-differentiated THP-1 cells were incubated with LMW-APM (1 µg/ml) or HMW-APM (10 µg/ml) for 1 h. LPS (1 µg/ml) was added, and cells were cultivated for an additional 20 and 40 min. NF-{kappa}B p65, phosphorylated STAT-1 (pSTAT-1), STAT-1, and ß-actin were determined in nuclear extracts isolated from these cells. (F) Primary monocytes isolated from six probands were incubated with LMW-APM (1 µg/ml) or HMW-APM (10 µg/ml) for 24 h. IL-6 was determined in the supernatants. Significance was calculated by paired, two-tailed Student’s t-test and is indicated by an asterisk for values of P < 0.05.

 
IKKß was reduced in cells incubated with LMW-APM and LPS for 40 or 60 min when compared with HMW-APM-treated cells (Fig. 3D) . Nuclear NF-{kappa}B p65 was lower in LMW-APM-treated, LPS-stimulated THP-1 cells when compared with HMW-APM incubation for 40 min. In contrast, Western blot analysis of nuclear STAT-1 or STAT-1 phosphorylated at Ser727 revealed that expression of these factors was not altered (Fig. 3E) . Akt phosphorylated at Ser473 was not reduced in THP-1 cells treated with LMW-APM and LPS when compared with LPS-activated cells solely (data not shown).

HMW-APM induces IL-6 in primary monocytes
The influence of LMW-APM and HMW-APM was also investigated in PMA-differentiated THP-1 cells and primary monocytes from six healthy individuals. Constitutive secretion of IL-6 in THP-1 is below the detection limit of the ELISA. Whereas LMW-APM did not induce IL-6 secretion, HMW-APM induced IL-6 release to 3.4 ± 0.4 pg/ml (P=0.005) in THP-1 cells (not shown). IL-6 release from primary cells was 5.6 ± 2.4 pg/ml, LMW-APM-treated cells release similar amounts of IL-6, and 71.3 ± 34 pg/ml IL-6 was secreted from HMW-APM-incubated monocytes (P<0.0001; Fig. 3F ). LPS (1 µg/ml) induced a secretion of 8902 ± 5370.9 pg/ml IL-6 in the identical monocytes (not shown), a nearly 130-fold higher amount of IL-6.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our studies demonstrate that baculovirus-derived APM most likely forms trimers and that HMW isoforms are not present. However, APM expressed in a mouse cell line is composed of dimers and HMW oligomers. Several studies describe anti-inflammatory effects of APM in activated monocytes, but most of these experiments have been performed with APM resembling LMW and HMW isoforms [5 , 7 , 26 ]. Our experiments show that only LMW-APM suppresses LPS-induced IL-6 secretion and enhances IL-10 release, whereas HMW-APM did not alter the secretion of these cytokines. The LMW-APM down-regulates IKKß in PMA-differentiated THP-1 cells in the presence and absence of LPS. Reduced abundance of IKKß hinders NF-{kappa}B p65 translocation to the nucleus, most likely because I{kappa}B is not efficiently phosphorylated and degraded [8 ]. Mice lacking IKKß in myeloid cells are protected from systemic insulin resistance [27 ], and the finding that LMW-APM down-regulates IKKß in monocytic cells may partly explain the insulin-sensitizing effects of APM. LPS also activates the transcription factor STAT-1 through the intermediate production of IFN-{gamma} [28 ], and secretion of IFN-{gamma} was shown to be reduced by APM [7 ], but neither STAT-1 nor pSTAT-1 was reduced by the rAPM isoforms.

HMW-APM, but not LMW-APM, induces the secretion of IL-6 in differentiated THP-1 cells and primary monocytes, although to a much lesser extent compared with LPS. HMW-APM purified from human plasma suppresses phagocytosis of apoptotic cells and IL-8 secretion in nonactivated monocytes but augmented both in the presence of LPS [29 ]. Lappas et al. [30 ] described the release of proinflammatory cytokines in APM-incubated placenta and maternal adipose tissue explants. In this study, the globular fragment of APM was used, and cytokine release was inhibited by a NF-{kappa}B inhibitor. In addition, globular APM induces TNF and IL-6 in human macrophages [31 ].

The data from the literature combined with our experiments indicate that the effects of APM depend on its high order structure and on the particular cells analyzed and their activation status.

Most studies published so far indicate that HMW-APM is the most active form of APM and that reduced, circulating HMW is associated with insulin resistance, type 2 diabetes mellitus, and atherosclerosis in mice and humans [32 ]. In vitro studies demonstrate potent, biologic effects of all of the different APM isoforms and that mutant APM, unable to form HMW complexes, was even more active in reducing glucose production in hepatocytes than APM with HMW forms [33 ]. Pajvani et al. [33 ] speculate that the HMW forms serve as a precursor pool and that proteolytic cleavage generates an active but short-lived trimer. This would explain why elevated HMW complexes are associated with better metabolic markers in association studies [12 , 33 ].

Besides exerting distinct effects, APM isoforms also share common effects on monocytic cells. LMW-APM and HMW-APM induce mainly mitochondrial-mediated apoptosis in nondifferentiated THP-1 cells, whereas the survival of primary macrophages or PMA-differentiated THP-1 cells was not impaired by LMW-APM or HMW-APM. In differentiated THP-1 cells, both isoforms suppress MSR mRNA expression and induce the phosphorylation of AMPK, a cellular energy sensor, which switches on catabolic pathways that generate ATP while turning off ATP-consuming processes [34 ].

As APM is regarded increasingly as a potential target for therapeutic approaches in various metabolic and inflammatory diseases, such as diabetes mellitus type 2 and atherosclerosis, detailed knowledge of the distinct effects of APM isoforms is essential for the development of novel treatment strategies in these disorders.


   ACKNOWLEDGEMENTS
 
This work was partly supported by grants from the Deutsche Diabetes Gesellschaft and by the Deutsche Forschungsgemeinschaft (BU 1141/3-2). The expert technical assistance of Kerstin Winkler is greatly appreciated.

Received September 20, 2005; revised October 31, 2005; accepted November 16, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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