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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2005-0467
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 12 6707-6713
Copyright © 2005 by The Endocrine Society

Adiponectin Is Functionally Active in Human Islets but Does Not Affect Insulin Secretory Function or ß-Cell Lipoapoptosis

K. Staiger1, N. Stefan1, H. Staiger, M. D. Brendel, D. Brandhorst, R. G. Bretzel, F. Machicao, M. Kellerer, M. Stumvoll, A. Fritsche and H.-U. Häring

Department of Internal Medicine (K.S., N.S., H.S., F.M., M.K., A.F., H.-U.H.), Division of Endocrinology, Metabolism and Pathobiochemistry, University of Tübingen, 72076 Tübingen, Germany; Third Medical Department (M.D.B., D.B., R.G.B.), Justus-Liebig-University of Giessen, 35390 Giessen, Germany; Marienhospital Stuttgart (M.K.), Department of Internal Medicine I, 70199 Stuttgart, Germany; and Third Medical Department (M.S.), University of Leipzig, 04109 Leipzig, Germany

Address all correspondence and requests for reprints to: Hans-Ulrich Häring, M.D., Department of Internal Medicine, University of Tübingen, Otfried-Müller Strasse 10, 72076 Tübingen, Germany. E-mail: hans-ulrich.haering{at}med.uni-tuebingen.de.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: The adipokine adiponectin has insulin-sensitizing, antiatherogenic, and antiinflammatory properties. Mouse and human adiponectin receptor-1 and -2 have been cloned, both of which are expressed in various tissues and mediate effects of globular and full-length adiponectin. Whether adiponectin affects insulin secretion and ß-cell apoptosis and whether plasma adiponectin is associated with ß-cell function in humans is under investigation.

Design and Methods: In human islets from multiorgan donors, we investigated expression of adiponectin receptor-1 and -2. Furthermore, glucose-stimulated insulin secretion was determined by RIA. In addition, we investigated fatty acid-induced ß-cell apoptosis by terminal dUTP nick end labeling and flow-cytometric cell cycle analysis (sub-G1 formation). In humans in vivo, insulin secretory function was measured during hyperglycemic clamps in 65 normal glucose-tolerant subjects. We determined first and second phase of glucose-stimulated, glucagon-like peptide-1-stimulated, and arginine-stimulated insulin secretion.

Results: Adiponectin receptor-1 and -2 are expressed in human islets at the mRNA and protein level. Moreover, full-length adiponectin induces phosphorylation of acetyl coenzyme A carboxylase. However, adiponectin did not affect basal or glucose-stimulated insulin secretion or basal or fatty acid-induced ß-cell apoptosis. In vivo, fasting plasma adiponectin concentrations were not associated with glucose-stimulated first- and second-phase insulin secretion or with glucagon-like peptide-1- or arginine-stimulated insulin secretion (all P > 0.42).

Conclusions: These data support a regulatory role of adiponectin in human islets; however, adiponectin does not seem to affect insulin secretion or basal/fatty acid-induced ß-cell apoptosis in humans.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
IN THE NATURAL history of type 2 diabetes, insulin secretory function starts to decline while adiposity and insulin resistance increase (1). Mechanisms mediating impairment of insulin secretion include gluco- and lipotoxicity (2, 3) and possibly the increase of secretion of the adipokine TNF-{alpha}, which negatively affects ß-cell function (4, 5). In contrast, leptin, which also increases in plasma with increase in adiposity, was shown to protect from nonesterified fatty acid (NEFA)-induced ß-cell apoptosis (6). However, it has an inhibitory effect on insulin secretory function (4). In contrast to TNF-{alpha} and leptin, plasma concentrations of adiponectin, the adipokine that plays an important role in glucose metabolism, lipid metabolism, and inflammation, declines with increasing adiposity (7, 8, 9, 10, 11). Whether this may also mediate the decrease in insulin secretory function that ultimately occurs when adiposity and insulin resistance persist is not known. To address this hypothesis it is necessary to resolve whether adiponectin plays a role in insulin secretory function. Both leptin and adiponectin suppress the activity of acetyl-coenzyme A carboxylase (ACC), thereby stimulating oxidation of NEFA in muscle and liver (12, 13, 14). ACC also plays an important role in lipid oxidation in ß-cells and affects insulin secretion (15). Because leptin modulates insulin secretion possibly by acting on ACC, it can be hypothesized that adiponectin may also act through phosphorylation of ACC to increase lipid oxidation in ß-cells and, thus, affect insulin secretion. Moreover, mouse and human adiponectin receptor-1 and -2 (AdipoR1 and -R2) have been cloned (16). Both receptors mediate effects of globular and/or full-length adiponectin. Expression of AdipoR1 and AdipoR2 was also detected in rat and human ß-cells (17).

There is increasing evidence that decreased functional ß-cell mass is a main characteristic of type 2 diabetes. Mechanisms involved in this process include increased ß-cell apoptosis (18). Adiponectin was shown to affect apoptosis in endothelial cells (19, 20) and to protect from NEFA- and cytokine-induced apoptosis in a ß-cell line (21). Therefore, it can be hypothesized that adiponectin plays a role in ß-cell apoptosis and that the decline in plasma adiponectin with increasing adiposity contributes to the decline in insulin secretion observed under this condition.

To address this issue and to determine the role of adiponectin in human insulin secretion, we conducted the following in vitro and in vivo experiments. First, we investigated whether adiponectin receptors are expressed in islets isolated from human donors. Second, we tested whether adiponectin is functional in these islets by determining adiponectin-mediated phosphorylation of ACC. Third, we investigated whether adiponectin affects basal and/or glucose-stimulated insulin secretion and basal and/or NEFA-induced ß-cell apoptosis. In humans in vivo, we analyzed whether plasma concentrations of adiponectin are associated with insulin secretory function. We included 65 normal glucose-tolerant individuals who had undergone a modified hyperglycemic clamp with additional glucagon-like peptide (GLP)-1-stimulated and arginine-stimulated insulin secretion. With the latter, insulin secretion can be stimulated on average 8-fold compared with glucose, and this challenge can be used as a rough estimate of ß-cell mass.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Isolation and culture of human islets

Islets were isolated from the pancreas of eight multiorgan donors (four women and four men) as previously described (22). Mean donor age was 52 ± 5 yr, and mean donor body mass index (BMI) was 25.2 ± 2.2 kg/m2. Islet volume and purity were determined by microscopic sizing on a grid after staining with diphenylthiocarbazone (23). In selected samples, determination of purity was confirmed by histomorphology. Isolated islets were formalin fixed and paraffin embedded, and sections were cut and stained with hormone-specific antibodies (insulin, glucagon, and somatostatin). In parallel, measurement of insulin and amylase content after cell sonication was performed (data not shown). Islet purity averaged 75% (range, 50–90%). Viability was assessed by membrane integrity testing (trypan blue exclusion) and in all cases exceeded 90%. Islets were cultured in Connaught Medical Research Laboratories 1066 medium supplemented with 5 mM glucose, 0.05 mM dithiothreitol, 2 mM glutamine, 1 mM sodium pyruvate, 20 µg/ml ciprofloxacine, 100 IU/ml penicillin, 100 µg/ml streptomycin, 10 mM HEPES (pH 7.4), 0.01 mM hydrocortisone, and 10% fetal calf serum at 37 C. In some experiments, cells were treated with recombinant human full-length adiponectin (R&D Systems, Wiesbaden, Germany), recombinant human globular adiponectin (tebu-bio, Offenbach, Germany), and/or NEFA. NEFA (Sigma-Aldrich, Taufkirchen, Germany) were bound to NEFA-free BSA as described previously (24). BSA-containing control medium was prepared analogously. In palmitate-containing medium, the final BSA concentrations were 2.5% (1 mM palmitate) and 1.25% (0.5 mM palmitate). In stearate-containing medium, the final BSA concentrations were 5% (1 mM stearate) and 2.5% (0.5 mM stearate).

RT-PCR

RNA from freshly isolated islets was purified with the RNeasy Protect Mini Kit according to the manufacturer’s instructions (QIAGEN, Hilden, Germany). Total RNA treated with RNase-free DNase I was transcribed into cDNA using avian myeloblastosis virus reverse transcriptase and the first-strand cDNA kit from Roche Diagnostics (Mannheim, Germany). Quantitative PCR was performed with SYBR Green I dye on a high-speed thermal cycler with integrated microvolume fluorometer according to the instructions of the manufacturer (Roche). The following primers were obtained from Invitrogen (Karlsruhe, Germany): AdipoR1 forward, 5'-ATTGAGGTACCAGCCAGATG-3'; AdipoR1 reverse, 5'-GAGGTCTATGACCATGTAGC-3'; AdipoR2 forward, 5'-GATTGTCATCTGTGTGCTGG-3'; and AdipoR2 reverse, 5'-CTGGAGACTGGTAGGTATCA-3'. The PCR conditions used were the following: for AdipoR1 mRNA, 66 C annealing temperature for 45 cycles with 4 mM MgCl2; for AdipoR2 mRNA, 64 C annealing temperature for 45 cycles with 4 mM MgCl2 and 5% (vol/vol) dimethylsulfoxide. Measurements were performed in triplicate. AdipoR1 and AdipoR2 mRNA levels are given in fg/µg total RNA.

Insulin secretion

Four days after plating, when most islets were attached and had begun to spread, cells were trypsinized and seeded on collagen I-coated six-well plates (Biocoat; BD Biosciences, Heidelberg, Germany). Two days later, cells were pretreated for 16 h as indicated and then washed three times with Krebs-Ringer-HEPES (KRH) buffer (125 mM NaCl, 4.74 mM KCl, 1 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 5 mM NaHCO3, 25 mM HEPES, 0.1% BSA, pH 7.4) containing 2.8 mM glucose. After 2 h of preincubation with KRH buffer containing 2.8 mM glucose, cells were incubated for 1 h with KRH buffer containing 2.8 mM glucose. Supernatant was collected and cells were stimulated with KRH buffer containing 5 mM glucose for 1 h. Again, the supernatant was collected. This procedure was repeated with KRH buffer containing 10 mM and 15 mM glucose. For acute stimulation with adiponectin, adiponectin was applied during the 1-h incubation period at the given glucose concentration. At the end, the cell monolayer was scraped off in 10 mM Tris/HCl (pH 7.5), 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml aprotinin, and cells were lysed by sonication. Insulin in the supernatant and lysate was determined by RIA (Linco Research, St. Charles, MO). Insulin in the supernatant and lysate was normalized to total cell protein content, as measured by the Bradford method.

Terminal dUTP nick end labeling (TUNEL) assay

Four days after plating, cells were trypsinized and seeded on collagen I-coated chamber slides (Biocoat; BD Biosciences). Two days later, cells were treated as indicated. Then, TUNEL assay was performed according to the manufacturer’s instructions (in situ cell death detection kit fluorescein; Roche Molecular Biochemicals, Mannheim, Germany). Insulin staining was performed as described earlier (24). Cells were examined using confocal laser microscopy (Leica, Heidelberg, Germany).

Cell cycle analysis

Four days after plating, cells were trypsinized and seeded on collagen I-coated six-well plates (Biocoat; BD Biosciences). Two days later, cells were treated as indicated. After treatment, detached cells were harvested from the supernatant by centrifugation and added to the nondetached cells harvested by trypsinization. Cells were washed with PBS, fixed in 70% ice-cold ethanol, centrifuged, and washed with PBS. After staining with propidium iodide (50 µg/ml) diluted in PBS containing RNase A (100 µg/ml), cells were subjected to flow cytometric analysis of DNA content using a Becton Dickinson FACScalibur cytometer (BD Biosciences). Percentages of cells in the different cell cycle phases were calculated by CellQuest software (BD Biosciences).

Immunoblotting

For the detection of ACC protein and phosphorylation, freshly isolated islets were cultured for 4 d and then stimulated as indicated. Cells were lysed in ice-cold lysis buffer [50 mM HEPES (pH 7.2), 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% (vol/vol) glycerol, 1% (vol/vol) Triton X-100, 100 mM NaF, 10 mM sodium pyrophosphate, 100 µM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml aprotinin]. Cleared crude cell lysates (20 min/12,000 g) were analyzed on 10% SDS-PAGE (100 µg protein per lane). Separated proteins were transferred to nitrocellulose membranes by semidry electroblotting [transfer buffer, 48 mM Tris/HCl (pH 7.5), 0.0004% (wt/vol) SDS, 39 mM glycine, and 20% (vol/vol) methanol]. After transfer, the membranes were blocked with NET buffer [150 mM NaCl, 5 mM EDTA, 50 mM Tris, 0.05% (vol/vol) Triton X-100, and 0.25% (wt/vol) gelatin, pH 7.4] for 1 h. Subsequently, membranes were incubated with the primary antibody (anti-phospho-ACC and anti-ACC; Cell Signaling, New England Biolabs, Frankfurt/Main, Germany) overnight at 4 C. The membranes were washed four times with NET buffer before incubating with horseradish peroxidase-conjugated antirabbit IgG for 1 h at room temperature. Visualization of immunocomplexes was performed by enhanced chemiluminescence. For detection of AdipoR1 and AdipoR2 protein, cells were trypsinized 4 d after plating and seeded on collagen I-coated six-well plates (Biocoat; BD Biosciences). Two days later, cells were lysed. Immunoblotting was performed as described above (primary antibody, anti-AdipoR1 and anti-AdipoR2; Autogen Bioclear, Calne, UK).

In vivo data

We analyzed data of 65 normal glucose-tolerant subjects (25) who participated in the Tübingen Family Study for type 2 diabetes. The participants did not take any medication known to affect glucose tolerance or insulin sensitivity. Tests were done at 0800 h after an overnight fast of 10 h. The subjects also were asked to refrain from smoking for the same period.

We previously investigated whether adiponectin was associated with indices of insulin secretion derived from the oral glucose tolerance test (OGTT) in 685 normal glucose-tolerant subjects from our Tübingen Family Study (26). Here we reanalyzed these data to determine whether there was an interaction of adiponectin with percent body fat and/or serum free NEFA to affect insulin secretion.

Informed written consent was obtained from all participants, and the local medical ethics committee had approved the protocol. The characteristics of the 65 subjects who underwent the clamp are shown in Table 1Go.


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  		TABLE 1. Subject characteristics

 
OGTT

All subjects underwent an OGTT. After an overnight fast, they ingested a solution containing 75 g dextrose, and venous blood samples were obtained at 0, 30, 60, 90, and 120 min for determination of plasma glucose.

Body composition and body fat distribution

BMI was calculated as weight divided by the square of height (kg/m2). Waist and hip circumferences were measured in the supine position, and waist-to-hip ratio was calculated as an index of body fat distribution.

Hyperglycemic clamp

After an overnight fast and after baseline samples had been obtained, a hyperglycemic clamp was performed as previously described (27, 28). In brief, an iv bolus of 20% glucose over 1 min was given to instantaneously raise blood glucose to 10 mM [bolus dose (mg) x body weight (kg) x desired increase in blood glucose (mg/dl) x 1.5]. Subsequently, a glucose infusion was adjusted to maintain blood glucose at 10 mM. After 120 min, GLP-1 [human GLP-1 (7–36) amide; Poly Peptide, Wolfenbüttel, Germany] was given as a primed-continuous infusion (0.6 pmol/kg; 1.5 pmol·kg–1·min–1) during the next 80 min. At 180 min, a bolus of 5 g arginine hydrochloride (Pharmacia & Upjohn, Erlangen, Germany) was injected over 45 sec while the GLP-1 infusion was continued.

Calculations

Insulin sensitivity (insulin sensitivity index) from the hyperglycemic clamp was determined by relating the glucose infusion rate to the plasma insulin concentration during the second hour (27). Phases of insulin secretion based on C-peptide concentrations during the hyperglycemic clamp were calculated as follows: first phase, mean of 2.5–10 min; second phase, mean of 80–120 min; first GLP phase, mean of 125–130 min; second GLP phase (plateau), mean of 160–180 min; and maximal insulin secretion, mean of 182.5–190 min (27, 28, 29).

Analytical procedures

Blood glucose was determined using a bedside glucose analyzer (glucose-oxidase method; Yellow Springs Instruments, Yellow Springs, CO). Plasma insulin was determined by microparticle enzyme immunoassay (Abbott Laboratories, Tokyo, Japan) and plasma C-peptide by RIA (Byk-Sangtec, Dietzenbach, Germany). Serum samples were frozen immediately and stored at –20 C for determination of adiponectin by RIA (Linco Research) and free NEFA with an enzymatic method (WAKO Chemicals, Neuss, Germany).

Statistical analyses

Parameters were logarithmically transformed to approximate linearity if necessary. Effects of adiponectin on insulin secretion or ß-cell apoptosis were tested by ANOVA and the unpaired Student’s t test. The secretion indexes of the hyperglycemic clamp were adjusted for insulin sensitivity, BMI, age, and sex in multivariate linear regression analyses. A P value of <0.05 was considered to be statistically significant. The statistical software package JMP (SAS Institute, Cary, NC) was used.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Evidence of functional adiponectin receptors in human islets

Cellular mRNA expression of AdipoR1 and AdipoR2 was determined in intact human islets freshly isolated from pancreata obtained from three multiorgan donors. AdipoR1 mRNA amounts reached a mean value of 369 ± 66.5 fg/µg total RNA (means ± SE) and individually varied between 269 and 495 fg/µg total RNA. AdipoR2 mRNA levels ranged around 852 ± 300 fg/µg total RNA (means ± SE) and individually varied between 259 and 1229 fg/µg total RNA. Furthermore, we investigated the expression of AdipoR1 and AdipoR2 at the protein level by immunoblotting. In freshly isolated islets, AdipoR1 and AdipoR2 were not detectable (Fig. 1AGo), probably because of collagenase treatment during the islet isolation procedure. By contrast, after culturing islet cells for 6 d (including one trypsinization step on d 4), both AdipoR1 and AdipoR2 were found at the protein level (Fig. 1AGo).



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  		FIG. 1. Evidence of functional adiponectin receptors in human islets. A, Human islets express AdipoR1 and AdipoR2 proteins. Freshly isolated islets (lanes 1 and 3) or for 6-d cultured islet cells (lanes 2 and 4) were lysed, and immunoblotting was performed as described in Subjects and Methods. The figure is representative of two similar experiments. B, Adiponectin induces phosphorylation of ACC in human islets. Human islets were left untreated (lane 1) or treated with full-length adiponectin (5 µg/ml for 15 min) (lane 2). As control, 5-amino-4-imidazolecarboxamide riboside-stimulated human islets (1 mM for 15 min) (lane 3) and full-length adiponectin-treated human myotubes (5 µg/ml for 15 min) (lane 4) were used. ACC protein and ACC phosphorylation were visualized by immunoblotting as described in Subjects and Methods. The figure is representative of two similar experiments.

 
Next, we studied whether commercially available human recombinant full-length adiponectin is functionally active in human islets. Adiponectin exerts most of its effects via activation of AMP-activated protein kinase, which in turn phosphorylates and thereby inactivates ACC. Therefore, we tested the effect of full-length adiponectin on phosphorylation of ACC in human islets. Treatment of human islets with full-length adiponectin (5 µg/ml, 15 min) markedly increased phosphorylation of ACC as shown by immunoblotting (Fig. 1BGo).

Effects of adiponectin on basal and NEFA-induced ß-cell apoptosis

We could show recently that saturated NEFA induce apoptosis in human ß-cells (24). Here, we tested whether adiponectin protects human islets from NEFA-induced apoptosis (lipoapoptosis). Human islets were incubated with palmitate or stearate (Fig. 2AGo, 1 mM for 72 h; Fig. 2BGo, 0.5 mM for 5 d) in the absence or presence of a commonly used (5 µg/ml, not shown) and a high (20 µg/ml, Fig. 2Go, A and B) concentration of adiponectin. At the lower NEFA concentration, the longer incubation time was necessary to significantly induce apoptotic effects. Adiponectin, at both concentrations tested, had no significant effect on basal apoptosis or lipoapoptosis, as shown in Fig. 2Go, A and B, for 20 µg/ml adiponectin.



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  		FIG. 2. Adiponectin does not prevent lipoapoptosis of human islets. A and B, Apoptosis determined by cell cycle analysis. Islet cells were left untreated (white bars) or were pretreated (black bars) with full-length adiponectin (20 µg/ml for 30 min) before palmitate or stearate was added (A, 1 mM fatty acid for 72 h; B, 0.5 mM fatty acid for 5 d). Sub-G1 formation was assessed as described in Subjects and Methods. *, Significantly different from untreated control; **, significantly different from adiponectin-treated control (P < 0.05; n = 3). C, Isolated human islet cells were incubated for 72 h with stearate (1 mM) in the absence or presence of full-length adiponectin (5 µg/ml pretreatment for 30 min) or globular (glob.) adiponectin (1 µg/ml pretreatment for 30 min). Apoptotic ß-cells were identified by double staining with TUNEL (green) and insulin antibodies (orange). Representative pictures of three independent experiments are shown.

 
Because islets contain various cell types, we confirmed these results by confocal laser microscopy. Isolated islet cells were incubated with palmitate or stearate (1 mM) for 72 h in the presence or absence of adiponectin (5 µg/ml) or globular adiponectin (1 µg/ml). Apoptotic ß-cells were identified by double staining with TUNEL, indicating DNA strand breaks in apoptotic cells, and with an insulin antibody. Adiponectin neither protected human ß-cells from lipoapoptosis nor influenced basal apoptosis. Representative images for stearate-treated human islets are shown in Fig. 2CGo.

Effects of adiponectin on insulin secretion in vitro

To examine whether adiponectin exerts long-term effects on basal or glucose-stimulated insulin secretion, human islets were preincubated (16 h) with recombinant human full-length adiponectin (5 or 20 µg/ml) or recombinant human globular adiponectin (1 µg/ml) before glucose stimulation. Neither globular nor full-length adiponectin, at both concentrations tested, affected basal or glucose-stimulated insulin secretion, as shown in Fig. 3Go for 20 µg/ml full-length adiponectin. To test for acute effects of adiponectin on insulin secretion, human islets cells were incubated with full-length adiponectin (5 or 20 µg/ml) or globular adiponectin (1 µg/ml). At any glucose concentration tested, full-length as well as globular adiponectin (data not shown) did not alter insulin secretion compared with control conditions, as shown in Fig. 3Go for 20 µg/ml full-length adiponectin. In addition, chronic (16 h) or acute (1 h) incubation of islet cells with adiponectin did not influence intracellular insulin content (data not shown).



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  		FIG. 3. Effects of adiponectin on insulin secretion in vitro. For chronic treatment, human islets were preincubated (16 h) with recombinant human full-length adiponectin (20 µg/ml) before glucose stimulation. To test for acute effects of adiponectin on insulin secretion, human islets were incubated with full-length adiponectin (20 µg/ml) for 1 h at the indicated glucose (Glc) concentrations. Insulin secretion was determined as described in Subjects and Methods. Insulin secretion is given as fold increase over basal (2.8 mM glucose, 0 µg/ml adiponectin).

 
Associations between adiponectin and insulin secretion in vivo

Subject characteristics are shown in Table 1Go. In this population, fasting plasma adiponectin concentrations were negatively associated with BMI (r = –0.27; P = 0.03) after adjustment for age and sex but not with insulin sensitivity (r = 0.11; P = 0.36) after adjustment for age, sex, and BMI in multiple linear regression models. Neither the first phase (Fig. 4Go) nor the second phase of glucose-stimulated insulin secretion nor first GLP-1 phase- or second GLP-1 phase-stimulated insulin secretion were associated with fasting plasma adiponectin concentrations before (all P > 0.08) and after adjustment for age, sex, BMI, and insulin sensitivity. In addition, there was no association between plasma adiponectin and maximal (arginine-stimulated) insulin secretion (Table 2Go). Adiponectin levels were also not associated with insulin secretion when data were analyzed in men and women separately (men, n = 29, all P > 0.06; women, n = 36, all P > 0.11).



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  		FIG. 4. Association between plasma adiponectin and insulin secretion in vivo. The association between plasma adiponectin concentrations and first phase of insulin secretion in 65 subjects who underwent the hyperglycemic clamp is depicted. Insulin secretion was adjusted for age, gender, BMI, and insulin sensitivity (clamp) in general linear models.

 

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  		TABLE 2. Associations between different phases of insulin secretion in vivo and adiponectin after adjustment for age, sex, BMI, and insulin sensitivity

 
To account for a potential impact of adiposity or serum free NEFA on these relationships, we investigated whether the association between adiponectin and insulin secretion is different under different stages of adiposity or serum free NEFA concentrations. Because of the relatively small number of subjects in the clamp group, this was tested in 685 subjects who were included in a previous analysis (26). There was no significant correlation of plasma adiponectin with percent body fat or serum free NEFA to affect the 30-min C-peptide plasma concentrations during the OGTT and/or the first phase of glucose-stimulated insulin secretion after adjustment for age, sex, percent body fat, and insulin sensitivity (all P for interaction > 0.36; data not shown).


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Because of the emerging evidence that adiponectin may play an outstanding role in the development of type 2 diabetes, it is necessary to thoroughly characterize its site of action. Even though the direct proof for the existence of adiponectin receptors in human ß-cells is still lacking, the identification of adiponectin receptors in human islets and insulinoma cell lines (17) implies that it may modulate ß-cell function. To address this issue we conducted three experiments. First, we investigated whether adiponectin affects glucose-stimulated insulin secretion in human islets. Second, we determined whether adiponectin modulates NEFA-induced ß-cell apoptosis. Third, we investigated in humans in vivo whether adiponectin was associated with glucose-, GLP-1-, and/or arginine-stimulated insulin secretion. Using these tools, physiologically relevant effects of adiponectin on insulin secretory function and/or ß-cell lipoapoptosis may become apparent. Particularly for our in vitro experiments, we hypothesized that an effect of adiponectin on ACC phosphorylation in human islets confirms that adiponectin may be active here. An inhibitory effect of adiponectin on ACC activity was previously shown for muscle and liver (12, 13). In muscle, this effect was associated with increased NEFA oxidation and glucose uptake, whereas in liver it was associated with reduced gluconeogenesis (13). Consistent with these reports we found that adiponectin phosphorylated ACC also in human islets. However, we did not detect effects of adiponectin on insulin secretion. Moreover, adiponectin had no protective effect on basal and/or NEFA-induced ß-cell apoptosis. Because human islets are more resistant to lipoapoptosis then insulinoma cells in vitro, the use of high up to supraphysiological concentrations of NEFA are necessary to study lipoapoptotic effects in human islets (24, 30, 31). Therefore, we cannot totally exclude that these conditions are too toxic to see protective effects of adiponectin. However, we tested two different NEFA concentrations and even at the lower NEFA concentration (500 µM) that can be reached in vivo in the fasting state, we were not able to detect any protective effect of adiponectin on lipoapoptosis. Furthermore, we have no data on a role of adiponectin in apoptotic events induced by other stimuli, and additional studies are necessary to address this issue.

There are conflicting results regarding effects of adiponectin on insulin secretory function in vitro. Recently, the group of Kadowaki (32) found that adiponectin increased insulin secretion in vitro by stimulation of exocytosis of insulin granules at 5 mM but not at higher glucose concentrations. Rakatzi et al. (21) reported that adiponectin decreased glucose/forskolin-induced insulin secretion but reversed NEFA-induced impairment of insulin secretion. In addition, it protected from NEFA- and cytokine-induced apoptosis. Winzell et al. (33) reported a dual action of adiponectin in insulin-resistant mice. It decreased insulin secretion under 2.8 mM but increased insulin secretion under 16.7 mM glucose. However, no effect of adiponectin on insulin secretion in islets from normal mice was found. This is in agreement with our findings in islets from healthy human donors. In contrast to data obtained with ß-cell lines, we consider that our data more closely reflect human physiology.

Some reports in the literature suggest that adiponectin is associated with insulin secretory function in vivo (34, 35). In the study by Bacha et al. (34), fasting proinsulin-to-insulin ratio was used as a surrogate marker of ß-cell function. However, this ratio rather reflects insulin resistance. In the study by Retnakaran et al. (35) in women with gestational diabetes, the product of insulin sensitivity and secretion, derived from the OGTT, was used. They found that this disposition index was positively associated with adiponectin levels. A limitation of that study is that insulin sensitivity, which shows a strong association with adiponectin levels, is an important determinant of this index and thus probably affects the relationship between this parameter and adiponectin levels. To further clarify whether adiponectin levels are associated with ß-cell function, we analyzed data from normal glucose-tolerant subjects who underwent the hyperglycemic clamp using three insulin secretagogues. Plasma adiponectin levels were not associated with glucose-, GLP-1-, or arginine-induced insulin secretion. We also investigated whether plasma NEFA and/or percent body fat modulate the association between adiponectin and insulin secretion. We did not find a statistically significant interaction of plasma NEFA and/or percent body fat with adiponectin on insulin secretion.

In conclusion, because we found that adiponectin phosphorylates ACC in human islets, we consider it a factor possibly modulating NEFA oxidation in human islets. However, it appears that this effect is not related to glucose-stimulated insulin secretion or protection from basal and/or NEFA-induced ß-cell apoptosis in humans.


   Acknowledgments
 
We thank all the research volunteers for their participation. We gratefully acknowledge the superb technical assistance of Elisabeth Metzinger, Elke Maerker, Anna Teigeler, Heike Luz, Melanie Weisser, Alke Guirguis, Claudia Peterfi, and Carina Haas. We also thank all participants for their cooperation.


   Footnotes
 
This work was supported by a grant from the Deutsche Forschungsgemeinschaft (KFO 114/1), the National Institute of Diabetes and Digestive and Kidney Diseases (Grant DK 56962 to R.G.B.), and the European Community’s FP6 EUGENE2 (LSHM-CT-2004-512013).

First Published Online October 4, 2005

1 K.S. and N.S. contributed equally to this work. Back

Abbreviations: ACC, Acetyl-coenzyme A carboxylase; AdipoR1, adiponectin receptor-1; BMI, body mass index; GLP-1, glucagon-like peptide-1; KRH, Krebs-Ringer-HEPES; NEFA, nonesterified fatty acids; OGTT, oral glucose tolerance test; TUNEL, terminal dUTP nick end labeling.

Received March 3, 2005.

Accepted September 22, 2005.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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