1 Department of Internal Medicine I, Medical University of Lübeck, 23538 Lübeck; 2 Department of Internal Medicine III, University of Leipzig, 04103 Leipzig, Germany; 3 Joslin Diabetes Center and Harvard Medical School, and 4 Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215; and 5 Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain
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ABSTRACT |
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Cross talk
between adrenergic and insulin signaling systems may represent a
fundamental molecular basis of insulin resistance. We have
characterized a newly established
3-adrenoceptor-deficient (
3-KO) brown
adipocyte cell line and have used it to selectively investigate the
potential role of novel-state and typical
-adrenoceptors (
-AR) on
insulin signaling and action. The novel-state
1-AR agonist CGP-12177 strongly induced uncoupling protein-1 in
3-KO brown adipocytes as opposed to the
3-selective agonist CL-316,243. Furthermore, CGP-12177
potently reduced insulin-induced glucose uptake and glycogen synthesis.
Neither the selective
1- and
2-antagonists metoprolol and ICI-118,551 nor the
nonselective antagonist propranolol blocked these effects. The
classical
1-AR agonist dobutamine and the
2-AR agonist clenbuterol also considerably diminished insulin-induced glucose uptake. In contrast to CGP-12177 treatment, these negative effects were completely abrogated by metoprolol and
ICI-118,551. Stimulation with CGP-12177 did not impair insulin receptor
kinase activity but decreased insulin receptor substrate-1 binding to
phosphatidylinositol (PI) 3-kinase and activation of protein kinase B. Thus the present study characterizes a novel cell system to selectively
analyze molecular and functional interactions between novel and
classical
-adrenoceptor types with insulin action. Furthermore, it
indicates insulin receptor-independent, but PI 3-kinase-dependent,
potent negative effects of the novel
1-adrenoceptor
state on diverse biological end points of insulin action.
-adrenoceptor; adipose tissue; insulin resistance; CGP-12177
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INTRODUCTION |
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INTERACTION BETWEEN THE
SYMPATHETIC nervous system and insulin plays an important role in
the pathogenesis of obesity, insulin resistance, and their related
cardiovascular complications (24). The
3-adrenergic receptor (
3-AR) is
predominantly expressed in brown and white adipose tissue and
represents a potential target for antiobesity drug therapy (25,
26). Selective activation of this adrenoceptor results in an
increase in lipolysis and energy expenditure. Regulation of energy
balance in rodents is mediated by activation of uncoupling protein-1
(UCP-1), which dissipates energy stored in the proton gradient across
the inner mitochondrial membrane, thereby uncoupling mitochondrial
respiration from ATP production (19, 20). Previous studies
in brown adipocytes have shown that insulin inhibits
3-adrenergic stimulation of UCP-1 (15) and
that
3-adrenergic stimulation leads to decreased activation of the insulin-signaling system (16). These
findings indicate a functionally relevant interplay between adrenergic and insulin-signaling systems.
From a variety of studies using the aryloxypropanolamine CGP-12177 in
both 3-knockout (
3-KO) and wild-type
mice, the concept of a novel atypical adrenergic receptor, initially
dubbed
4-AR, in adipose tissue (3, 6, 23),
heart (12), and gut (21) has been developed.
CGP-12177-induced effects that can be neither mimicked by
3-AR-selective phenethanolamine agonists nor abolished by
1- and
2-AR blockade indicated a novel
atypical
-AR (11). However, most recent studies using
recombinant
-AR subtypes (17, 18, 22) and knockout mice
(9) have demonstrated that the
4-AR is a
novel state of the
1-AR (7, 17). Thus
Konkar et al. (17) found that CGP-12177-mediated
activation of brown fat adenylyl cyclase in
3-KO mice
occurs through a receptor that is pharmacologically identical to
recombinant
1-AR. This receptor phenotype is absent in
1-KO mice, indicating that the observed agonist effects
of CGP-12177 are mediated by the
1-AR.
Furthermore, studies in
1- and
1-/
2-KO mice showed an obligatory role of the
1-AR for putative
4-AR effects
(9). CGP-12177-induced effects have been shown to be
coupled to activation of the Gs protein-adenylyl cyclase
system (10). In brown adipose tissue, CGP-mediated
induction of oxygen consumption has been shown (8, 23).
All of these effects can be completely accounted for by the notion of
an atypical state of the
1-AR.
To further dissect molecular and functional aspects of this atypical
receptor state from the atypical 3-AR, we have created a
brown adipocyte cell line from
3-KO mice. In the present
report, we characterize this novel cell line and use it to study the
novel CGP-12177-activated receptor state and the classical
1- and
2-ARs as potential candidates for
the induction of insulin resistance. We demonstrate a multidirectional
inhibitory role in cross talk with insulin.
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MATERIALS AND METHODS |
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Materials.
The partial 3-receptor agonist CGP-12177 was purchased
from Tocris Cookson (Bristol, UK), and the selective
3-receptor agonist CL-316,243 was a generous gift of Dr.
Kurt Steiner (Wyeth-Ayerst Research, Princeton, NJ). Adenosine
deaminase was obtained from Roche Molecular Biochemicals (Mannheim,
Germany). Antibodies used for immunoprecipitation and immunoblotting
included anti-insulin receptor substrate (IRS)-1, anti-IRS-2, and
anti-phosphatidylinositol (PI) 3-kinase p85 (Upstate Biotechnology,
Lake Placid, NY), anti-
3-AR and anti-UCP-1 (Alpha
Diagnostic International, San Antonio, TX), anti-phosphospecific
Ser473-PKB, anti-phosphospecific mitogen-activated protein
(MAP) kinase (New England Biolabs, Beverly, MA), and
anti-phosphotyrosine (Transduction Laboratories, Lexington, KY).
Peroxidase-coupled antibodies came from Dako (Glostrup, Denmark);
recombinant IRS-1 was from Upstate Biotechnology;
125I-labeled Tyr-A14-monoiodoinsulin was
purchased from Amersham-Pharmacia (Freiburg, Germany); and
[
-32P]ATP, 2-deoxy-[3H]glucose, and
D-[1-14C]glucose were from NEN Life
Technologies (Dreieich, Germany). Protein G-Sepharose was from
ImmunoPure (Pierce, Rockford, IL), and nitrocellulose was from
Schleicher & Schuell, (Dassel, Germany). All other materials were from
Sigma (Deisenhofen, Germany).
Cell isolation and culture.
Brown adipocytes and their precursor cells were isolated from newborn
3-AR KO mice by collagenase digestion as described previously (16). Briefly, preadipocytes were immortalized
by infection with the retroviral vector pBabe, encoding SV40 T antigen (kindly provided by J. DeCaprio, Dana Farber Cancer Institute, Boston,
MA) and selected with puromycin (1 µg/ml). Preadipocytes were grown
to confluence in culture medium supplemented with 20 nM insulin and 1 nM triiodothyronine (T3) (differentiation medium). Adipocyte differentiation was induced by treating confluent cells for
24 h in differentiation medium further supplemented with 0.5 mM
isobutylmethylxanthine, 0.5 µM dexamethasone, and 0.125 mM indomethacin. After this induction period (24 h), cells were changed back to differentiation medium for 4-5 days until exhibiting a fully differentiated phenotype with massive accumulation of
multilocular fat droplets. The different stimulation experiments were
carried out after the cells were starved in serum-free medium for
48 h and pretreatment with adenosine deaminase (ADA; 2 U/ml).
Western blot analysis.
Western blotting was performed as previously described
(4). Briefly, after the stimulation period, cells were
washed twice with ice-cold PBS and harvested in lysis buffer (in mM: 50 HEPES, 137 NaCl, 1 MgCl2, 1 CaCl2, 10 Na4P2O7, 10 NaF, 2 EDTA, and 2 vanadate, and 10% glycerol, 1% Igepal CA-630, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 2 mM phenylmethylsulfonyl fluoride, pH 7.4).
Lysates were clarified by centrifugation at 12,000 rpm for 10 min at
4°C. Protein content was determined by the method of Bradford
(1). Equal amounts of protein (100 and 500 µg,
respectively) were either solubilized directly in Laemmli sample buffer
or immunoprecipitated for 2 h at 4°C with the indicated antibodies.
Immunocomplexes were collected by adding 50 µl of protein G-Sepharose
for 2 h at 4°C, washed in lysis buffer, and solubilized in
Laemmli sample buffer. Proteins were resolved by SDS-PAGE, transferred
to nitrocellulose membranes, blocked for 1 h, and immunoblotted
with the appropriate antibodies for 2 h. Specifically bound
primary antibodies were detected with peroxidase-coupled secondary
antibody and enhanced chemiluminescence.
Insulin receptor kinase and binding activities.
Insulin receptor binding and kinase activity were measured essentially
as described (13). Briefly, 40 µl of cell lysate were
added to microwells coated with anti-insulin receptor antibodies for
16 h at 4°C. Wells were washed, and receptor-mediated
32P incorporation into recombinant IRS-1 was measured at a
concentration of 240 nmol/l [-32P]ATP and 2.3 µg/ml recombinant IRS-1. Insulin receptor kinase activity
was expressed as attomoles 32P incorporated into IRS-1
per minute per femtomole insulin-binding capacity (14).
Furthermore, [125I]Tyr-A14-monoiodoinsulin
binding to immobilized insulin receptors was also measured in the wells
as described. Insulin-binding capacity was defined as the amount of
specifically bound insulin at a concentration of 8.7 nmol/l
(14).
Glucose uptake assay. Cells were assayed for glucose uptake essentially as described (5). After a serum-free starvation period of 48 h and pretreatment with ADA (2 U/ml), fully differentiated monolayers of brown adipocytes were treated with insulin (100 nM) for 30 min. At the end of the stimulation period, cells were exposed to 50 µl of 2-deoxy-[3H]glucose (0.5 µCi/ml final concentration) for 4 min, and the incorporated radioactivity was determined by liquid scintillation counting. Nonspecific uptake was measured in the presence of 10 µM cytochalasin B and subtracted from all measured values.
Glycogen synthesis assay. Fully differentiated monolayers of brown adipocytes were starved in serum-free medium for 48 h, washed two times with Krebs-Ringer-HEPES buffer, and then pretreated with ADA (2 U/ml). After the pretreatment period with the indicated agonists and antagonists, insulin (100 nM) was added for 30 min. Cells were exposed for 1 h to 50 µl of D-[1-14C]glucose (0.2 µCi/ml final concentration) and then washed two times with ice-cold PBS. After an incubation with 30% KOH for 15 min at 37°C, lysates were transferred to new vials prefilled with 50 µl of glycogen (40 mg/ml), and samples were boiled for 30 min. After addition of 95% ethanol, samples were centrifuged for 5 min at 13,000 rpm. The supernatant was removed and the pellet redissolved in 500 µl of distilled water. The incorporated radioactivity was determined by liquid scintillation counting.
Statistical analysis. Results are indicated as means ± SE. Unpaired Student's t-tests were used for analysis of differences between various cell treatments. P values <0.05 are considered significant and <0.01 highly significant.
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RESULTS |
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3-KO adipocytes can be fully differentiated and are
insulin sensitive.
As expected, immunoblot analyses failed to detect the
3-AR in brown preadipocytes derived from the
3-KO mouse (Fig.
1A). By use of a
differentiation protocol with triiodothyronine, insulin, dexamethasone, indomethacin, and isobutylmethylxanthine, both wild-type
and
3-KO cells massively accumulated fat in typical multilocular droplets over a period of 10 days, which was demonstrated by the fat-specific Oil red O stain (Fig. 1B). No major
difference was noted between wild-type and
3-KO cells
with regard to kinetics and extent of maximal differentiation. Insulin
stimulation of fully differentiated
3-KO cells
demonstrated a remarkable increase in tyrosine phosphorylation of the
insulin receptor, IRS-1, and IRS-2 (Fig. 1C). Insulin also
induced a robust augmentation in binding of the regulatory PI 3-kinase
subunit p85 to IRS-1 and IRS-2 (Fig. 1C). Finally, a
strong insulin-induced activation of the major downstream signaling
intermediates PKB and MAP kinase was found in differentiated
3-KO cells, thus demonstrating a high level of insulin
sensitivity in these cells (Fig. 1C).
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CGP-12177 induces UCP-1 protein expression in 3-KO
adipocytes.
Fully differentiated
3-AR-deficient brown
adipocytes were stimulated with 10 µM partial
3-AR
agonist CGP-12177 for 18 h. Western blot analysis with a
UCP-1-specific antibody showed a fivefold increase of UCP-1 protein
levels (Fig. 2). In contrast, the
3-selective agonist CL-316,243 had no effect on basal
UCP-1 protein expression (Fig. 2). The observed CGP-12177 effect was dose dependent, with small increases detectable at concentrations as
low as 1 nM (Fig. 2).
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CGP-12177 reduces insulin-induced glucose uptake and glycogen
synthesis independently of 1- and
2-ARs.
Glucose uptake and glycogen synthesis represent two important
functional end points of the insulin-signaling cascade. Insulin treatment (100 nM) stimulated a 6.5-fold increase in basal glucose uptake and an 11-fold increase in glycogen synthesis in differentiated
3-KO adipocytes (Fig. 3,
A and B). When cells were pretreated with
CGP-12177 at a concentration of 1 µM 30 min before insulin stimulation, the insulin-induced glucose uptake was reduced by 65%
(P < 0.01; Fig. 3A). Similarly,
insulin-induced glycogen synthesis was decreased by 50%
(P < 0.01; Fig. 3B). These effects were
dose responsive, with decreases already detectable at a CGP-12177
concentration of 10 nM (Fig. 3, A and B).
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Dobutamine and clenbuterol inhibit insulin-induced glucose uptake
via 1- and
2-ARs.
Furthermore, we determined whether activation of the classical
1- and
2-AR also inhibits insulin-induced
glucose uptake. When
3-KO adipocytes were pretreated for
30 min with the
1-AR agonist dobutamine (1 µM) or the
2-AR agonist clenbuterol (10 nM), insulin-induced
glucose uptake was diminished by maximally 70% and 85%, respectively
(P < 0.01; Fig. 4,
A and B). As expected, and in contrast to
CGP-12177 treatment, the
1-AR-mediated negative effects
of dobutamine were diminished by pretreatment of cells with the
selective
1-antagonist metoprolol in a
dose-dependent manner and could be almost completely abolished at
metoprolol concentrations of 1 µM (P < 0.01; Fig.
4C). Similarly, the
2-AR-mediated effects
could be fully blocked by pretreatment with the selective
2-antagonist ICI-118,551 at concentrations as low as 10 nM (Fig. 4D).
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CGP-12177 impairs insulin-stimulated binding of IRS-1 and IRS-2 to
PI 3-kinase and PKB activation.
Because the observed reductions in insulin-induced glucose uptake and
glycogen synthesis by CGP-12177 stimulation might be caused by
alterations at a proximal level of insulin signaling, we first measured
the activity of the insulin receptor kinase toward IRS-1. Insulin
stimulation induced an 11-fold increase in receptor kinase activity
(Fig. 5A). However, this
increase was not altered by CGP-12177 pretreatment (Fig.
5A). Furthermore, insulin binding to its receptor showed no
difference between CGP-12177-treated and nontreated cells (data not
shown). To investigate whether CGP-12177 treatment impairs the
insulin-signaling cascade distal from the insulin receptor, tyrosine
phosphorylation of IRS-1 and IRS-2, IRS-1/2 binding to the p85
regulatory subunit of PI 3-kinase and the phosphorylation of
Ser473-PKB and MAP kinase were measured. CGP-12177
pretreatment tended to diminish insulin-induced tyrosine
phosphorylation of IRS-1 and IRS-2 by 10%, but this change did not
reach the level of statistical significance (data not shown).
Furthermore, insulin-induced binding of p85 to IRS-1 and IRS-2 was
reduced by ~25 and 35%, respectively, after CGP-12177 (1 µM)
pretreatment (P < 0.05; Fig. 5, B and
C). Moreover, CGP-12177 treatment led to a small but highly
significant decrease in insulin-mediated Ser473-PKB
phosphorylation by 20% (P < 0.01; Fig.
5D), whereas the phosphorylation of MAP kinase was not
impaired (data not shown).
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DISCUSSION |
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In this study, we characterize a newly established brown adipocyte
line derived from the 3-AR knockout mouse and use it to dissect a potent inhibitory role of the atypical
1-AR
state and the classical
1- and
2-AR in
the interplay with insulin signaling and action.
We have previously described an adipocyte cell system that allows for
the generation of cell lines from a single, late fetal or newborn mouse
from a broad variety of different mouse models, including those with
perinatal mortality (4, 5, 15, 16). We have now used the
same approach to establish SV40T-immortalized brown adipocytes from the
3-AR knockout mouse (27). As we demonstrate here, immortalized
3-KO brown adipocytes differentiate
normally and are highly insulin sensitive. Therefore, the
3-KO adipocytes provide an ideal tool for separating
biological effects of the novel CGP-12177-activated
1-AR
state from
3-adrenergic effects and for characterizing
its role in cross talk with insulin.
As expected, the highly selective 3-AR agonist
CL-316,243 fails to induce UCP-1 protein expression in the
3-AR-deficient cell line, confirming the absence of the
3-AR (27). The aryloxypropanolamine CGP-12177 has been used in previous studies to infer the existence of a
novel atypical
-AR. Although the actions of CGP-12177 at the
respective receptor site are incompletely understood, a number of
studies provide compelling evidence for the notion of a novel state of
the
1-AR mediating this compound's effects (17,
18, 22). We show that, in contrast to CL-316,243, CGP-12177
induces UCP-1 protein expression in a dose-dependent manner in the
3-KO cells. This most likely provides the molecular
basis for previous findings demonstrating an increase in oxygen
consumption in adipose tissue of
3-KO mice after
CGP-12177 treatment (8, 23).
Recently, we have delineated cross talk between the sympathetic nervous
system and the insulin signaling cascade. Thus, on the one hand,
3-adrenergic stimulation with the selective
3-AR phenethanolamine agonist CL-316,243 inhibits the
insulin signaling cascade on several levels and results in a reduction
of insulin-induced glucose uptake (16). On the other hand,
insulin impacts on
-adrenergic regulation of UCP-1 expression
(15). This cross talk between adrenergic and insulin
signaling systems may play an important role in the pathogenesis of
insulin resistance and the insulin resistance syndrome. We now compared
effects of CGP-12177 on important biological end points of insulin
action to those of classical
1- and
2-AR
agonists and antagonists. When cells were pretreated with the
1-AR agonist dobutamine, insulin-induced glucose uptake was almost completely abolished. These negative effects could be
blocked by pretreatment with the classical selective
1-AR antagonist metoprolol, indicating that the observed
inhibition is mediated via the
1-AR. In parallel,
2-adrenergic stimulation with clenbuterol dramatically
decreased insulin-induced glucose uptake, and this inhibition could be
completely abolished by pretreatment with the selective
2-AR antagonist ICI-118,551. The negative CGP-12177
effects on insulin action were similar in their extent compared with
1- and
2-AR agonists, but in contrast to
these classical
-ARs, the CGP-12177-mediated inhibition of insulin action in the
3-KO adipocytes could be blocked neither
by selective
1- and
2-antagonists nor by
nonselective
-blockade, thus fulfilling the functional criteria for
an atypical
-adrenoceptor, i.e., 1) stimulation by
CGP-12177, 2) resistance to
-blockade, and 3)
lack of activation by
3-selective agonists like
CL-316,243. This can be taken to indicate a concurrent negative role of
both the novel
1-AR state and the classical
1- and
2-ARs in controlling insulin
action with respect to both glucose uptake and glycogen synthesis. Our
study characterizes for the first time the role of this novel atypical
receptor state and of the classical
1- and
2-AR in the interplay with insulin action in brown
adipocytes. It has to be noted, however, that actions of CGP-12177 at
the
1-AR site are incompletely understood. Due to the
present lack of detailed information on the molecular properties of the
novel
1-AR state, conclusions as to its effects using
this compound remain indirect.
The observed reduction of insulin-induced glucose uptake by
conventional 1-,
2-, and the novel
atypical
1-receptor state is similar to the one seen
after
3-adrenergic stimulation in brown adipocytes from
wild-type mice (16). In line with these previous findings,
it appears safe to assume a signaling pathway from a stimulatory
-adrenergic G protein subunit that activates adenylyl cyclase and
protein kinase A to exert the negative effect on both glucose uptake
and, as shown here for the first time, glycogen synthesis. Konkar et
al. (17) found that CGP-12177 activated brown fat adenylyl
cyclase in
3-KO mice. Furthermore, increases of
intracellular cAMP levels, enhanced activity of cAMP-dependent protein
kinase, and potentiation of effects by the phosphodiesterase inhibitor isobutylmethylxanthine have been demonstrated for
stimulation with CGP-12177 (10). Yet, interestingly, in
our study, the insulin receptor kinase activity and the insulin-binding
capacity were not impaired after CGP-12177 stimulation, whereas
p85-binding to IRS-1 and IRS-2 was diminished. These findings may
indicate a negative regulation of the novel
1-AR state
on an insulin receptor-independent proximal level of the insulin
signaling cascade. This appears to be in contrast to the
3-AR, where reduced tyrosine phosphorylation of IRS-1/2
and the associated PI 3-kinase activity were correlated with decreased
phosphorylation of the insulin receptor (16). Thus the
novel atypical adrenoceptor may employ different signaling pathways to
interact with insulin action. Potential signaling candidates could be
protein kinase C- and p38 MAP kinase-dependent pathways. In line with
this concept, qualitatively different effects of different
conformational
1-AR states in mouse cardiac muscle have
been suggested (9). In this context, it should also be mentioned that switching from Gs to Gi protein
coupling of receptors, depending on specific receptor phosphorylation
sites, has been described for the
2-AR (2).
This could well be another intriguing signaling mechanism of the novel
1-AR state. Nevertheless, similar to the
3-AR, a relatively small inhibition of PKB, a key
element for metabolic insulin signaling, by the novel-state
1-AR appears to be sufficient to cause a considerable
inhibition of glucose uptake and glycogen synthesis.
Taken together, the newly established 3-KO brown
adipocyte cell line provides an ideal tool for investigating
adrenoceptor-insulin cross talk and its impact on adipose tissue
insulin resistance. The novel state
1-AR in
3-KO brown adipocytes increases UCP-1 protein expression
and reduces insulin-induced glucose uptake and glycogen synthesis by
postreceptor alterations of proximal insulin signaling elements.
Stimulation of classical
1- and
2-AR results in the same negative effects on insulin action. These interactions may play a role in the development of the insulin resistance syndrome.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge Dr. James DeCaprio (Dana Farber Cancer
Institute, Boston, MA) for providing us with the SV40T-encoding retroviral pBabe vector. We are indebted to Dr. Kurt Steiner
(Wyeth-Ayerst Research, Princeton, NJ) for the generous gift of the
selective 3-adrenoceptor agonist CL-316,243.
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FOOTNOTES |
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This work was supported by grants from the Deutsche Forschungsgemeinschaft (Kl1131/2-1) and the Deutsche Diabetes Gesellschaft to J. Klein. P. Jost is a recipient of a scholarship from the Studienstiftung des Deutschen Volkes.
Address for reprint requests and other correspondence: J. Klein, Dept. of Internal Medicine I, Medical Univ. of Lübeck, Ratzeburger Allee 160, 23538 Lubeck, Germany (E-mail: johannes.klein{at}medinf.mu-luebeck.de).
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.
First published March 5, 2002;10.1152/ajpendo.00531.2001
Received 27 November 2001; accepted in final form 27 February 2002.
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