Glucagon-like peptide-1 improves insulin and
proinsulin binding on RINm5F cells and human monocytes
Martin
Ebinger1,
Daniela
R.
Jehle2,
Rolf D.
Fussgaenger3,
Hans C.
Fehmann4, and
Peter M.
Jehle2
1 Children's Hospital Mannheim, University of Heidelberg,
68735 Mannheim; Departments of Internal Medicine 3 I and
2 II, University of Ulm, 89070 Ulm; and 4 Department of
Internal Medicine, Philipp's University of Marburg, 35033 Marburg,
Germany
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ABSTRACT |
Glucagon-like peptide-1-(7---36) amide (GLP-1) is a potent incretin
hormone secreted from distal gut. It stimulates basal and glucose-induced insulin secretion and proinsulin gene expression. The
present study tested the hypothesis that GLP-1 may modulate insulin
receptor binding. RINm5F rat insulinoma cells were incubated with GLP-1
(0.01-100 nM) for different periods (1 min-24 h). Insulin receptor
binding was assessed by competitive ligand binding studies. In
addition, we investigated the effect of GLP-1 on insulin receptor binding on monocytes isolated from type 1 and type 2 diabetes patients
and healthy volunteers. In RINm5F cells, GLP-1 increased the capacity
and affinity of insulin binding in a time- and concentration-dependent manner. The GLP-1 receptor agonist exendin-4 showed similar effects, whereas the receptor antagonist exendin-(9---39) amide inhibited the
GLP-1-induced increase in insulin receptor binding. The GLP-1 effect
was potentiated by the adenylyl cyclase activator forskolin and the
stable cAMP analog
Sp-5,6-dichloro-1-
-D-ribofuranosyl-benzimidazole-3',5'-monophosphorothioate but was antagonized by the intracellular Ca2+ chelator
1,2-bis(0-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM.
Glucagon, gastric inhibitory peptide (GIP), and GIP-(1---30) did not
affect insulin binding. In isolated monocytes, 24 h incubation with 100 nM GLP-1 significantly (P < 0.05) increased
the diminished number of high-capacity/low-affinity insulin binding
sites per cell in type 1 diabetics (9,000 ± 3,200 vs. 18,500 ± 3,600) and in type 2 diabetics (15,700 ± 2,100 vs. 28,900 ± 1,800) compared with nondiabetic control subjects (25,100 ± 2,700 vs. 26,200 ± 4,200). Based on our previous experiments in
IEC-6 cells and IM-9 lymphoblasts indicating that the
low-affinity/high-capacity insulin binding sites may be more specific
for proinsulin (Jehle, PM, Fussgaenger RD, Angelus NK, Jungwirth RJ,
Saile B, and Lutz MP. Am J Physiol Endocrinol Metab
276: E262-E268, 1999 and Jehle, PM, Lutz MP, and Fussgaenger RD.
Diabetologia 39: 421-432, 1996), we further
investigated the effect of GLP-1 on proinsulin binding in RINm5F cells
and monocytes. In both cell types, GLP-1 induced a significant increase
in proinsulin binding. We conclude that, in RINm5F cells and in
isolated human monocytes, GLP-1 specifically increases the number of
high-capacity insulin binding sites that may be functional proinsulin receptors.
diabetes mellitus; incretin hormones; insulin resistance; insulin
secretion
 |
INTRODUCTION |
AS EARLY AS 1906, the
treatment of diabetes mellitus with an acid extract of duodenal mucous
membrane was proposed (33). This idea was further pursued
in 1929 by Zunz and LaBarre (48), who coined the term
"incretin" for a humoral factor from the intestinal tract that
releases insulin or potentiates the glucose-induced insulin release.
The ability of an oral glucose load to release more insulin than an
intravenous glucose infusion, despite a similar increase in the blood
glucose level, is due to the release of incretin hormones
(10). In recent years, evidence has accumulated that
glucagon-like peptide-1 (GLP-1) is an important incretin hormone
(3, 8, 26,
35-38). GLP-1 is derived from the posttranslational processing of proglucagon [proglucagon-(78---107) amide; see Ref. 2]
and is secreted from the distal parts of the jejunum, ileum, and colon
in response to mixed meals (11, 26). Despite
the physiological importance of the enteroinsular axis, disruption of
GLP-1 is associated with only modest glucose intolerance in GLP-1
receptor
/
mice (44). These animals exhibit
compensatory changes in the enteroinsulinar axis via increased
glucose-dependent insulinotropic polypeptide (GIP) secretion and GIP
action (40). Although GLP-1 is supposed to play an
important role in the regulation of food intake (19),
available data suggest that GLP-1 signaling may not be essential for
the regulation of satiety or body weight (6).
In clinical studies in diabetics of both types, GLP-1 has been shown to
considerably improve diabetes control (9, 18, 33). In contrast to other drugs for the treatment of type
2 diabetes, GLP-1 possesses a "glucagonostatic" effect that is of high therapeutic relevance (11, 34). The
mechanisms responsible for the actions of GLP-1 in peripheral
tissues, such as fat, liver, and muscle, remain unclear. In the
present study, we tested the hypothesis that GLP-1 may specifically
modulate insulin receptor binding, as suggested by our preliminary
findings in RINm5F insulinoma cells (23). The present
study extended these experiments and investigated the effects of GLP-1
agonistic and antagonistic peptides and structurally related
gastrointestinal hormones. We further addressed the question whether
GLP-1 may also modulate insulin receptors in other cell types and
determined insulin receptor binding in monocytes isolated from healthy
subjects and diabetic patients. Finally, additional experiments are
provided, showing that GLP-1 increases the binding of proinsulin but
not insulin-like growth factor I (IGF-I).
 |
MATERIALS AND METHODS |
Reagents.
GLP-1-(7---36) amide, GLP-1-(7---37), exendin-4, exendin-(9---39)
amide, GIP-(1---42), truncated porcine GIP-(1---30), forskolin, and human recombinant IGF-I were obtained from Saxon (Hannover, Germany). Human biosynthetic insulin, proinsulin, and glucagon were from Lilly
(Indianapolis, IN),
Sp-5,6-dichloro-1-
-D-ribofuranosyl-benzimidazole-3',5'-monophosphorothioate (cBiMPS) was from Biolog (Bremen, Germany), and 1,2-bis(0-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM was from
Calbiochem (Bad Soden/Taunus, Germany). Human biosynthetic
[125I]TyrA14-insulin (specific activity 360 µCi/µg) was labeled and HPLC was purified by A. Liebe (Hoechst,
Frankfurt, Germany). Human recombinant [125I]Tyr-A14
proinsulin (specific activity 233 µCi/µg) was labeled by the
lactoperoxidase method and subsequently purified by HPLC, as previously
described (24). [125I]IGF-I (specific
activity 250 µCi/µg) was purchased from Amersham (Braunschweig,
Germany). [125I]GLP-1-(7---36) amide was labeled using
the chloramine T method and was purified by HPLC as described
(17). The binding assay buffer consisted of 50 mmol/l
HEPES, 10 mmol/l dextrose, 15 mmol/l sodium acetate, 5 mmol/l KCl, 120 mmol/l NaCl, 1.2 mmol/l MgSO4, 10 mmol/l CaCl2,
and 0.1% BSA, pH 7.8 adjusted with NaOH. All chemicals were purchased
from Merck (Darmstadt, Germany). Forskolin and cBiMPs were solved in
DMSO and then diluted in HEPES assay buffer. All other reagents were
solved in HEPES assay buffer.
RINm5F cell culture.
RINm5F cells were kindly provided by H. P. T. Ammon
(Tübingen, Germany). This established insulinoma cell line
expresses insulin and GLP-1 receptors (14,
16). Cells were grown in RPMI 1640 (Biochrom, Berlin,
Germany) and were supplemented with 25 mM HEPES buffer, 10% FCS
(Seromed, Munich, Germany), 200 mM glutamine, 100 U/ml penicillin G,
and 10 µg/ml streptomycin, pH 7.4. Medium was changed every 3 days.
Cells were divided one time per week by trypsination.
Isolation of human monocytes.
After informed consent from healthy nondiabetic volunteers
{n = 1/3 [females (F)/males (M)]; age 28.0 ± 4.1 (SD) yr; body mass index 22.9 ± 1.5 kg/m2},
type 1 diabetes patients [n = 1/3 (F/M); age 36.0 ± 4.0 yr; body mass index 25.6 ± 2.3; duration of diabetes
mellitus 10.3 ± 5.6 yr; fasting blood glucose 9.8 ± 1.4 mmol/l; HbA1c 9.0 ± 3.1%], and type 2 diabetes
patients [n = 2/2 (F/M); age 56.8 ± 5.6 yr; body
mass index 30.6 ± 3.2; duration of diabetes mellitus 11.8 ± 6.9 yr; fasting blood glucose 13.4 ± 1.1 mmol/l;
HbA1c 11.3 ± 2.6%], 40 ml venous blood were
collected. Mononuclear cells were isolated by Ficoll-Hypaque density
gradient sedimentation (Sigma, Deisenhofen, Germany), as previously
described (21). The mononuclear cell layer was removed and
diluted in HEPES assay buffer to a final concentration of
107 mononuclear cells/ml. Viability as assessed by trypan
blue exclusion was always >95%.
Receptor binding studies.
Freshly isolated monocytes were incubated for 24 h with indicated
concentrations of GLP-1 in binding assay buffer at 37°C. The cell
suspension was washed two times and adjusted to a cell number of
106 cells/ml in binding assay buffer. The binding assays
with RINm5F cells were performed with confluent cell monolayers grown
to a density of 1.5 × 106/well. Both cell types were
incubated in assay buffer and solvent with GLP-1 or other agents or
solvent alone for the indicated time periods. After removing the
incubation buffer and washing the cells two times, specific binding of
insulin, proinsulin, IGF-I, and GLP-1 was determined from competitive
binding studies as previously described (21). Briefly,
cells were incubated in assay buffer with 10 pM of
125I-labeled peptides and various concentrations (10 pM-1
µM) of unlabeled homologous peptides for 2 h at 15°C. Cell
bound and free intact activities were measured in an automatic gamma
counter with 70% efficiency. Degradation of labeled peptides was
determined by measuring the ability of intact tracer peptides to
precipitate in ice-cold 5% TCA. The percentage of degraded tracer was
determined after centrifugation (2,000 g force, 10 min) from
the increase in TCA-soluble radioactivity over that observed in control
wells containing buffer but no cells. 125I-labeled insulin,
proinsulin, and IGF-I were >98% precipitable before and >90%
precipitable at the end of each experiment. 125I-GLP-1 was
>97% precipitable before and >85% precipitable at the end of each
experiment. Specific 125I peptide binding was determined by
subtracting the amount of radioactivity bound nonspecifically in the
presence of 1 µM unlabeled homologous peptide. All binding data were
corrected for nonspecific binding and tracer degradation. Peptide
binding capacity (%specific tracer binding) and affinity
(IC50 values indicating half-maximal tracer displacement)
were calculated from computer-assisted competition-inhibition curves.
The number of binding sites per cell and dissociation constant
(Kd) values of binding affinity were estimated
by computer-assisted Scatchard analysis (21,
24, 42).
Data analysis.
If not otherwise indicated, data are expressed as mean values ± SE of 3-17 independent experiments using separate sets of cells.
Unpaired Student's t-test was used for statistical
comparisons. A P value <0.05 was considered statistically significant.
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RESULTS |
Binding of 125I-GLP-1-(7---36) amide to RINm5F cells
and monocytes.
RINm5F cells displayed specific binding of
125I-GLP-1-(7---36) amide (4.6%/106 cells;
n = 3) that was competed by unlabeled GLP-1 with high affinity (IC50: 0.2 nM; Kd: 0.3 nM).
Using the same tracer in freshly isolated monocytes of two healthy
subjects, only weak binding of 125I-GLP-1-(7---36)-amide
could be measured (0.44 ± 0.11%/106 cells) without
displacement by unlabeled GLP-1 given from 10 pM to 1 µM. Increasing
the number of monocytes up to 4 × 107 cells or
performing the binding studies at 4°C did not change the negative results.
Effect of GLP-1 and related peptides on insulin binding in RINm5F
cells.
As shown in Fig. 1, GLP-1-(7---36) amide
induced a time-dependent increase of specific insulin binding with
significant effects already after 5 min. A twofold higher insulin
binding was observed after 24 h incubation without further
increases by longer incubation periods (up to 72 h). GLP-1 yielded
a dose-dependent stimulation of insulin binding, with half-maximal
effects at 0.1 nM given for 24 h (Fig.
2). We next investigated whether
structurally or effect-related hormones may also modulate insulin
receptor binding. RINm5F cells were incubated for 2 h with 1 nM of
GLP-1 alone or in combination with the GLP-1 receptor agonist exendin-4
or the GLP-1 receptor antagonist exendin-(9---39) amide
(8, 17). GLP-1-(7---36) amide alone increased
insulin binding from 100% (control) to 142 ± 6.3%
(n = 17). Similar effects were observed with
GLP-(7---37), which increased insulin binding to 141.7 ± 18.4%
(n = 3). Incubation with exendin-4 (1 nM) alone
stimulated insulin binding from 100% (control) to 161 ± 6%
(P < 0.001; n = 6). The GLP-1
antagonist exendin-(9---39) amide alone showed no significant effects
(116 ± 8%; n = 6). The GLP-1-induced increase in
insulin binding (150 ± 10%) could be effectively blocked by 100 nM of exendin-(9---39) amide (116 ± 9%, P < 0.05 vs. GLP-1 alone; n = 6). As shown in Fig.
3, the dose-dependent increase in insulin
binding observed with GLP-1-(7---36) amide was not seen with glucagon,
GIP, or GIP-(1---30).

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Fig. 1.
Time dependency of the effect of 1 nM glucagon-like
peptide (GLP)-1 on specific 125I-labeled insulin binding in
RINm5F cells. Specific 125I-insulin binding was determined
from competitive binding studies by subtracting the amount of
radioactivity bound nonspecifically in the presence of 1 µM unlabeled
insulin, which was <3%. All binding data were corrected for
nonspecific binding and tracer degradation. Data are means ± SE
of 3 independent experiments. * P < 0.05 and
** P < 0.005.
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Fig. 2.
Dose dependency of the GLP-1 effect on specific
125I-insulin binding in RINm5F cells. Cells were incubated
for 12 h with different concentrations of GLP-1. Specific
125I-insulin binding was determined as described in
MATERIALS AND METHODS and Fig. 1. Data are means ± SE
of 6 independent experiments. * P < 0.05 and
** P < 0.01.
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Fig. 3.
Effects of GLP-1 and related peptides on
125I-insulin binding in RINm5F cells. Cells were incubated
for 2 h with 1 and 100 nM of GLP-1, glucagon, GIP, and
GIP-(1---30). Specific 125I-insulin binding was determined
from competitive binding studies as described in MATERIALS AND
METHODS. Mean values ± SE of 6-17 separate experiments
are shown. ** P < 0.001.
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Mechanism of GLP-1-induced increase of insulin binding in RINm5F
cells.
Competitive binding data revealed that GLP-1 increased
125I-insulin binding due to an increased receptor binding
affinity and capacity. Figure 4
demonstrates a marked left shift of the competition-inhibition curves
after 24 h incubation with GLP-1, indicating the improved binding
affinity (IC50: 0.12 vs. 1.5 nM; Kd:
1.2 vs. 3.2 nM; GLP-1 vs. control, P < 0.05). As shown
by Scatchard analysis (Fig. 5), the total
number of insulin receptors concomitantly increased by 2.5-fold.
Performing Northern blot analysis using our previously described
protocol (12), we were not able to detect an increase of
insulin receptor mRNA after GLP-1 incubation. In control cells, the
Scatchard plot of insulin binding was nearly linear, whereas a marked
curvilinearity was observed after incubation with GLP-1. Because it
appeared that GLP-1 did not modulate insulin receptor mRNA but
influences negative cooperativity of insulin receptors and/or
heterogeneity of binding sites, we further compared the effect of GLP-1
on specific binding of insulin and proinsulin in RINm5F cells. As shown
in Fig. 6, GLP-1 induced a dose- and time-dependent increase in the amount of proinsulin binding.

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Fig. 4.
Competition-inhibition curves of 125I-insulin
binding to RINm5F cells incubated with 1 nM GLP-1 or solvent for
24 h. Cells were incubated with 10 pM of 125I-insulin
and various concentrations (10 pM-1 µM) of unlabeled insulin in assay
buffer for 2 h at 15°C. GLP-1 induced a marked increase in
insulin binding affinity. Data are means ± SE of 3 independent
experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001.
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Fig. 5.
Scatchard analysis of 125I-insulin binding in
RINm5F cells incubated with 1 nM GLP-1 or solvent for 24 h.
Binding data from Fig. 4 were analyzed according to Scatchard analysis
using a computer-assisted curve-fitting program. Two classes of binding
sites were obtained after incubation with GLP-1, with a significant
(P < 0.05) increase in the number of
low-affinity/high-capacity sites. Mean values ± SE of 3 separate
experiments are shown.
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Fig. 6.
Effect of GLP-1 on specific 125I-proinsulin
binding in RINm5F cells. Specific 125I-proinsulin binding
was determined by subtracting the amount of radioactivity bound
nonspecifically in the presence of 1 µM unlabeled proinsulin. Cells
were preincubated with different concentrations of GLP-1 for different
periods. Binding data were corrected for nonspecific binding and tracer
degradation. Data are means ± SE of 3 independent experiments.
* P < 0.01 and ** P < 0.001.
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Finally, we assessed which intracellular pathway might be responsible
for the GLP-1-induced increase in insulin binding. Therefore, we
incubated RINm5F cells in HEPES assay buffer with the adenylyl cyclase
activator forskolin (28), the stable cAMP analog cBiMPS (41), or the intracellular Ca2+ chelator
BAPTA-AM (45). Control cells were incubated with the same
volume of solvent alone. Two hours of incubation with forskolin (1 µM) or cBiMPS (100 nM) increased insulin binding capacity to 156 ± 15% or 145 ± 10% (P < 0.01 vs. controls;
n = 6). In contrast, BAPTA-AM (10 µM) did not
increase insulin binding (91 ± 5%; n = 6).
Coincubation of BAPTA-AM with GLP-1 (1 nM) completely antagonized the
stimulatory effect of GLP-1 on insulin receptor binding (92 ± 2%, P < 0.01 vs. GLP-1 alone; n = 6).
Effect of GLP-1 on insulin binding in monocytes.
To address the question whether GLP-1 may affect insulin receptor
binding in peripheral non-insulin-dependent tissues, we performed
binding studies with mononuclear cells isolated from type 1 and type 2 diabetics and healthy control subjects. Scatchard analysis of binding
data indicated two classes of binding sites (high-affinity/low-capacity
and low-affinity/high-capacity), as in previous reports
(5, 21, 24). As shown in Table
1, type 1 and type 2 diabetics showed a
significantly lower number of high-capacity sites than control subjects
(P < 0.05). Interestingly, GLP-1 (100 nM) yielded a
significant increase in the number of high-capacity sites in both types
of diabetes patients (P < 0.05). In isolated
monocytes, we further compared the effects of 100 nM GLP-1 given for
24 h on the binding of insulin, proinsulin, and IGF-I. As shown in
Table 2, proinsulin binding was
significantly increased in all groups by GLP-1, whereas insulin binding
increased in type 2 diabetics only. Interestingly, GLP-1 did not
influence IGF-I binding.
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Table 1.
Effect of GLP-1 on number and affinity of insulin receptors on
monocytes isolated from healthy subjects, type 1 diabetics, and type 2 diabetics
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Table 2.
Effects of GLP-1 on binding of insulin, proinsulin, or IGF-I on
monocytes isolated from healthy subjects, type 1 diabetics, and type 2 diabetics
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DISCUSSION |
GLP-1 regulates several functions in
-cells (6,
11). In the presence of elevated glucose levels, the
peptide potently augments insulin secretion. Insulin production is
increased by a direct effect on proinsulin gene transcription, and
glucose-insensitive
-cells are rendered responsive by the peptide.
GLP-1 regulates function and number of its own receptors on
-cells.
Other hormones, e.g., somatostatin and leptin, have been shown to
inhibit the effects of GLP-1. The present study demonstrates for the
first time that GLP-1 improves insulin and proinsulin binding in
insulinoma cells and in monocytes isolated from healthy and diabetic
subjects. In RINm5F cells, GLP-1 exerts time- and dose-dependent
effects, with 30% of the maximum already at concentrations of
20-50 pM, which are found in humans under physiological conditions
(37). GLP-1 significantly increased both capacity and
affinity of insulin binding. The stimulatory effect of GLP-1 on insulin
binding occured even within minutes. This suggests that GLP-1 may act
on insulin receptors mainly located within or near the cell membrane
rather than stimulating receptor de novo synthesis. From the changes in
the shape of the Scatchard plots, it appeared that GLP-1 may lead to a
conformational change of the insulin receptor that may be responsible
for the improved binding affinity and capacity (5,
29, 47). GLP-1 increased the number of
low-affinity/high-capacity sites, which in terms of their biological
role are still a matter of debate [e.g., sparing receptors, insulin
receptors mediating the mitogenic effects of insulin (5),
binding sites for proinsulin/C peptide (20,
24)]. GLP-1 also induced a significant increase in
proinsulin binding. This is in accordance with our previous findings in
IM-9 lymphoblasts (24) and the small intestinal crypt cell
line IEC-6 (20), indicating that the
low-affinity/high-capacity insulin binding sites are receptors for
proinsulin. In isolated pancreatic islets of normal hamsters,
proinsulin was shown to be more potent than insulin to decrease insulin
secretion (7). In epidemiological studies, elevated
proinsulin levels have been correlated with
-cell injury, obesity,
type 2 diabetes, and cardiovascular mortality (13,
25). Although the role of proinsulin in atherogenesis is
not clear, in should be noted that proinsulin stimulates the synthesis
of plasminogen-activator inhibitor type 1 from vascular endothelial
cells with higher potency than insulin (43). Therefore, it
would make sense that GLP-1 increases not only insulin but also
proinsulin binding on
-cells to prevent proinsulin secretion in the
postprandial state. This hypothesis needs to be confirmed in further
studies, e.g., by revealing the molecular structure and biological
relevance of the proinsulin receptor and using pancreatic
-cell-specific knock out of the insulin receptor (27).
The increase in insulin binding capacity was specific for GLP-1 and was
not found for the related peptides GIP or glucagon. Exendin-4, a known
agonist at the GLP-1 receptor (17), induced GLP-1 like
effects, whereas exendin-(9---39) amide, an antagonist with a small
intrinsic activity in long-term experiments (17), blocked
the GLP-1 effect by ~75%. We therefore assessed which pathways may
be involved in mediating the stimulatory effects of GLP-1 on insulin
receptor binding. On pancreatic
-cells, GLP-1 interacts with
specific G protein-coupled receptors, thereby facilitating insulin
exocytosis by raising intracellular levels of cAMP and Ca2+
(11, 30-32). Our data in RINm5F cells
revealed that forskolin and cBiMPS imitated the GLP-1 effect, whereas
chelation of intracellular Ca2+ by BAPTA-AM completely
antagonized the GLP-1 effect. The results indicate that the effect of
GLP-1 on insulin binding is mediated by cAMP and Ca2+ as
second messengers. It should be noted that GLP-1, GIP, and glucagon
activate the same pathway. In our experiments, we did not determine the
abundance of receptors for glucagon, GIP, or GLP-1 to rule out the
possibility that differences in receptor expression may account for the
GLP-1-specific effect. There are alternative explanations for our
observation that GLP-1 but neither GIP nor glucagon modulated insulin
receptor binding in insulinoma cells and monocytes. First, GIP and
glucagon receptors may be expressed at a lower level than GLP-1
receptors; second, within the
Gs-cAMP-protein kinase A pathway,
Ca2+ influx, Ca2+ mobilization, and
phosphorylation of downstream signaling proteins could show different
kinetics when induced by GLP-1, GIP, and glucagon; third, the cross
talk between G protein-coupled pathways and tyrosine kinases (e.g.,
insulin receptor) may be different for GLP-1, GIP, or glucagon
(15, 46).
Our observation that GLP-1 but not GIP increases insulin binding may be
of clinical relevance and may explain why GLP-1 preserved its activity
as an incretin hormone in patients with type 2 diabetes mellitus,
whereas GIP did not (33). GLP-1 not only acts as an incretin hormone stimulating postprandial insulin secretion but also
enhances peripheral glucose metabolism. The mechanisms underlying the
latter effect of GLP-1 remain unclear. It has been suggested that GLP-1
may improve insulin sensitivity in healthy subjects and in diabetic
patients (4, 18). Hyperinsulinemic,
euglycemic clamp studies in healthy humans showed that GLP-1-(7---36)
amide administered for 3 h, leading to circulating levels within
the physiological range, does not affect insulin sensitivity
(39). It is possible that the conventional techniques for
assessing insulin sensitivity in vivo are not sensitive to detect the
effect of GLP-1 on insulin binding. Although GLP-1 receptors are
reported in a wide variety of tissues (6,
11), it could be possible that the effect of GLP-1 is
limited to pancreatic
-cells and not to tissues that comprise the
bulk of measured whole body insulin sensitivity. As recently discussed,
some controversy remains with regard to the expression of GLP-1
receptors in peripheral tissues (6). Structural variants
of the GLP-1 receptor or a second closely related receptor may be
expressed in different tissues. Our binding data in monocytes do not
completely exclude the existence of GLP-1 receptors. It should be noted
that peptide degradation was underestimated by the TCA method, which is
suitable to demonstrate gross degradation of peptides but will not give
any indication of degradation limited to only a few amino acids.
Fragmented GLP-1 may have disturbed the ability of unlabeled GLP-1 to
displace the radioligand. GLP-1 is especially susceptible to
degradation by dipeptidyl peptidase IV (present on monocytes), which
removes a dipeptide from the NH2-terminus of the molecule.
The resultant metabolite can still bind to the GLP-1 receptor but is
unable to transduce a signal because of the loss of the
NH2-terminal dipeptide. This may explain why in monocytes
higher concentrations of GLP-1 were needed than in RINm5F cells to
modulate insulin and proinsulin binding.
The binding studies in isolated monocytes, a model for the human
insulin receptor on peripheral tissues (1,
21, 22), are consistent with the hypothesis
that GLP-1 modulates insulin receptor binding. We demonstrated that
incubation of isolated monocytes with GLP-1 differentially affected the
two types of insulin binding sites. According to our previous findings
(21, 22), the low-affinity/high-capacity
binding sites were diminished in type 1 and type 2 diabetics but were
significantly increased after incubation with GLP-1. It has been
elucidated that this binding type is mainly involved in signaling the
metabolic activity of insulin (5). The type 2 diabetics
were poorly matched in terms of age and body mass index. However, when
we investigated insulin binding in a larger cohort of subjects
(unpublished observations), we found no significant influence of age,
whereas a high body mass index was associated with lower insulin
binding. Our data further demonstrate that, in monocytes of control
subjects and diabetics, GLP-1 significantly increases proinsulin
binding but not IGF-I binding. This underlines the specificity of GLP-1
action and indicates that the high-capacity/low-affinity insulin
binding sites are not functional IGF-I receptors.
In summary, our findings in insulinoma cells and in circulating
mononuclear cells demonstrate that GLP-1 specifically increases the
number of high-capacity/low-affinity insulin binding sites that may be
functional proinsulin receptors. Further studies are necessary to
elcuidate whether the feedback regulation of insulin and proinsulin
secretion and the increase of insulin-independent glucose disposal
could be attributed to the GLP-1 effect on receptor binding of insulin
and proinsulin.
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ACKNOWLEDGEMENTS |
We thank E. Rüber for excellent technical assistance.
 |
FOOTNOTES |
This work was supported by the Landesforschungsschwerpunkt
Baden-Württemberg: Spätkomplikationen bei Diabetes Mellitus
(to R. D. Fussgaenger) and the Landesforschungsschwerpunkt
Baden-Württemberg: Modulation von Wachstumsfaktoren als
Therapieprinzip (to P. M. Jehle).
Address for reprint requests and other correspondence: P. M. Jehle, Div. of Nephrology, Dept. of Internal Medicine II, Univ. of
Ulm, Robert-Koch-Str. 8, 89070 Ulm, Germany (E-mail:
peter.jehle{at}medizin.uni-ulm.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
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in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 18 August 1999; accepted in final form 8 February 2000.
 |
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