Urocortin III
Stimulates Insulin and Glucagon Secretion
Urocortin III Is Expressed in Pancreatic
Cells and Stimulates Insulin and Glucagon Secretion
Urocortin (Ucn) III, or stresscopin, is a high affinity ligand
for the type 2 corticotropin-releasing factor (CRFR2) receptor
recently identified in rodents and human. Ucn III was initially
identified as a neuropeptide expressed in discrete areas in
the brain. In the present study, we demonstrate that Ucn III
is expressed in pancreatic ß-cells and in a mouse ß-cell line,
MIN6. Ucn III secretion from the cells was measured using a
highly specific RIA, and we found that high potassium, forskolin,
or high glucose can stimulate Ucn III secretion from these cells.
In vivo studies showed that rats receiving an iv Ucn III injection
had a significant elevation of plasma glucagon followed by plasma
glucose levels compared with rats receiving vehicle. Ucn III
injections also result in an increase in plasma insulin levels.
The observed effects of Ucn III were blocked by pretreatment
with a CRFR2 antagonist, astressin2-B. Furthermore,
Ucn III stimulated glucagon and insulin release from isolated
rat islets, and astressin2-B abolished the effects
of Ucn III, in keeping with a CRFR2-mediated mechanism.Taken
together, the present studies suggest pancreatic Ucn III acting
through CRFR2 is involved in the local regulation ofglucagon
and insulin secretion. Chien Li, et
al. Endocrinology Vol. 2003,144(7), 3216-3224
(see remaining paper at the end of this
page).
Selective
Agonist for the Type 2 Corticotropin-Releasing Factor Receptor
Human Urocortin II, a Selective Agonist for the Type 2 Corticotropin-Releasing
Factor Receptor, Decreases Feeding and Drinking in the Rat Corticotropin-releasing factor (CRF) has been hypothesized
to modulate consummatory behavior through the Type 2 CRF (CRF(2))
receptor. However, behavioral functions subserved by the CRF(2)
receptor remain poorly understood. Recently, human urocortin
II (hUcn II), a selective CRF(2) receptor agonist, was identified.
To study the effects of this neuropeptide on ingestive behavior,
we examined the effects of centrally infused hUcn II (i.c.v.
0, 0.01, 0.1, 1.0, 10.0 &mgr;g) on the microstructure of
nose-poke responding for food and water in nondeprived, male
rats. Malaise-inducing properties of the peptide were monitored
using conditioned taste aversion (CTA) testing. To identify
potential sites of action, central induction of Fos protein
expression was examined. hUcn II dose dependently reduced the
quantity and duration of responding for food and water at doses
lower (0.01-1.0 &mgr;g) than that forming a CTA (10 &mgr;g).
Effects were most evident during hours 4 to 6 of the dark cycle.
Meal pattern analysis showed that hUcn II potently (0.1 &mgr;g)
increased the satiating value of food. Rats ate and drank smaller
and shorter meals without changing meal frequency. Rats also
ate more slowly. hUcn II induced Fos in regions involved in
visceral sensory processing and autonomic/neuroendocrine regulation
and resembling those activated by appetite suppressants. hUcn
II is a promising neuropeptide for investigating the role of
the CRF(2) receptor in ingestive behavior. Inoue
K,et al. J Pharmacol Exp Ther 2003 Apr 1;305(1):385-393
Human Urocortin II (Urocortin Related Peptide,
URP) is identical with Stresscopin Related Peptide (SRP) (6-43)-NH2
(Human).
References: Lewis, K. et al. Proc. Natl. Acad. Sci. USA 98, 7570-7575,
2001 (June 19) Hsu, S.Y. & Hsueh, A.J.W. Nature Medicine, 7 605-611,
2001 Review by Jun Yang on June 21, 2001
Human urocortin II, a new CRF-related peptide, displays selective
CRF(2)-mediated action on gastric transit in rats
Human urocortin (hUcn) II is a new member of the corticotropin-releasing
factor (CRF) family that selectively binds to the CRF(2) receptor.
We investigated the CRF receptors involved in mediating the
effects of hUcn II and human/rat CRF (h/rCRF) on gut transit.
Gastric emptying, 4 h after a solid meal, and distal colonic
transit (bead expulsion time) were monitored simultaneously
in conscious rats. CRF antagonists were given subcutaneously
30 min before intravenous injection of peptides or partial
restraint (for 90 min). hUcn II (3 or 10 microg/kg i.v.) inhibited
gastric emptying (by 45% and 55%, respectively) and did not
influence distal colonic transit. The CRF(2) peptide antagonist
astressin(2)-B blocked hUcn II action. h/rCRF, rat Ucn, and
restraint delayed gastric emptying while accelerating distal
colonic transit. The gastric response to intravenous h/rCRF
and restraint was blocked by the CRF(2) antagonist but not
by the CRF(1) antagonist CP-154,526, whereas the colonic response
was blocked only by CP-154,526. None of the CRF antagonists
influenced postprandial gut transit. These data show that
intravenous h/rCRF and restraint stress-induced delayed gastric
emptying involve CRF(2) whereas stimulation of distal colonic
transit involves CRF(1). The distinct profile of hUcn II,
only on gastric transit, is linked to its CRF(2) selectivity.
Million M, et al. Am J Physiol Gastrointest
Liver Physiol 2002 Jan;282(1):G34-40
Differential actions of peripheral corticotropin-releasing
factor (CRF), urocortin II, and urocortin III on gastric emptying
and colonic transit in mice: role of CRF receptor subtypes
1 and 2
Peripheral CRF inhibits gastric emptying and stimulates colonic
motor function in rats. We investigated the role of CRF(1)
and CRF(2) receptors in i.p. CRF-induced alterations of gut
transit in conscious mice using selective CRF(1) and CRF(2)
ligands injected i.p. Gastric emptying 2 h after ingestion
of a solid chow meal and colonic transit (time to expel a
bead inserted into the distal colon) were determined simultaneously.
Rat/human (r/h)CRF, which has CRF(1) > CRF(2) binding affinity,
decreased distal colonic transit time at lower doses (6-12
microg/kg) than those inhibiting gastric emptying (20-60 microg/kg).
Ovine CRF, a preferential CRF(1) receptor agonist (6-60 microg/kg),
reduced significantly the colonic transit time without altering
gastric emptying, whereas the selective CRF(2) receptor agonists
mouse urocortin II (20-60 microg/kg) and urocortin III (120
microg/kg) inhibited significantly gastric emptying without
modifying colonic transit. The CRF(1)/CRF(2) receptor antagonist,
astressin (30-120 microg/kg), dose dependently prevented r/hCRF
(20 microg/kg)-induced inhibition of gastric emptying and
reduction of colonic transit time. The selective CRF(1) receptor
antagonists, NBI-27914 (C(18)H(20)Cl(4)N(4)C(7)H(8)O(3)S)
and CP-154,526 (butyl-[2,5-dimethyl-7-(2,4,6-trimethylphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]e
thylamine) (5-30 mg/kg), dose dependently blocked r/hCRF action
on the colon without influencing the gastric response, whereas
the CRF(2) receptor antagonist, antisauvagine-30 (30-100 microg/kg),
dose dependently abolished r/hCRF-induced delayed gastric
emptying and had no effect on colonic response. These data
show that i.p. r/hCRF-induced opposite actions on upper and
lower gut transit in conscious mice are mediated by different
CRF receptor subtypes: the activation of CRF(1) receptors
stimulates colonic propulsive activity, whereas activation
of CRF(2) receptors inhibits gastric emptying.
Martinez V,et al. J Pharmacol Exp Ther
2002 May;301(2):611-7
Urocortin-Related Peptides Increase Interleukin-6 Output
via Cyclic Adenosine 5'-Monophosphate-Dependent Pathways in
A7r5 Aortic Smooth Muscle Cells Corticotropin-releasing factor receptor type 2ß, expressedin the rodent cardiovascular system, is a member of the
G protein-coupledreceptor family. This receptor
is coupled positively to adenylatecyclase and is
bound preferentially by the urocortin (Ucn)-relatedpeptides
(Uncs): Ucn, Ucn II, and Ucn III. In the present study,we investigated the effects of Ucns on IL-6 levels in
A7r5 aorticsmooth muscle cells. In this cell line,
both Ucn and Ucn IIinduced accumulation of intracellular
cAMP via corticotropin-releasingfactor receptor
type 2?and also caused a significantincrease in
IL-6 output levels. The adenylate cyclase inhibitor,MDL-12330A,
inhibited this Ucn- or Ucn II-induced increase inIL-6
levels. Although H89 (10 ?font size="-2">M), a protein kinase
Ainhibitor, had no effect on the increase in IL-6
concentration,bisindolylmaleimide I (10 nM),
a protein kinase C inhibitor,was found to significantly
inhibit IL-6 output levels. Blockadeof Ucn- or Ucn
II-induced increases in IL-6 levels by SB203580(100
nM), a p38 MAPK inhibitor, suggested
that the p38 MAPKpathway was involved in this regulation.
The cAMP-mediated increasein IL-6 levels was suppressed
synergistically by both bisindolylmaleimideI and
SB203580. These findings demonstrate that both proteinkinase C and p38 MAPK signaling cascades are involved
downstreamof the Ucns-cAMP pathway in A7r5 aortic
smooth muscle cells. Kazunori
Kageyama and Toshihiro
Suda. Endocrinology 2003, 144(6) 2234-2241
Figure 1. Expression and localization of Ucn III
in mouse pancreatic islets and MIN6 cells. A, Representative
electrophoretic analysis of the RT-PCR products of
Ucn, Ucn II, and Ucn III in mouse pancreas and MIN6
cells. S16 RNA was used as positive control. -RT,
RNA samples were used for PCR without RT; -cDNA, no
RT product added to the PCR. B, Representative autoradiogram
showing the detection of Ucn II and Ucn III mRNAs
by RNase protection assay. Total RNA isolated from
mouse brain stem (for Ucn II), hypothalamus (for Ucn
III), or whole pancreas was hybridized with antisense
probes specific to mouse Ucn II or Ucn III and mouse
glyceraldehyde-3-phosphate dehydrogenase. Ucn II probe
protects a 592-nt band in the brain stem and the pancreas
and Ucn III probe protects a 414-nt band in the hypothalamus
and the pancreas. C, Representative dark-field photomicrograph
showing a mouse pancreas section probed with mouse
Ucn III riboprobe. Ucn III mRNA-positive signals (silver
grain clusters) were found in the pancreatic islets.
D, Bright-field photomicrograph of panel C with nissl
staining showing the islets. *, Blood vessel. E and
F, Representative photomicrographs showing Ucn III
immunostaining in the mouse pancreatic sections without
(E) or with (F) preabsorption of the anti-Ucn III
serum with 30 ?font size="-2">M of mouse Ucn III.
Ucn III-positive cells (dark purple staining)
were observed only in the islets. Scale bar,
50 µm. Chien
Li, et al. Endocrinology
Vol. 2003,144(7), 3216-3224
Figure 2. Colocalization
of Ucn III and insulin in mouse pancreatic islets.
Stacked serial confocal images of insulin (A) and
Ucn III (B) cells in the islet. C, Visualization
of both substances simultaneously showing the majority
of insulin and Ucn III colocalize in the same cells.
High magnification of confocal images showing insulin
(D) and Ucn III (E) in a single ß-cell. F, Combined
image of panels D and E to visualize insulin and
Ucn III simultaneously. Note that little colocalization
is observed. G–I, Stacked serial confocal
images of glucagon (G), Ucn III (H), and combined
image (I) of panels G and H showing no colocalization
of the two substances. Scale bar, 25 µm for
A–C and G–I and 1 µm for D–F.
Chien
Li, et al. Endocrinology
Vol. 2003,144(7), 3216-3224
Table 2. Effects of Ucn III
and rat/human CRF on glucagon and insulin secretion
from isolated rat islets
Ucn III
Rat/human CRF
0.1
1
10
100
(nM)
1
10
100
(nM)
Glucagon
103.6
?38a
157.3
?18a
169.7
?26a1
284.1
?771
119.5
?29a
95.3
?15a1
281.9
?791
Insulin
180.8
?66d
296.4
?51e
231.8
?39e
258.3
?43e
132.6
?24d
113.3
?14d
362.7
?87e
Data are expressed as percent of control (2.8
mM glucose). Values in
same row with different superscripts (a,
c, d, e) are significantly different with
P < 0.05. 1 Significantly
different from each other with P < 0.05.
Chien Li,
et al. Endocrinology Vol. 2003,144(7), 3216-3224
Figure 3. Displacement
of 125I-labeled mUcn III binding to rabbit
anti-mUcn III by mUcn III ()
and partially purified rat pancreatic ()
or isolated rat islet ()
extract. Rat pancreas or isolated rat islets were
acid-extracted and partially purified using octadecyl
silica cartridges. Human Unc III (h Ucn III, )
shows approximately 10% cross-reactivity. Closely
related family peptides including rat Ucn (r Ucn,
),
rat CRF (r CRF, ),
or mouse Ucn II (m Ucn II, )
showed little or no cross-reactivity. B/B0,
Bound to maximum-bound ratio.
Chien Li, et al. Endocrinology Vol. 2003,144(7),
3216-3224
Figure 4. Ucn
III-like ir in secreted media from MIN6 cells. MIN6
cells (106 cells per well) were incubated
in media containing various concentrations of glucose,
30 ?font size="-2">M forskolin, or 30 mM
KCl and supplemented with BSA (0.2% wt/vol) for
4 h. Cultured supernatants were collected and assayed
for Ucn III. Cells were treated in triplicate, and
the average of duplicate experiments is shown. *,
P < 0.05 vs. 5 mM
glucose; **, P < 0.01 vs. 5 mM
glucose.
Chien Li, et al. Endocrinology Vol. 2003,144(7),
3216-3224
Figure 6. Plasma
glucose (A) and glucagon (B) levels in male rats
treated with vehicle or Ucn III (9 or 90 nmol/kg).
Basal glucose and glucagon concentrations are 5.1
± 0.2 mmol/liter and 52.3 ± 3.2 pg/ml, respectively.
C, Glucagon levels at 5 min after vehicle or Ucn
III (90 nmol/kg) injection with or without Ast2-B
(90 nmol/kg) pretreatment. B, *, P < 0.05 vs.
vehicle. C, *, P < 0.05 vs. the rest of groups.
Chien Li, et al.
Endocrinology Vol. 2003,144(7), 3216-3224
Figure 5. The
effects of either vehicle or Ucn III (0.09–9
nmol/kg, iv) on plasma glucose (A) and insulin (B)
levels in male rats. C, Insulin levels at 30 min
after iv vehicle or Ucn III (90 nmol/kg) with or
without Ast2-B (90 nmol/kg) pretreatment.
Basal glucose level, 4.25 ± 0.18 mmol/liter. B,
For 9 nmol/kg group: +, P < 0.05 vs.
vehicle; for 90 nmol/kg group: *, P <
0.05; **, P < 0.01 vs. vehicle.
C, *, P < 0.05 vs. the rest of
groups.
Chien Li,
et al. Endocrinology Vol. 2003,144(7), 3216-3224
Figure 8. Effects of a glucagon
antagonist on Ucn III-induced insulin secretion
from isolated rat islets. Ucn III (1 nM)
and 10 nM glucagon stimulate
insulin secretion. Pretreatment with 1 ?font size="-2">M
des-glucagon abolishes the effect of glucagon but
not Ucn III on insulin secretion. Values from islets
incubated in medium with 2.8 mM
glucose serve as controls. *, P < 0.05
compared with control. +, P < 0.05 vs.
control and glucagon plus des-glucagon.
Chien Li,
et al. Endocrinology Vol. 2003,144(7), 3216-3224
An illustrated scheme of the pathways
involved in Ucn/Ucn II, cAMP induction, and IL-6
output. Ucns increase intracellular cAMP levels
via the stimulation of adenylate cyclase. To increase
IL-6 output, both PKC and p38 MAPK signaling cascades
are involved downstream of the Ucn/Ucn II-cAMP pathway
in A7r5 cells.
Kazunori Kageyama
and Toshihiro Suda. Endocrinology 2003,
144(6) 2234-2241
Effects of CRF family peptides on
interleukin-6 levels in A7r5 aortic smooth muscle
cells. Cells were incubated for 48 h with medium
alone (control) or with medium containing CRF (open
triangle), Ucn (open circle), Ucn II
(open square), antisauvagine-30 (closed
triangle), 100 nM of
Ucn with increasing concentrations of antisauvagine-30
(closed circle), and 100 nM
of Ucn II with increasing concentrations of antisauvagine-30
(closed square). IL-6 levels in the medium
were measured by ELISA. Results shown are representative
of three independent experiments. Statistical analyses
were performed using one-way ANOVA, followed by
Scheffé’s F post hoc test.
)
and partially purified rat pancreatic (
)
or isolated rat islet (
)
extract. Rat pancreas or isolated rat islets were
acid-extracted and partially purified using octadecyl
silica cartridges. Human Unc III (h Ucn III, 
)
shows approximately 10% cross-reactivity. Closely
related family peptides including rat Ucn (r Ucn,
),
rat CRF (r CRF, 
),
or mouse Ucn II (m Ucn II, 
)
showed little or no cross-reactivity. B/B0,
Bound to maximum-bound ratio. 
