RECÉM-LANÇADO, ESTA REVISTA MÉDICA É DE ALTO NÍVEL E ESTÁ COM OS PRIMEIROS NÚMEROS DE GRAÇA. APROVEITEM!! ESSE ARTIGO CAI MUITO BEM NO NOSSO BLOG SOBRE SULFONILURÉIAS. Journal of Diabetes Investigation Volume 1 Issue 1/2 February/April 2010

1. COMMENTARY
Sulfonylurea action re-revisited
family, and showed for the first time that exert its action through protein phosphor-
Sulfonylureas (SU), commonly used the b-cell KATP channel is composed of ylation by protein kinase A (PKA). How-
in the treatment of type 2 diabetes Kir6.2 and SUR1.4 The KATP channel is a ever, a novel cAMP-binding protein
mellitus (T2DM), stimulate insulin hetero-octameric complex comprising family, termed Epac (exchange protein
secretion by inhibiting adenosine two subunits: a pore-forming subunit activated by cAMP) or cAMP-GEF
triphosphate (ATP)-sensitive K+ (KATP) Kir6.x (Kir6.1 or Kir6.2) and a regula- (cAMP-regulated guanine nucleotide
tory subunit SURx (SUR1, SUR2A or exchange factor) has been identified8.
channels in pancreatic b-cells. SU are
SUR2B)5. Different combinations of There are two members of the Epac fam-
now known to also activate cyclic
Kir6.1 or Kir6.2 and SUR1 or a SUR2 ily, Epac1 and Epac2, both of which pos-
adenosine monophosphate (cAMP) variant (mix and match) form KATP sess guanine nucleotide exchange factor
sensor Epac2 (cAMP-GEFII) to Rap1 channels with differing nucleotides and (GEF) activity towards Rap1, the small
signaling, which promotes insulin SU sensitivities that play distinct physio- molecular weight GTP-binding protein, in
secretion. The different effects of logical and pathophysiological roles in a cAMP-dependent manner. We showed
various SU on Epac2 ⁄ Rap1 signaling, different tissues5,6. While Kir6.2 plus that Epac2 is involved in the potentiation
as well as KATP channels in different SUR1 constitutes pancreatic b-cell KATP of cAMP-dependent, PKA-independent
tissues, underlie the diverse pancre- channels, Kir6.2 plus SUR2A constitutes insulin secretion9. By studying Epac2 null
atic and extra-pancreatic actions of cardiac and skeletal muscle KATP chan- mice, we recently found that Epac2 ⁄ Rap1
SU. (J Diabetes Invest, doi: 10.1111 ⁄ nels. Kir6.2 plus SUR2B constitutes signaling is especially important in early
j.2040-1124.2010.00014.x, 2010) smooth muscle KATP channels and Kir6.1 phase (first phase) potentiation by cAMP
plus SUR2B constitutes vascular smooth of glucose-stimulated insulin granule exo-
muscle KATP channels, both of which are cytosis10. We have proposed a model in
Although earlier studies have suggested somewhat ATP-insensitive, nucleotide which Epac2 ⁄ Rap1 signaling regulates
various mechanisms of sulfonylurea (SU) diphosphate-activated and glibenclamide- cAMP-induced insulin granule exocytosis
action, the discovery of KATP channels by sensitive K+ channels. SU actions were by controlling the size of a readily releas-
electrophysiology brought a breakthrough revisited after the cloning of the various able pool (RPP), most likely through the
in the understanding of the mechanism KATP channels7. regulation of granule density near the
of the action of SU as well as the mecha- Mice lacking KATP channels (Kir6.2 plasma membrane10.
nism of glucose-stimulated insulin secre- null mice and SUR1 null mice) were gen- In the course of the studies of Epac2-
tion. KATP channels were first reported in erated6. Neither glucose nor tolbutamide mediated mechanisms of insulin secre-
cardiac cell membranes and were later stimulation elicited any change in [Ca2+]i tion, we developed a fluorescence
described in many other tissues including in Kir6.2 null b-cells. Importantly, neither resonance energy transfer (FRET)-based
pancreatic islet cells1. In 1985, Sturgess glucose nor tolbutamide stimulation Epac2 sensor (termed C-Epac2-Y) in
et al. found that tolbutamide inhibits caused a significant insulin secretion in which the full-length Epac2 is fused
KATP channels in pancreatic b-cells, sug- Kir6.2 null mice. Examination of SUR1 amino-terminally to enhanced cyan fluo-
gesting that the channels are the target of null mice also confirmed that both glu- rescent protein (ECFP) and carboxyl-ter-
SU2. In 1995, Aguilar-Bryan et al. cloned cose-stimulated and sulfonylurea-stimu- minally to enhanced yellow fluorescent
the SU receptor (now called SUR1) from lated insulin secretion depend critically protein (EYFP)11. Epac2 is a closed form
the pancreatic b-cell cDNA libraries3. on the activity of b-cell KATP channels. in the inactive state8, so that ECFP and
SUR1 belongs to members of the adeno- Based on these findings, it is generally EYFP are located very closely to each
sine triphosphate (ATP)-binding cassette accepted that the primary target of SU is other (within 10 nm), which causes
(ABC) protein superfamily. At almost the SUR1 and that action of SU is mediated FRET. On cAMP binding to Epac2,
same time, we cloned Kir6.24, a member by closure of the KATP channels through Epac2 changes its conformation to an
of the inwardly rectifying K+ channel binding to SUR1. open form. As a result, ECFP and EYFP
Cyclic adenosine monophosphate separate away, so that FRET does not
(cAMP) is a universal intracellular second occur (active state)8. Utilizing this princi-
*Corresponding author. Susumu Seino messenger involved in the regulation of ple, we are able to monitor the activation
Tel.: +81-78-382-5860 Fax: +81-78-382-6762
E-mail address: seino@med.kobe-u.ac.jp various cellular functions in many cell status of Epac2. In the search for agents
Received 19 January 2010; accepted 22 January 2010 types. cAMP has long been considered to that activate Epac2 using this FRET
ª 2010 Asian Association for the Study of Diabetes and Blackwell Publishing Asia Pty Ltd Journal of Diabetes Investigation Volume 1 Issue 1/2 February/April 2010 37

2. Seino et al.
binds specifically to the A-site of SUR1;
the second group (which includes gliben-
clamide and glimepiride) binds to the
B-sites of both SUR1 and SUR2A as well
as the A-site of SUR1; and the third
group (which includes meglinitide and
repaglinide) binds to the B-site of SUR1
and SUR2A. In addition, SU, with the
exception of gliclazide, activate Epac2 ⁄
Rap1 signaling, whereas glinides do not.
Thus, different SU and glinides have dif-
ferent mechanisms of action in insulin
secretion in terms of specificities for
SUR1 and Epac2.
Mutations of Kir6.2 have recently
been shown to cause neonatal diabetes
Figure 1 | Model of sulfonylurea (SU) action in insulin secretion. Closure of KATP channels is essen- mellitus (ND) with varying degrees of
tial for SU to stimulate insulin secretion. Activation of Epac2 ⁄ Rap1 signaling is required for SU to severity12. In most ND patients, insulin
exert their full effect on insulin secretion. cAMP, cyclic adenosine monophosphate; GIP, glucose- injection can be replaced by high-dose
dependent insulinotropic polypeptide; GLP-1, glucagon-like peptide 1; PKA, protein kinase A; RRP, SU orally. Studies by Zhang et al. sug-
readily releasable pool; SUR, SU receptor; VDDC, voltage-dependent Ca2+ channels. gest that the effectiveness of SU in the
treatment of ND patients might vary,
depending on the properties of the
sensor, we found that tolbutamide, gli- mice was significantly less than that in SU11.
benclamide, chlorpropamide, acetohexa- wild-type mice. Incretin-related drugs such as analogs
mide and glipizide significantly decreased As described above, it is well estab- of glucagon-like peptide 1 (GLP-1) and
the FRET response in COS-1 cells trans- lished that SU stimulate insulin secretion dipeptidyl peptidase IV (DPP-IV) inhibi-
fected with the Epac2 FRET sensor in dif- by eliciting a series of ionic events includ- tors, which potentiate insulin secretion
ferent degrees and varying kinetics, ing closure of KATP channels, opening through cAMP signaling in pancreatic
suggesting strongly that these SU activate of voltage-dependent Ca2+ channels b-cells, are currently being used as new
Epac2. However, gliclazide, another SU, (VDCC), and Ca2+ influx into the b-cells. hypoglycemic agents to treat T2DM.
did not decrease the FRET response. Although closure of the KATP channels is Because Epac2 is also required for poten-
Direct binding of SU to Epac2 was con- a prerequisite for SU to stimulate insulin tiation of insulin secretion by cAMP, it is
firmed by specific binding of radiolabeled secretion, the activation of Epac2 ⁄ Rap1 a target of both SU and incretin-related
glibenclamide to Epac2 expressed in signaling is required for SU to exert their drugs. There are many basic and clinical
COS-1 cells. We also found that tolbuta- full effects in insulin secretion (except in questions yet to be answered. Where is
mide and glibenclamide activate Rap1 in the case of gliclazide). Considering the the SU binding site in Epac2? Is there
clonal pancreatic b-cells (MIN6 cells), role of Epac2 ⁄ Rap1 signaling in insulin any additive or synergistic effect of cAMP
but gliclazide does not. In addition, tol- granule exocytosis10, SU might increase and SU on activation of Epac2 ⁄ Rap1 sig-
butamide-stimulated insulin secretion the size of a readily releasable pool of naling? Is there any accessory protein that
and glibenclamide-stimulated insulin insulin granules near the plasma mem- might facilitate direct interaction of SU
secretion from isolated pancreatic islets brane (Figure 1). and Epac2? Is Epac2 ⁄ Rap1 signaling
of Epac2 null mice were significantly A two-site (A-site and B-site) model involved in the extrapancreatic effects
reduced, compared with those of wild- for the interaction of SU and glinides of SU and incretin-related drugs and
type mice. However, there was no sig- with SUR has been proposed6. The A-site which SU has the least adverse effect? Is
nificant difference in insulin secretion in is located on the eighth (between trans- Epac2 ⁄ Rap1 signaling involved in the sec-
response to gliclazide. Furthermore, the membrane segment (TM) 15 and 16) ondary failure of SU and incretin-related
insulin response to the oral administra- cytosolic loop, which is specific for SUR1, drugs, and which SU shows least second-
tion of tolbutamide alone or concomi- and the B-site involves the third (between ary failure? What is the best combination
tant administration of glucose and TM 5 and 6) cytosolic loop, which is very of SU and incretin-related drugs for the
tolbutamide in Epac2 null mice was sig- similar in all SUR. Based on this model, most beneficial effect for treatment of
nificantly reduced, compared with that SU and glinides can be divided into three T2DM in terms of insulin secretion,
in wild-type mice, and the glucose low- groups. The first group (which includes glycemic control and adverse effects?
ering effect of tolbutamide in Epac2 null tolbutamide, gliclazide and nateglinide) Answers to these questions are required
38 Journal of Diabetes Investigation Volume 1 Issue 1/2 February/April 2010 ª 2010 Asian Association for the Study of Diabetes and Blackwell Publishing Asia Pty Ltd

3. Sulfonylurea action
to provide a basis of beneficial treatment 2. Sturgess NC, Cook DL, Ashford ML, 7. Gribble FM, Reimann F. Sulphonylurea
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ª 2010 Asian Association for the Study of Diabetes and Blackwell Publishing Asia Pty Ltd Journal of Diabetes Investigation Volume 1 Issue 1/2 February/April 2010 39