Rui Liu,1 Haitao Wang,1,2 Baofeng Xu,1,2 Wenliang Chen,1,2,3 Ekaterina Turlova,1,2

Nan Dong,1 Christopher L.F. Sun,1 Yangqingqin Lu,1 Hanhui Fu,1,2 Ranran Shi,1,2

Andrew Barszczyk,1 Dongzi Yang,4 Tianru Jin,1,5 Edoardo Mannucci,6

Zhong-Ping Feng,1 and Hong-Shuo Sun1,2,3,5

Cerebrovascular Safety ofSulfonylureas: The Role of KATPChannels in Neuroprotection andthe Risk of Stroke in Patients WithType 2 DiabetesDiabetes 2016;65:2795–2809 | DOI: 10.2337/db15-1737

Sulfonylureas are ATP-sensitive potassium (KATP) chan-nel blockers commonly used in the treatment of type2 diabetes mellitus (T2DM). Activation of KATP channelsplays a neuroprotective role in ischemia; thus, whethersulfonylureas affect the outcomes of stroke in patientswith T2DM needs to be further studied. In our study,streptozotocin (STZ)-induced diabetic mice subjectedto transient middle cerebral artery occlusion (MCAO)showed larger areas of brain damage and poorer behav-ioral outcomes. Blocking the KATP channel by tolbutamideincreased neuronal injury induced by oxygen-glucosedeprivation (OGD) in vitro and permanent MCAO (pMCAO)in vivo. Activating the KATP channel by diazoxide reducedthe effects of both the OGD and pMCAO. Western blotanalysis in STZ mouse brains indicated an early increasein protein levels of N-methyl-D-aspartate receptor 2Band postsynaptic density protein-95, followed by a de-crease in phosphorylation of glycogen synthase kinase3b. Our systematic meta-analysis indicated that patientswith T2DM treated with sulfonylureas had a higher oddsratio for stroke morbidity than those who received com-parator drugs. Taken together, these results suggestthat sulfonylurea treatment in patients with T2DM mayinhibit the neuroprotective effects of KATP channels andincrease the risk of stroke.

Sulfonylureas, as monotherapy or combined with otherdrugs, have been prescribed to millions of patients withdiabetes for more than 60 years. Although the adminis-tration of sulfonylureas has decreased in recent years,they are still one of the most widely used oral antidiabetesdrugs because of their high efficacy, at least in the short-term, and the low cost (1). In fact, sulfonylureas are stillreported in many guidelines and treatment algorithmsamong possible options as an add-on to metformin inpatients in whom monotherapy is failing. Type 2 diabetesmellitus (T2DM) is associated with a two- to threefoldincreased risk for cardiovascular (CV) disease (2) and isviewed as an independent risk factor for stroke (3). There-fore, prevention of CV morbidity and mortality is an im-portant goal for diabetes treatment.

The ATP-sensitive potassium (KATP) channel is a plasmamembrane protein that plays important roles in controllingcellular functions and regulating metabolism. The KATP

channel is composed of four pore-forming Kir6.x and fourregulatory sulfonylurea receptor SURx subunits that form ahetero-octameric complex (Kir/SUR) (4). Opening and clos-ing of KATP channels are facilitated by ADP and ATP mol-ecules, respectively. The KATP channel was originally foundin cardiac myocytes (5) and later in pancreatic b-cells (6)

1Department of Physiology, Faculty of Medicine, University of Toronto, Toronto,Ontario, Canada2Department of Surgery, Faculty of Medicine, University of Toronto, Toronto,Ontario, Canada3Department of Pharmacology, Faculty of Medicine, University of Toronto, Tor-onto, Ontario, Canada4Department of Obstetrics and Gynecology, Memorial Hospital of Sun Yat-SenUniversity, Guangzhou, Guangdong, China5Institute of Medical Science, Faculty of Medicine, University of Toronto, Toronto,Ontario, Canada6Diabetology, Careggi Hospital, University of Florence, Florence, Italy

Corresponding authors: Zhong-Ping Feng, zp.feng@utoronto.ca, and Hong-Shuo Sun,hss.sun@utoronto.ca.

Received 21 December 2015 and accepted 11 May 2016.

R.L., H.W., and B.X. contributed equally to this work.

© 2016 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, andthe work is not altered. More information is available at http://diabetesjournals.org/site/license.

See accompanying article, p. 2479.

Diabetes Volume 65, September 2016 2795

PHARMACOLOGYAND

THERAPEUTIC

S

and neuronal cells (7). Sulfonylureas bind to SUR subunitsand block the channel (8,9), thus stimulating insulin secre-tion through the depolarization of pancreatic b-cells (10).

In the brain, the KATP channel is highly expressed in thehippocampus and cortex (7,11–14). Our previous studiesdemonstrated that focal ischemia (transient middle cere-bral artery [MCA] occlusion [tMCAO] model) in the brainled to larger cortical infarcts and more severe neurolog-ical deficits in adult Kir6.2 knockout mice (14). Ourrecent work showed that KATP channels mediated hypoxicpreconditioning–induced neuroprotection in a hypoxic-ischemic brain injury model in neonatal mice (15). Inhibi-tion of KATP channels by tolbutamide increased infarctvolumes and opening of KATP channels by diazoxide-reduced infarct volumes (14,15). Opening of the KATP

channel hyperpolarizes the neurons and stabilizes the rest-ing membrane potential during ischemia when energyfailure reduces the ATP-to-ADP ratio (11,14,16). We pre-viously reported that this hypoxia/ischemia-induced mem-brane hyperpolarization mediated by KATP channels is acellular mechanism underlying neuroprotection againststroke (14,15). These findings indicate that KATP channelsmay be considered as a potential therapeutic target for thetreatment of stroke. However, the role of KATP channels incerebral ischemia has not been tested in a diabetic modeland a permanent (p)MCAO model in mice.

CV safety is a major issue for glucose-lowering drugs.After meta-analytical data suggesting a possible increase inthe risk of myocardial infarction with rosiglitazone (17),the U.S. Food and Drug Administration issued a guidelinerequiring postmarketing CV safety trials for all novel drugs.However, no formal study on the CV safety of older drugswas required. In particular, the CV safety of sulfonylureashas been widely debated. The use of glibenclamide maycounter the effect of preconditioning through the blockingof KATP channel activity (18). The University Group Diabe-tes Program (UGDP) study reported that the use of tolbu-tamide increased CV mortality in patients with T2DMcompared with other nonsulfonylurea agents (19,20). Anincreased mortality, despite a similar risk of major CVevents, was also found in a meta-analysis of randomizedtrials with sulfonylureas (21).

Because sulfonylureas prevent KATP channel activationthat provides neuroprotection in stroke, their use may po-tentially increase the incidence of stroke in patients withdiabetes (21). Currently, the relationship between sulfonyl-urea treatment and stroke incidence in patients with dia-betes is still unclear. In this study, we present our in vitroand in vivo findings from animal models as well as a sys-tematic meta-analysis of randomized clinical trials.

RESEARCH DESIGN AND METHODS

Animal Studies

AnimalsThe animal protocol was approved by the University ofToronto Animal Care Committee and conformed to Canadian

Council on Animal Care guidelines. Adult male C57BL/6Jmice (9–10 weeks old, body weight 23–26 g) were pur-chased from Charles River Laboratories (Sherbrooke,Quebec, Canada).

MaterialsTolbutamide, diazoxide, 2,3,5-triphenyltetrazolium chlo-ride (TTC), 0.25% trypsin-EDTA solution, propidium iodide(PI), and streptozotocin (STZ) were purchased from Sigma-Aldrich Canada; and GlutaMax B-27 supplement, penicillinstreptomycin, Neurobasal medium, and Hanks’ balancedsalt solution were purchased from Gibco by Life Technol-ogies. Anti–N-methyl-D-aspartate (NMDA) receptor 2B(NR2B) and anti-postsynaptic density protein 95 (PSD95)were from Abcam (Cambridge, MA); anti-phosphorylatedglycogen synthase kinase 3b (p-GSK3b), anti-total GSK3b(t-GSK3b), and anti-GAPDH were obtained from Cell Sig-naling Technology (Danvers, MA).

STZ Administration and tMCAO Animal ModelFor the diabetic model, adult male C57BL/6J mice (6 weeksold; body weight, 17–21 g) were fasted before injection for4 hours. STZ (50 mg/kg) or Na-citrate solution was in-traperitoneally administered to the mice once daily for5 consecutive days. Mice were confirmed hyperglycemic($16 mmol/L) (22) at 1, 2, 4, and 5 weeks after injection(Glucometer, Roche Diagnostics GmbH). Body weightswere measured at 1, 2, 4, and 5 weeks after injection.

For the tMCAO model (14), mice were anesthetizedwith 2.5% isoflurane in oxygen. A heating pad was usedto maintain body temperature at 376 0.2°C. A 20-mm-longmonofilament suture with silicon-coated tip (cat#1620A5;Beijing SUNBIO Biotech Co., Ltd.) was inserted through theright external carotid artery into the internal carotid arteryto block the origin of the MCA. After 90 min, the suture wasremoved to reestablish the perfusion, and animals wereallowed to recover for 24 h until histological and behavioralevaluations.

pMCAOSimilar to the tMCAO procedure, the suture was insertedthrough the right common carotid artery into the internalcarotid artery to block the origin of MCA and left in placefor 24 h until the next procedure. Because the pMCAOprocedure causes larger brain damage and may affectsurvival after the procedure, it was not performed on miceadministered STZ.

Measurement of Infarct VolumeThe MCAO brains were sectioned coronally after 24 h into1-mm slices and incubated in 2% TTC in PBS at 37°C for30 min. Infarct volumes were calculated by summing therepresentative areas in all sections and multiplying by theslice thickness. After correcting for edema, the volumes ofinfarction were calculated: Corrected infarct volume (CIV)(%) = [contralateral hemisphere volume – (ipsilateral hemi-sphere volume – infarct volume)]/contralateral hemispherevolume 3 100 (15).

2796 KATP Channels in Stroke Diabetes Volume 65, September 2016

Neurobehavioral TestsBehavioral tests were conducted 24 h after pMCAO ortMCAO using a standard 6-point scale (23). Scale ofscores: 0–no neurological deficit; 1–retracts left forepawwhen lifted by the tail; 2–circles to the left; 3–falls whilewalking; 4–does not walk spontaneously; 5–dead.

Primary Cortical CulturePrimary cortical cultures were prepared from E16 CD1mice as previously described (24). Cells were plated onpoly-D-lysine coated plates using Neurobasal culture mediumat the desired density (1 3 105 per well) and maintainedat 37°C in a humidified 5% CO2 atmosphere.

Oxygen and Glucose DeprivationCortical cultures were incubated with tolbutamide ordiazoxide in oxygen and glucose deprivation (OGD) solu-tion for 30 min, followed by incubation in an anaerobicchamber flushed with 5% CO2 and 95% N2 (v/v) at 37°Cfor 90 min (25). Cells were then incubated in normoxicconditions for an additional 24 h.

Immunocytochemistry and Confocal ImagingCell damage was determined by quantitative measurementsof PI fluorescence with a multiwell plate Synergy H1fluorescence reader (BioTek, Winooski, VT) (25). In brief,cells were incubated with PI (5 mg/mL) for 20 min. Fluo-rescence intensity was read under an excitation wavelengthof 488 nm and an emission wavelength of 630 nm. PIenters the cell and stains DNA in the nucleus when cellmembrane integrity is compromised during OGD. Greaterfluorescence intensity indicates greater cell damage withOGD. The fraction of OGD-induced dead cells in each cul-ture was calculated as: fraction dead = (Ft – Fo)/(FNMDA –Fo), where Fo is the initial fluorescence reading of the platebefore OGD, Ft is the maximum fluorescence reading afterOGD, and FNMDA is the PI fluorescence of sister cultures24 h after a 60 min exposure to NMDA (1 mmol/L) at 37°C(26,27). The relative assessments of neuronal cell deathwere normalized by comparison with 100% cell death in-duced by NMDA. The result represents OGD-induced dam-age as a percentage of NMDA-induced cell death (28).

Images of PI staining were also taken to show damagedcells. In brief, cells were stained with PI for 20 min andthen fixed with 4% paraformaldehyde in PBS for 20 min.Images were taken using a Zeiss LSM 710 Confocal mic-roscope, and quantitative analysis was performed by count-ing PI-positive cells in five random fields with a 310objective per coverslip.

Western BlotTo determine NR2B, PSD95, p-GSK3b, and t-GSK3b pro-tein levels in the brains of mice with STZ administrationor vehicle treatment, the brain tissues were homogenizedin radioimmunoprecipitation assay buffer containing aproteinase inhibitor cocktail. Proteins in equal amountswere separated by SDS-PAGE gels. Protein was transferredto nitrocellulose membranes (Pall, Pensacola, FL). The blots

were incubated with appropriate horseradish peroxidase-conjugated secondary antibody accordingly (1:7,500;Jackson ImmunoResearch Laboratories) and analyzedby exposure to film (HyBlot CL).

Data AnalysisStatistical analysis was performed with SigmaPlot soft-ware (Jandel Scientific). Data are presented as mean 6SEM. Differences between experimental groups were eval-uated using Student t test for two groups and one-wayANOVA, followed by the Fisher least significant differencemethod for multiple experimental groups. Value of P, 0.05was considered statistically significant. All experiments wereperformed in a blinded manner to all treatment conditionsand assessments.

Meta-analysis of Randomized Trials

Search Strategy, Inclusion Criteria, and Data ExtractionThe meta-analysis was performed following the PreferredReporting Items for Systematic Reviews and Meta-Anal-yses (PRISMA) guidelines (29). We systemically conductedan extensive search in Medline, Web of Science, Library ofCongress, Embase, and the Cochrane Library, using thesearch terms “sulfonylureas” or “glyburide” or “glibencla-mide” or “tolbutamide” or “acetohexamide” or “gliclazide”or “glipizide” or “chlorpropamide” or “glimepiride,” incombination with “stroke” or “cerebral infarction” or“cerebral ischemia” (or “ischaemia”) or “cerebrovasculardisease” or “cerebrovascular attack” or “brain ischemia”(or “ischaemia”). We collected all randomized controlledtrials comparing sulfonylureas with placebo or other anti-diabetic drugs up to 9 February 2016. Results of unpubli-shed trials were retrieved if they were available onwww.clinicaltrials.gov, www.clinicalstudyresults.org, orwww.controlled-trials.com.

Studies on the relationship between oral sulfonylureas andthe risk of stroke were selected, and only studies reportingraw data on morbidity during sulfonylureas treatment wereincluded (Table 1). Other entry criteria included 1) publi-cations written in English; 2) studies performed on pa-tients with T2DM; 3) randomized trials with a controlgroup; and 4) follow-up duration of at least 24 weeks.

The quality of the extracted studies was assessed bythe two experimenters (Y.L. and H.F.) independentlyusing the criteria for judging risk of bias as suggestedby the Cochrane Handbook of Reviews of Assessing Riskof Bias. Disagreements were resolved by discussionbetween the two experimenters or the third person(R.L.) by referencing the original reports.

Outcome Measures and Data AnalysisThe outcome of the meta-analysis was the effect of sulfo-nylureas, compared with other antidiabetes drugs, on theincidence of stroke in T2DM; the odds ratio (OR) and 95%CI were used as the operational measures. All ORs werepooled using random-effects models.

A sensitivity analysis was performed including onlytrials in which stroke was part of the primary end point.

diabetes.diabetesjournals.org Liu and Associates 2797

Tab

le1—

Bas

elinech

arac

teristicsan

doutco

mes

forea

chstud

yinclud

edin

themeta-an

alys

is

Firstau

thor,

year

(ref.)

Study

duration

(wee

k)SU

NCom

parator

N

Strok

e(SU/C

,n)

Rac

e(SU/C

,%

ofwhite)

Sex

(SU/C

,%of

male)

Mea

n

Adjudication

(Yes

/No)

Age

(SU/C

,ye

ar)

Diabetes

duration

(SU/C

,ye

ar)

HbA

1c

(SU/C

,%

)BMI

(SU/C

,kg

/m2)

Bas

eline

End

point

Arech

avaleta

2011

(52)

30Glim

epiride+

metform

in51

9Sita

gliptin

+metform

in51

62/0

57.4/57.6

53.8/55.0

56.2/56.3

6.7/6.8

7.5/7.5

7.1/7.0

30.2/29.7

No

Bak

ris20

06(53)

32Glyburide+

metform

in18

0Ros

iglitaz

one+

metform

in19

41/0

76.0/78.0

69.0/63.0

58.8/60.0

7.6/8.0

8.3/8.3

74/7.8

31.8/31.6

No

Cha

rbon

nel2

005(54)

52Gliclazide+

metform

in31

3Pioglita

zone

+metform

in31

73/2

NR/N

R49

.2/50.8

57.0/56.0

5.5/5.8

8.5/8.7

7.9/7.8

32.6/32.6

No

Ferran

nini

2009

(55)

52Glim

epiride

1,39

3Vild

aglip

tin1,39

67/0

85.2/86.3

54.1/52.8

57.5/57.5

5.8/5.7

7.3/7.3

6.6/6.8

31.7/31.8

Yes

Gallwitz

2012

(56)

104

Glim

epiride

775

Lina

gliptin

776

11/3

85.0/85.0

61.0/60.0

59.8/59.8

NR/N

R7.7/7.7

7.3/7.5

30.3/30.2

Yes

Gerstein20

10(57)

78Glip

izide

339

Ros

iglitaz

one

333

1/5

NR/N

R65

.8/70.0

60.2/61.8

4.6/5.0

7.2/7.1

7.0/6.8

29.8/29.3

Yes

Gök

e20

10(58)

52Glip

izide+

metform

in43

0Sax

aglip

tin+

metform

in42

81/0

84.2/82.2

54.0/49.5

57.6/57.5

5.4/5.5

7.7/7.7

6.7/6.7

31.3/31.5

No

Hom

e20

09(59)

286

SU

+metform

in1,10

5Ros

iglitaz

one+

metform

in1,11

732

/25

98.4/98.9

52.9/53.8

57.2/57.0

6.3/6.1

7.8/7.8

7.7/7.5

32.7/32.8

Yes

Hon

g20

13(60)

156

Glip

izide

148

Metform

in15

615

/10

NR/N

R77

.0/78.2

63.8/62.8

5.6/5.6

7.6/7.6

7.1/7.0

25.1/25.2

Yes

Kah

n20

06(61)

260

Glyburide

1,44

1Ros

iglitaz

one

1,45

617

/16

89.0/87.2

58.0/55.7

56.4/56.3

NR/N

R7.4/7.4

7.6/7.1

32.2/32.2

Yes

Glyburide

1,44

1Metform

in1,45

417

/19

89.0/89.1

58.0/59.4

56.4/57.9

NR/N

R7.4/7.4

7.6/7.3

32.2/32.1

Matthew

s20

05(62)

52Gliclazide

313

Pioglita

zone

317

5/4

100.0/99

.449

.2/50.8

57.0/56.0

5.5/5.8

8.5/8.7

7.3/7.2

32.6/32.6

No

Maz

zone

2006

(63)

72Glim

epiride

228

Pioglita

zone

230

0/1

65.4/59.6

62.7/63.5

59.9/59.3

7.5/8.0

7.4/7.4

7.2/7.2

31.9/32.0

Yes

Nisse

n20

08(64)

78Glim

epiride

273

Pioglita

zone

270

1/0

80.6/83.3

65.9/68.9

59.7/60.0

5.9/5.8

7.4/7.4

7.0/6.9

32.0/32.1

Yes

Riddle

1998

(65)

78Glim

epiride+

insu

lin72

Place

bo+insu

lin73

1/0

57.0/58.0

62.5/54.8

58.0/58.0

7.0/7.0

9.7/9.9

7.6/7.7

32.2/33.7

Yes

Sullivan

2011

(66)

260

SU

1,63

2Metform

in1,74

659

/45

91.0/94.0

NR/N

R63

.6/61.1

5.0/3.0

6.7/6.7

NR/N

R28

.0/31.0

No

SU

1,63

2Diet

2,62

759

/63

91.0/95.0

NR/N

R63

.6/62.2

5.0/2.0

6.7/6.0

NR/N

R28

.0/29.0

No

Tolm

an20

09(67)

156

Glib

enclam

ide

1,04

6Pioglita

zone

1,05

19/10

62.1/59.8

55.5/57.2

55.0/54.0

5.6/5.9

9.5/9.5

NR/N

R32

.5/32.5

No

UKPDS19

98(68)

577

Chlorpropam

ide

619

Insu

lin91

133

/42

79.0/82.0

58.0/72.0

54.0/54.0

NR/N

R6.3/6.1

7.8/7.8

27.0/27.0

Yes

Chlorpropam

ide

619

Diet

896

33/47

79.0/83.0

58.0/61.9

54.0/54.0

NR/N

R6.3/6.2

7.8/8.6

27.0/27.5

Glib

enclam

ide

615

Insu

lin91

145

/42

84.0/82.0

61.9/72.0

54.0/54.0

NR/N

R6.3/6.1

8.4/7.8

27.4/27.0

Glib

enclam

ide

615

Diet

896

45/47

84.0/83.0

61.9/61.9

54.0/54.0

NR/N

R6.3/6.2

8.4/8.6

27.4/27.5

Relev

antda

tainclud

edthefirstau

thor’s

name,

year

ofpub

lication,

follo

w-upduration,

charac

teris

ticsof

thestud

ypo

pulation(ra

ce,s

ex,m

eanag

e,mea

ndurationof

diab

etes

,mea

nBMI,

andinitialmea

nHbA

1c).Th

enu

mber

ofev

ents

forthe

morbidity

ofstroke

was

also

abstracted

.Ifs

imilars

tudieswerereportedin

morethan

onepub

lication,

weex

trac

teddatafrom

themos

trece

ntartic

le.S

tudies

wereex

clud

edifthese

arch

term

sus

edin

these

arch

strategies

wereno

tcon

tained

intheab

stract

(filte

redus

ingRefWorks

).Allof

theinform

ationwas

summarized

inastan

dardized

dataex

trac

tiontable.C,co

mparator;NR,no

treported;SU,su

lfony

lurea.

2798 KATP Channels in Stroke Diabetes Volume 65, September 2016

Further sensitivity analyses were performed: 1) includingonly trials in which sulfonylureas was used as monother-apy; 2) excluding trials in which the quality estimationwas “unclear”; 3) including only trials in which events ofstroke were formally adjudicated, irrespective of the factthat they were part of the principal end points.

Funnel plots (30) and the Harbord modified test (31)were used to estimate possible publication bias. Funnel plotsprovide a graphic representation of treatment effect, plottedversus trial sample size; an asymmetry in favor of a morebeneficial effect of the experimental treatment in smallertrials suggests a publication bias, based on the assumptionthat larger studies have a greater chance of being publishedeven when the results are unfavorable. The Harbord testprovides a statistical support to data reported in the funnelplot, based on the same principle. Subgroup analyses wereperformed for different comparators (diet, metformin,dipeptidyl peptidase 4 inhibitor [plus metformin], thiazoli-dinediones [plus metformin]), and insulin [plus placebo]),follow-up duration (,72 or $72 weeks), mean age (,60or $60 years), percentage of men (,60 or $60%), meandiabetes duration (,5, $5 years, or not reported), meanbaseline HbA1c (,7.5 or $7.5%), mean baseline BMI (,30or $30 kg/m2), and generation of sulfonylureas (first, sec-ond, third, or not reported). All of those analyses wereperformed using Stata 12.0 and RevMan 5.2 software.

RESULTS

Effect of STZ-Induced Diabetes in StrokeThe group with STZ-induced diabetes showed larger infarctvolumes compared with the vehicle-treated control group(Fig. 1). Infarct volume in the STZ group (50.39 6 7.66%,n = 8) was significantly larger than that of the vehicle-treatedgroup (32.44 6 2.93%, n = 8; P = 0.046) (Fig. 1B). Theneurological deficit score in STZ mice (4.13 6 0.30, n = 8)was significantly higher than that of the vehicle-treatedgroup (3.00 6 0.42, n = 8; P = 0.047) (Fig. 1C). Theseresults indicate that stroke is more severe in diabetic micethan in vehicle-treated mice. (STZ-induced diabetes wasconfirmed by lower body weights and higher blood glucoselevels shown in Fig. 1D and E.)

Diabetes Upregulated the Protein Levels of NR2B andPSD95 but Decreased p-GSk3b in DiabetesTo explore the role of stroke-related proteins in the mousebrain of STZ-induced diabetes, we compared the proteinlevels of p-GSK3b, NR2B, and PSD95 in STZ and vehiclegroups by Western blot. We found that levels of p-GSK3b,rather than the t-GSK3b, were significantly decreased inthe STZ group (P , 0.05) (Fig. 2A and B). We also foundthat protein levels of NR2B and PSD95, which mediatebrain damage in stroke, were upregulated in mice withSTZ-induced diabetes (P , 0.05) (Fig. 2A–D). These resultswere consistent with our findings that stroke is more se-vere in the group with STZ-induced diabetes. These find-ings are likely to underlie a potential mechanism for higherincidence of stroke in diabetes.

KATP Channels Protect Against OGD-Induced PrimaryCortical Neuronal Injury In VitroAt 24 h after cortical cultures were exposed to 90 min ofOGD, cells treated with 500 mmol/L tolbutamide showedsignificantly higher neuronal cell death (61.93 6 3.15%,n = 5) compared with vehicle-treated cells (39.78 61.74%, n = 5; P , 0.001) (Fig. 3A). Conversely, neuronstreated with 750 mmol/L diazoxide showed less cell death(21.78 6 3.38%, n = 5) than vehicle-treated neurons(38.07 6 4.68%, n = 5; P = 0.004) (Fig. 4A).

PI fluorescence images also indicated that PI-positivecell counts increased significantly in the tolbutamidegroup (313.8 6 34.77, n = 5; P , 0.05) (Fig. 3B and C),but decreased significantly in the diazoxide group (79.92612.71, n = 5; P , 0.05) (Fig. 4B and C) compared withvehicle-control groups (175.52 6 24.42 and 162.44 614.67, respectively, n = 5 per group). These results indicatethat activation of the KATP channel protects against OGD-induced neuronal cell injury in vitro.

KATP Channels Provide Neuroprotection AgainstStroke In VivoTo investigate the effect of KATP channels in neuroprotec-tion, the KATP channel blocker, tolbutamide (100 mg/kg,i.p.), KATP channel opener, diazoxide (20 mg/kg, i.p.), orvehicle (DMSO) was administrated 20 min before pMCAO (inmice not administered STZ). The infarct volume was in-creased significantly in the tolbutamide group (52.99 64.46%, n = 5; P , 0.05) (Fig. 5A and B) but was decreasedsignificantly in the diazoxide group (25.01 6 2.39%, n = 5;P , 0.05) (Fig. 6A and B), compared with the vehicle-controlgroups (38.126 3.85% and 42.436 3.78%, respectively, n =4 per group). These results indicate that activation of neuro-nal KATP channels provides neuroprotection to stroke in vivo.

Effects of KATP Channel Modulators in BehavioralOutcomesThe neurological deficit score was higher in the tolbuta-mide group (n = 5, P = 0.193) but was significantly lowerin the diazoxide group (n = 5, P = 0.046) compared withthe vehicle-control group (Figs. 5C and 6C).

Meta-analysis Showed That Use of SulfonylureasPoses a Greater Risk of Stroke for Patients With T2DMFigure 7A shows the study selection process and the cri-teria for exclusion. We selected 17 studies (Table 1) fromthe 1,954 articles that were retrieved. The total number ofpatients was 27,705, including 11,441 receiving sulfonyl-urea treatment (11 trials with monotherapy and 6 withcombination therapy) and 16,264 receiving comparatordrugs or placebo. The baseline characteristics and the num-ber of stroke events recorded are reported in Table 1. Thelength of follow-up ranged from 30 to 577 weeks, mean ageranged from 54.0 to 63.8 years, and diabetes durationranged from 2.0 to 8.0 years. All of the studies providedthe number of dropouts and the reasons as a total or thenumber of individuals per reason. No major asymmetryappeared in the funnel plot (Fig. 7B1), and the Harbord

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modified test showed that there was no publication bias(P = 0.31) (Fig. 7B2). Among the 17 trials, all acceptedblinded method (UK Prospective Diabetes Study [UKPDS]blinded outcome assessment method) and 11 accepteddouble-blinded (blinding of participants and personnel as

well as outcome assessment). We used the Cochrane sys-tem to estimate the quality of trials, which is shown in Fig.7C. The evaluation contained three levels: high risk, un-clear, and low risk. Among the 17 studies, 41.18% werein the low-risk level, 58.82% were in the unclear level, and

Figure 1—Effects of STZ-induced diabetes in increasing infarct volume in the mouse tMCAO model. A: Representative images of TTCstaining show that normal brain tissue is stained red and the infarcted brain tissue is stained white. B: The effects of STZ-induced diabeteson brain infarct volume are shown compared with that of the vehicle-treated control animals. Mice with STZ-induced diabetes showedincreased brain damage after the tMCAO compared with the vehicle-treated control group (n = 8 per group). *P = 0.046 by Student t test.C: The 6-point standard scale is used for neurological deficit score in mice 24 h after tMCAO. The STZ-administered mice with tMCAOexhibited significantly higher neurological deficit scores than the vehicle-treated group. *P = 0.047. D: Body weights of mice before and 1, 2,4, and 5 weeks after STZ or vehicle injections. Mean body weight was significantly reduced in animals 2 weeks after STZ injectioncompared with the controls (Cont). *P < 0.05. E: Blood glucose in mice 1, 2, 4, and 5 weeks after STZ or vehicle injection. The bloodglucose level in STZ-injected mice is significantly higher than that of the control animals, indicating successful induction of the diabeticmouse model. *P < 0.01.

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no studies were high risk. The total quality of the includedarticles was high.

Data were combined using random-effects models. Figure7D shows the OR for stroke morbidity for the 17 includedtrials. Patients who received sulfonylurea treatment had ahigher OR for stroke morbidity of 1.39 (95% CI 1.16–1.65) than those who received comparator drugs. The I2

statistic for heterogeneity between trials was 0.0% (P =0.66), suggesting that there was no obvious heterogene-ity. In a sensitivity analysis, we included only trials inwhich stroke events were formally adjudicated. This didnot reduce the between-study heterogeneity (I2 = 24.5%)and did not change the OR estimation (1.35, 95% CI1.08–1.69) (Fig. 7E1) for stroke morbidity of those pa-tients using sulfonylurea treatment. In addition, weassessed monotherapy only. This did not reduce thebetween-study heterogeneity (I2 = 14.3%) and did notchange the OR estimation (1.37, 95% CI 1.14–1.66) forstroke morbidity of those patients using sulfonylureatreatment (Fig. 7E2). In another sensitivity analysis, afterexcluding the studies in which quality assessments were“unclear,” the heterogeneity and OR estimation (1.41,95% CI 1.09–1.81) did not vary to a relevant degree(Fig. 7E3).

Subgroup analysis was performed for further investi-gation of the effect of sulfonylureas on stroke incidencein patients with T2DM with different characteristics.

The OR did not differ significantly by sex, age, durationof diabetes, BMI, or the HbA1c level (Fig. 7F). In directcomparisons, the increase of risk with sulfonylureasreached statistical significance versus dipeptidyl peptidase4 inhibitors.

DISCUSSION

With the increasing diabetes epidemic around the world, athorough knowledge of the effects of individual glucose-lowering medications on the incidence of CV diseases iscritical. The main focus is on CV risk assessment ratherthan extension of glucose control before new drug approval(32). Some studies have explored the CV risk of treatmentwith sulfonylureas in T2DM (33,34), but very few haveassessed the risk of stroke. Our results suggest that sulfo-nylureas may increase the risk of stroke in patients withT2DM.

Diabetes may increase risk of stroke. In our in vivostudy, STZ-induced diabetic mice with hyperglycemia andweight loss showed exacerbated brain damage in the strokemodel, suggesting that diabetes could have a negative ef-fect on the outcome of stroke. This result is in line withfindings from epidemiological studies reporting that dia-betes is associated with a greater lethality of stroke (35).An increased morbidity and mortality for stroke in pa-tients with T2DM could be a result of the effect of hyper-glycemia (21,36–38) or of the treatment of diabetes (39),

Figure 2—Molecules involved in the destructive effect of STZ-induced diabetes in mice. A: Representative Western blot images show theprotein levels of NR2B, PSD95, p-GSK3b, and t-GSK3b from brain tissues from STZ-administered and vehicle-treated groups. B: Relativelevels of p-GSK3b vs. t-GSK3b were determined by densitometry of the blots. The ratio of p-GSK3b and t-GSK3b proteins was significantlydecreased in the STZ-administered group compared with that of the vehicle-treated control (CTL) group (n = 6 per group). *P < 0.05.Relative levels of NR2B, PSD95, and GAPDH were determined by densitometry of the blots, and NR2B-to-GAPDH (C) and PSD95-to-GAPDH (D) ratios were calculated. Protein levels of NR2B and PSD95 in the brains were upregulated in the STZ-administered micecompared with the vehicle-treated group (n = 4 per group). *P < 0.05.

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or both. In particular, sulfonylureas could enhance ana-tomical and functional damage caused by cerebral ische-mia, with a potential effect on stroke incidence (39).Experimental data also support the notion that inhibition

of the KATP channel increases ischemic brain damage andthat activation of the KATP channel confers neuroprotec-tion in cerebral ischemia (14,15).

Excessive calcium entry into neurons through NMDAreceptors is thought to be one of the triggers of neuronal

Figure 4—Effect of diazoxide in OGD-induced neuronal cell injury.Diazoxide (100, 500, and 750 mmol/L) or 0.5N NaOH (vehicle-treatedcontrol group) was applied 30 min before OGD. Cell damage wasassessed 24 h after OGD. A: Quantitative measurements of PI fluo-rescence of cortical cultures 24 h after OGD showed diazoxide(750 mmol/L) decreased the fraction dead compared with the con-trol group. *P < 0.05. B: Confocal fluorescence imaging showsthat the number of PI-positive cells was significantly lower in thediazoxide-treated group compared with the control group. C: Quan-titative analysis was performed by counting five random fields witha 310 objective per coverslip (n = 5, for each sample we chose5 slices). Scale bars: 50 mm (310 objective).

Figure 3—Effect of tolbutamide in OGD-induced neuronal cell in-jury. Tolbutamide (50, 100, and 500 mmol/L) or DMSO (0.5%)(vehicle-treated control group) was applied 30 min before OGD. Celldamage was assessed 24 h after OGD. A: Quantitative measure-ments of PI fluorescence of cortical cultures 24 h after OGD showedthat the fraction dead was significantly higher in the tolbutamide-treated group than in the control group. *P < 0.05. B: Confocalfluorescence imaging shows a significantly higher number ofPI-positive cells in the tolbutamide-treated group than in the controlgroup.C: Quantitative analysis was performed by counting five randomfields with a310 objective per coverslip (n = 5, with 5 slices included ineach sample). Scale bars: 50 mm (310 objective).

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Figure 6—Effects of diazoxide in decreasing infarct volume in mousepMCAO model. Diazoxide (20 mg/kg in DMSO) or DMSO (0.1%) alone(vehicle-treated control group) was intraperitoneally administered to themice 20 min before the onset of pMCAO. Infarct volume was assessed24 h after MCAO. A: Representative TTC images. B: The effects ofdiazoxide (20 mg/kg i.p.) are shown on brain infarct volume comparedwith the vehicle-treated control animals. *P = 0.002 by Student t test.C:The 6-point standard scale was used for the neurological deficit scorein mice 24 h after the pMCAO. The diazoxide-treated mice (n = 5) withpMCAO exhibited significantly lower neurological deficit scores com-pared with the vehicle-treated group (n = 4). *P = 0.046.

Figure 5—Effects of tolbutamide in increasing infarct volumein mouse pMCAO model. Tolbutamide (100 mg/kg in DMSO) orDMSO (0.1%) alone (vehicle-treated control group) was intraperito-neally administered to the mice 20 min before the onset of pMCAO.Infarct volume was assessed 24 h after MCAO. A: RepresentativeTTC images. B: Effects of tolbutamide (100 mg/kg i.p.) are shownon brain infarct volume compared with the vehicle-treated controlanimals. *P = 0.044 by Student t test. C: The 6-point standard scalewas used for the neurological deficit score in mice 24 h afterpMCAO. The tolbutamide-treated mice (n = 5) with pMCAOexhibited higher neurological deficit scores than the vehicle-treatedgroup (n = 4), but the difference was not statistically significant (P =0.193).

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Figure 7—A systematic meta-analysis of 17 randomized control trials (RCT). A: Study selection process. B: Funnel plot and the Harbordmodified test for the 17 included trials to visualize potential publication bias. B1: The shape of funnel plots did not reveal obvious evidence ofasymmetry. B2: The P value for the Harbord modified test was 0.31, suggesting publication bias was not observed. C: Quality of the includedarticles was assessed using the criteria for judging risk of bias in the “risk of bias” assessment tool from the Cochrane Library. There are threelevels in these criteria: low risk, high risk, and unclear. D: The individual and pooled OR with 95% CI of the incidence of stroke during treatment:any sulfonylurea (SU) treatment (single use and combination) vs. any other treatment without SU. The pooled OR and 95% CI for the 17 trialswere 1.39 (95% CI 1.16–1.65; P = 0.659). E1: Results of a sensitivity analysis in which we included only trials in which events of stroke wereformally adjudicated show the between-study heterogeneity (I2 = 24.5%) and the OR estimation (1.35, 95% CI 1.08–1.69). E2: Results of asensitivity analysis in which we assessed monotherapy only show the between-study heterogeneity (I2 = 14.3%) and the OR estimation (1.37,95% CI 1.14–1.66). E3: Results of a sensitivity analysis excluding trials in which the quality estimation was “unclear” show the between-studyheterogeneity (I2 = 19.3) and the OR estimation (1.41, 95% CI 1.09–1.81). All of these sensitivity analyses did not reduce the between-studyheterogeneity and did not change the OR estimation for stroke morbidity of those patients using SU treatment. F: Subgroup analyses ofrelative risks for incidence of stroke during treatment: any SU treatment (single use and combination) vs. any treatment without SU. Randomeffects analysis was used. DPP-IV inh, dipeptidyl peptidase-4 inhibitor; LL, lower limit; NR, not recorded; UL, upper limit.

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Figure 7—Continued.

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Figure 7—Continued.

2806 KATP Channels in Stroke Diabetes Volume 65, September 2016

injury, where the NR2A subunit mediates cell survivalsignaling and the NR2B subunit links cell death signaling(40). PSD95 is a postsynaptic protein that interacts withNR2B. Disruption of the NR2B/PSD95 complex inhibitsNMDA receptor–mediated neuronal death after stroke(41). Thus, increased levels of NR2B or PSD95 may predicta higher risk of stroke and poorer neurological outcomes.The current study mainly focused on protein expression ofthese two molecules, which may provide an insight into thereason patients with diabetes show a greater risk of stroke.Our results confirm our hypothesis that the NR2B andPSD95 proteins were both upregulated in the brain afterSTZ-induced diabetes.

GSK3b is also highly expressed in the brain and regu-lates a variety of neuronal functions, including regulationof glucose metabolism and neuronal apoptosis. Our re-search confirms that p-GSK3b was decreased in diabetes.Because GSK3b is negatively regulated by phosphorylationat Ser9 (42), inhibition of GSK3b was neuroprotective andameliorated stroke-induced neurological impairments. To-gether with the increased NR2B and PSD95 expressionsin the diabetic mouse brain, targeting NR2B/PSD95 andGSK3b may be a potential strategy to reduce the risk ofstroke and improve stroke outcomes.

Previous studies showed that KATP channels are ex-pressed in CA1, CA3, and cortical neurons (14,43), inter-neurons, and granule cells (7). The neuronal KATP channelsare important in neuroprotection against ischemic injury,in focal and global ischemia (14,44,45), and in neonatalhypoxic-ischemic brain injury (15). Neuronal KATP channelsare activated during hypoxia and ischemia when there is adramatic decrease in the ATP-to-ADP ratio in the neurons.Opening of the KATP channels hyperpolarizes the cell mem-brane and suppresses neuronal excitability, providing cyto-protection (11,14–16).

In this study, we further confirm the neuroprotectiverole of KATP channels in vivo and in vitro by using KATP

blocker and opener. Brain damage in neuronal injury in-duced by pMCAO and OGD is increased by blocking theKATP channel by tolbutamide and decreased by opening theKATP channel by diazoxide. KATP channels in neurons andpancreatic b-cells share the same isoform, Kir6.2/SUR1(10). It is important to note that the STZ-induced dia-betes model in the animals may not fully resemble theT2DM model; however, the STZ-induced diabetes modelshares the essential common feature, hyperglycemia, ofthe T2DM model and is a commonly used animal model.Future testing of the brain damage in response to sulfo-nylureas treatment in mouse models of diabetes driven bydiet or genetic defect will be desirable.

It is possible that sulfonylureas block the neuronal KATP

channels and thus increase the likelihood of stroke in pa-tients with diabetes. To test our hypothesis, we furtherperformed a meta-analysis using clinical data to evaluatethe effect of sulfonylureas on the risk of stroke in diabetes.

Our systematic meta-analysis comparing the effect ofsulfonylurea and nonsulfonylurea treatments on the risk

of stroke in patients with T2DM found that patients whoused sulfonylureas had a higher risk of stroke. Notably,this result was obtained by summarizing available ran-domized trials, where there are no risk-confoundingfactors. In addition, the increased risk was confirmed byseveral sensitivity analyses with different criteria for trialinclusion. This strengthens the result, which cannot beattributed to the choice of any particular set of inclusionor exclusion criteria.

Although 20% of strokes may be associated with dia-betes (46), the mechanisms linking stroke and diabetesare not clearly understood. Epidemiological studies haveshowed that diabetes is a critical risk factor for ischemicstroke and that stroke is the second leading cause ofdeath and disability in humans (47–49). Moreover, ische-mic injury as a complication of diabetes leads to increasedneuronal damage and poor functional recovery (50,51);therefore, exploring the mechanisms of ischemic braininjury and developing better neuroprotective measuresin the population with diabetes will be a major focus ofmedical research.

Taken together, this study in our mouse stroke modelshowed, first, that STZ administration and blocking KATP

channels increased brain damage. It is very possible thatsulfonylureas block the neuronal KATP channels in thebrain and thus increase the likelihood of stroke in pa-tients with diabetes. Second, we further tested our hy-pothesis by performing a meta-analysis using clinicaldata to evaluate the effect of sulfonylureas on the riskof stroke in diabetes. Our meta-analysis also showed thatpatients with diabetes receiving sulfonylurea treatmenthave a higher relative risk for stroke morbidity than thosereceiving comparator drugs.

In summary, our study shows:

1. STZ-induced diabetes increased brain damage afterstroke. STZ-administered mouse brains also showedincreased expression of NR2B/PSD95 proteins, indicatingpotentially further increase stroke risk in diabetes.

2. The KATP channel blocker tolbutamide increased brainand neuronal injury in vivo and in vitro. The KATP

channel opener diazoxide reduced brain and neuronalinjury in vivo and in vitro.

3. The meta-analysis indicates patients with T2DM receiv-ing sulfonylurea treatment have a higher relative risk forstroke morbidity than those receiving comparator drugs.

Our findings suggest that the KATP channel is a poten-tial target for neuroprotection against stroke and thatantidiabetic therapy using sulfonylureas should be consid-ered carefully or even avoided in the long-term manage-ment of T2DM.

Funding. This research was supported by China Scholarship Council Fellow-ships for R.L. and B.X., a Canadian Institutes of Health Research (CIHR) Student-ship for E.T. (CIHR-CGS-M), Ontario Graduate Studentships for E.T., N.D.,

diabetes.diabetesjournals.org Liu and Associates 2807

C.L.F.S., and A.B., and operating grants to Z.-P.F. from the National Sciencesand Engineering Research Council of Canada (NSERC-249962-09) and to H.-S.S.from the Heart and Stroke Foundation of Canada (G-13-0003069) and CanadianInstitutes of Health Research China–Canada Joint Health Research Initiative(CIHR, FRN #132571).Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. R.L., H.W., and B.X. performed experiments. R.L.,Y.L., and H.F. performed the meta-analysis. H.W., B.X., W.C., E.T., N.D., C.L.F.S.,and A.B. performed experimental data analysis. All authors contributed tomanuscript preparation, discussed the results, analyzed data, and commented onthe manuscript. Z.-P.F. and H.-S.S. developed the concepts and designedthe study. Z.-P.F. and H.-S.S. are the guarantors of this work and, as such, had fullaccess to all the data in the study and take responsibility for the integrity of the dataand the accuracy of the data analysis.

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