Hindawi Publishing CorporationJournal of Diabetes ResearchVolume 2013 Article ID 405284 8 pageshttpdxdoiorg1011552013405284

Research ArticleAngiotensin-Converting Enzyme 2 DeficiencyAggravates Glucose Intolerance via Impairment of IsletMicrovascular Density in Mice with High-Fat Diet

Li Yuan Ying Wang Chunli Lu and Xiaoya Li

Department of Endocrinology Union Hospital Tongji Medical College of HuaZhong University of Science amp TechnologyWuhan 430022 China

Correspondence should be addressed to Li Yuan yuanli18cnyahoocomcn

Received 15 January 2013 Accepted 20 February 2013

Academic Editor Raffaele Marfella

Copyright copy 2013 Li Yuan et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The aim of this study was to evaluate the effects of angiotensin-converting enzyme 2 (ACE2) on glucose homeostasis and islet func-tion in mice Male wildtype (WT) and ACE2 knockout (ACE2 KO)mice were divided into chow diet group and long-term high-fatdiet (HFD) group After 16 weeks of feeding the islet function of the animals was evaluated by intraperitoneal glucose tolerancetest (IPGTT) and intraperitoneal insulin releasing test (IPIRT) The pancreas was immunohistochemically stained to analyze therelative content of insulin (IRC) vascular endothelial growth factor (VEGF) and microvessel density (MVD) in islets There wasno difference of body weight area under curve of glucose (AUCG) area under curve of insulin from 0 to 5min (AUGI

0minus5) MVD

and RVC (relative content of VEGF) between WT and ACE2 KO mice with regular chow diet Under the condition of long-termHFD the AUCGof ACE2KOmice was increased obviously in comparisonwith theWTmice with decreased IRCMVD AUGI

0minus5

AUCI0minus30

and RVC (all 119875 lt 005) In conclusion these results show that ACE2 deficiency deteriorates islet function of mice withlong-term HFD via impairment of islet microvasculature

1 Introduction

The classical renin-angiotensin system (RAS) ACE-Ang II-AT1 receptor axis is a coordinated hormonal cascade facil-itating the dynamic control of perfusion in both health anddisease [1] In 2000 a new member of the RAS ACE2 wasdiscovered by two independent groups [2 3] ACE2 is capableof metabolizing Ang II to generate Ang (1-7) [4] Ang (1-7)interactingwith its receptorMas elicits numerous actions thatcounterbalance those of Ang II [5] Accumulating evidencehave indicated that ACE2-Ang (1-7)-Mas axis acts as anegative regulator of the ACE-Ang II-AT1 receptor axis [6]

During the past decades the existence of a local RAS hasbeen confirmed in both the endocrine and exocrine pancreas[7ndash9] In 1991 an intrinsic RAS in the pancreas was firstlydescribed by Chappell et al [10] Lau et al [11] identified theexpression of several RAS components in mouse pancreaticislet such as angiotensinogen ACE AT1 and AT2 receptorsThe evidence for the existence of these above componentswere also observed in human pancreas [12] In addition

ACE2 mRNA and protein have been identified in the pan-creatic islets [13] Canine pancreatic Ang (1-7) [10] and ratpancreatic Mas receptor have also been confirmed [14]

Previous studies [15 16] have provided additional evi-dence for the close association of RAS with pancreatic endo-crine physiology and its pathophysiology Islet angiotensino-gen andAT1 receptor expressionwere increased in the Zuckerdiabetic fatty rats [17] Islet RAS activation is responsible forthe islet dysfunction through exacerbation of impairment ofislet blood flow [8] promoting islet cell destruction fibrosisand apoptosis [18] Treatment with ACEI or ARB amelioratesthe impairment of glucose tolerance [19] reduces new-onsetdiabetes [20] and prevents the development of diabetic com-plications [21] To date there have been limited studies onwhether ACE2 plays a key role in islet function Wong et al[22] reported that there was no difference between the plasmaglucose concentrations in the ACE2 KO mice and WT miceThe exact functions ofACE2 in the regulation of islet functionremain controversial and as yet inadequately elucidatedThus the present study aimed to investigate the potential role

2 Journal of Diabetes Research

of ACE2 in glucose homeostasis and explore the mechanismsof ACE2 in islet function

2 Materials and Methods

21 Animals Male ACE2 KO mice and their age matchedmale wildtype littermates (C57BL6J) at the age of 5 weekswere purchased from the Institute of Laboratory Animal Sci-ence Chinese Academy of Medical Sciences ACE2 KO micewere maintained on the C57BL6J background Mice wereindividually housed under specific pathogen-free conditionswith a 12 hours light-dark cycle ambient temperature of 22∘Cand allowed free access to food and water After 2 weeks ofacclimatization the mice were randomly divided into twogroups Groups of mice were fed regular rodent chow diet(20 protein 70 carbohydrate and 10 fat) or high-fat diet(20 protein 20 carbohydrate and 60 fat) for 16 weeksThe body weight of each mice was measured weekly duringthe study The institutional animal care and use committeeapproved all procedures

22 Intraperitoneal Glucose Tolerance Test (IPGTT) Micewere fasted for 6 hours and were injected intraperitoneallywith glucose at 20 gkg body weight Blood was collectedfrom the tail Blood glucose was analyzed at 0 15 30 60and 120min after the injection The values of AUC werecalculated The area under the curve (AUC) was calculatedusing the formulaAUCG= 75G

0+ 15G

15+ 225G

30+ 45G

60+

30G120

where G0 G15 G30 G60 and G

120were blood glucose

level at each time point during IPGTT Blood glucose wasmeasured using an automatic glucometer (One Touch Byer)

23 Intraperitoneal Insulin Releasing Test (IPIRT) For insulinsecretion test mice were fasted for 6 hours and glucose(30 gkg body weight) was injected intraperitoneally Bloodsamples were collected from the orbital venous plexus at 0 25 15 and 30min Insulin AUC was calculated by the formulaAUCI

0minus30= I0+ 25I2+65I

5+ 125I

15+ 75I30TheAUCI from

0min to 5min (AUCI0minus5

) which represented the first stageinsulin secretion was calculated as follows AUCI

0minus5= I0+

25I2+ 15I5 I0 I2 I5 I15 and I

30represented the insulin level

at 0 2 5 15 30min respectively Insulin levels were measuredwith themouse insulin enzyme-linked immunosorbent assaykit

24 Insulin Tolerance Test (ITTs) For insulin tolerance testmice were fasted for 6 hours and then injected insulin(05Ukg body weight) (Novolog) intraperitoneally Glucoselevels were measured at 0 15 30 60 and 90min after theinjection Results are shown as percentage of glucose levelsat the time of injection

25 Islet Immunohistochemistry Pancreas specimens werefixed in 4 chilled paraformaldehyde and embedded inparaffin Sections were mounted on glass slides and pro-cessed for immunohistochemistry staining The pancreaticislets were stained for insulin CD31 and vascular endothe-lial growth factor (VEGF) using anti-rat primary antibodywith suitable concentration The slides were then incubated

overnight at 4∘C After three rinses with PBS a secondaryantibody (Sigma)was applied for 30min at 37∘CA third anti-body (Sigma) was applied as the same way and DABH

2O2

staining was used at last Five discontinuous sections fromeach animal were stained and each section was analyzedrandomly and double-blindly using light microscopy theresults were captured and evaluated by a computer imageanalysis system (Motic Wetzlar Germany)

26 Islet Morphology and Insulin Expression Insulin stainingwas used to assess the change of islets morphology andinsulin concentration in 120573 cells The reciprocal of grayscale of intraislet insulin-positive staining was computed thenatural logarithm was calculated as intraislet insulin relativeconcentration (IRC) which represented the insulin reserve inendochylema of 120573 cells Insulin-positive cell density (ICD)which was the ratio of intraislet insulin-positive nuclearquantity to insulin-positive staining area was used to reflectthe amount of beta cells intraislets

27 Islet Microvascular Vessel Density (MVD) The MVD ofislets was calculated by CD31-positive cells as reported [23]Any brown-stained endothelial cell or endothelial cell clusterin islets was regarded as a microvessel for counting Six dis-continuous sections from each animal were stained and 8ndash10islets in each section were selected randomlyThe percentageof the area for CD31 positive cell was counted divided bythe islet area to obtain MVD All counts were performedby two investigators simultaneously and independently Thereciprocal of gray scales of intraislet VEGF positive stainingwas computed and the natural logarithm was calculated topresent the relative content of vascular endothelial growthfactor (VRC)

28 Statistical Analysis All statistical analyses were per-formed using SPSS 115 Data are expressed as mean plusmn SEMStatistical significance of differences was assessed using one-way analysis of variance and unpaired Studentrsquos t-test asappropriate A 119875 value lt 005 was considered significant

3 Results

31 Body Weight The body weight of mice was shown inFigure 1 There was no obvious difference in body weightamong the four groups at the beginning of the study Thebody weights ofWT and KO (ACE2 KO) groups were similarat end of the study When fed with high-fat diet the bodyweights of WH (WT with high-fat diet) and KH (ACE2 KOwith high-fat diet) groups gradually increased After 16 weekshigh-fat diet the body weights of WH and KH groups weresignificantly higher than those in corresponding chow dietgroups (both 119875 lt 005) When the study was completedno significant difference of body weight was observed in KHgroup as compared with those in WH group (Table 1)

32 Islet Function The islets function was evaluated by intra-peritoneal glucose tolerance test (IPGTT) and intraperitonealinsulin releasing test (IPIRT) Fasting blood glucose levelswere not significantly different among those four groups In

Journal of Diabetes Research 3

Table 1 Body weight blood glucose blood insulin and islet function (mean plusmn SD)

WT WH KO KHBody weight (g) (beginning) 2260 plusmn 065 2273 plusmn 055 2248 plusmn 033 2256 plusmn 033Body weight (g) (ending) 2905 plusmn 083 3424 plusmn 145lowast 2921 plusmn 069 3441 plusmn 069lowast

Fasting blood glucose (mmolL) 818 plusmn 072 838 plusmn 042 860 plusmn 046 863 plusmn 055Fasting blood insulin (ngmL) 084 plusmn 002 097 plusmn 002lowast 087 plusmn 001 085 plusmn 003

AUCI0-5 ((ngmL) lowastmin) 965 plusmn 013 931 plusmn 006lowast 958 plusmn 015 740 plusmn 015lowast

AUCI0-30 ((ngmL) lowastmin) 4382 plusmn 021 6161 plusmn 056lowast 4431 plusmn 031 4493 plusmn 071

WT wild type mice WH wild type mice with high-fat diet KO ACE2 knockout mice KH ACE2 knockout mice with high-fat diet versus WT lowast119875 lt 005versus KO

119875 lt 005 and versus WH 119875 lt 005

0

5

10

15

20

25

30

35

0 2 4 6 8 10 12 14 16Time (week)

WT WH

KOKH

Body

wei

ght (

g)

Figure 1 The curve of body weight WT wildtype mice WHwildtype mice with high-fat diet KO ACE2 knockout mice KHACE2 KO mice with high-fat diet

IPGTT blood glucose level in WT group peaked at approx-imately 229mmolL and approached baseline levels by120min Similar glucose profiles were observed in KO inresponse to glucose challenge The values of 30 and 60minglucose of WH group were significantly higher than those inWT group (both 119875 lt 005) However blood glucose concen-tration in KH group peaked at approximately 309mmolLand remained elevated We took the area under the IPGTTcurve (AUCG) to quantify glucose tolerance There was nosignificant difference of AUCG between WT mice and KOmiceThe AUCG inWHmice was significantly elevated thanthat in WT mice (119875 lt 005) The AUCG in KH groups wasobviously increased than that in KO (119875 lt 005) Interestinglywe observed increased AUCG in KH mice compared withWH mice (119875 lt 005) These data indicated that deletion ofACE2 leads to more severe glucose intolerance induced byhigh-fat diet (Figure 2 Table 1)

The fating blood insulin between WT group and KOgroup was not different However the fasting blood insulinlevels in WH group were significantly higher than those inWT and KH groups (both 119875 lt 005) In the IPIRT insulinpeak climbed rapidly to about 4-fold as basic level in 2minin WT group after glucose challenge The levels of insulinrelease in the KO group stimulated by glucose infusion were

almost the same as those in the WT group The AUCI0minus30

and AUCI0minus5

were not different between WT and KO groupCompared with WT group the insulin secretion of WHgroup increased slowly with a delayed and increased peakWe observed a decreased AUCI

0minus5and increased AUCI

0minus30

in WH group compared with those in WT group (both119875 lt 005) indicating the impairment of first stage insulinreleasing and a compensatory enhancement of insulin releaseunder the condition of high-fat diet The AUCI

0minus30in KH

group was not increased when compared with that in KOgroup In addition the insulin secretion in KH and WHgroup climbed to the peak at the same time whereas theformer exhibited a lower peak releasing after glucose loadThis finding suggested that ACE2 deficiency inhibits thecompensatory enhancement of insulin secretion in responseto the increased metabolic demand (Figure 3 Table 1)

33 Insulin Tolerance Test In IPITT blood glucose level inWT group dropped obviously and approached bottom levelsby 30min Similar glucose disappearance rate was observedin KO mice After an intraperitoneal injection of insulin theglucose disappearance rate of 15 and 30min was higher inWH and KH mice than those in corresponding mice withchow diet (both 119875 lt 005) There was no difference ofthe glucose disappearance rate between KH and WH mice(Figure 4)

34 IRC and ICD of Islets Islets in WT group were regularwith high expression of insulin There was no significantdifference of IRC and ICD between WT and KO groups Theislet area in the pancreases of WH group was larger than thatin WT group and the IRC dropped obviously (119875 lt 005)indicating the reduction of insulin reserve in WH group Incomparison with KO mice KH mice exhibited a decreasedIRC (119875 lt 005) Moreover a decreased IRC was noted in KHgroup in comparison with WH group (119875 lt 005) implyingmore severe impairment of insulin reserve in KH group TheICD in the WH group was lower than that in the WT groupbut the difference was not significant No difference of ICDwas observed between KH group and KO group There wasno difference of ICD between KH group and WH group(Figure 5 Table 2)

35 MVD in Islets The MVD of islet was slightly decreasedin KOmice than that in WTmice but the difference was notsignificant Staining of the pancreas using anti-CD31 antibody

4 Journal of Diabetes Research

0

5

10

15

20

25

30

35

0 30 60 90 120Time (min)

Plas

ma g

luco

se (m

mol

L)

WT

WH

KO

KH

(a)

0

500

1000

1500

2000

2500

3000

WT WH KO KH

lowast

lowast

AUCG

(minlowast

(mm

olL

))

(b)

Figure 2The curve of glucose tolerance test (a) Glucose curve (b) area under curve of glucose (AUCG) WT wildtype mice WH wildtypemice with high-fat diet KO ACE2 knockout mice KH ACE2 KO mice with high-fat diet versus WT lowast119875 lt 005 versus KO

119875 lt 005 andversus WH 998779119875 lt 005

0

05

1

15

2

25

3

0 5 10 15 20 25 30Time (min)

Plas

ma i

nsul

in (n

gm

L)

WT

WHKO

KH

Figure 3 The curve of insulin releasing test WT wildtype miceWH wildtype mice with high-fat diet KO ACE2 knockout miceKH ACE2 KO mice with high-fat diet

demonstrated enhancedMVD inWH group thanWT group(119875 lt 005) which suggested higher islet vascularizationafter high-fat diet However the KH mice did not showan increased MVD in islet when compared with KO miceMoreover there was reduced MVD in islet from KH groupwhen compared with WH group (119875 lt 005) These findingsindicated that loss of ACE2 decreases the compensation ofislet vascularization in response to high-fat diet (Figure 5Table 2)

36 VRC Intraislets The VRC was similar between the WTand KO group In comparison with WT group VRC of WH

0

02

04

06

08

1

12

0 20 40 60 80 100Time (min)

Glu

cose

( in

itial

)

WT

WH

KO

KH

Figure 4 The curve of insulin tolerance test WT wildtype miceWH wildtype mice with high-fat diet KO ACE2 knockout miceKH ACE2 KO mice with high-fat diet

group was increased (119875 lt 005) However there was nodifference of VRC between KO group and KH group TheVRC in KH group was decreased when compared with WHgroup (119875 lt 005) (Figure 5 Table 2)

4 Discussion

The RAS has an important role in the endocrine pancreas[7] Many projects have focused on the effects of ACE-AngII-AT1 axis A homolog of ACE ACE2 cleaves the terminalphenylalanine residue from Ang II to synthesis Ang (1-7)thereby functioning as a negative regulator of the RAS In this

Journal of Diabetes Research 5

Table 2 Data of immunohistochemistry (mean plusmn SD)

WT WH KO KHInsulin relative concentration minus400 plusmn 005 minus463 plusmn 006lowast minus410 plusmn 004 minus507 plusmn 006lowast

Insulin-positive cell density 235 plusmn 002 230 plusmn 001 232 plusmn 002 228 plusmn 001Microvascular density 1352 plusmn 048 1709 plusmn 052lowast 1187 plusmn 045 789 plusmn 046lowast

Relative content of VEGF minus467 plusmn 011 minus399 plusmn 005lowast minus491 plusmn 004 minus511 plusmn 004lowast

WTwild typemiceWHwild typemice with high-fat diet KO ACE2 knockoutmice KH ACE2 knockoutmice with high-fat diet VEGF vascular endothelialgrowth factor versus WT lowast119875 lt 005 versus KO

119875 lt 005 and versus WH 119875 lt 005

WT WH KO KH

Insulin

CD31

VEGF

Figure 5 ImmunohistochemistryWT wildtypemiceWH wildtypemice with high-fat diet KO ACE2 knockout mice KH ACE2 KOmicewith high-fat diet VEGF vascular endothelial growth factor

current study we demonstrated that high-fat-diet-inducedACE2 KO mice exhibit a progressive impairment of glucosetolerance and a reduction of insulin secretion in response toglucose compared with WT mice In the high-fat-inducedACE2 KO mice loss of ACE2 triggered a greater decreasein islet 120573 cell and islet MVD Our study supported a pivotalrole of ACE2 in the regulation of 120573 cell function and isletvasculature

As the major bioactive component of the classical RASAng II mainly interacts with the AT1 receptor to exertvarious actions including vasoconstriction activation of pro-inflammation fibrosis and oxidative stress ACE2 is a mon-ocarboxypeptidase cleaving Ang II to form Ang (1-7) As aclass of G-protein-coupled receptorMas have been identifiedas a receptor of Ang (1-7) Ang (1-7) interacting with Mas

elicits protective actions such as vasodilatation and nitricoxide generation that counterbalance those of Ang II [6]TheACE2-Ang (1-7)-Mas axis may act as a negative modulatorof the classical RAS effects [24] There is emerging evidencefor a functional ACE2-Ang (1-7)-Mas axis which is more thanregulation of cardiovascular system [25]

The association of the classic RAS with the endocrinesystem has been identified in diabetes and metabolic syn-drome [26] A large number of clinical trials have indicatedthat inhibitors of the RAS such as ACEI and ARB reducethe incidence of new-onset T2DM in high-risk individualsand protect against the development of T2DM [19 20]Furthermore inhibition of RAS has been also reported toexert protective action to glucose homeostasis in animalmodels of T2DM [27] On the other hand ACE2 may be

6 Journal of Diabetes Research

a potential protective enzyme for glucose homeostasis [9]but the role of ACE2 in the regulation of islet function isincompletely understood In the present study there were ondifferences in fasting blood glucose and glucose tolerance inmale ACE2 KOmice with standard chow diet compared withcorresponding WT mice This observation was consistentwith a previous study by Wong et al [22] who found thatglycemic control was not worsened by the absence of ACE2in the Akita diabetic miceTherefore we supposed that ACE2ablation does not seem to be sufficient to alter glucose statusin the condition of regular chow diet However an interestingand important finding of the current study was that theconsequence of ACE2 absence became apparent after high-fat diet Under the condition of high-fat diet there weremuchmore reductions of the first-stage insulin secretion andglucose tolerance in KH mice compared with WHmice Notsurprisingly these changes were linked to decreased 120573 cellmass It is noteworthy that this finding was partly supportedby the report of gene therapy in obese diabetic mice Bindomet al [28] found that ACE2 gene therapy was able to improvefasting blood glucose and glucose tolerance in obese diabeticmice Consistent with the improvement of glycemic controlACE2 gene therapy leaded to enhanced first-stage insulinsecretion and reduced 120573 cell apoptosis Our current studyfor the first time provided evidence that ACE2 absence leadsto more severe effects in 120573 cell dysfunction in obese micewith high-fat diet Taken together it is hypothesized that lossof ACE2 renders the pancreatic islet more susceptible to thepathological actions of obesity induced by high-fat diet

It is known that islet vascularization is essential for theislet to respond to fluctuations in blood glucose andmodulateinsulin release as needed Defects in the vascularization ofpancreatic islets can lead to deleterious effects on glucose tol-erance and islet insulin secretion In our study an increasedislet vascularization in high-fat diet WT mice was observedcompared with chow diet WT mice which represented acompensatory response to support higher vascularizationduring 120573 cell expansion In contrast no increased isletvascularization was observed in KH mice compared withKO mice Furthermore KH mice exhibited defects of isletvascularization compared with WH mice These findingssuggested that deletion of ACE2 reduces the compensationof islet vascularization in response to high-fat diet Thereis an intimate connection between islet beta cell and isletvascularization On the one hand islet vascularization hasbeen regarded as playing a crucial role in maintaining betacell survival and function On the other hand beta cellssecrete several factors such as VEGF to promote vasculardevelopment VEGF which is highly expressed in islet hasbeen identified as an important factor to be involved in theregulation of islet vascularization [23 29] Vascular endothe-lial cells migrate to the source of VEGF-A and proliferateand form blood vessels in response to VEGF [30] Previousstudies [31 32] suggested that mice with specific deletionof the VEGF in 120573 cells had reduced islet vascularizationand impaired glucose tolerance These findings were in linewith observations in the transplanted pancreatic islets It hasbeen reported that reduction of VEGF-A by 120573 cells not only

reduces the number of intraislet endothelial cells participat-ing in graft vessel formation but also limits recruitment ofhost endothelial cell and their invasion into the graft [31 33]In our study we found an increase of islet VEGF in WHmice which was consistent with the enhancement of isletvascularization after high-fat diet However with the deletionof ACE2 this compensatory increase of VEGF in islet wasdisappeared after high-fat diet This was parallel to the islet120573 cell impairment and islet vascularization reduction Withthe defects of the islet vascularization the beta cell wasimpairedThe deteriorated beta cell might lead to a reductionof VEGF expression which in turn accelerates the loss ofvascularization Taken together we speculated that underthe condition of high-fat diet ACE2 might play a criticalrole in the glucose intolerance through impairment of isletvascularization

ACE2 not only functions as a carboxypeptidase cleavinga single residue from Ang I generating Ang1-9 and asingle residue from Ang II to generate Ang 1-7 but alsocleaves several other biological peptides such as apelinACE2 hydrolyzes apelin with high catalytic efficiency [34]Apelin is the endogenous ligand of the G-protein coupledapj receptor The effects of apelin on the pancreas focusedon the regulation of insulin secretion [35] Winzell et al[36] reported that apelin-36 inhibited the glucose-stimulatedinsulin secretion in obese insulin resistant high-fat fed miceMoreover apelin-13 was shown to inhibit insulin secretionstimulated by high glucose concentrations in INS-1 cells [37]Accordingly we hypothesized that with the deletion of ACE2the hydrolyzation of apelin is inhibited which leads to theinhibition of insulin secretion

Peripheral Insulin sensitivity has long been regarded asplaying a crucial role in glucose homeostasis However theeffect of ACE2 on insulin sensitivity is unclear In this studywe took insulin tolerance test to evaluate peripheral insulinsensitivity We found no significant difference of peripheralinsulin sensitivity between KO mice and WT mice Theperipheral insulin sensitivity betweenWHmice andKHmicewas similar too So we concluded that loss of ACE2 hasno effects on peripheral insulin sensitivity More accuratemeasures such as hyperinsulinemic euglycemic clamp shouldbe needed in further studies It should be noted that therewas no difference of the body weight betweenWT and ACE2KO mice neither with chow diet nor with high-fat dietAccordingly we speculated that ACE2was not involved in theregulation of body weight

In summary the results obtained in this study show thatloss of ACE2 in mice with long-term high-fat diet leadsto impairment of glucose tolerance and insulin secretionThe primary mechanisms involved in these effects appear toinclude a reduction in islet 120573 cell and islet vascularizationOur data suggested that the absence of ACE2 weakened theadaptive changes of islet 120573 cell function and islet vasculariza-tion due to high-fat diet ACE2 seems to be a potential thera-peutic target in the treatment of islet dysfunction induced byhigh-fat diet

Journal of Diabetes Research 7

Acknowledgment

This project was supported by the National Natural ScienceFoundation of China (no 81070615)

References

[1] J R A Skipworth G Szabadkai S W M Olde Damink PS Leung S E Humphries and H E Montgomery ldquoReviewarticle pancreatic renin-angiotensin systems in health anddiseaserdquo Alimentary Pharmacology amp Therapeutics vol 34 no8 pp 840ndash852 2011

[2] S R Tipnis N M Hooper R Hyde E Karran G Christieand A J Turner ldquoA human homolog of angiotensin-convertingenzyme cloning and functional expression as a captopril-insensitive carboxypeptidaserdquo Journal of Biological Chemistryvol 275 no 43 pp 33238ndash33243 2000

[3] M Donoghue F Hsieh E Baronas et al ldquoA novel angiotensin-converting enzyme-related carboxypeptidase (ACE2) convertsangiotensin I to angiotensin 1-9rdquo Circulation Research vol 87no 5 pp e1ndashe9 2000

[4] G I Rice D A Thomas P J Grant A J Turner and N MHooper ldquoEvaluation of angiotensin-converting enzyme (ACE)its homologue ACE2 and neprilysin in angiotensin peptide me-tabolismrdquo Biochemical Journal vol 383 no 1 pp 45ndash51 2004

[5] C M Ferrario ldquoACE2 more of Ang-(1ndash7) or less Ang IIrdquo Cur-rent Opinion in Nephrology and Hypertension vol 20 no 1 pp1ndash6 2011

[6] R A S Santos A J Ferreira and A C Simoes E Silva ldquoRecentadvances in the angiotensin-converting enzyme 2-angiotens-in(1ndash7)-Mas axisrdquo Experimental Physiology vol 93 no 5 pp519ndash527 2008

[7] Q Cheng and P S Leung ldquoAn update on the islet renin-angiot-ensin systemrdquo Peptides vol 32 no 5 pp 1087ndash1095 2011

[8] P S Leung ldquoThe physiology of a local renin-angiotensin systemin the pancreasrdquo Journal of Physiology vol 580 no 1 pp 31ndash372007

[9] D Batlle M J Soler and M Ye ldquoACE2 and diabetes ACE ofACEsrdquo Diabetes vol 59 no 12 pp 2994ndash2996 2010

[10] M C Chappell A Millsted D I Diz K B Brosnihan and CM Ferrario ldquoEvidence for an intrinsic angiotensin system inthe canine pancreasrdquo Journal of Hypertension vol 9 no 8 pp751ndash759 1991

[11] T Lau P O Carlsson and P S Leung ldquoEvidence for a localangiotensin-generating system and dose-dependent inhibitionof glucose-stimulated insulin release by angiotensin II in iso-lated pancreatic isletsrdquoDiabetologia vol 47 no 2 pp 240ndash2482004

[12] K Y Lam and P S Leung ldquoRegulation and expression of arenin-angiotensin system in human pancreas and pancreaticendocrine tumoursrdquo European Journal of Endocrinology vol146 no 4 pp 567ndash572 2002

[13] C Tikellis M E Cooper andM CThomas ldquoRole of the renin-angiotensin system in the endocrine pancreas implications forthe development of diabetesrdquo International Journal of Biochem-istry and Cell Biology vol 38 no 5-6 pp 737ndash751 2006

[14] S M Bindom and E Lazartigues ldquoThe sweeter side of ACE2physiological evidence for a role in diabetesrdquo Molecular andCellular Endocrinology vol 302 no 2 pp 193ndash202 2009

[15] P S Leung and P O Carlsson ldquoPancreatic islet renin angiot-ensin system its novel roles in islet function and in diabetesmellitusrdquo Pancreas vol 30 no 4 pp 293ndash298 2005

[16] P S Leung ldquoMechanisms of protective effects induced by block-ade of the renin-angiotensin system novel role of the pancreaticislet angiotensin-generating system inType 2 diabetesrdquoDiabeticMedicine vol 24 no 2 pp 110ndash116 2007

[17] C Tikellis P J Wookey R Candido S Andrikopoulos M CThomas and M E Cooper ldquoImproved islet morphology afterblockade of the renin-angiotensin system in the ZDF ratrdquo Dia-betes vol 53 no 4 pp 989ndash997 2004

[18] L Yuan X Li J Li H l Li and S S Cheng ldquoEffects of renin-angiotensin system blockade on the islet morphology and func-tion in rats with long-term high-fat dietrdquo Acta Diabetologica Inpress

[19] A J Scheen ldquoRenin-angiotensin system inhibition preventstype 2 diabetes mellitusmdashpart 1 a meta-analysis of randomisedclinical trialsrdquo Diabetes and Metabolism vol 30 no 6 pp 487ndash496 2004

[20] E L Gillespie C M White M Kardas M Lindberg and C IColeman ldquoThe impact of ACE inhibitors or angiotensin II type1 receptor blockers on the development of new-onset type 2diabetesrdquo Diabetes Care vol 28 no 9 pp 2261ndash2266 2005

[21] P Vejakama A Thakkinstian D Lertrattananon A IngsathitC Ngarmukos and J Attia ldquoReno-protective effects of renin-angiotensin system blockade in type 2 diabetic patients a sys-tematic review and network meta-analysisrdquo Diabetologia vol55 no 3 pp 566ndash578 2011

[22] D W Wong G Y Oudit H Reich et al ldquoLoss of Angiotensin-converting enzyme-2 (Ace2) accelerates diabetic kidney injuryrdquoAmerican Journal of Pathology vol 171 no 2 pp 438ndash451 2007

[23] E M Akirav M T Baquero L W Opare-Addo et al ldquoGlucoseand inflammation control islet vascular density and 120573-cell func-tion in NOD mice control of islet vasculature and vascularendothelial growth factor by glucoserdquo Diabetes vol 60 no 3pp 876ndash883 2011

[24] M Iwai and M Horiuchi ldquoDevil and angel in the renin-angiot-ensin system ACE-angiotensin II-AT1 receptor axis vs ACE2-angiotensin-(1ndash7)-Mas receptor axisrdquo Hypertension Researchvol 32 no 7 pp 533ndash536 2009

[25] M C Chappell ldquoEmerging evidence for a functional angiotens-in-converting enzyme 2-angiotensin-(1ndash7)-Mas receptor axismore than regulation of blood pressurerdquoHypertension vol 50no 4 pp 596ndash599 2007

[26] K Putnam R Shoemaker F Yiannikouris and L Cassis ldquoTherenin-angiotensin system a target of and contributor to dyslip-idemias altered glucose homeostasis and hypertension of themetabolic syndromerdquo American Journal of Physiology vol 15no 302 pp H1219ndashH1230 2012

[27] V Souza-Mello B M Gregorio F S Cardoso-De-Lemos LDe Carvalho M B Aguila and C A Mandarim-De-LacerdaldquoComparative effects of telmisartan sitagliptin and metforminalone or in combination on obesity insulin resistance and liverand pancreas remodelling in C57BL6 mice fed on a very high-fat dietrdquo Clinical Science vol 119 no 6 pp 239ndash250 2010

[28] S M Bindom C P Hans H Xia A H Boulares and E Lazar-tigues ldquoAngiotensin I-converting enzyme type 2 (ACE2) genetherapy improves glycemic control in diabetic micerdquo Diabetesvol 59 no 10 pp 2540ndash2548 2010

[29] J Agudo E Ayuso V Jimenez et al ldquoVascular endothelialgrowth factor-mediated islet hypervascularization and inflam-mation contribute to progressive reduction of 120573-cell massrdquoDia-betes vol 61 no 11 pp 2851ndash2861 2012

[30] O Cleaver and Y Dor ldquoVascular instruction of pancreas devel-opmentrdquo Development vol 139 no 16 pp 2833ndash2843 2012

8 Journal of Diabetes Research

[31] M Brissova A Shostak M Shiota et al ldquoPancreatic islet pro-duction of vascular endothelial growth factor-A is essential forislet vascularization revascularization and functionrdquoDiabetesvol 55 no 11 pp 2974ndash2985 2006

[32] N Jabs L FranklinM B Brenner et al ldquoReduced insulin secre-tion and content in VEGF-A deficient mouse pancreatic isletsrdquoExperimental and Clinical Endocrinology and Diabetes vol 116no 1 pp S46ndashS49 2008

[33] M Brissova M Fowler P Wiebe et al ldquoIntraislet endothelialcells contribute to revascularization of transplanted pancreaticisletsrdquo Diabetes vol 53 no 5 pp 1318ndash1325 2004

[34] C Vickers P Hales V Kaushik et al ldquoHydrolysis of biologicalpeptides by human angiotensin-converting enzyme-related car-boxypeptidaserdquo Journal of Biological Chemistry vol 277 no 17pp 14838ndash14843 2002

[35] I Castan-laurell C Dray C Knauf O Kunduzova and P ValetldquoApelin a promising target for type 2 diabetes treatmentrdquoTrends in Endocrinology amp Metabolism vol 23 no 5 pp 234ndash241 2012

[36] M SWinzell CMagnusson and B Ahren ldquoThe apj receptor isexpressed in pancreatic islets and its ligand apelin inhibitsinsulin secretion in micerdquo Regulatory Peptides vol 131 no 1ndash3pp 12ndash17 2005

[37] L Guo Q Li W Wang et al ldquoApelin inhibits insulin secretionin pancreatic 120573-cells by activation of PI3-kinase-phosphodies-terase 3Brdquo Endocrine Research vol 34 no 4 pp 142ndash154 2009

Journal of Diabetes Research 3

Table 1 Body weight blood glucose blood insulin and islet function (mean plusmn SD)

WT WH KO KHBody weight (g) (beginning) 2260 plusmn 065 2273 plusmn 055 2248 plusmn 033 2256 plusmn 033Body weight (g) (ending) 2905 plusmn 083 3424 plusmn 145lowast 2921 plusmn 069 3441 plusmn 069lowast

Fasting blood glucose (mmolL) 818 plusmn 072 838 plusmn 042 860 plusmn 046 863 plusmn 055Fasting blood insulin (ngmL) 084 plusmn 002 097 plusmn 002lowast 087 plusmn 001 085 plusmn 003

AUCI0-5 ((ngmL) lowastmin) 965 plusmn 013 931 plusmn 006lowast 958 plusmn 015 740 plusmn 015lowast

AUCI0-30 ((ngmL) lowastmin) 4382 plusmn 021 6161 plusmn 056lowast 4431 plusmn 031 4493 plusmn 071

WT wild type mice WH wild type mice with high-fat diet KO ACE2 knockout mice KH ACE2 knockout mice with high-fat diet versus WT lowast119875 lt 005versus KO

119875 lt 005 and versus WH 119875 lt 005

0

5

10

15

20

25

30

35

0 2 4 6 8 10 12 14 16Time (week)

WT WH

KOKH

Body

wei

ght (

g)

Figure 1 The curve of body weight WT wildtype mice WHwildtype mice with high-fat diet KO ACE2 knockout mice KHACE2 KO mice with high-fat diet

IPGTT blood glucose level in WT group peaked at approx-imately 229mmolL and approached baseline levels by120min Similar glucose profiles were observed in KO inresponse to glucose challenge The values of 30 and 60minglucose of WH group were significantly higher than those inWT group (both 119875 lt 005) However blood glucose concen-tration in KH group peaked at approximately 309mmolLand remained elevated We took the area under the IPGTTcurve (AUCG) to quantify glucose tolerance There was nosignificant difference of AUCG between WT mice and KOmiceThe AUCG inWHmice was significantly elevated thanthat in WT mice (119875 lt 005) The AUCG in KH groups wasobviously increased than that in KO (119875 lt 005) Interestinglywe observed increased AUCG in KH mice compared withWH mice (119875 lt 005) These data indicated that deletion ofACE2 leads to more severe glucose intolerance induced byhigh-fat diet (Figure 2 Table 1)

The fating blood insulin between WT group and KOgroup was not different However the fasting blood insulinlevels in WH group were significantly higher than those inWT and KH groups (both 119875 lt 005) In the IPIRT insulinpeak climbed rapidly to about 4-fold as basic level in 2minin WT group after glucose challenge The levels of insulinrelease in the KO group stimulated by glucose infusion were

almost the same as those in the WT group The AUCI0minus30

and AUCI0minus5

were not different between WT and KO groupCompared with WT group the insulin secretion of WHgroup increased slowly with a delayed and increased peakWe observed a decreased AUCI

0minus5and increased AUCI

0minus30

in WH group compared with those in WT group (both119875 lt 005) indicating the impairment of first stage insulinreleasing and a compensatory enhancement of insulin releaseunder the condition of high-fat diet The AUCI

0minus30in KH

group was not increased when compared with that in KOgroup In addition the insulin secretion in KH and WHgroup climbed to the peak at the same time whereas theformer exhibited a lower peak releasing after glucose loadThis finding suggested that ACE2 deficiency inhibits thecompensatory enhancement of insulin secretion in responseto the increased metabolic demand (Figure 3 Table 1)

33 Insulin Tolerance Test In IPITT blood glucose level inWT group dropped obviously and approached bottom levelsby 30min Similar glucose disappearance rate was observedin KO mice After an intraperitoneal injection of insulin theglucose disappearance rate of 15 and 30min was higher inWH and KH mice than those in corresponding mice withchow diet (both 119875 lt 005) There was no difference ofthe glucose disappearance rate between KH and WH mice(Figure 4)

34 IRC and ICD of Islets Islets in WT group were regularwith high expression of insulin There was no significantdifference of IRC and ICD between WT and KO groups Theislet area in the pancreases of WH group was larger than thatin WT group and the IRC dropped obviously (119875 lt 005)indicating the reduction of insulin reserve in WH group Incomparison with KO mice KH mice exhibited a decreasedIRC (119875 lt 005) Moreover a decreased IRC was noted in KHgroup in comparison with WH group (119875 lt 005) implyingmore severe impairment of insulin reserve in KH group TheICD in the WH group was lower than that in the WT groupbut the difference was not significant No difference of ICDwas observed between KH group and KO group There wasno difference of ICD between KH group and WH group(Figure 5 Table 2)

35 MVD in Islets The MVD of islet was slightly decreasedin KOmice than that in WTmice but the difference was notsignificant Staining of the pancreas using anti-CD31 antibody

4 Journal of Diabetes Research

0

5

10

15

20

25

30

35

0 30 60 90 120Time (min)

Plas

ma g

luco

se (m

mol

L)

WT

WH

KO

KH

(a)

0

500

1000

1500

2000

2500

3000

WT WH KO KH

lowast

lowast

AUCG

(minlowast

(mm

olL

))

(b)

Figure 2The curve of glucose tolerance test (a) Glucose curve (b) area under curve of glucose (AUCG) WT wildtype mice WH wildtypemice with high-fat diet KO ACE2 knockout mice KH ACE2 KO mice with high-fat diet versus WT lowast119875 lt 005 versus KO

119875 lt 005 andversus WH 998779119875 lt 005

0

05

1

15

2

25

3

0 5 10 15 20 25 30Time (min)

Plas

ma i

nsul

in (n

gm

L)

WT

WHKO

KH

Figure 3 The curve of insulin releasing test WT wildtype miceWH wildtype mice with high-fat diet KO ACE2 knockout miceKH ACE2 KO mice with high-fat diet

demonstrated enhancedMVD inWH group thanWT group(119875 lt 005) which suggested higher islet vascularizationafter high-fat diet However the KH mice did not showan increased MVD in islet when compared with KO miceMoreover there was reduced MVD in islet from KH groupwhen compared with WH group (119875 lt 005) These findingsindicated that loss of ACE2 decreases the compensation ofislet vascularization in response to high-fat diet (Figure 5Table 2)

36 VRC Intraislets The VRC was similar between the WTand KO group In comparison with WT group VRC of WH

0

02

04

06

08

1

12

0 20 40 60 80 100Time (min)

Glu

cose

( in

itial

)

WT

WH

KO

KH

Figure 4 The curve of insulin tolerance test WT wildtype miceWH wildtype mice with high-fat diet KO ACE2 knockout miceKH ACE2 KO mice with high-fat diet

group was increased (119875 lt 005) However there was nodifference of VRC between KO group and KH group TheVRC in KH group was decreased when compared with WHgroup (119875 lt 005) (Figure 5 Table 2)

4 Discussion

The RAS has an important role in the endocrine pancreas[7] Many projects have focused on the effects of ACE-AngII-AT1 axis A homolog of ACE ACE2 cleaves the terminalphenylalanine residue from Ang II to synthesis Ang (1-7)thereby functioning as a negative regulator of the RAS In this

Journal of Diabetes Research 5

Table 2 Data of immunohistochemistry (mean plusmn SD)

WT WH KO KHInsulin relative concentration minus400 plusmn 005 minus463 plusmn 006lowast minus410 plusmn 004 minus507 plusmn 006lowast

Insulin-positive cell density 235 plusmn 002 230 plusmn 001 232 plusmn 002 228 plusmn 001Microvascular density 1352 plusmn 048 1709 plusmn 052lowast 1187 plusmn 045 789 plusmn 046lowast

Relative content of VEGF minus467 plusmn 011 minus399 plusmn 005lowast minus491 plusmn 004 minus511 plusmn 004lowast

WTwild typemiceWHwild typemice with high-fat diet KO ACE2 knockoutmice KH ACE2 knockoutmice with high-fat diet VEGF vascular endothelialgrowth factor versus WT lowast119875 lt 005 versus KO

119875 lt 005 and versus WH 119875 lt 005

WT WH KO KH

Insulin

CD31

VEGF

Figure 5 ImmunohistochemistryWT wildtypemiceWH wildtypemice with high-fat diet KO ACE2 knockout mice KH ACE2 KOmicewith high-fat diet VEGF vascular endothelial growth factor

current study we demonstrated that high-fat-diet-inducedACE2 KO mice exhibit a progressive impairment of glucosetolerance and a reduction of insulin secretion in response toglucose compared with WT mice In the high-fat-inducedACE2 KO mice loss of ACE2 triggered a greater decreasein islet 120573 cell and islet MVD Our study supported a pivotalrole of ACE2 in the regulation of 120573 cell function and isletvasculature

As the major bioactive component of the classical RASAng II mainly interacts with the AT1 receptor to exertvarious actions including vasoconstriction activation of pro-inflammation fibrosis and oxidative stress ACE2 is a mon-ocarboxypeptidase cleaving Ang II to form Ang (1-7) As aclass of G-protein-coupled receptorMas have been identifiedas a receptor of Ang (1-7) Ang (1-7) interacting with Mas

elicits protective actions such as vasodilatation and nitricoxide generation that counterbalance those of Ang II [6]TheACE2-Ang (1-7)-Mas axis may act as a negative modulatorof the classical RAS effects [24] There is emerging evidencefor a functional ACE2-Ang (1-7)-Mas axis which is more thanregulation of cardiovascular system [25]

The association of the classic RAS with the endocrinesystem has been identified in diabetes and metabolic syn-drome [26] A large number of clinical trials have indicatedthat inhibitors of the RAS such as ACEI and ARB reducethe incidence of new-onset T2DM in high-risk individualsand protect against the development of T2DM [19 20]Furthermore inhibition of RAS has been also reported toexert protective action to glucose homeostasis in animalmodels of T2DM [27] On the other hand ACE2 may be

6 Journal of Diabetes Research

a potential protective enzyme for glucose homeostasis [9]but the role of ACE2 in the regulation of islet function isincompletely understood In the present study there were ondifferences in fasting blood glucose and glucose tolerance inmale ACE2 KOmice with standard chow diet compared withcorresponding WT mice This observation was consistentwith a previous study by Wong et al [22] who found thatglycemic control was not worsened by the absence of ACE2in the Akita diabetic miceTherefore we supposed that ACE2ablation does not seem to be sufficient to alter glucose statusin the condition of regular chow diet However an interestingand important finding of the current study was that theconsequence of ACE2 absence became apparent after high-fat diet Under the condition of high-fat diet there weremuchmore reductions of the first-stage insulin secretion andglucose tolerance in KH mice compared with WHmice Notsurprisingly these changes were linked to decreased 120573 cellmass It is noteworthy that this finding was partly supportedby the report of gene therapy in obese diabetic mice Bindomet al [28] found that ACE2 gene therapy was able to improvefasting blood glucose and glucose tolerance in obese diabeticmice Consistent with the improvement of glycemic controlACE2 gene therapy leaded to enhanced first-stage insulinsecretion and reduced 120573 cell apoptosis Our current studyfor the first time provided evidence that ACE2 absence leadsto more severe effects in 120573 cell dysfunction in obese micewith high-fat diet Taken together it is hypothesized that lossof ACE2 renders the pancreatic islet more susceptible to thepathological actions of obesity induced by high-fat diet

It is known that islet vascularization is essential for theislet to respond to fluctuations in blood glucose andmodulateinsulin release as needed Defects in the vascularization ofpancreatic islets can lead to deleterious effects on glucose tol-erance and islet insulin secretion In our study an increasedislet vascularization in high-fat diet WT mice was observedcompared with chow diet WT mice which represented acompensatory response to support higher vascularizationduring 120573 cell expansion In contrast no increased isletvascularization was observed in KH mice compared withKO mice Furthermore KH mice exhibited defects of isletvascularization compared with WH mice These findingssuggested that deletion of ACE2 reduces the compensationof islet vascularization in response to high-fat diet Thereis an intimate connection between islet beta cell and isletvascularization On the one hand islet vascularization hasbeen regarded as playing a crucial role in maintaining betacell survival and function On the other hand beta cellssecrete several factors such as VEGF to promote vasculardevelopment VEGF which is highly expressed in islet hasbeen identified as an important factor to be involved in theregulation of islet vascularization [23 29] Vascular endothe-lial cells migrate to the source of VEGF-A and proliferateand form blood vessels in response to VEGF [30] Previousstudies [31 32] suggested that mice with specific deletionof the VEGF in 120573 cells had reduced islet vascularizationand impaired glucose tolerance These findings were in linewith observations in the transplanted pancreatic islets It hasbeen reported that reduction of VEGF-A by 120573 cells not only

reduces the number of intraislet endothelial cells participat-ing in graft vessel formation but also limits recruitment ofhost endothelial cell and their invasion into the graft [31 33]In our study we found an increase of islet VEGF in WHmice which was consistent with the enhancement of isletvascularization after high-fat diet However with the deletionof ACE2 this compensatory increase of VEGF in islet wasdisappeared after high-fat diet This was parallel to the islet120573 cell impairment and islet vascularization reduction Withthe defects of the islet vascularization the beta cell wasimpairedThe deteriorated beta cell might lead to a reductionof VEGF expression which in turn accelerates the loss ofvascularization Taken together we speculated that underthe condition of high-fat diet ACE2 might play a criticalrole in the glucose intolerance through impairment of isletvascularization

ACE2 not only functions as a carboxypeptidase cleavinga single residue from Ang I generating Ang1-9 and asingle residue from Ang II to generate Ang 1-7 but alsocleaves several other biological peptides such as apelinACE2 hydrolyzes apelin with high catalytic efficiency [34]Apelin is the endogenous ligand of the G-protein coupledapj receptor The effects of apelin on the pancreas focusedon the regulation of insulin secretion [35] Winzell et al[36] reported that apelin-36 inhibited the glucose-stimulatedinsulin secretion in obese insulin resistant high-fat fed miceMoreover apelin-13 was shown to inhibit insulin secretionstimulated by high glucose concentrations in INS-1 cells [37]Accordingly we hypothesized that with the deletion of ACE2the hydrolyzation of apelin is inhibited which leads to theinhibition of insulin secretion

Peripheral Insulin sensitivity has long been regarded asplaying a crucial role in glucose homeostasis However theeffect of ACE2 on insulin sensitivity is unclear In this studywe took insulin tolerance test to evaluate peripheral insulinsensitivity We found no significant difference of peripheralinsulin sensitivity between KO mice and WT mice Theperipheral insulin sensitivity betweenWHmice andKHmicewas similar too So we concluded that loss of ACE2 hasno effects on peripheral insulin sensitivity More accuratemeasures such as hyperinsulinemic euglycemic clamp shouldbe needed in further studies It should be noted that therewas no difference of the body weight betweenWT and ACE2KO mice neither with chow diet nor with high-fat dietAccordingly we speculated that ACE2was not involved in theregulation of body weight

In summary the results obtained in this study show thatloss of ACE2 in mice with long-term high-fat diet leadsto impairment of glucose tolerance and insulin secretionThe primary mechanisms involved in these effects appear toinclude a reduction in islet 120573 cell and islet vascularizationOur data suggested that the absence of ACE2 weakened theadaptive changes of islet 120573 cell function and islet vasculariza-tion due to high-fat diet ACE2 seems to be a potential thera-peutic target in the treatment of islet dysfunction induced byhigh-fat diet

Journal of Diabetes Research 7

Acknowledgment

This project was supported by the National Natural ScienceFoundation of China (no 81070615)

References

[1] J R A Skipworth G Szabadkai S W M Olde Damink PS Leung S E Humphries and H E Montgomery ldquoReviewarticle pancreatic renin-angiotensin systems in health anddiseaserdquo Alimentary Pharmacology amp Therapeutics vol 34 no8 pp 840ndash852 2011

[2] S R Tipnis N M Hooper R Hyde E Karran G Christieand A J Turner ldquoA human homolog of angiotensin-convertingenzyme cloning and functional expression as a captopril-insensitive carboxypeptidaserdquo Journal of Biological Chemistryvol 275 no 43 pp 33238ndash33243 2000

[3] M Donoghue F Hsieh E Baronas et al ldquoA novel angiotensin-converting enzyme-related carboxypeptidase (ACE2) convertsangiotensin I to angiotensin 1-9rdquo Circulation Research vol 87no 5 pp e1ndashe9 2000

[4] G I Rice D A Thomas P J Grant A J Turner and N MHooper ldquoEvaluation of angiotensin-converting enzyme (ACE)its homologue ACE2 and neprilysin in angiotensin peptide me-tabolismrdquo Biochemical Journal vol 383 no 1 pp 45ndash51 2004

[5] C M Ferrario ldquoACE2 more of Ang-(1ndash7) or less Ang IIrdquo Cur-rent Opinion in Nephrology and Hypertension vol 20 no 1 pp1ndash6 2011

[6] R A S Santos A J Ferreira and A C Simoes E Silva ldquoRecentadvances in the angiotensin-converting enzyme 2-angiotens-in(1ndash7)-Mas axisrdquo Experimental Physiology vol 93 no 5 pp519ndash527 2008

[7] Q Cheng and P S Leung ldquoAn update on the islet renin-angiot-ensin systemrdquo Peptides vol 32 no 5 pp 1087ndash1095 2011

[8] P S Leung ldquoThe physiology of a local renin-angiotensin systemin the pancreasrdquo Journal of Physiology vol 580 no 1 pp 31ndash372007

[9] D Batlle M J Soler and M Ye ldquoACE2 and diabetes ACE ofACEsrdquo Diabetes vol 59 no 12 pp 2994ndash2996 2010

[10] M C Chappell A Millsted D I Diz K B Brosnihan and CM Ferrario ldquoEvidence for an intrinsic angiotensin system inthe canine pancreasrdquo Journal of Hypertension vol 9 no 8 pp751ndash759 1991

[11] T Lau P O Carlsson and P S Leung ldquoEvidence for a localangiotensin-generating system and dose-dependent inhibitionof glucose-stimulated insulin release by angiotensin II in iso-lated pancreatic isletsrdquoDiabetologia vol 47 no 2 pp 240ndash2482004

[12] K Y Lam and P S Leung ldquoRegulation and expression of arenin-angiotensin system in human pancreas and pancreaticendocrine tumoursrdquo European Journal of Endocrinology vol146 no 4 pp 567ndash572 2002

[13] C Tikellis M E Cooper andM CThomas ldquoRole of the renin-angiotensin system in the endocrine pancreas implications forthe development of diabetesrdquo International Journal of Biochem-istry and Cell Biology vol 38 no 5-6 pp 737ndash751 2006

[14] S M Bindom and E Lazartigues ldquoThe sweeter side of ACE2physiological evidence for a role in diabetesrdquo Molecular andCellular Endocrinology vol 302 no 2 pp 193ndash202 2009

[15] P S Leung and P O Carlsson ldquoPancreatic islet renin angiot-ensin system its novel roles in islet function and in diabetesmellitusrdquo Pancreas vol 30 no 4 pp 293ndash298 2005

[16] P S Leung ldquoMechanisms of protective effects induced by block-ade of the renin-angiotensin system novel role of the pancreaticislet angiotensin-generating system inType 2 diabetesrdquoDiabeticMedicine vol 24 no 2 pp 110ndash116 2007

[17] C Tikellis P J Wookey R Candido S Andrikopoulos M CThomas and M E Cooper ldquoImproved islet morphology afterblockade of the renin-angiotensin system in the ZDF ratrdquo Dia-betes vol 53 no 4 pp 989ndash997 2004

[18] L Yuan X Li J Li H l Li and S S Cheng ldquoEffects of renin-angiotensin system blockade on the islet morphology and func-tion in rats with long-term high-fat dietrdquo Acta Diabetologica Inpress

[19] A J Scheen ldquoRenin-angiotensin system inhibition preventstype 2 diabetes mellitusmdashpart 1 a meta-analysis of randomisedclinical trialsrdquo Diabetes and Metabolism vol 30 no 6 pp 487ndash496 2004

[20] E L Gillespie C M White M Kardas M Lindberg and C IColeman ldquoThe impact of ACE inhibitors or angiotensin II type1 receptor blockers on the development of new-onset type 2diabetesrdquo Diabetes Care vol 28 no 9 pp 2261ndash2266 2005

[21] P Vejakama A Thakkinstian D Lertrattananon A IngsathitC Ngarmukos and J Attia ldquoReno-protective effects of renin-angiotensin system blockade in type 2 diabetic patients a sys-tematic review and network meta-analysisrdquo Diabetologia vol55 no 3 pp 566ndash578 2011

[22] D W Wong G Y Oudit H Reich et al ldquoLoss of Angiotensin-converting enzyme-2 (Ace2) accelerates diabetic kidney injuryrdquoAmerican Journal of Pathology vol 171 no 2 pp 438ndash451 2007

[23] E M Akirav M T Baquero L W Opare-Addo et al ldquoGlucoseand inflammation control islet vascular density and 120573-cell func-tion in NOD mice control of islet vasculature and vascularendothelial growth factor by glucoserdquo Diabetes vol 60 no 3pp 876ndash883 2011

[24] M Iwai and M Horiuchi ldquoDevil and angel in the renin-angiot-ensin system ACE-angiotensin II-AT1 receptor axis vs ACE2-angiotensin-(1ndash7)-Mas receptor axisrdquo Hypertension Researchvol 32 no 7 pp 533ndash536 2009

[25] M C Chappell ldquoEmerging evidence for a functional angiotens-in-converting enzyme 2-angiotensin-(1ndash7)-Mas receptor axismore than regulation of blood pressurerdquoHypertension vol 50no 4 pp 596ndash599 2007

[26] K Putnam R Shoemaker F Yiannikouris and L Cassis ldquoTherenin-angiotensin system a target of and contributor to dyslip-idemias altered glucose homeostasis and hypertension of themetabolic syndromerdquo American Journal of Physiology vol 15no 302 pp H1219ndashH1230 2012

[27] V Souza-Mello B M Gregorio F S Cardoso-De-Lemos LDe Carvalho M B Aguila and C A Mandarim-De-LacerdaldquoComparative effects of telmisartan sitagliptin and metforminalone or in combination on obesity insulin resistance and liverand pancreas remodelling in C57BL6 mice fed on a very high-fat dietrdquo Clinical Science vol 119 no 6 pp 239ndash250 2010

[28] S M Bindom C P Hans H Xia A H Boulares and E Lazar-tigues ldquoAngiotensin I-converting enzyme type 2 (ACE2) genetherapy improves glycemic control in diabetic micerdquo Diabetesvol 59 no 10 pp 2540ndash2548 2010

[29] J Agudo E Ayuso V Jimenez et al ldquoVascular endothelialgrowth factor-mediated islet hypervascularization and inflam-mation contribute to progressive reduction of 120573-cell massrdquoDia-betes vol 61 no 11 pp 2851ndash2861 2012

[30] O Cleaver and Y Dor ldquoVascular instruction of pancreas devel-opmentrdquo Development vol 139 no 16 pp 2833ndash2843 2012

8 Journal of Diabetes Research

[31] M Brissova A Shostak M Shiota et al ldquoPancreatic islet pro-duction of vascular endothelial growth factor-A is essential forislet vascularization revascularization and functionrdquoDiabetesvol 55 no 11 pp 2974ndash2985 2006

[32] N Jabs L FranklinM B Brenner et al ldquoReduced insulin secre-tion and content in VEGF-A deficient mouse pancreatic isletsrdquoExperimental and Clinical Endocrinology and Diabetes vol 116no 1 pp S46ndashS49 2008

[33] M Brissova M Fowler P Wiebe et al ldquoIntraislet endothelialcells contribute to revascularization of transplanted pancreaticisletsrdquo Diabetes vol 53 no 5 pp 1318ndash1325 2004

[34] C Vickers P Hales V Kaushik et al ldquoHydrolysis of biologicalpeptides by human angiotensin-converting enzyme-related car-boxypeptidaserdquo Journal of Biological Chemistry vol 277 no 17pp 14838ndash14843 2002

[35] I Castan-laurell C Dray C Knauf O Kunduzova and P ValetldquoApelin a promising target for type 2 diabetes treatmentrdquoTrends in Endocrinology amp Metabolism vol 23 no 5 pp 234ndash241 2012

[36] M SWinzell CMagnusson and B Ahren ldquoThe apj receptor isexpressed in pancreatic islets and its ligand apelin inhibitsinsulin secretion in micerdquo Regulatory Peptides vol 131 no 1ndash3pp 12ndash17 2005

[37] L Guo Q Li W Wang et al ldquoApelin inhibits insulin secretionin pancreatic 120573-cells by activation of PI3-kinase-phosphodies-terase 3Brdquo Endocrine Research vol 34 no 4 pp 142ndash154 2009

4 Journal of Diabetes Research

0

5

10

15

20

25

30

35

0 30 60 90 120Time (min)

Plas

ma g

luco

se (m

mol

L)

WT

WH

KO

KH

(a)

0

500

1000

1500

2000

2500

3000

WT WH KO KH

lowast

lowast

AUCG

(minlowast

(mm

olL

))

(b)

Figure 2The curve of glucose tolerance test (a) Glucose curve (b) area under curve of glucose (AUCG) WT wildtype mice WH wildtypemice with high-fat diet KO ACE2 knockout mice KH ACE2 KO mice with high-fat diet versus WT lowast119875 lt 005 versus KO

119875 lt 005 andversus WH 998779119875 lt 005

0

05

1

15

2

25

3

0 5 10 15 20 25 30Time (min)

Plas

ma i

nsul

in (n

gm

L)

WT

WHKO

KH

Figure 3 The curve of insulin releasing test WT wildtype miceWH wildtype mice with high-fat diet KO ACE2 knockout miceKH ACE2 KO mice with high-fat diet

demonstrated enhancedMVD inWH group thanWT group(119875 lt 005) which suggested higher islet vascularizationafter high-fat diet However the KH mice did not showan increased MVD in islet when compared with KO miceMoreover there was reduced MVD in islet from KH groupwhen compared with WH group (119875 lt 005) These findingsindicated that loss of ACE2 decreases the compensation ofislet vascularization in response to high-fat diet (Figure 5Table 2)

36 VRC Intraislets The VRC was similar between the WTand KO group In comparison with WT group VRC of WH

0

02

04

06

08

1

12

0 20 40 60 80 100Time (min)

Glu

cose

( in

itial

)

WT

WH

KO

KH

Figure 4 The curve of insulin tolerance test WT wildtype miceWH wildtype mice with high-fat diet KO ACE2 knockout miceKH ACE2 KO mice with high-fat diet

group was increased (119875 lt 005) However there was nodifference of VRC between KO group and KH group TheVRC in KH group was decreased when compared with WHgroup (119875 lt 005) (Figure 5 Table 2)

4 Discussion

The RAS has an important role in the endocrine pancreas[7] Many projects have focused on the effects of ACE-AngII-AT1 axis A homolog of ACE ACE2 cleaves the terminalphenylalanine residue from Ang II to synthesis Ang (1-7)thereby functioning as a negative regulator of the RAS In this

Journal of Diabetes Research 5

Table 2 Data of immunohistochemistry (mean plusmn SD)

WT WH KO KHInsulin relative concentration minus400 plusmn 005 minus463 plusmn 006lowast minus410 plusmn 004 minus507 plusmn 006lowast

Insulin-positive cell density 235 plusmn 002 230 plusmn 001 232 plusmn 002 228 plusmn 001Microvascular density 1352 plusmn 048 1709 plusmn 052lowast 1187 plusmn 045 789 plusmn 046lowast

Relative content of VEGF minus467 plusmn 011 minus399 plusmn 005lowast minus491 plusmn 004 minus511 plusmn 004lowast

WTwild typemiceWHwild typemice with high-fat diet KO ACE2 knockoutmice KH ACE2 knockoutmice with high-fat diet VEGF vascular endothelialgrowth factor versus WT lowast119875 lt 005 versus KO

119875 lt 005 and versus WH 119875 lt 005

WT WH KO KH

Insulin

CD31

VEGF

Figure 5 ImmunohistochemistryWT wildtypemiceWH wildtypemice with high-fat diet KO ACE2 knockout mice KH ACE2 KOmicewith high-fat diet VEGF vascular endothelial growth factor

current study we demonstrated that high-fat-diet-inducedACE2 KO mice exhibit a progressive impairment of glucosetolerance and a reduction of insulin secretion in response toglucose compared with WT mice In the high-fat-inducedACE2 KO mice loss of ACE2 triggered a greater decreasein islet 120573 cell and islet MVD Our study supported a pivotalrole of ACE2 in the regulation of 120573 cell function and isletvasculature

As the major bioactive component of the classical RASAng II mainly interacts with the AT1 receptor to exertvarious actions including vasoconstriction activation of pro-inflammation fibrosis and oxidative stress ACE2 is a mon-ocarboxypeptidase cleaving Ang II to form Ang (1-7) As aclass of G-protein-coupled receptorMas have been identifiedas a receptor of Ang (1-7) Ang (1-7) interacting with Mas

elicits protective actions such as vasodilatation and nitricoxide generation that counterbalance those of Ang II [6]TheACE2-Ang (1-7)-Mas axis may act as a negative modulatorof the classical RAS effects [24] There is emerging evidencefor a functional ACE2-Ang (1-7)-Mas axis which is more thanregulation of cardiovascular system [25]

The association of the classic RAS with the endocrinesystem has been identified in diabetes and metabolic syn-drome [26] A large number of clinical trials have indicatedthat inhibitors of the RAS such as ACEI and ARB reducethe incidence of new-onset T2DM in high-risk individualsand protect against the development of T2DM [19 20]Furthermore inhibition of RAS has been also reported toexert protective action to glucose homeostasis in animalmodels of T2DM [27] On the other hand ACE2 may be

6 Journal of Diabetes Research

a potential protective enzyme for glucose homeostasis [9]but the role of ACE2 in the regulation of islet function isincompletely understood In the present study there were ondifferences in fasting blood glucose and glucose tolerance inmale ACE2 KOmice with standard chow diet compared withcorresponding WT mice This observation was consistentwith a previous study by Wong et al [22] who found thatglycemic control was not worsened by the absence of ACE2in the Akita diabetic miceTherefore we supposed that ACE2ablation does not seem to be sufficient to alter glucose statusin the condition of regular chow diet However an interestingand important finding of the current study was that theconsequence of ACE2 absence became apparent after high-fat diet Under the condition of high-fat diet there weremuchmore reductions of the first-stage insulin secretion andglucose tolerance in KH mice compared with WHmice Notsurprisingly these changes were linked to decreased 120573 cellmass It is noteworthy that this finding was partly supportedby the report of gene therapy in obese diabetic mice Bindomet al [28] found that ACE2 gene therapy was able to improvefasting blood glucose and glucose tolerance in obese diabeticmice Consistent with the improvement of glycemic controlACE2 gene therapy leaded to enhanced first-stage insulinsecretion and reduced 120573 cell apoptosis Our current studyfor the first time provided evidence that ACE2 absence leadsto more severe effects in 120573 cell dysfunction in obese micewith high-fat diet Taken together it is hypothesized that lossof ACE2 renders the pancreatic islet more susceptible to thepathological actions of obesity induced by high-fat diet

It is known that islet vascularization is essential for theislet to respond to fluctuations in blood glucose andmodulateinsulin release as needed Defects in the vascularization ofpancreatic islets can lead to deleterious effects on glucose tol-erance and islet insulin secretion In our study an increasedislet vascularization in high-fat diet WT mice was observedcompared with chow diet WT mice which represented acompensatory response to support higher vascularizationduring 120573 cell expansion In contrast no increased isletvascularization was observed in KH mice compared withKO mice Furthermore KH mice exhibited defects of isletvascularization compared with WH mice These findingssuggested that deletion of ACE2 reduces the compensationof islet vascularization in response to high-fat diet Thereis an intimate connection between islet beta cell and isletvascularization On the one hand islet vascularization hasbeen regarded as playing a crucial role in maintaining betacell survival and function On the other hand beta cellssecrete several factors such as VEGF to promote vasculardevelopment VEGF which is highly expressed in islet hasbeen identified as an important factor to be involved in theregulation of islet vascularization [23 29] Vascular endothe-lial cells migrate to the source of VEGF-A and proliferateand form blood vessels in response to VEGF [30] Previousstudies [31 32] suggested that mice with specific deletionof the VEGF in 120573 cells had reduced islet vascularizationand impaired glucose tolerance These findings were in linewith observations in the transplanted pancreatic islets It hasbeen reported that reduction of VEGF-A by 120573 cells not only

reduces the number of intraislet endothelial cells participat-ing in graft vessel formation but also limits recruitment ofhost endothelial cell and their invasion into the graft [31 33]In our study we found an increase of islet VEGF in WHmice which was consistent with the enhancement of isletvascularization after high-fat diet However with the deletionof ACE2 this compensatory increase of VEGF in islet wasdisappeared after high-fat diet This was parallel to the islet120573 cell impairment and islet vascularization reduction Withthe defects of the islet vascularization the beta cell wasimpairedThe deteriorated beta cell might lead to a reductionof VEGF expression which in turn accelerates the loss ofvascularization Taken together we speculated that underthe condition of high-fat diet ACE2 might play a criticalrole in the glucose intolerance through impairment of isletvascularization

ACE2 not only functions as a carboxypeptidase cleavinga single residue from Ang I generating Ang1-9 and asingle residue from Ang II to generate Ang 1-7 but alsocleaves several other biological peptides such as apelinACE2 hydrolyzes apelin with high catalytic efficiency [34]Apelin is the endogenous ligand of the G-protein coupledapj receptor The effects of apelin on the pancreas focusedon the regulation of insulin secretion [35] Winzell et al[36] reported that apelin-36 inhibited the glucose-stimulatedinsulin secretion in obese insulin resistant high-fat fed miceMoreover apelin-13 was shown to inhibit insulin secretionstimulated by high glucose concentrations in INS-1 cells [37]Accordingly we hypothesized that with the deletion of ACE2the hydrolyzation of apelin is inhibited which leads to theinhibition of insulin secretion

Peripheral Insulin sensitivity has long been regarded asplaying a crucial role in glucose homeostasis However theeffect of ACE2 on insulin sensitivity is unclear In this studywe took insulin tolerance test to evaluate peripheral insulinsensitivity We found no significant difference of peripheralinsulin sensitivity between KO mice and WT mice Theperipheral insulin sensitivity betweenWHmice andKHmicewas similar too So we concluded that loss of ACE2 hasno effects on peripheral insulin sensitivity More accuratemeasures such as hyperinsulinemic euglycemic clamp shouldbe needed in further studies It should be noted that therewas no difference of the body weight betweenWT and ACE2KO mice neither with chow diet nor with high-fat dietAccordingly we speculated that ACE2was not involved in theregulation of body weight

In summary the results obtained in this study show thatloss of ACE2 in mice with long-term high-fat diet leadsto impairment of glucose tolerance and insulin secretionThe primary mechanisms involved in these effects appear toinclude a reduction in islet 120573 cell and islet vascularizationOur data suggested that the absence of ACE2 weakened theadaptive changes of islet 120573 cell function and islet vasculariza-tion due to high-fat diet ACE2 seems to be a potential thera-peutic target in the treatment of islet dysfunction induced byhigh-fat diet

Journal of Diabetes Research 7

Acknowledgment

This project was supported by the National Natural ScienceFoundation of China (no 81070615)

References

[1] J R A Skipworth G Szabadkai S W M Olde Damink PS Leung S E Humphries and H E Montgomery ldquoReviewarticle pancreatic renin-angiotensin systems in health anddiseaserdquo Alimentary Pharmacology amp Therapeutics vol 34 no8 pp 840ndash852 2011

[2] S R Tipnis N M Hooper R Hyde E Karran G Christieand A J Turner ldquoA human homolog of angiotensin-convertingenzyme cloning and functional expression as a captopril-insensitive carboxypeptidaserdquo Journal of Biological Chemistryvol 275 no 43 pp 33238ndash33243 2000

[3] M Donoghue F Hsieh E Baronas et al ldquoA novel angiotensin-converting enzyme-related carboxypeptidase (ACE2) convertsangiotensin I to angiotensin 1-9rdquo Circulation Research vol 87no 5 pp e1ndashe9 2000

[4] G I Rice D A Thomas P J Grant A J Turner and N MHooper ldquoEvaluation of angiotensin-converting enzyme (ACE)its homologue ACE2 and neprilysin in angiotensin peptide me-tabolismrdquo Biochemical Journal vol 383 no 1 pp 45ndash51 2004

[5] C M Ferrario ldquoACE2 more of Ang-(1ndash7) or less Ang IIrdquo Cur-rent Opinion in Nephrology and Hypertension vol 20 no 1 pp1ndash6 2011

[6] R A S Santos A J Ferreira and A C Simoes E Silva ldquoRecentadvances in the angiotensin-converting enzyme 2-angiotens-in(1ndash7)-Mas axisrdquo Experimental Physiology vol 93 no 5 pp519ndash527 2008

[7] Q Cheng and P S Leung ldquoAn update on the islet renin-angiot-ensin systemrdquo Peptides vol 32 no 5 pp 1087ndash1095 2011

[8] P S Leung ldquoThe physiology of a local renin-angiotensin systemin the pancreasrdquo Journal of Physiology vol 580 no 1 pp 31ndash372007

[9] D Batlle M J Soler and M Ye ldquoACE2 and diabetes ACE ofACEsrdquo Diabetes vol 59 no 12 pp 2994ndash2996 2010

[10] M C Chappell A Millsted D I Diz K B Brosnihan and CM Ferrario ldquoEvidence for an intrinsic angiotensin system inthe canine pancreasrdquo Journal of Hypertension vol 9 no 8 pp751ndash759 1991

[11] T Lau P O Carlsson and P S Leung ldquoEvidence for a localangiotensin-generating system and dose-dependent inhibitionof glucose-stimulated insulin release by angiotensin II in iso-lated pancreatic isletsrdquoDiabetologia vol 47 no 2 pp 240ndash2482004

[12] K Y Lam and P S Leung ldquoRegulation and expression of arenin-angiotensin system in human pancreas and pancreaticendocrine tumoursrdquo European Journal of Endocrinology vol146 no 4 pp 567ndash572 2002

[13] C Tikellis M E Cooper andM CThomas ldquoRole of the renin-angiotensin system in the endocrine pancreas implications forthe development of diabetesrdquo International Journal of Biochem-istry and Cell Biology vol 38 no 5-6 pp 737ndash751 2006

[14] S M Bindom and E Lazartigues ldquoThe sweeter side of ACE2physiological evidence for a role in diabetesrdquo Molecular andCellular Endocrinology vol 302 no 2 pp 193ndash202 2009

[15] P S Leung and P O Carlsson ldquoPancreatic islet renin angiot-ensin system its novel roles in islet function and in diabetesmellitusrdquo Pancreas vol 30 no 4 pp 293ndash298 2005

[16] P S Leung ldquoMechanisms of protective effects induced by block-ade of the renin-angiotensin system novel role of the pancreaticislet angiotensin-generating system inType 2 diabetesrdquoDiabeticMedicine vol 24 no 2 pp 110ndash116 2007

[17] C Tikellis P J Wookey R Candido S Andrikopoulos M CThomas and M E Cooper ldquoImproved islet morphology afterblockade of the renin-angiotensin system in the ZDF ratrdquo Dia-betes vol 53 no 4 pp 989ndash997 2004

[18] L Yuan X Li J Li H l Li and S S Cheng ldquoEffects of renin-angiotensin system blockade on the islet morphology and func-tion in rats with long-term high-fat dietrdquo Acta Diabetologica Inpress

[19] A J Scheen ldquoRenin-angiotensin system inhibition preventstype 2 diabetes mellitusmdashpart 1 a meta-analysis of randomisedclinical trialsrdquo Diabetes and Metabolism vol 30 no 6 pp 487ndash496 2004

[20] E L Gillespie C M White M Kardas M Lindberg and C IColeman ldquoThe impact of ACE inhibitors or angiotensin II type1 receptor blockers on the development of new-onset type 2diabetesrdquo Diabetes Care vol 28 no 9 pp 2261ndash2266 2005

[21] P Vejakama A Thakkinstian D Lertrattananon A IngsathitC Ngarmukos and J Attia ldquoReno-protective effects of renin-angiotensin system blockade in type 2 diabetic patients a sys-tematic review and network meta-analysisrdquo Diabetologia vol55 no 3 pp 566ndash578 2011

[22] D W Wong G Y Oudit H Reich et al ldquoLoss of Angiotensin-converting enzyme-2 (Ace2) accelerates diabetic kidney injuryrdquoAmerican Journal of Pathology vol 171 no 2 pp 438ndash451 2007

[23] E M Akirav M T Baquero L W Opare-Addo et al ldquoGlucoseand inflammation control islet vascular density and 120573-cell func-tion in NOD mice control of islet vasculature and vascularendothelial growth factor by glucoserdquo Diabetes vol 60 no 3pp 876ndash883 2011

[24] M Iwai and M Horiuchi ldquoDevil and angel in the renin-angiot-ensin system ACE-angiotensin II-AT1 receptor axis vs ACE2-angiotensin-(1ndash7)-Mas receptor axisrdquo Hypertension Researchvol 32 no 7 pp 533ndash536 2009

[25] M C Chappell ldquoEmerging evidence for a functional angiotens-in-converting enzyme 2-angiotensin-(1ndash7)-Mas receptor axismore than regulation of blood pressurerdquoHypertension vol 50no 4 pp 596ndash599 2007

[26] K Putnam R Shoemaker F Yiannikouris and L Cassis ldquoTherenin-angiotensin system a target of and contributor to dyslip-idemias altered glucose homeostasis and hypertension of themetabolic syndromerdquo American Journal of Physiology vol 15no 302 pp H1219ndashH1230 2012

[27] V Souza-Mello B M Gregorio F S Cardoso-De-Lemos LDe Carvalho M B Aguila and C A Mandarim-De-LacerdaldquoComparative effects of telmisartan sitagliptin and metforminalone or in combination on obesity insulin resistance and liverand pancreas remodelling in C57BL6 mice fed on a very high-fat dietrdquo Clinical Science vol 119 no 6 pp 239ndash250 2010

[28] S M Bindom C P Hans H Xia A H Boulares and E Lazar-tigues ldquoAngiotensin I-converting enzyme type 2 (ACE2) genetherapy improves glycemic control in diabetic micerdquo Diabetesvol 59 no 10 pp 2540ndash2548 2010

[29] J Agudo E Ayuso V Jimenez et al ldquoVascular endothelialgrowth factor-mediated islet hypervascularization and inflam-mation contribute to progressive reduction of 120573-cell massrdquoDia-betes vol 61 no 11 pp 2851ndash2861 2012

[30] O Cleaver and Y Dor ldquoVascular instruction of pancreas devel-opmentrdquo Development vol 139 no 16 pp 2833ndash2843 2012

8 Journal of Diabetes Research

[31] M Brissova A Shostak M Shiota et al ldquoPancreatic islet pro-duction of vascular endothelial growth factor-A is essential forislet vascularization revascularization and functionrdquoDiabetesvol 55 no 11 pp 2974ndash2985 2006

[32] N Jabs L FranklinM B Brenner et al ldquoReduced insulin secre-tion and content in VEGF-A deficient mouse pancreatic isletsrdquoExperimental and Clinical Endocrinology and Diabetes vol 116no 1 pp S46ndashS49 2008

[33] M Brissova M Fowler P Wiebe et al ldquoIntraislet endothelialcells contribute to revascularization of transplanted pancreaticisletsrdquo Diabetes vol 53 no 5 pp 1318ndash1325 2004

[34] C Vickers P Hales V Kaushik et al ldquoHydrolysis of biologicalpeptides by human angiotensin-converting enzyme-related car-boxypeptidaserdquo Journal of Biological Chemistry vol 277 no 17pp 14838ndash14843 2002

[35] I Castan-laurell C Dray C Knauf O Kunduzova and P ValetldquoApelin a promising target for type 2 diabetes treatmentrdquoTrends in Endocrinology amp Metabolism vol 23 no 5 pp 234ndash241 2012

[36] M SWinzell CMagnusson and B Ahren ldquoThe apj receptor isexpressed in pancreatic islets and its ligand apelin inhibitsinsulin secretion in micerdquo Regulatory Peptides vol 131 no 1ndash3pp 12ndash17 2005

[37] L Guo Q Li W Wang et al ldquoApelin inhibits insulin secretionin pancreatic 120573-cells by activation of PI3-kinase-phosphodies-terase 3Brdquo Endocrine Research vol 34 no 4 pp 142ndash154 2009

Journal of Diabetes Research 5

Table 2 Data of immunohistochemistry (mean plusmn SD)

WT WH KO KHInsulin relative concentration minus400 plusmn 005 minus463 plusmn 006lowast minus410 plusmn 004 minus507 plusmn 006lowast

Insulin-positive cell density 235 plusmn 002 230 plusmn 001 232 plusmn 002 228 plusmn 001Microvascular density 1352 plusmn 048 1709 plusmn 052lowast 1187 plusmn 045 789 plusmn 046lowast

Relative content of VEGF minus467 plusmn 011 minus399 plusmn 005lowast minus491 plusmn 004 minus511 plusmn 004lowast

WTwild typemiceWHwild typemice with high-fat diet KO ACE2 knockoutmice KH ACE2 knockoutmice with high-fat diet VEGF vascular endothelialgrowth factor versus WT lowast119875 lt 005 versus KO

119875 lt 005 and versus WH 119875 lt 005

WT WH KO KH

Insulin

CD31

VEGF

Figure 5 ImmunohistochemistryWT wildtypemiceWH wildtypemice with high-fat diet KO ACE2 knockout mice KH ACE2 KOmicewith high-fat diet VEGF vascular endothelial growth factor

current study we demonstrated that high-fat-diet-inducedACE2 KO mice exhibit a progressive impairment of glucosetolerance and a reduction of insulin secretion in response toglucose compared with WT mice In the high-fat-inducedACE2 KO mice loss of ACE2 triggered a greater decreasein islet 120573 cell and islet MVD Our study supported a pivotalrole of ACE2 in the regulation of 120573 cell function and isletvasculature

As the major bioactive component of the classical RASAng II mainly interacts with the AT1 receptor to exertvarious actions including vasoconstriction activation of pro-inflammation fibrosis and oxidative stress ACE2 is a mon-ocarboxypeptidase cleaving Ang II to form Ang (1-7) As aclass of G-protein-coupled receptorMas have been identifiedas a receptor of Ang (1-7) Ang (1-7) interacting with Mas

elicits protective actions such as vasodilatation and nitricoxide generation that counterbalance those of Ang II [6]TheACE2-Ang (1-7)-Mas axis may act as a negative modulatorof the classical RAS effects [24] There is emerging evidencefor a functional ACE2-Ang (1-7)-Mas axis which is more thanregulation of cardiovascular system [25]

The association of the classic RAS with the endocrinesystem has been identified in diabetes and metabolic syn-drome [26] A large number of clinical trials have indicatedthat inhibitors of the RAS such as ACEI and ARB reducethe incidence of new-onset T2DM in high-risk individualsand protect against the development of T2DM [19 20]Furthermore inhibition of RAS has been also reported toexert protective action to glucose homeostasis in animalmodels of T2DM [27] On the other hand ACE2 may be

6 Journal of Diabetes Research

a potential protective enzyme for glucose homeostasis [9]but the role of ACE2 in the regulation of islet function isincompletely understood In the present study there were ondifferences in fasting blood glucose and glucose tolerance inmale ACE2 KOmice with standard chow diet compared withcorresponding WT mice This observation was consistentwith a previous study by Wong et al [22] who found thatglycemic control was not worsened by the absence of ACE2in the Akita diabetic miceTherefore we supposed that ACE2ablation does not seem to be sufficient to alter glucose statusin the condition of regular chow diet However an interestingand important finding of the current study was that theconsequence of ACE2 absence became apparent after high-fat diet Under the condition of high-fat diet there weremuchmore reductions of the first-stage insulin secretion andglucose tolerance in KH mice compared with WHmice Notsurprisingly these changes were linked to decreased 120573 cellmass It is noteworthy that this finding was partly supportedby the report of gene therapy in obese diabetic mice Bindomet al [28] found that ACE2 gene therapy was able to improvefasting blood glucose and glucose tolerance in obese diabeticmice Consistent with the improvement of glycemic controlACE2 gene therapy leaded to enhanced first-stage insulinsecretion and reduced 120573 cell apoptosis Our current studyfor the first time provided evidence that ACE2 absence leadsto more severe effects in 120573 cell dysfunction in obese micewith high-fat diet Taken together it is hypothesized that lossof ACE2 renders the pancreatic islet more susceptible to thepathological actions of obesity induced by high-fat diet

It is known that islet vascularization is essential for theislet to respond to fluctuations in blood glucose andmodulateinsulin release as needed Defects in the vascularization ofpancreatic islets can lead to deleterious effects on glucose tol-erance and islet insulin secretion In our study an increasedislet vascularization in high-fat diet WT mice was observedcompared with chow diet WT mice which represented acompensatory response to support higher vascularizationduring 120573 cell expansion In contrast no increased isletvascularization was observed in KH mice compared withKO mice Furthermore KH mice exhibited defects of isletvascularization compared with WH mice These findingssuggested that deletion of ACE2 reduces the compensationof islet vascularization in response to high-fat diet Thereis an intimate connection between islet beta cell and isletvascularization On the one hand islet vascularization hasbeen regarded as playing a crucial role in maintaining betacell survival and function On the other hand beta cellssecrete several factors such as VEGF to promote vasculardevelopment VEGF which is highly expressed in islet hasbeen identified as an important factor to be involved in theregulation of islet vascularization [23 29] Vascular endothe-lial cells migrate to the source of VEGF-A and proliferateand form blood vessels in response to VEGF [30] Previousstudies [31 32] suggested that mice with specific deletionof the VEGF in 120573 cells had reduced islet vascularizationand impaired glucose tolerance These findings were in linewith observations in the transplanted pancreatic islets It hasbeen reported that reduction of VEGF-A by 120573 cells not only

reduces the number of intraislet endothelial cells participat-ing in graft vessel formation but also limits recruitment ofhost endothelial cell and their invasion into the graft [31 33]In our study we found an increase of islet VEGF in WHmice which was consistent with the enhancement of isletvascularization after high-fat diet However with the deletionof ACE2 this compensatory increase of VEGF in islet wasdisappeared after high-fat diet This was parallel to the islet120573 cell impairment and islet vascularization reduction Withthe defects of the islet vascularization the beta cell wasimpairedThe deteriorated beta cell might lead to a reductionof VEGF expression which in turn accelerates the loss ofvascularization Taken together we speculated that underthe condition of high-fat diet ACE2 might play a criticalrole in the glucose intolerance through impairment of isletvascularization

ACE2 not only functions as a carboxypeptidase cleavinga single residue from Ang I generating Ang1-9 and asingle residue from Ang II to generate Ang 1-7 but alsocleaves several other biological peptides such as apelinACE2 hydrolyzes apelin with high catalytic efficiency [34]Apelin is the endogenous ligand of the G-protein coupledapj receptor The effects of apelin on the pancreas focusedon the regulation of insulin secretion [35] Winzell et al[36] reported that apelin-36 inhibited the glucose-stimulatedinsulin secretion in obese insulin resistant high-fat fed miceMoreover apelin-13 was shown to inhibit insulin secretionstimulated by high glucose concentrations in INS-1 cells [37]Accordingly we hypothesized that with the deletion of ACE2the hydrolyzation of apelin is inhibited which leads to theinhibition of insulin secretion

Peripheral Insulin sensitivity has long been regarded asplaying a crucial role in glucose homeostasis However theeffect of ACE2 on insulin sensitivity is unclear In this studywe took insulin tolerance test to evaluate peripheral insulinsensitivity We found no significant difference of peripheralinsulin sensitivity between KO mice and WT mice Theperipheral insulin sensitivity betweenWHmice andKHmicewas similar too So we concluded that loss of ACE2 hasno effects on peripheral insulin sensitivity More accuratemeasures such as hyperinsulinemic euglycemic clamp shouldbe needed in further studies It should be noted that therewas no difference of the body weight betweenWT and ACE2KO mice neither with chow diet nor with high-fat dietAccordingly we speculated that ACE2was not involved in theregulation of body weight

In summary the results obtained in this study show thatloss of ACE2 in mice with long-term high-fat diet leadsto impairment of glucose tolerance and insulin secretionThe primary mechanisms involved in these effects appear toinclude a reduction in islet 120573 cell and islet vascularizationOur data suggested that the absence of ACE2 weakened theadaptive changes of islet 120573 cell function and islet vasculariza-tion due to high-fat diet ACE2 seems to be a potential thera-peutic target in the treatment of islet dysfunction induced byhigh-fat diet

Journal of Diabetes Research 7

Acknowledgment

This project was supported by the National Natural ScienceFoundation of China (no 81070615)

References

[1] J R A Skipworth G Szabadkai S W M Olde Damink PS Leung S E Humphries and H E Montgomery ldquoReviewarticle pancreatic renin-angiotensin systems in health anddiseaserdquo Alimentary Pharmacology amp Therapeutics vol 34 no8 pp 840ndash852 2011

[2] S R Tipnis N M Hooper R Hyde E Karran G Christieand A J Turner ldquoA human homolog of angiotensin-convertingenzyme cloning and functional expression as a captopril-insensitive carboxypeptidaserdquo Journal of Biological Chemistryvol 275 no 43 pp 33238ndash33243 2000

[3] M Donoghue F Hsieh E Baronas et al ldquoA novel angiotensin-converting enzyme-related carboxypeptidase (ACE2) convertsangiotensin I to angiotensin 1-9rdquo Circulation Research vol 87no 5 pp e1ndashe9 2000

[4] G I Rice D A Thomas P J Grant A J Turner and N MHooper ldquoEvaluation of angiotensin-converting enzyme (ACE)its homologue ACE2 and neprilysin in angiotensin peptide me-tabolismrdquo Biochemical Journal vol 383 no 1 pp 45ndash51 2004

[5] C M Ferrario ldquoACE2 more of Ang-(1ndash7) or less Ang IIrdquo Cur-rent Opinion in Nephrology and Hypertension vol 20 no 1 pp1ndash6 2011

[6] R A S Santos A J Ferreira and A C Simoes E Silva ldquoRecentadvances in the angiotensin-converting enzyme 2-angiotens-in(1ndash7)-Mas axisrdquo Experimental Physiology vol 93 no 5 pp519ndash527 2008

[7] Q Cheng and P S Leung ldquoAn update on the islet renin-angiot-ensin systemrdquo Peptides vol 32 no 5 pp 1087ndash1095 2011

[8] P S Leung ldquoThe physiology of a local renin-angiotensin systemin the pancreasrdquo Journal of Physiology vol 580 no 1 pp 31ndash372007

[9] D Batlle M J Soler and M Ye ldquoACE2 and diabetes ACE ofACEsrdquo Diabetes vol 59 no 12 pp 2994ndash2996 2010

[10] M C Chappell A Millsted D I Diz K B Brosnihan and CM Ferrario ldquoEvidence for an intrinsic angiotensin system inthe canine pancreasrdquo Journal of Hypertension vol 9 no 8 pp751ndash759 1991

[11] T Lau P O Carlsson and P S Leung ldquoEvidence for a localangiotensin-generating system and dose-dependent inhibitionof glucose-stimulated insulin release by angiotensin II in iso-lated pancreatic isletsrdquoDiabetologia vol 47 no 2 pp 240ndash2482004

[12] K Y Lam and P S Leung ldquoRegulation and expression of arenin-angiotensin system in human pancreas and pancreaticendocrine tumoursrdquo European Journal of Endocrinology vol146 no 4 pp 567ndash572 2002

[13] C Tikellis M E Cooper andM CThomas ldquoRole of the renin-angiotensin system in the endocrine pancreas implications forthe development of diabetesrdquo International Journal of Biochem-istry and Cell Biology vol 38 no 5-6 pp 737ndash751 2006

[14] S M Bindom and E Lazartigues ldquoThe sweeter side of ACE2physiological evidence for a role in diabetesrdquo Molecular andCellular Endocrinology vol 302 no 2 pp 193ndash202 2009

[15] P S Leung and P O Carlsson ldquoPancreatic islet renin angiot-ensin system its novel roles in islet function and in diabetesmellitusrdquo Pancreas vol 30 no 4 pp 293ndash298 2005

[16] P S Leung ldquoMechanisms of protective effects induced by block-ade of the renin-angiotensin system novel role of the pancreaticislet angiotensin-generating system inType 2 diabetesrdquoDiabeticMedicine vol 24 no 2 pp 110ndash116 2007

[17] C Tikellis P J Wookey R Candido S Andrikopoulos M CThomas and M E Cooper ldquoImproved islet morphology afterblockade of the renin-angiotensin system in the ZDF ratrdquo Dia-betes vol 53 no 4 pp 989ndash997 2004

[18] L Yuan X Li J Li H l Li and S S Cheng ldquoEffects of renin-angiotensin system blockade on the islet morphology and func-tion in rats with long-term high-fat dietrdquo Acta Diabetologica Inpress

[19] A J Scheen ldquoRenin-angiotensin system inhibition preventstype 2 diabetes mellitusmdashpart 1 a meta-analysis of randomisedclinical trialsrdquo Diabetes and Metabolism vol 30 no 6 pp 487ndash496 2004

[20] E L Gillespie C M White M Kardas M Lindberg and C IColeman ldquoThe impact of ACE inhibitors or angiotensin II type1 receptor blockers on the development of new-onset type 2diabetesrdquo Diabetes Care vol 28 no 9 pp 2261ndash2266 2005

[21] P Vejakama A Thakkinstian D Lertrattananon A IngsathitC Ngarmukos and J Attia ldquoReno-protective effects of renin-angiotensin system blockade in type 2 diabetic patients a sys-tematic review and network meta-analysisrdquo Diabetologia vol55 no 3 pp 566ndash578 2011

[22] D W Wong G Y Oudit H Reich et al ldquoLoss of Angiotensin-converting enzyme-2 (Ace2) accelerates diabetic kidney injuryrdquoAmerican Journal of Pathology vol 171 no 2 pp 438ndash451 2007

[23] E M Akirav M T Baquero L W Opare-Addo et al ldquoGlucoseand inflammation control islet vascular density and 120573-cell func-tion in NOD mice control of islet vasculature and vascularendothelial growth factor by glucoserdquo Diabetes vol 60 no 3pp 876ndash883 2011

[24] M Iwai and M Horiuchi ldquoDevil and angel in the renin-angiot-ensin system ACE-angiotensin II-AT1 receptor axis vs ACE2-angiotensin-(1ndash7)-Mas receptor axisrdquo Hypertension Researchvol 32 no 7 pp 533ndash536 2009

[25] M C Chappell ldquoEmerging evidence for a functional angiotens-in-converting enzyme 2-angiotensin-(1ndash7)-Mas receptor axismore than regulation of blood pressurerdquoHypertension vol 50no 4 pp 596ndash599 2007

[26] K Putnam R Shoemaker F Yiannikouris and L Cassis ldquoTherenin-angiotensin system a target of and contributor to dyslip-idemias altered glucose homeostasis and hypertension of themetabolic syndromerdquo American Journal of Physiology vol 15no 302 pp H1219ndashH1230 2012

[27] V Souza-Mello B M Gregorio F S Cardoso-De-Lemos LDe Carvalho M B Aguila and C A Mandarim-De-LacerdaldquoComparative effects of telmisartan sitagliptin and metforminalone or in combination on obesity insulin resistance and liverand pancreas remodelling in C57BL6 mice fed on a very high-fat dietrdquo Clinical Science vol 119 no 6 pp 239ndash250 2010

[28] S M Bindom C P Hans H Xia A H Boulares and E Lazar-tigues ldquoAngiotensin I-converting enzyme type 2 (ACE2) genetherapy improves glycemic control in diabetic micerdquo Diabetesvol 59 no 10 pp 2540ndash2548 2010

[29] J Agudo E Ayuso V Jimenez et al ldquoVascular endothelialgrowth factor-mediated islet hypervascularization and inflam-mation contribute to progressive reduction of 120573-cell massrdquoDia-betes vol 61 no 11 pp 2851ndash2861 2012

[30] O Cleaver and Y Dor ldquoVascular instruction of pancreas devel-opmentrdquo Development vol 139 no 16 pp 2833ndash2843 2012

8 Journal of Diabetes Research

[31] M Brissova A Shostak M Shiota et al ldquoPancreatic islet pro-duction of vascular endothelial growth factor-A is essential forislet vascularization revascularization and functionrdquoDiabetesvol 55 no 11 pp 2974ndash2985 2006

[32] N Jabs L FranklinM B Brenner et al ldquoReduced insulin secre-tion and content in VEGF-A deficient mouse pancreatic isletsrdquoExperimental and Clinical Endocrinology and Diabetes vol 116no 1 pp S46ndashS49 2008

[33] M Brissova M Fowler P Wiebe et al ldquoIntraislet endothelialcells contribute to revascularization of transplanted pancreaticisletsrdquo Diabetes vol 53 no 5 pp 1318ndash1325 2004

[34] C Vickers P Hales V Kaushik et al ldquoHydrolysis of biologicalpeptides by human angiotensin-converting enzyme-related car-boxypeptidaserdquo Journal of Biological Chemistry vol 277 no 17pp 14838ndash14843 2002

[35] I Castan-laurell C Dray C Knauf O Kunduzova and P ValetldquoApelin a promising target for type 2 diabetes treatmentrdquoTrends in Endocrinology amp Metabolism vol 23 no 5 pp 234ndash241 2012

[36] M SWinzell CMagnusson and B Ahren ldquoThe apj receptor isexpressed in pancreatic islets and its ligand apelin inhibitsinsulin secretion in micerdquo Regulatory Peptides vol 131 no 1ndash3pp 12ndash17 2005

[37] L Guo Q Li W Wang et al ldquoApelin inhibits insulin secretionin pancreatic 120573-cells by activation of PI3-kinase-phosphodies-terase 3Brdquo Endocrine Research vol 34 no 4 pp 142ndash154 2009

6 Journal of Diabetes Research

a potential protective enzyme for glucose homeostasis [9]but the role of ACE2 in the regulation of islet function isincompletely understood In the present study there were ondifferences in fasting blood glucose and glucose tolerance inmale ACE2 KOmice with standard chow diet compared withcorresponding WT mice This observation was consistentwith a previous study by Wong et al [22] who found thatglycemic control was not worsened by the absence of ACE2in the Akita diabetic miceTherefore we supposed that ACE2ablation does not seem to be sufficient to alter glucose statusin the condition of regular chow diet However an interestingand important finding of the current study was that theconsequence of ACE2 absence became apparent after high-fat diet Under the condition of high-fat diet there weremuchmore reductions of the first-stage insulin secretion andglucose tolerance in KH mice compared with WHmice Notsurprisingly these changes were linked to decreased 120573 cellmass It is noteworthy that this finding was partly supportedby the report of gene therapy in obese diabetic mice Bindomet al [28] found that ACE2 gene therapy was able to improvefasting blood glucose and glucose tolerance in obese diabeticmice Consistent with the improvement of glycemic controlACE2 gene therapy leaded to enhanced first-stage insulinsecretion and reduced 120573 cell apoptosis Our current studyfor the first time provided evidence that ACE2 absence leadsto more severe effects in 120573 cell dysfunction in obese micewith high-fat diet Taken together it is hypothesized that lossof ACE2 renders the pancreatic islet more susceptible to thepathological actions of obesity induced by high-fat diet

It is known that islet vascularization is essential for theislet to respond to fluctuations in blood glucose andmodulateinsulin release as needed Defects in the vascularization ofpancreatic islets can lead to deleterious effects on glucose tol-erance and islet insulin secretion In our study an increasedislet vascularization in high-fat diet WT mice was observedcompared with chow diet WT mice which represented acompensatory response to support higher vascularizationduring 120573 cell expansion In contrast no increased isletvascularization was observed in KH mice compared withKO mice Furthermore KH mice exhibited defects of isletvascularization compared with WH mice These findingssuggested that deletion of ACE2 reduces the compensationof islet vascularization in response to high-fat diet Thereis an intimate connection between islet beta cell and isletvascularization On the one hand islet vascularization hasbeen regarded as playing a crucial role in maintaining betacell survival and function On the other hand beta cellssecrete several factors such as VEGF to promote vasculardevelopment VEGF which is highly expressed in islet hasbeen identified as an important factor to be involved in theregulation of islet vascularization [23 29] Vascular endothe-lial cells migrate to the source of VEGF-A and proliferateand form blood vessels in response to VEGF [30] Previousstudies [31 32] suggested that mice with specific deletionof the VEGF in 120573 cells had reduced islet vascularizationand impaired glucose tolerance These findings were in linewith observations in the transplanted pancreatic islets It hasbeen reported that reduction of VEGF-A by 120573 cells not only

reduces the number of intraislet endothelial cells participat-ing in graft vessel formation but also limits recruitment ofhost endothelial cell and their invasion into the graft [31 33]In our study we found an increase of islet VEGF in WHmice which was consistent with the enhancement of isletvascularization after high-fat diet However with the deletionof ACE2 this compensatory increase of VEGF in islet wasdisappeared after high-fat diet This was parallel to the islet120573 cell impairment and islet vascularization reduction Withthe defects of the islet vascularization the beta cell wasimpairedThe deteriorated beta cell might lead to a reductionof VEGF expression which in turn accelerates the loss ofvascularization Taken together we speculated that underthe condition of high-fat diet ACE2 might play a criticalrole in the glucose intolerance through impairment of isletvascularization

ACE2 not only functions as a carboxypeptidase cleavinga single residue from Ang I generating Ang1-9 and asingle residue from Ang II to generate Ang 1-7 but alsocleaves several other biological peptides such as apelinACE2 hydrolyzes apelin with high catalytic efficiency [34]Apelin is the endogenous ligand of the G-protein coupledapj receptor The effects of apelin on the pancreas focusedon the regulation of insulin secretion [35] Winzell et al[36] reported that apelin-36 inhibited the glucose-stimulatedinsulin secretion in obese insulin resistant high-fat fed miceMoreover apelin-13 was shown to inhibit insulin secretionstimulated by high glucose concentrations in INS-1 cells [37]Accordingly we hypothesized that with the deletion of ACE2the hydrolyzation of apelin is inhibited which leads to theinhibition of insulin secretion

Peripheral Insulin sensitivity has long been regarded asplaying a crucial role in glucose homeostasis However theeffect of ACE2 on insulin sensitivity is unclear In this studywe took insulin tolerance test to evaluate peripheral insulinsensitivity We found no significant difference of peripheralinsulin sensitivity between KO mice and WT mice Theperipheral insulin sensitivity betweenWHmice andKHmicewas similar too So we concluded that loss of ACE2 hasno effects on peripheral insulin sensitivity More accuratemeasures such as hyperinsulinemic euglycemic clamp shouldbe needed in further studies It should be noted that therewas no difference of the body weight betweenWT and ACE2KO mice neither with chow diet nor with high-fat dietAccordingly we speculated that ACE2was not involved in theregulation of body weight

In summary the results obtained in this study show thatloss of ACE2 in mice with long-term high-fat diet leadsto impairment of glucose tolerance and insulin secretionThe primary mechanisms involved in these effects appear toinclude a reduction in islet 120573 cell and islet vascularizationOur data suggested that the absence of ACE2 weakened theadaptive changes of islet 120573 cell function and islet vasculariza-tion due to high-fat diet ACE2 seems to be a potential thera-peutic target in the treatment of islet dysfunction induced byhigh-fat diet

Journal of Diabetes Research 7

Acknowledgment

This project was supported by the National Natural ScienceFoundation of China (no 81070615)

References

[1] J R A Skipworth G Szabadkai S W M Olde Damink PS Leung S E Humphries and H E Montgomery ldquoReviewarticle pancreatic renin-angiotensin systems in health anddiseaserdquo Alimentary Pharmacology amp Therapeutics vol 34 no8 pp 840ndash852 2011

[2] S R Tipnis N M Hooper R Hyde E Karran G Christieand A J Turner ldquoA human homolog of angiotensin-convertingenzyme cloning and functional expression as a captopril-insensitive carboxypeptidaserdquo Journal of Biological Chemistryvol 275 no 43 pp 33238ndash33243 2000

[3] M Donoghue F Hsieh E Baronas et al ldquoA novel angiotensin-converting enzyme-related carboxypeptidase (ACE2) convertsangiotensin I to angiotensin 1-9rdquo Circulation Research vol 87no 5 pp e1ndashe9 2000

[4] G I Rice D A Thomas P J Grant A J Turner and N MHooper ldquoEvaluation of angiotensin-converting enzyme (ACE)its homologue ACE2 and neprilysin in angiotensin peptide me-tabolismrdquo Biochemical Journal vol 383 no 1 pp 45ndash51 2004

[5] C M Ferrario ldquoACE2 more of Ang-(1ndash7) or less Ang IIrdquo Cur-rent Opinion in Nephrology and Hypertension vol 20 no 1 pp1ndash6 2011

[6] R A S Santos A J Ferreira and A C Simoes E Silva ldquoRecentadvances in the angiotensin-converting enzyme 2-angiotens-in(1ndash7)-Mas axisrdquo Experimental Physiology vol 93 no 5 pp519ndash527 2008

[7] Q Cheng and P S Leung ldquoAn update on the islet renin-angiot-ensin systemrdquo Peptides vol 32 no 5 pp 1087ndash1095 2011

[8] P S Leung ldquoThe physiology of a local renin-angiotensin systemin the pancreasrdquo Journal of Physiology vol 580 no 1 pp 31ndash372007

[9] D Batlle M J Soler and M Ye ldquoACE2 and diabetes ACE ofACEsrdquo Diabetes vol 59 no 12 pp 2994ndash2996 2010

[10] M C Chappell A Millsted D I Diz K B Brosnihan and CM Ferrario ldquoEvidence for an intrinsic angiotensin system inthe canine pancreasrdquo Journal of Hypertension vol 9 no 8 pp751ndash759 1991

[11] T Lau P O Carlsson and P S Leung ldquoEvidence for a localangiotensin-generating system and dose-dependent inhibitionof glucose-stimulated insulin release by angiotensin II in iso-lated pancreatic isletsrdquoDiabetologia vol 47 no 2 pp 240ndash2482004

[12] K Y Lam and P S Leung ldquoRegulation and expression of arenin-angiotensin system in human pancreas and pancreaticendocrine tumoursrdquo European Journal of Endocrinology vol146 no 4 pp 567ndash572 2002

[13] C Tikellis M E Cooper andM CThomas ldquoRole of the renin-angiotensin system in the endocrine pancreas implications forthe development of diabetesrdquo International Journal of Biochem-istry and Cell Biology vol 38 no 5-6 pp 737ndash751 2006

[14] S M Bindom and E Lazartigues ldquoThe sweeter side of ACE2physiological evidence for a role in diabetesrdquo Molecular andCellular Endocrinology vol 302 no 2 pp 193ndash202 2009

[15] P S Leung and P O Carlsson ldquoPancreatic islet renin angiot-ensin system its novel roles in islet function and in diabetesmellitusrdquo Pancreas vol 30 no 4 pp 293ndash298 2005

[16] P S Leung ldquoMechanisms of protective effects induced by block-ade of the renin-angiotensin system novel role of the pancreaticislet angiotensin-generating system inType 2 diabetesrdquoDiabeticMedicine vol 24 no 2 pp 110ndash116 2007

[17] C Tikellis P J Wookey R Candido S Andrikopoulos M CThomas and M E Cooper ldquoImproved islet morphology afterblockade of the renin-angiotensin system in the ZDF ratrdquo Dia-betes vol 53 no 4 pp 989ndash997 2004

[18] L Yuan X Li J Li H l Li and S S Cheng ldquoEffects of renin-angiotensin system blockade on the islet morphology and func-tion in rats with long-term high-fat dietrdquo Acta Diabetologica Inpress

[19] A J Scheen ldquoRenin-angiotensin system inhibition preventstype 2 diabetes mellitusmdashpart 1 a meta-analysis of randomisedclinical trialsrdquo Diabetes and Metabolism vol 30 no 6 pp 487ndash496 2004

[20] E L Gillespie C M White M Kardas M Lindberg and C IColeman ldquoThe impact of ACE inhibitors or angiotensin II type1 receptor blockers on the development of new-onset type 2diabetesrdquo Diabetes Care vol 28 no 9 pp 2261ndash2266 2005

[21] P Vejakama A Thakkinstian D Lertrattananon A IngsathitC Ngarmukos and J Attia ldquoReno-protective effects of renin-angiotensin system blockade in type 2 diabetic patients a sys-tematic review and network meta-analysisrdquo Diabetologia vol55 no 3 pp 566ndash578 2011

[22] D W Wong G Y Oudit H Reich et al ldquoLoss of Angiotensin-converting enzyme-2 (Ace2) accelerates diabetic kidney injuryrdquoAmerican Journal of Pathology vol 171 no 2 pp 438ndash451 2007

[23] E M Akirav M T Baquero L W Opare-Addo et al ldquoGlucoseand inflammation control islet vascular density and 120573-cell func-tion in NOD mice control of islet vasculature and vascularendothelial growth factor by glucoserdquo Diabetes vol 60 no 3pp 876ndash883 2011

[24] M Iwai and M Horiuchi ldquoDevil and angel in the renin-angiot-ensin system ACE-angiotensin II-AT1 receptor axis vs ACE2-angiotensin-(1ndash7)-Mas receptor axisrdquo Hypertension Researchvol 32 no 7 pp 533ndash536 2009

[25] M C Chappell ldquoEmerging evidence for a functional angiotens-in-converting enzyme 2-angiotensin-(1ndash7)-Mas receptor axismore than regulation of blood pressurerdquoHypertension vol 50no 4 pp 596ndash599 2007

[26] K Putnam R Shoemaker F Yiannikouris and L Cassis ldquoTherenin-angiotensin system a target of and contributor to dyslip-idemias altered glucose homeostasis and hypertension of themetabolic syndromerdquo American Journal of Physiology vol 15no 302 pp H1219ndashH1230 2012

[27] V Souza-Mello B M Gregorio F S Cardoso-De-Lemos LDe Carvalho M B Aguila and C A Mandarim-De-LacerdaldquoComparative effects of telmisartan sitagliptin and metforminalone or in combination on obesity insulin resistance and liverand pancreas remodelling in C57BL6 mice fed on a very high-fat dietrdquo Clinical Science vol 119 no 6 pp 239ndash250 2010

[28] S M Bindom C P Hans H Xia A H Boulares and E Lazar-tigues ldquoAngiotensin I-converting enzyme type 2 (ACE2) genetherapy improves glycemic control in diabetic micerdquo Diabetesvol 59 no 10 pp 2540ndash2548 2010

[29] J Agudo E Ayuso V Jimenez et al ldquoVascular endothelialgrowth factor-mediated islet hypervascularization and inflam-mation contribute to progressive reduction of 120573-cell massrdquoDia-betes vol 61 no 11 pp 2851ndash2861 2012

[30] O Cleaver and Y Dor ldquoVascular instruction of pancreas devel-opmentrdquo Development vol 139 no 16 pp 2833ndash2843 2012

8 Journal of Diabetes Research

[31] M Brissova A Shostak M Shiota et al ldquoPancreatic islet pro-duction of vascular endothelial growth factor-A is essential forislet vascularization revascularization and functionrdquoDiabetesvol 55 no 11 pp 2974ndash2985 2006

[32] N Jabs L FranklinM B Brenner et al ldquoReduced insulin secre-tion and content in VEGF-A deficient mouse pancreatic isletsrdquoExperimental and Clinical Endocrinology and Diabetes vol 116no 1 pp S46ndashS49 2008

[33] M Brissova M Fowler P Wiebe et al ldquoIntraislet endothelialcells contribute to revascularization of transplanted pancreaticisletsrdquo Diabetes vol 53 no 5 pp 1318ndash1325 2004

[34] C Vickers P Hales V Kaushik et al ldquoHydrolysis of biologicalpeptides by human angiotensin-converting enzyme-related car-boxypeptidaserdquo Journal of Biological Chemistry vol 277 no 17pp 14838ndash14843 2002

[35] I Castan-laurell C Dray C Knauf O Kunduzova and P ValetldquoApelin a promising target for type 2 diabetes treatmentrdquoTrends in Endocrinology amp Metabolism vol 23 no 5 pp 234ndash241 2012

[36] M SWinzell CMagnusson and B Ahren ldquoThe apj receptor isexpressed in pancreatic islets and its ligand apelin inhibitsinsulin secretion in micerdquo Regulatory Peptides vol 131 no 1ndash3pp 12ndash17 2005

[37] L Guo Q Li W Wang et al ldquoApelin inhibits insulin secretionin pancreatic 120573-cells by activation of PI3-kinase-phosphodies-terase 3Brdquo Endocrine Research vol 34 no 4 pp 142ndash154 2009

Journal of Diabetes Research 7

Acknowledgment

This project was supported by the National Natural ScienceFoundation of China (no 81070615)

References

[1] J R A Skipworth G Szabadkai S W M Olde Damink PS Leung S E Humphries and H E Montgomery ldquoReviewarticle pancreatic renin-angiotensin systems in health anddiseaserdquo Alimentary Pharmacology amp Therapeutics vol 34 no8 pp 840ndash852 2011

[2] S R Tipnis N M Hooper R Hyde E Karran G Christieand A J Turner ldquoA human homolog of angiotensin-convertingenzyme cloning and functional expression as a captopril-insensitive carboxypeptidaserdquo Journal of Biological Chemistryvol 275 no 43 pp 33238ndash33243 2000

[3] M Donoghue F Hsieh E Baronas et al ldquoA novel angiotensin-converting enzyme-related carboxypeptidase (ACE2) convertsangiotensin I to angiotensin 1-9rdquo Circulation Research vol 87no 5 pp e1ndashe9 2000

[4] G I Rice D A Thomas P J Grant A J Turner and N MHooper ldquoEvaluation of angiotensin-converting enzyme (ACE)its homologue ACE2 and neprilysin in angiotensin peptide me-tabolismrdquo Biochemical Journal vol 383 no 1 pp 45ndash51 2004

[5] C M Ferrario ldquoACE2 more of Ang-(1ndash7) or less Ang IIrdquo Cur-rent Opinion in Nephrology and Hypertension vol 20 no 1 pp1ndash6 2011

[6] R A S Santos A J Ferreira and A C Simoes E Silva ldquoRecentadvances in the angiotensin-converting enzyme 2-angiotens-in(1ndash7)-Mas axisrdquo Experimental Physiology vol 93 no 5 pp519ndash527 2008

[7] Q Cheng and P S Leung ldquoAn update on the islet renin-angiot-ensin systemrdquo Peptides vol 32 no 5 pp 1087ndash1095 2011

[8] P S Leung ldquoThe physiology of a local renin-angiotensin systemin the pancreasrdquo Journal of Physiology vol 580 no 1 pp 31ndash372007

[9] D Batlle M J Soler and M Ye ldquoACE2 and diabetes ACE ofACEsrdquo Diabetes vol 59 no 12 pp 2994ndash2996 2010

[10] M C Chappell A Millsted D I Diz K B Brosnihan and CM Ferrario ldquoEvidence for an intrinsic angiotensin system inthe canine pancreasrdquo Journal of Hypertension vol 9 no 8 pp751ndash759 1991

[11] T Lau P O Carlsson and P S Leung ldquoEvidence for a localangiotensin-generating system and dose-dependent inhibitionof glucose-stimulated insulin release by angiotensin II in iso-lated pancreatic isletsrdquoDiabetologia vol 47 no 2 pp 240ndash2482004

[12] K Y Lam and P S Leung ldquoRegulation and expression of arenin-angiotensin system in human pancreas and pancreaticendocrine tumoursrdquo European Journal of Endocrinology vol146 no 4 pp 567ndash572 2002

[13] C Tikellis M E Cooper andM CThomas ldquoRole of the renin-angiotensin system in the endocrine pancreas implications forthe development of diabetesrdquo International Journal of Biochem-istry and Cell Biology vol 38 no 5-6 pp 737ndash751 2006

[14] S M Bindom and E Lazartigues ldquoThe sweeter side of ACE2physiological evidence for a role in diabetesrdquo Molecular andCellular Endocrinology vol 302 no 2 pp 193ndash202 2009

[15] P S Leung and P O Carlsson ldquoPancreatic islet renin angiot-ensin system its novel roles in islet function and in diabetesmellitusrdquo Pancreas vol 30 no 4 pp 293ndash298 2005

[16] P S Leung ldquoMechanisms of protective effects induced by block-ade of the renin-angiotensin system novel role of the pancreaticislet angiotensin-generating system inType 2 diabetesrdquoDiabeticMedicine vol 24 no 2 pp 110ndash116 2007

[17] C Tikellis P J Wookey R Candido S Andrikopoulos M CThomas and M E Cooper ldquoImproved islet morphology afterblockade of the renin-angiotensin system in the ZDF ratrdquo Dia-betes vol 53 no 4 pp 989ndash997 2004

[18] L Yuan X Li J Li H l Li and S S Cheng ldquoEffects of renin-angiotensin system blockade on the islet morphology and func-tion in rats with long-term high-fat dietrdquo Acta Diabetologica Inpress

[19] A J Scheen ldquoRenin-angiotensin system inhibition preventstype 2 diabetes mellitusmdashpart 1 a meta-analysis of randomisedclinical trialsrdquo Diabetes and Metabolism vol 30 no 6 pp 487ndash496 2004

[20] E L Gillespie C M White M Kardas M Lindberg and C IColeman ldquoThe impact of ACE inhibitors or angiotensin II type1 receptor blockers on the development of new-onset type 2diabetesrdquo Diabetes Care vol 28 no 9 pp 2261ndash2266 2005

[21] P Vejakama A Thakkinstian D Lertrattananon A IngsathitC Ngarmukos and J Attia ldquoReno-protective effects of renin-angiotensin system blockade in type 2 diabetic patients a sys-tematic review and network meta-analysisrdquo Diabetologia vol55 no 3 pp 566ndash578 2011

[22] D W Wong G Y Oudit H Reich et al ldquoLoss of Angiotensin-converting enzyme-2 (Ace2) accelerates diabetic kidney injuryrdquoAmerican Journal of Pathology vol 171 no 2 pp 438ndash451 2007

[23] E M Akirav M T Baquero L W Opare-Addo et al ldquoGlucoseand inflammation control islet vascular density and 120573-cell func-tion in NOD mice control of islet vasculature and vascularendothelial growth factor by glucoserdquo Diabetes vol 60 no 3pp 876ndash883 2011

[24] M Iwai and M Horiuchi ldquoDevil and angel in the renin-angiot-ensin system ACE-angiotensin II-AT1 receptor axis vs ACE2-angiotensin-(1ndash7)-Mas receptor axisrdquo Hypertension Researchvol 32 no 7 pp 533ndash536 2009

[25] M C Chappell ldquoEmerging evidence for a functional angiotens-in-converting enzyme 2-angiotensin-(1ndash7)-Mas receptor axismore than regulation of blood pressurerdquoHypertension vol 50no 4 pp 596ndash599 2007

[26] K Putnam R Shoemaker F Yiannikouris and L Cassis ldquoTherenin-angiotensin system a target of and contributor to dyslip-idemias altered glucose homeostasis and hypertension of themetabolic syndromerdquo American Journal of Physiology vol 15no 302 pp H1219ndashH1230 2012

[27] V Souza-Mello B M Gregorio F S Cardoso-De-Lemos LDe Carvalho M B Aguila and C A Mandarim-De-LacerdaldquoComparative effects of telmisartan sitagliptin and metforminalone or in combination on obesity insulin resistance and liverand pancreas remodelling in C57BL6 mice fed on a very high-fat dietrdquo Clinical Science vol 119 no 6 pp 239ndash250 2010

[28] S M Bindom C P Hans H Xia A H Boulares and E Lazar-tigues ldquoAngiotensin I-converting enzyme type 2 (ACE2) genetherapy improves glycemic control in diabetic micerdquo Diabetesvol 59 no 10 pp 2540ndash2548 2010

[29] J Agudo E Ayuso V Jimenez et al ldquoVascular endothelialgrowth factor-mediated islet hypervascularization and inflam-mation contribute to progressive reduction of 120573-cell massrdquoDia-betes vol 61 no 11 pp 2851ndash2861 2012

[30] O Cleaver and Y Dor ldquoVascular instruction of pancreas devel-opmentrdquo Development vol 139 no 16 pp 2833ndash2843 2012

8 Journal of Diabetes Research

[31] M Brissova A Shostak M Shiota et al ldquoPancreatic islet pro-duction of vascular endothelial growth factor-A is essential forislet vascularization revascularization and functionrdquoDiabetesvol 55 no 11 pp 2974ndash2985 2006

[32] N Jabs L FranklinM B Brenner et al ldquoReduced insulin secre-tion and content in VEGF-A deficient mouse pancreatic isletsrdquoExperimental and Clinical Endocrinology and Diabetes vol 116no 1 pp S46ndashS49 2008

[33] M Brissova M Fowler P Wiebe et al ldquoIntraislet endothelialcells contribute to revascularization of transplanted pancreaticisletsrdquo Diabetes vol 53 no 5 pp 1318ndash1325 2004

[34] C Vickers P Hales V Kaushik et al ldquoHydrolysis of biologicalpeptides by human angiotensin-converting enzyme-related car-boxypeptidaserdquo Journal of Biological Chemistry vol 277 no 17pp 14838ndash14843 2002

[35] I Castan-laurell C Dray C Knauf O Kunduzova and P ValetldquoApelin a promising target for type 2 diabetes treatmentrdquoTrends in Endocrinology amp Metabolism vol 23 no 5 pp 234ndash241 2012

[36] M SWinzell CMagnusson and B Ahren ldquoThe apj receptor isexpressed in pancreatic islets and its ligand apelin inhibitsinsulin secretion in micerdquo Regulatory Peptides vol 131 no 1ndash3pp 12ndash17 2005

[37] L Guo Q Li W Wang et al ldquoApelin inhibits insulin secretionin pancreatic 120573-cells by activation of PI3-kinase-phosphodies-terase 3Brdquo Endocrine Research vol 34 no 4 pp 142ndash154 2009

8 Journal of Diabetes Research

[31] M Brissova A Shostak M Shiota et al ldquoPancreatic islet pro-duction of vascular endothelial growth factor-A is essential forislet vascularization revascularization and functionrdquoDiabetesvol 55 no 11 pp 2974ndash2985 2006

[32] N Jabs L FranklinM B Brenner et al ldquoReduced insulin secre-tion and content in VEGF-A deficient mouse pancreatic isletsrdquoExperimental and Clinical Endocrinology and Diabetes vol 116no 1 pp S46ndashS49 2008

[33] M Brissova M Fowler P Wiebe et al ldquoIntraislet endothelialcells contribute to revascularization of transplanted pancreaticisletsrdquo Diabetes vol 53 no 5 pp 1318ndash1325 2004

[34] C Vickers P Hales V Kaushik et al ldquoHydrolysis of biologicalpeptides by human angiotensin-converting enzyme-related car-boxypeptidaserdquo Journal of Biological Chemistry vol 277 no 17pp 14838ndash14843 2002

[35] I Castan-laurell C Dray C Knauf O Kunduzova and P ValetldquoApelin a promising target for type 2 diabetes treatmentrdquoTrends in Endocrinology amp Metabolism vol 23 no 5 pp 234ndash241 2012

[36] M SWinzell CMagnusson and B Ahren ldquoThe apj receptor isexpressed in pancreatic islets and its ligand apelin inhibitsinsulin secretion in micerdquo Regulatory Peptides vol 131 no 1ndash3pp 12ndash17 2005

[37] L Guo Q Li W Wang et al ldquoApelin inhibits insulin secretionin pancreatic 120573-cells by activation of PI3-kinase-phosphodies-terase 3Brdquo Endocrine Research vol 34 no 4 pp 142ndash154 2009