the classical renin - angiotensin system ( ras ) , ace - ang ii - at1 receptor axis , is a coordinated hormonal cascade facilitating the dynamic control of perfusion in both health and disease . in 2000 , a new member of the ras , ace2 , was discovered by two independent groups [ 2 , 3 ] . ace2 is capable of metabolizing ang ii to generate ang ( 1 - 7 ) . ang ( 1 - 7 ) interacting with its receptor mas elicits numerous actions that counterbalance those of ang ii . accumulating evidence have indicated that ace2-ang ( 1 - 7)-mas axis acts as a negative regulator of the ace - ang ii - at1 receptor axis . during the past decades , the existence of a local ras has been confirmed in both the endocrine and exocrine pancreas [ 79 ] . in 1991 , identified the expression of several ras components in mouse pancreatic islet , such as angiotensinogen , ace , at1 , and at2 receptors . the evidence for the existence of these above components were also observed in human pancreas . canine pancreatic ang ( 1 - 7 )   and rat pancreatic mas receptor have also been confirmed . previous studies [ 15 , 16 ] have provided additional evidence for the close association of ras with pancreatic endocrine physiology and its pathophysiology . islet angiotensinogen and at1 receptor expression were increased in the zucker diabetic fatty rats . islet ras activation is responsible for the islet dysfunction through exacerbation of impairment of islet blood flow , promoting islet cell destruction , fibrosis , and apoptosis . treatment with acei or arb ameliorates the impairment of glucose tolerance , reduces new - onset diabetes , and prevents the development of diabetic complications . to date , there have been limited studies on whether ace2 plays a key role in islet function . wong et al .   reported that there was no difference between the plasma glucose concentrations in the ace2 ko mice and wt mice . the exact functions of ace2 in the regulation of islet function remain controversial and as yet inadequately elucidated . thus , the present study aimed to investigate the potential role of ace2 in glucose homeostasis and explore the mechanisms of ace2 in islet function . male ace2 ko mice and their age matched male wildtype littermates ( c57bl/6j ) at the age of 5 weeks were purchased from the institute of laboratory animal science , chinese academy of medical sciences . mice were individually housed under specific pathogen - free conditions with a 12 hours light - dark cycle , ambient temperature of 22c , and allowed free access to food and water . 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 weeks . the body weight of each mice was measured weekly during the study . the institutional animal care and mice were fasted for 6 hours and were injected intraperitoneally with glucose at 2.0  g / kg body weight . blood glucose was analyzed at 0 ,  15 ,  30 ,  60 , and 120  min after the injection . the area under the curve ( auc ) was calculated using the formula aucg = 7.5g0 + 15g15 + 22.5g30 + 45g60 + 30g120 where g0 , g15 , g30 , g60 , and g120 were blood glucose level at each time point during ipgtt . blood glucose was measured using an automatic glucometer ( one touch , byer ) . for insulin secretion test , mice were fasted for 6 hours , and glucose ( 3.0  g / kg body weight ) was injected intraperitoneally . blood samples were collected from the orbital venous plexus at 0 , 2 , 5 , 15 , and 30  min . the auci from 0  min to 5  min ( auci05 ) which represented the first stage insulin secretion was calculated as follows : auci05 = i0 + 2.5i2 + 1.5i5 . i0 , i2 , i5 , i15 , and i30 represented the insulin level at 0 , 2 , 5 , 15 , 30  min respectively . insulin levels were measured with the mouse insulin enzyme - linked immunosorbent assay kit .   for insulin tolerance test , mice were fasted for 6 hours and then injected insulin ( 0.5  u / kg body weight ) ( novolog ) intraperitoneally . glucose levels were measured at 0 , 15 , 30 , 60 , and 90  min after the injection . the pancreatic islets were stained for insulin , cd31 , and vascular endothelial growth factor ( vegf ) using anti - rat primary antibody with suitable concentration . after three rinses with pbs , a secondary antibody ( sigma ) was applied for 30  min at 37c . a third antibody ( sigma ) was applied as the same way , and dab / h2o2 staining was used at last . five discontinuous sections from each animal were stained and each section was analyzed randomly and double - blindly , using light microscopy , the results were captured and evaluated by a computer image analysis system ( motic , wetzlar , germany ) . insulin staining was used to assess the change of islets morphology and insulin concentration in  cells . the reciprocal of gray scale of intraislet insulin - positive staining was computed , the natural logarithm was calculated as intraislet insulin relative concentration ( irc ) , which represented the insulin reserve in endochylema of  cells . insulin - positive cell density ( icd ) , which was the ratio of intraislet insulin - positive nuclear quantity to insulin - positive staining area , was used to reflect the amount of beta cells intraislets . the mvd of islets was calculated by cd31-positive cells as reported . any brown - stained endothelial cell or endothelial cell cluster in islets six discontinuous sections from each animal were stained and 810 islets in each section were selected randomly . the percentage of the area for cd31 positive cell was counted divided by the islet area to obtain mvd . the reciprocal of gray scales of intraislet vegf positive staining was computed , and the natural logarithm was calculated to present the relative content of vascular endothelial growth factor ( vrc ) . statistical significance of differences was assessed using one - way analysis of variance , and unpaired student 's t - test , as appropriate . there was no obvious difference in body weight among the four groups at the beginning of the study . the body weights of wt and ko ( ace2 ko ) groups were similar at end of the study . when fed with high - fat diet , the body weights of wh ( wt with high - fat diet ) and kh ( ace2 ko with high - fat diet ) groups gradually increased . after 16 weeks high - fat diet , the body weights of wh and kh groups were significantly higher than those in corresponding chow diet groups ( both p < 0.05 ) . when the study was completed , no significant difference of body weight was observed in kh group as compared with those in wh group ( table 1 ) . the islets function was evaluated by intraperitoneal glucose tolerance test ( ipgtt ) and intraperitoneal insulin releasing test ( ipirt ) . fasting blood glucose levels were not significantly different among those four groups . in ipgtt , the values of 30 and 60  min glucose of wh group were significantly higher than those in wt group ( both p < 0.05 ) . however , blood glucose concentration in kh group peaked at approximately 30.9 we took the area under the ipgtt curve ( aucg ) to quantify glucose tolerance . the aucg in wh mice was significantly elevated than that in wt mice ( p < 0.05 ) . the aucg in kh groups was obviously increased than that in ko ( p < 0.05 ) . interestingly , we observed increased aucg in kh mice compared with wh mice ( p < 0.05 ) . these data indicated that deletion of ace2 leads to more severe glucose intolerance induced by high - fat diet ( figure 2 , table 1 ) . however , the fasting blood insulin levels in wh group were significantly higher than those in wt and kh groups ( both , p < 0.05 ) . in the ipirt , insulin peak climbed rapidly to about 4-fold as basic level in 2  min in wt group after glucose challenge . the levels of insulin release in the ko group stimulated by glucose infusion were almost the same as those in the wt group . the auci030 and auci05 were not different between wt and ko group . compared with wt group , we observed a decreased auci05 and increased auci030 in wh group compared with those in wt group ( both p < 0.05 ) , indicating the impairment of first stage insulin releasing and a compensatory enhancement of insulin release under the condition of high - fat diet . the auci030 in kh group was not increased when compared with that in ko group . in addition , the insulin secretion in kh and wh group climbed to the peak at the same time , whereas , the former exhibited a lower peak releasing after glucose load . this finding suggested that ace2 deficiency inhibits the compensatory enhancement of insulin secretion in response to the increased metabolic demand ( figure 3 , table 1 ) . in ipitt , blood glucose level in wt group dropped obviously and approached bottom levels by 30  min . after an intraperitoneal injection of insulin , the glucose disappearance rate of 15 and 30  min was higher in wh and kh mice than those in corresponding mice with chow diet ( both p < 0.05 ) . there was no difference of the glucose disappearance rate between kh and wh mice ( figure 4 ) . the islet area in the pancreases of wh group was larger than that in wt group , and the irc dropped obviously ( p < 0.05 ) , indicating the reduction of insulin reserve in wh group . in comparison with ko mice , kh mice exhibited a decreased irc ( p < 0.05 ) . moreover , a decreased irc was noted in kh group in comparison with wh group ( p < 0.05 ) , implying more severe impairment of insulin reserve in kh group . the icd in the wh group was lower than that in the wt group , but the difference was not significant . there was no difference of icd between kh group and wh group ( figure 5 , table 2 ) . the mvd of islet was slightly decreased in ko mice than that in wt mice , but the difference was not significant . staining of the pancreas using anti - cd31 antibody demonstrated enhanced mvd in wh group than wt group ( p < 0.05 ) , which suggested higher islet vascularization after high - fat diet . however , the kh mice did not show an increased mvd in islet when compared with ko mice . moreover , there was reduced mvd in islet from kh group when compared with wh group ( p < 0.05 ) . these findings indicated that loss of ace2 decreases the compensation of islet vascularization in response to high - fat diet ( figure 5 , table 2 ) . the vrc was similar between the wt and ko group . in comparison with wt group , vrc of wh group was increased ( p < 0.05 ) . however , there was no difference of vrc between ko group and kh group . the vrc in kh group was decreased when compared with wh group ( p < 0.05 ) ( figure 5 , table 2 ) . many projects have focused on the effects of ace - ang ii - at1 axis . a homolog of ace , ace2 cleaves the terminal phenylalanine residue from ang ii to synthesis ang ( 1 - 7 ) , thereby functioning as a negative regulator of the ras . in this current study , we demonstrated that high - fat - diet - induced ace2 ko mice exhibit a progressive impairment of glucose tolerance and a reduction of insulin secretion in response to glucose , compared with wt mice . in the high - fat - induced ace2 ko mice , loss of ace2 triggered a greater decrease in islet  cell and islet mvd . our study supported a pivotal role of ace2 in the regulation of  cell function and islet vasculature . as the major bioactive component of the classical ras , ang ii mainly interacts with the at1 receptor to exert various actions including vasoconstriction , activation of pro - inflammation , fibrosis and oxidative stress . ace2 is a monocarboxypeptidase cleaving ang ii to form ang ( 1 - 7 ) . as a class of g - protein - coupled receptor , ang ( 1 - 7 ) interacting with mas elicits protective actions such as vasodilatation and nitric oxide generation that counterbalance those of ang ii . the ace2-ang ( 1 - 7)-mas axis may act as a negative modulator of the classical ras effects . there is emerging evidence for a functional ace2-ang ( 1 - 7)-mas axis which is more than regulation of cardiovascular system . the association of the classic ras with the endocrine system has been identified in diabetes and metabolic syndrome . a large number of clinical trials have indicated that inhibitors of the ras , such as acei and arb , reduce the incidence of new - onset t2 dm in high - risk individuals and protect against the development of t2 dm [ 19 , 20 ] . furthermore , inhibition of ras has been also reported to exert protective action to glucose homeostasis in animal models of t2 dm . on the other hand , ace2 may be a potential protective enzyme for glucose homeostasis , but the role of ace2 in the regulation of islet function is incompletely understood . in the present study , there were on differences in fasting blood glucose and glucose tolerance in male ace2 ko mice with standard chow diet compared with corresponding wt mice . this observation was consistent with a previous study by wong et al . , who found that glycemic control was not worsened by the absence of ace2 in the akita diabetic mice . therefore , we supposed that ace2 ablation does not seem to be sufficient to alter glucose status in the condition of regular chow diet . however , an interesting and important finding of the current study was that the consequence of ace2 absence became apparent after high - fat diet . under the condition of high - fat diet , there were much more reductions of the first - stage insulin secretion and glucose tolerance in kh mice compared with wh mice . it is noteworthy that this finding was partly supported by the report of gene therapy in obese diabetic mice . bindom et al .   found that ace2 gene therapy was able to improve fasting blood glucose and glucose tolerance in obese diabetic mice . consistent with the improvement of glycemic control , ace2 gene therapy leaded to enhanced first - stage insulin secretion and reduced  cell apoptosis . our current study for the first time provided evidence that ace2 absence leads to more severe effects in  cell dysfunction in obese mice with high - fat diet . taken together , it is hypothesized that loss of ace2 renders the pancreatic islet more susceptible to the pathological actions of obesity induced by high - fat diet . it is known that islet vascularization is essential for the islet to respond to fluctuations in blood glucose and modulate insulin release as needed . defects in the vascularization of pancreatic islets can lead to deleterious effects on glucose tolerance and islet insulin secretion . in our study , an increased islet vascularization in high - fat diet wt mice was observed compared with chow diet wt mice , which represented a compensatory response to support higher vascularization during  cell expansion . in contrast , no increased islet vascularization was observed in kh mice compared with ko mice . furthermore , kh mice exhibited defects of islet vascularization compared with wh mice . these findings suggested that deletion of ace2 reduces the compensation of islet vascularization in response to high - fat diet . there is an intimate connection between islet beta cell and islet vascularization . on the one hand , islet vascularization has been regarded as playing a crucial role in maintaining beta cell survival and function . on the other hand , vegf , which is highly expressed in islet , has been identified as an important factor to be involved in the regulation of islet vascularization [ 23 , 29 ] . vascular endothelial cells migrate to the source of vegf - a and proliferate and form blood vessels in response to vegf . previous studies [ 31 , 32 ] suggested that mice with specific deletion of the vegf in  cells had reduced islet vascularization and impaired glucose tolerance . it has been reported that reduction of vegf - a by  cells not only reduces the number of intraislet endothelial cells participating in graft vessel formation but also limits recruitment of host endothelial cell and their invasion into the graft [ 31 , 33 ] . in our study , we found an increase of islet vegf in wh mice , which was consistent with the enhancement of islet vascularization after high - fat diet . however , with the deletion of ace2 , this compensatory increase of vegf in islet was disappeared after high - fat diet . this was parallel to the islet  cell impairment and islet vascularization reduction . with the defects of the islet vascularization the deteriorated beta cell might lead to a reduction of vegf expression which in turn accelerates the loss of vascularization . taken together , we speculated that under the condition of high - fat diet , ace2 might play a critical role in the glucose intolerance through impairment of islet vascularization . ace2 not only functions as a carboxypeptidase , cleaving a single residue from ang i , generating ang1 - 9 , and a single residue from ang ii to generate ang 1 - 7 but also cleaves several other biological peptides such as apelin . winzell et al .   reported that apelin-36 inhibited the glucose - stimulated insulin secretion in obese insulin resistant high - fat fed mice . moreover , apelin-13 was shown to inhibit insulin secretion stimulated by high glucose concentrations in ins-1 cells . accordingly , we hypothesized that with the deletion of ace2 , the hydrolyzation of apelin is inhibited , which leads to the inhibition of insulin secretion . peripheral insulin sensitivity has long been regarded as playing a crucial role in glucose homeostasis . we found no significant difference of peripheral insulin sensitivity between ko mice and wt mice . so , we concluded that loss of ace2 has no effects on peripheral insulin sensitivity . it should be noted that there was no difference of the body weight between wt and ace2 ko mice neither with chow diet nor with high - fat diet . accordingly , we speculated that ace2 was not involved in the regulation of body weight . in summary , the results obtained in this study show that loss of ace2 in mice with long - term high - fat diet leads to impairment of glucose tolerance and insulin secretion . the primary mechanisms involved in these effects appear to include a reduction in islet  cell and islet vascularization . our data suggested that the absence of ace2 weakened the adaptive changes of islet  cell function and islet vascularization due to high - fat diet . ace2 seems to be a potential therapeutic target in the treatment of islet dysfunction induced by high - fat diet .
the aim of this study was to evaluate the effects of angiotensin - converting enzyme 2 ( ace2 ) on glucose homeostasis and islet function in mice . 
 male wildtype ( wt ) and ace2 knockout ( ace2 ko ) mice were divided into chow diet group and long - term high - fat diet ( hfd ) group . 
 after 16 weeks of feeding , the islet function of the animals was evaluated by intraperitoneal glucose tolerance test ( ipgtt ) and intraperitoneal insulin releasing test ( ipirt ) . 
 the pancreas was immunohistochemically stained to analyze the relative content of insulin ( irc ) , vascular endothelial growth factor ( vegf ) , and microvessel density ( mvd ) in islets . 
 there was no difference of body weight , area under curve of glucose ( aucg ) , area under curve of insulin from 0 to 5  min ( augi05 ) , mvd , and rvc ( relative content of vegf ) between wt and ace2 ko mice with regular chow diet . under the condition of long - term hfd 
 , the aucg of ace2 ko mice was increased obviously in comparison with the wt mice , with decreased irc , mvd , augi05 , auci030 , and rvc ( all p < 0.05 ) . in conclusion , these results show that ace2 deficiency deteriorates islet function of mice with long - term hfd via impairment of islet microvasculature .