Young capillary vessels rejuvenate agedpancreatic isletsJoana Almaçaa,b,c, Judith Molinac, Rafael Arrojo e Drigoc,d,e, Midhat H. Abdulredaa, Won Bae Jeonf, Per-Olof Berggrena,d,e,g,1,Alejandro Caicedoa,c,h,i,1, and Hong Gil Namb,1

aDiabetes Research Institute, University of Miami Miller School of Medicine, Miami, FL 33136; bCenter for Plant Aging Research, Institute for Basic Science, andDepartment of New Biology, Daegu Gyeongbuk Institute of Science and Technology, Daegu 711-873, Republic of Korea; cDivision of Endocrinology, Diabetesand Metabolism, Department of Medicine, University of Miami Miller School of Medicine, Miami, FL 33136; dLee Kong Chien School of Medicine, NanyangTechnical University, Singapore; eImperial College, London, United Kingdom; fDivision of NanoBio Technology, Daegu Gyeongbuk Institute of Science andTechnology, Daegu 711-873, Republic of Korea; gRolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Stockholm SE-17177,Sweden; and hDepartment of Physiology and Biophysics and iProgram in Neuroscience, University of Miami Miller School of Medicine, Miami, FL 33136

Edited* by Michael J. Berridge, The Babraham Institute, Cambridge, United Kingdom, and approved October 22, 2014 (received for review July 23, 2014)

Pancreatic islets secrete hormones that play a key role in regulat-ing blood glucose levels (glycemia). Age-dependent impairment ofislet function and concomitant dysregulation of glycemia aremajor health threats in aged populations. However, the majorcauses of the age-dependent decline of islet function are stilldisputed. Here we demonstrate that aging of pancreatic islets inmice and humans is notably associated with inflammation andfibrosis of islet blood vessels but does not affect glucose sensingand the insulin secretory capacity of islet beta cells. Accordingly,when transplanted into the anterior chamber of the eye of youngmice with diabetes, islets from old mice are revascularized withhealthy blood vessels, show strong islet cell proliferation, and fullyrestore control of glycemia. Our results indicate that beta cellfunction does not decline with age and suggest that islet functionis threatened by an age-dependent impairment of islet vascularfunction. Strategies to mitigate age-dependent dysregulation inglycemia should therefore target systemic and/or local inflamma-tion and fibrosis of the aged islet vasculature.

aging | pancreatic islets | glucose metabolism | vasculature |insulin secretion

Aging leads to progressive decline of various homeostaticprocesses in mammals, including a deteriorating regulation

of blood glucose levels. Pancreatic islets are small organs com-posed of endocrine cells that secrete the major hormones insulin,glucagon, and somatostatin, which play a key role in regulatingblood glucose levels. Age-dependent dysfunction of islets and theconcomitant dysregulation of blood glucose levels increase therisk for type 2 diabetes (1), which in turn contributes to otherage-related chronic diseases. In general, it has been assumed thataging causes an intrinsic dysfunction of the insulin-secreting betacells through reduced proliferative capacity and/or defective in-sulin secretion (1–9). However, there have been numerous reportsthat age-dependent impairment of glucose homeostasis is not justa result of intrinsic, age-dependent dysfunction of islets but is alsocaused by systemic factors. For example, islet function may becompromised by age-related increases in adiposity (10, 11) and bybloodborne factors (12), or it could be affected indirectly by age-related deficiencies in vascular remodeling (13). Thus, the repli-cative decline of old pancreatic beta cells can be attributed tosystemic factors (12). Recent studies identified factors present inyoung blood that reverse age-related cognitive impairments andinduce vascular remodeling and regeneration in the brain andskeletal muscle (14–16), but so far it has not been feasible todiscriminate systemic influences from aging factors intrinsic toislet endocrine cells. Here we address the long-standing questionof whether the age-dependent impairment of glucose homeostasisis caused by intrinsic, age-dependent dysfunction of islets or bysystemic aging factors.Our strategy to discern age-related intrinsic changes in islet

function was to study islets from young mature (2 mo) and aged

(18 mo) mice and to follow these same groups of islets in threedifferent environments: in vivo in the body of young and agedmice, in vitro after isolation, and again in vivo after trans-plantation into the anterior chamber of the eye in young mice(17). We also examined a large number of human islets fromyoung mature and old pancreatic donors (17–65 y of age). Wehypothesized that islets are affected by the systemic milieu, suchthat the effects the aged organism exerts on the islet can berescued in a young organism. We characterized islet structureand function at the molecular, anatomic, and physiologic levelsto distinguish intrinsic from systemic factors impinging on theislet as the organism ages. Our results reveal that aging of isletsinvolves little intrinsic decline of beta cell function but is ac-companied by malfunctioning blood vessels, suggesting that age-impaired glucose homeostasis is not caused by the intrinsic agingof beta cells but, rather, is a result of vascular aging that can bereversed by placing aged islets in a young environment.

ResultsBeta Cell Secretory Function Is Robust in Aged Mice and Humans.How does aging affect beta cell secretory function? To answerthis question, we used both mouse and human islets, as thesediffer in several aspects, such as cytoarchitecture, cellular plas-ticity, and turnover (18–20). Mouse islets were isolated fromyoung mature (2 mo) and old (18 mo) virgin male C57BL/6 mice

Significance

The regulation of blood glucose is a homeostatic process thatdeclines with age, but it is unknown whether this disturbanceis a consequence of intrinsic dysfunction of the regulatory or-gan, the pancreatic islet. In marked contrast to the widely heldnotion that the insulin-producing pancreatic beta cell losesfunction with wear and tear, and thus causes age-related dis-turbances in glucose homeostasis, we show that mouse andhuman beta cells are fully functional at advanced age. Thepancreatic islet as an organ, however, is threatened by vascularsenescence. Replacing the islet vasculature in aged islet graftsrejuvenates the islet and fully restores glucose homeostasis,indicating that islet blood vessels should be targeted to miti-gate frail glucose homeostasis associated with aging.

Author contributions: J.A., P.-O.B., A.C., and H.G.N. designed research; J.A. and J.M. per-formed research; M.H.A. contributed new reagents/analytic tools; J.A., R.A.e.D., W.B.J.,and A.C. analyzed data; and J.A., A.C., and H.G.N. wrote the paper.

Conflict of interest statement: P.-O.B. is one of the founders of the biotech companyBiocrine, which is going to use the anterior chamber of the eye as a commercial servicingplatform. A.C. holds a patent on this servicing platform. M.H.A. is a consultant of Biocrine.

*This Direct Submission article had a prearranged editor.1To whom correspondence may be addressed. Email: acaicedo@med.miami.edu, per-olof.berggren@ki.se, or nam@dgist.ac.kr.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1414053111/-/DCSupplemental.

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from the National Institute on Aging that do not develop di-abetes as they age (21). Human islets were obtained from non-diabetic donors (range 17–65 y). In perifusion experiments, isletsisolated from aged mice released significantly more insulin percell in response to high glucose levels (11 mM; Fig. 1 A and B).

The amount of insulin released by human islets did not showa statistically significant correlation with donor age [r2 = 0.041(P = 0.08) and r2 = 0.01 (P = 0.39), respectively; Fig. 1 C and Dand SI Appendix, Fig. S1]. Even when pooling insulin secretiondata from young human islets (donor age, 17–27 y; n = 10) andcomparing these data with those from islets of aged humans (donorage, 50–60 y; n = 25), no significant differences were observed. Wealso found that islets from old mice are capable of healthy buff-ering of [Ca2+]i in response to high glucose (SI Appendix, Fig. S1).These results indicate that as they age, mouse and human beta cellsremain glucose-sensitive, produce adequate responses to glucose,and have robust insulin secretion.Cytoarchitecture was similar in young and old mouse islets,

with alpha cells in the periphery and beta cells in the core (SIAppendix, Fig. S2), and islets retained similar proportions of eachcell type (SI Appendix, Fig. S2). We consistently observed thatislets in aged mice were larger, which was a result of a simulta-neous increase in beta cell number and size (SI Appendix, Fig.S2). Islet area, beta cell number, and size were not significantlydifferent between islets of young (age 15–25 y) and old (age 50–60 y) humans, showing that beta cell mass is preserved in humanswith advanced aging [SI Appendix, Fig. S2 (22)].

Glucose Homeostasis Is Impaired in Old Mice. The intrinsic ability ofbeta cells to produce and secrete insulin and the structuralproperties of islets did not deteriorate in old mice, but do agedmice have a normal glucose metabolism? We compared glucosemetabolism in young and aged mice and found that old mice wereinsulin-resistant (Fig. 1E). Plasma insulin levels were twice as highin old mice (Fig. 1F), and blood glucose levels in nonfastingconditions were lower in old mice (SI Appendix, Fig. S1). Theseresults suggest that old mice compensate for the increased de-mand for insulin, as indicated by insulin resistance, by increasingbeta cell mass and beta cell secretory function (Fig. 1 A and B andSI Appendix, Fig. S2). We further compared glucose tolerance inyoung and aged mice and found that blood glucose levels duringglucose tolerance tests did not differ when challenged with a con-ventional glucose load (2 g/kg; Fig. 1G). However, when micewere challenged with a larger glucose load (4 g/kg; Fig. 1G) orwith a conventional glucose load under stress conditions (Fig. 1H)(23), the recovery was delayed in old mice. These results indicatean age-dependent decline in glucose homeostasis in mice.

Aged Pancreatic Islets Have Inflamed and Fibrotic Blood Vessels.Given the lack of functional decline in beta cells of old mice,what, then, is leading to impaired glucose homeostasis? Islets arestrongly vascularized, as their ability to sense blood glucose andrelease insulin depends on close contact with blood vessels.Because advanced age is associated with vascular alterations andchronic inflammation (24), we tested whether the age-associatedimpairment in glucose homeostasis is caused by islet blood vesseldysfunction. Blood vessel density did not differ between youngand old mouse islets (14.5 ± 2.1% versus 14.8 ± 1.5%, re-spectively; n = 3 islets/pancreas, n = 3 pancreata/age). To ex-amine the inflammatory status of the islet, we immunostainedmacrophages in pancreatic sections of young and old mice andhumans (Fig. 2 A and B and SI Appendix, Fig. S3) and found thatislets in aged mice and humans contained twice as many mac-rophages (Fig. 2B). Macrophages were often associated withblood vessels expressing intercellular adhesion molecule 1(ICAM-1) (Fig. 2A), an adhesion molecule and inflammatorymarker (25) whose expression was increased in islets of agedmice (Fig. 2C). These findings indicate that blood vessels in agedislets are inflamed. This was further supported by increasedexpression of macrophage colony-stimulating factor receptor(CSFR1) and genes involved in immune cell recruitment such asICAM1 and vascular cell adhesion molecule 1 (VCAM1) in oldislets (Fig. 2D).

Fig. 1. Beta cells in aged mice and humans are functionally robust, but glu-cose tolerance is fragile. (A) Insulin secretion from islets isolated from young (2mo, green) and aged (18 mo, brown) C57BL/6 mice, stimulated with 11 mMglucose and KCl (25 mM; n = 4; insulin levels normalized to DNA concentra-tion). (B) Total amount of insulin released during high glucose (area under thecurve). (C and D) Insulin secretion in response to high glucose (11 mM) fromhuman islets from 82 cadaveric donors (ages, 17–65 y). Peak insulin secretion(C) denotes the peak of first-phase insulin secretion, and total insulin secretion(D) denotes the total amount of insulin released during 20 min glucose(expressed relative to responses to KCl). (E) Insulin tolerance tests performedwith young and old mice (0.75 units insulin/kg body weight, i.p.; n = 10 mice;glycemia normalized to value at t0). (F) Fed plasma insulin concentration (n =10 mice). (G and H) Glucose tolerance tests with young and aged mice withdifferent glucose loads (G: 2 g/kg, n =10 mice, open symbols; 4 g/kg, n = 5mice, filled symbols; one-way ANOVA, * P < 0.05) or in restrained mice (H:2 g/kg, n = 7–8 mice, area under the curve, 28,010 ± 581 for young mice versus36,350 ± 3399; *P = 0.02). Mean ± SEM are shown.

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Fibrosis is a hallmark of aging in many organs (26, 27). Wefound that blood vessels in islets of aged mice contained morelaminin, a biomarker of fibrosis (28) (Fig. 2 F–H). In addition,expression of the matrix metalloproteinase genes MMP2 andMMP9, which are also involved in fibrosis and remodeling of theextracellular matrix, increased in islets of aged mice (Fig. 2E). Thisresult is in line with the increased macrophage density in old mouseislets, as macrophages are key sources of matrix metalloproteinases(29). Extensive accumulation of fibrotic material was also observedin the extracellular matrix of islet blood vessels in pancreata fromold human donors, as assessed by laminin immunostaining (SIAppendix, Fig. S3). Fibrosis and macrophage infiltration was evenmore evident in islets from an old donor with diabetes (SI Ap-pendix, Fig. S3). Together, our data show that blood vessels in isletsof aged mice and humans are inflamed and exhibit fibrosis.

Aged Islet Grafts Functionally Recover in Young Recipient Mice After aProlonged Period. Our results show that different tissue componentsin islets take separate aging paths: in old islets, blood vessels

become inflamed and fibrotic, but beta cells remain functional.Because the vasculature is a systemic organ, we tested whethervascular defects observed in aged islets are caused by systemic orlocal pancreatic influences, rather than intrinsic aging of islets.To discriminate between these factors, we used a transplantation

Fig. 2. Aged pancreatic mouse and human islets have inflamed and fibroticblood vessels. (A) Maximal projection of confocal images showing macro-phages (green) and endothelial cells expressing the adhesion molecule ICAM-1(red) in an old islet. (Scale bar, 20 μm.) (B) Quantification of the number ofmacrophages per islet area (mm2) in islets in pancreatic sections from youngand old mice and humans. (C) Quantification of the fractional area of ICAM-1immunostaining inside islets in young and aged mice. (D) Transcript levels ofmacrophage colony-stimulating factor receptor gene (CSFR1) and genes in-volved in the recruitment of immune cells (ICAM1, VCAM1). (E) Expressionlevels of the extracellular matrix remodeling genes MMP2 and MMP9 in oldislets, relative to values in young islets (*P < 0.05, unpaired t test). 18S RNA wasused as an internal reference (n = 200 young or old islets, three replicates). (Fand G) Maximal projections of confocal images of islets labeled for laminin(green) and the endothelial cell marker PECAM (red) in pancreatic sectionsfrom young (F) and aged (G) mice. (Scale bars, 50 μm.) (H) Quantification ofthe fractional area of laminin immunostaining inside young and old islets.

Fig. 3. Aged islet grafts functionally recover after a prolonged period inyoung recipient mice. (A) Illustration of islet transplantation into the ante-rior chamber of the eye. (B) In the experimental strategy, young and oldislets were transplanted into the eye of young mice with diabetes and fol-lowed for 11 mo (gray arrow). Green and brown arrows, respectively, in-dicate the actual aging of young and old islet grafts. Dashed line representsthe survival curve of C57BL/6 mice and shows that aged islets were studiedbeyond the 50% survival age [28 mo old (36)]. (C) Fed glycemia in strepto-zotocin (STZ)-induced young mice with diabetes after transplantation ofyoung (green; n = 5 mice) and old (brown; n = 6 mice) islets (enucleation =removal of the islet graft-bearing eye). (D) Percentage of young mice withdiabetes (fed glycemia > 200 mg/dL) transplanted with young (green) or old(brown) islets and of old mice with diabetes transplanted with old islets(black). (E and F) Average glycemia during glucose tolerance tests performedat 3, 4.5, 7, and 10 mo after transplantation in young mice with young islets(E; n = 5 mice) or old islets (F; n = 5 mice). (G) Glycemia at 120 min (t120) afterglucose injection in mice with young (green) and old islets [brown; one-wayANOVA, *P < 0.05; dashed line, glycemia (t120) in young donor mice]. (H) Fedplasma insulin concentration in young and old islet recipient mice.

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strategy in which islets are transplanted into the anterior cham-ber of the mouse eye. Transplanted islets have been shown toengraft on the iris, reverse diabetes, and regulate glucose ho-meostasis in the recipient mice (17, 30, 31). We transplanted 200islets from young (2 mo) and aged (18 mo) mice into the eye ofyoung mice with diabetes (2 mo; Fig. 3A). This experimentalsetup allowed us to compare the function of transplanted youngand old islets in the same systemic environment and to follow inreal time and noninvasively the age-dependent changes in isletgrafts (Fig. 3B).In this experimental setup, it was evident that islets from aged

mice (“aged” islets) can reverse diabetes and maintain glucosehomeostasis in young recipient mice for prolonged periods oftime. Within 3 mo after transplantation, most of the recipientmice with diabetes transplanted with aged islets recovered nor-mal blood glucose levels similar to mice transplanted with isletsfrom young mice (“young” islets; Fig. 3 C and D). In contrast,aged islets transplanted into old hosts reversed diabetes only inhalf of the recipients, and only after a prolonged time (6 mo; Fig.3D). Within 7 mo after transplantation, glucose tolerance ofmice transplanted with aged islets was indistinguishable fromthat of young islet recipients (Fig. 3 E–G). Furthermore, plasmainsulin levels were similar between the two groups within 9 moafter transplantation (Fig. 3H). During this period, mice withaged islet grafts were able to grow and to gain weight at a ratesimilar to that of mice with young islets, albeit with some growthdelay at the initial stages after transplantation (SI Appendix, Fig.S4). These results show that after a prolonged period in younghosts, aged islet grafts become as functionally competent asyoung islet grafts.A reduced proliferation rate of old beta cells has been pro-

posed as one of the main causes of age-dependent loss of glucosehomeostasis (7). Because aged islets were able to rescue re-cipient mice from diabetes, similar to young islets, we examinedthe proliferative activity of aged and young islet grafts trans-planted into the eye of young recipients. The young recipientmouse continued to grow and gain weight in the period from 3 to7 mo after transplantation (SI Appendix, Fig. S4). During thisperiod, both aged and young islet grafts grew similarly (Fig. 4 Aand SI Appendix, Fig. S4). Growth of the islets grafts was in part

a result of beta cell proliferation (Fig. 4 B–D and SI Appendix,Fig. S4). Aged islet grafts mounted a stronger proliferative re-sponse most likely caused by a higher glycemic pressure in theinitial months after transplantation (Fig. 3 C and D) (32, 33).Our results show that given enough time to recover in a young

organism, aged islets can regulate glucose homeostasis, secreteinsulin, and mount a proliferative response that is as strong as inyoung islet grafts. Thus, aged islets are not intrinsically old, buttheir age-dependent impairment can be rescued by systemicfactors from a young host.

Functional Recovery of Aged Islet Grafts in a Young Host Is Associatedwith Appearance of New Blood Vessels. Although mice trans-planted with aged islets recovered from diabetes, they tooklonger than recipients of young islets to return to normoglycemia(3 versus 2 mo; Fig. 3 C and D). In addition, mice transplantedwith aged islets were less glucose-tolerant at 3 and 4.5 mo aftertransplantation, as shown by a delayed return to basal glycemiclevels 120 min after glucose load (Fig. 3 E–G). Up to 7 mo aftertransplantation, aged islet recipients also had diminished plasmainsulin levels (Fig. 3H).Islets from aged mice secreted more insulin (Fig. 1 A and B). It

was thus puzzling that islets from aged donors took longer toreverse diabetes and produced lower plasma insulin levels aftertransplantation. Because aged islets had defective blood vessels(Fig. 2), we monitored the engraftment of individual islets non-invasively and longitudinally (Fig. 5 A and B and SI Appendix,Fig. S5). We found that revascularization of aged islet grafts wasdelayed by 1 mo (SI Appendix, Fig. S5), which coincided with thedelay in diabetes reversal (Fig. 3 C and D). Aged islets had lowervessel densities than young islets in the first month after trans-plantation, but they showed noticeable revascularization withinthe following month (SI Appendix, Fig. S5).In addition to a slower initial revascularization, blood vessels

in aged islet grafts were larger and did not branch out as much asblood vessels in young islet grafts (Fig. 5B). The diameter of thecapillaries at the end of the revascularization period in youngislet grafts (7.5 ± 0.1 μm) was close to that measured in corrosioncasts of islets in rat pancreata [6 μm (34)], indicating that bloodvessels reached appropriate vascular sizes. Blood flow in largervessels was faster and more turbulent than that in smallercapillaries (SI Appendix, Fig. S5 and Movies S1 and S2), thus

Fig. 4. Old islet grafts in young recipient mice show strong proliferativeactivity. (A) Photograph of a mouse eye (*, pupil) showing two old isletgrafts 3 and 9 mo after transplantation. A marked increase in islet graft sizecan be seen. (Scale bar, 200 μm.) (B) Quantification of the fraction of pro-liferating beta cells (*P < 0.05; n = 6 young or old islet grafts). BrdU (1 mg/mL)was added for 21 d to the drinking water at 11 mo after transplantation.(C and D) Confocal images of a young (C ) and an aged (D) islet graftshowing BrdU-labeled, proliferating cells (green, BrdU; red, insulin; blue,DNA). Arrows point at beta cells, arrowhead indicates a nonbeta cell.(Scale bars, 50 μm.)

Fig. 5. Revascularization of old islet grafts. (A) Longitudinal in vivo imagesof blood vessels in an old islet graft. Capillaries with smaller diameters ap-pear in newly formed regions. (Scale bars, 100 μm.) (B) Distribution curves ofthe diameters of blood vessels in young and old islets grafts at 2 wk and 2and 10 mo after transplantation. (C and D) Quantification of the fractionalarea of laminin immunostaining (C) and of the number of macrophages perislet area (mm2, D) in young and old islet grafts 11 mo after transplantation(n = 3 islet grafts/eye; n = 2 recipient mice/age).

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diminishing the efficiency of transcapillary exchange (35). Dur-ing the last 4 mo, however, capillaries with small diametersappeared in newly formed regions of aged islet grafts (Fig. 5 Aand B and SI Appendix, Fig. S5). We conclude that the dys-functional vascular phenotype of aged islet grafts, characterizedby larger diameters and turbulent blood flow, contributed tolower plasma insulin levels and glucose intolerance during thefirst 7 mo after transplantation (Fig. 3 E–H), and appearance ofsmall capillaries in the following months likely favored thefunctional recovery of aged islets.Because aged islets in the pancreas displayed an inflamed and

fibrotic vascular phenotype (Fig. 2), we compared these pheno-types in blood vessels formed in young and aged islet grafts.Expression levels of laminin were similar in young and old isletgrafts 11 mo after transplantation (Fig. 5C), in contrast to theage-related differences we observed in the pancreas (Fig. 2H).Laminin expression in old islet grafts was heterogeneous, withsmaller vessels containing less laminin (SI Appendix, Fig. S6).The number of macrophages in young and aged islet grafts wasnot significantly different at this stage (Fig. 5D). These resultsshow that the young environment reverses the inflamed and fi-brotic nature of blood vessels, even though the actual age of oldislet grafts at the end was 29 mo.

DiscussionOur study demonstrates that the ability of beta cells to secreteinsulin does not decline with age in mice and humans (Fig. 1 A–D).The most notable feature of islets in aged mice and humans wasa deranged vasculature, characterized by increased inflammationand fibrosis of islet blood vessels (Fig. 2 and Fig. S3), which likelyled to dysfunction of aged islets (Fig. 1 E–H). After prolongedresidence in a young host, islets from aged mice display newblood vessels without inflammatory and fibrotic phenotypesand function similar to islets from young mice. This observa-tion is striking, given that the aged islets under examinationwere 29 mo old, which is near the end of the mouse’s life span[equivalent to an 80-y-old human (36); Fig. 3B]. Our resultsclearly indicate that beta cells in mice and humans show littlefunctional decline, but islets undergo an age-dependent de-cline in their vascular function.All organisms experience a slow physiological decline and in-

creased risk for disease with aging. Our results show that pan-creatic islets in mice are affected by systemic aging, and agedmice exhibit age-dependent deterioration of glucose homeo-stasis, despite beta cells being fully competent in advanced age.In particular, aged mice were insulin-resistant and glucose-intolerant, although intolerance was only observed when aged micewere forced to release more insulin; for example, after a higherglucose load (37, 38). Our results concur with those of severalprevious studies (3, 5, 6, 39) but contrast with studies reportinginsulin secretory defects with age (1, 2, 4, 8). The discrepancies inthe literature may be explained by the confounding influence ofthe systemic environment (10). For example, aging is associatedwith an increase in visceral fat, with higher levels of circulatingproinflammatory cytokines secreted by adipocytes, and with in-creased inflammation of tissues such as the local pancreaticenvironment that contribute to insulin resistance and disturbbeta cell proliferation and function (40, 41). However, whensystemic effects are compensated for, as we did here, aged isletsfunction similar to young islets, indicating there is little intrinsicage-related decline in beta cell function.Diabetic predisposition and added risk factors and epigenetic

regulation, however, may trump the resiliency of the beta cell (9,42). Our study now shows that the islet as an organ seems to bethreatened by factors that affect vascular function. The increasedfunctional demand aged islets face may lead to increased isletblood flow (43, 44), which in turn may trigger increased capillarypressure and compromise islet microcirculation (45). In addition,

with aging, the vascular phenotype makes a proinflammatoryshift that contributes to endothelial dysfunction (46). As demandfor insulin increases with age, beta cells also cosecrete more ATPand other molecules that are potentially proinflammatory andthat stimulate cytokine secretion and innate immune responses(47–49). The fibrosis associated with blood vessels in the agedislet likely diminishes hormone diffusion through the interstitialspace and disrupts hormone release into the circulation (35),delaying insulin delivery to target tissues and causing the fragileglucose tolerance we observed in aged mice (Fig. 1 E–H). Ofnote, local islet inflammation and fibrosis are further increasedin type 2 diabetes [SI Appendix, Fig. S3 (50)]. A dysfunctionalvasculature also helps explain why there is a discrepancy betweenin vitro data showing no deterioration of insulin secretion withage (5) and in vivo studies reporting lower plasma insulin levelsin response to hyperglycemia in aged mice (6, 10).The damaging effects of inflamed and fibrotic blood vessels

were exacerbated in the context of transplantation. Indeed, theaged islet showed delayed islet graft revascularization, likelybecause the inflamed and fibrotic vascular cells are transferredtogether with the donor islet and participate in early processes ofblood vessel formation (30, 51). With time, the young systemicenvironment reversed these effects by replacing the vasculaturewith healthy blood vessels with diameters close to those of pan-creatic capillaries (5–7 μm) and with regular blood flow [Fig. 5and Fig. S5 (34)], coinciding with restored glucose tolerance(Fig. 3).In mice, aged islets are able to adapt to increased demand for

insulin caused by insulin resistance, possibly through increasedislet size and increased insulin secretory capacity (Fig. 1 A and Band SI Appendix, Fig. S2), in agreement with previous reports(5). This was accompanied by increased beta cell numbers, in-dicating some beta cell proliferation during aging (SI Appendix,Fig. S2). In the transplantation experiment, we observed thataged islet grafts mounted an even stronger proliferative re-sponse to accommodate for the organismal growth and in-creased insulin demand of the young recipient mouse. Theseresults are remarkable because a diminished proliferative re-sponse is considered a hallmark of islet aging (7). However, ourstudy concurs with recent studies (32, 33) showing that betacells in old mice can increase their proliferative rate underparticular circumstances.Most studies on islet aging have focused on the age-related loss of

regenerative capacity of beta cells, presumably because of its ther-apeutic implications. Our results indicate that the functional prop-erties of beta cells in humans, as well as in mice, change little as theadult organism ages. Instead, bloodborne factors, low-grade chronicinflammation, and other factors affecting vascular function mayrepresent larger threats to islet health and glucose homeostasis.Potential strategies for mitigating age-related impairment in isletfunction should therefore target systemic or local inflammationand fibrosis within the islet. Although expanding beta cell massmay be desirable for future therapies, improving the local envi-ronment of the otherwise healthy aged beta cell could preventage-associated deterioration in glucose tolerance and promotehealthy aging.

MethodsWe used young mature (2 mo old) and old (18 mo old) virgin C57BL/6 micefrom the National Institute on Aging. Human islets (n = 82 preparations;age range: 17–65 y) were obtained from the Integrated Islet DistributionProgram of the National Institute of Diabetes and Digestive and KidneyDiseases. Human pancreatic tissue used was from young (15–25 y) and old(50–60 y) donors.

Islet isolation, transplantation into the anterior chamber of themouse eye,and in vivo islet imaging were performed as previously described (17, 31).Blood vessels were labeled by tail vein injection of 150,000 Da Dextran-FITC.Diabetes was induced in young mice with streptozotocin (200 mg/kg, i.v.)before 200 mouse islet equivalents from young and old donors were

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transplanted. BrdU (1 mg/mL) was added to the drinking water for 21d at the end of the study.

Assessment of Islet Function in Vivo. Islet function was monitored by mea-suring glycemia and plasma insulin under fed conditions, as well as duringglucose and insulin tolerance tests (17).

Assessment of Islet Function in Vitro. Perifusion and [Ca2+]I were performed aspreviously described (49). Insulin secretion from isolated mouse and humanislets (100 islets per column) was stimulated with 11 mM glucose or 25 mMKCl. Insulin content was normalized for DNA.

Statistical Analyses. Statistical tests were performed with Prism 5.0 software(GraphPad Software). Significance was considered when P < 0.05 (unpairedStudent t test or one-way ANOVA). Data presented as mean ± SEM.

A complete description of materials and methods is available in theSI Appendix.

ACKNOWLEDGMENTS. This work was funded by the Institute for BasicScience (IBS-R013-D1-2014-a00) and a Korean Ministry of Education andScience Technology grant (The National Honor Scientist Support Program2010-0020417) (to H.G.N.); Diabetes Research Institute Foundation (to P.-O.B);NIH Grants R56DK084321 and R01DK084321 (to A.C.); Daegu GyeongbukInstitute of Science and Technology Grant 14-NB-01 and Ministry of Sci-ence, Information & Communication Technology and Future PlanningGrant 2014R1A2A2A01005619 (to W.B.J.); the Juvenile Diabetes ResearchFoundation; the Swedish Research Council; the Novo Nordisk Foundation;the Swedish Diabetes Association; the Family Erling-Persson Foundation;the Skandia Insurance Company Ltd; Strategic Research Program in Diabe-tes at Karolinska Institutet; the Berth von Kantzow’s Foundation; VIBRANT(in vivo imaging of beta-cell receptors by applied nanotechnology) GrantFP7-2288933; the Knut and Alice Wallenberg Foundation; Funds of KarolinskaInstitutet, Diabetes and Wellness Foundation; the Stichting af Jochnick Foun-dation; and Lee Kong Chien School of Medicine, Nanyang Technical University,Singapore and Imperial College, London, United Kingdom ERC-2013-AdG338936-BetaImage. J.A. is a recipient of a postdoctoral fellowship from theAmerican Heart Association (14POST20380499).

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