Oestrogens improve human pancreatic islet transplantation in a mouse model of insulin deficient diabetes
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Pancreatic islet transplantation (PIT) offers a physiological treatment for type 1 diabetes, but the failure of islet engraftment hinders its application. The female hormone 17β-oestradiol (E2) favours islet survival and stimulates angiogenesis, raising the possibility that E2 may enhance islet engraftment following PIT.
To explore this hypothesis, we used an insulin-deficient model with xenotransplantation of a marginal dose of human islets in nude mice rendered diabetic with streptozotocin. This was followed by 4 weeks of treatment with vehicle, E2, the non-feminising oestrogen 17α-oestradiol (17α-E2), the oestrogen receptor (ER) α agonist propyl-pyrazole-triol (PPT), the ERβ agonist diarylpropionitrile (DPN) or the G protein-coupled oestrogen receptor (GPER) agonist G1.
Treatment with E2, 17α-E2, PPT, DPN or G1 acutely improved blood glucose and eventually promoted islet engraftment, thus reversing diabetes. The effects of E2 were retained in the presence of immunosuppression and persisted after discontinuation of E2 treatment. E2 produced an acute decrease in graft hypoxic damage and suppressed beta cell apoptosis. E2 also acutely suppressed hyperglucagonaemia without altering insulin secretion, leading to normalisation of blood glucose.
During PIT, E2 synergistic actions contribute to enhancing human islet-graft survival, revascularisation and functional mass. This study identifies E2 as a short-term treatment to improve PIT.
KeywordsDiabetes Engraftment Glucagon Islets Oestrogen Survival Transplantation
G protein-coupled oestrogen receptor
Juvenile Diabetes Research Foundation
Pancreatic islet transplantation
Thiobarbituric acid reactive substances
Replacing insulin-producing beta cells by human pancreatic islet transplantation (PIT) offers a physiological therapeutic approach for type 1 diabetes. PIT benefits recipients in a way that insulin therapy can never achieve [1, 2, 3]. Currently, most immunosuppressed type 1 diabetic recipients of human islets allotransplant [3, 4] can successfully attain insulin independence and protection from hypoglycaemia at 1 year. However, there are critical limitations to the widespread application of PIT. First, procuring sufficient islet yield requires several deceased human donors and novel strategies are needed to achieve insulin independence with fewer islets . Evidence suggests that as little as 20% of the islets within a pancreas are required to maintain euglycaemia . Still, the islets of one or two pancreases are needed to reverse diabetes following PIT, suggesting that a significant portion of the transplanted islets are destroyed or become non-functional. This early islet loss is believed to represent up to 70% of the transplanted islet mass [6, 7, 8]. The transplanted islets are avascular, and islet revascularisation takes 2–3 weeks to complete. During that period, the islets are vulnerable to hypoxic stress and may be destroyed before revascularisation and engraftment occur [9, 10]. Thus, in the immediate post-transplant period, apoptosis and failure of vascular engraftment pose a challenging problem. We must explore new therapeutic approaches promoting islet survival and revascularisation to enhance islet engraftment.
Recently, the female hormone, 17β-oestradiol (E2) has been shown to protect mouse and human islets from apoptosis induced by multiple injuries and to stimulate insulin biosynthesis via oestrogen receptors (ERs) present in beta cells [11, 12, 13, 14, 15, 16, 17, 18]. E2 also stimulates endometrial angiogenesis and promotes endothelial cell recovery after injury [19, 20, 21, 22, 23]. Together, these findings raise the prospect that E2 may be applied to improve islet functional mass and revascularisation during PIT. Here, we used an insulin-deficient mouse model with xenotransplantation of a marginal dose of human islets to test the hypothesis that oestrogens improve islet engraftment during PIT.
Animals and the induction of experimental diabetes
Diabetes was induced in 10-week-old male nude mice (C57BL/6; Harlan Laboratories, Indianapolis, IN, USA) or male mice null for Erα  by a single i.p. injection of 200 mg/kg streptozotocin (STZ; Sigma Aldrich, St Louis, MO, USA). Blood glucose was monitored with a One Touch Ultra Glucose Monitor (LifeScan, Milpitas, CA, USA). Mice with fed blood glucose exceeding 20.8 mmol/l were used as recipients. All animal experiments were approved by Northwestern University Animal Care and Use Committee.
Human islet transplantation and in vivo oestrogen treatment
Human islets were obtained through the Integrated Islet Distribution Program and cultured for 48 h in phenol-red-free CMRL-1066 medium (Sigma Aldrich) containing 10% (vol./vol.) charcoal-stripped FBS (Gemini Bio, West Sacramento, CA, USA). For in vivo treatment, E2, 17α-oestradiol (17α-E2) and G1 (0.18 mg/pellet), propyl-pyrazole-triol (PPT) and diarylpropionitrile (DPN) (3.6 mg/pellet) 60-day-release pellets (Innovative Research of America, Sarasota, FL, USA) were implanted subcutaneously immediately before islet transplantation. The dose of E2 was chosen to obtain serum concentrations within physiological limits (ranging from oestrus to pregnancy). The doses of 17α-E2 and G1, PPT and DPN were chosen according to the affinity of each agonist for its ER to achieve similar activation of ERs as caused by E2. A marginal dose of 1,000 islet equivalents (IEQ) of human islets were transplanted under the kidney capsule of recipient mice.
Immunosuppression was achieved as described in the Edmonton protocol, following adaptation to the mouse [1, 25]. Sirolimus (rapamycin; LC Laboratories, Woburn, MA, USA) was administered via i.p. injection every other day at the dose of 0.1 mg/kg for 4 weeks, starting from the day of islet transplantation. Tacrolimus (FK506; Cayman, Ann Arbor, MI, USA) was administered i.p. daily at 1 mg/kg, starting on the day of islet transplantation. Control animals received daily vehicle injections.
Kidneys bearing islet grafts were fixed overnight in 4% (wt/vol) paraformaldehyde at 4°C. The tissues were immersed in 30% (wt/vol) sucrose and embedded in tissue-freezing medium (Tissue-Tek; Sakura Finetek, Torrance, CA, USA). Sections, 5–10 μm, were mounted on charged slides. For immunohistochemical studies, the following primary antibodies were used: guinea pig anti-human insulin (1:1,000; Linco Research, Saint Charles, MO, USA); rat anti-mouse CD31 (1:400; BD Biosciences, San Jose, CA, USA); rabbit anti-mouse Ki67 (1:400; Novocastra, Newcastle Upon Tyne, UK); rat anti-mouse F4/80 (1:200; AbD Serotec, Raleigh, NC, USA); mouse anti-ERα (1D5; 1:100; Zymed Laboratories, South San Francisco, CA, USA); and goat anti-ERβ (Y-19; 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The secondary antibodies were: Cy3-conjugated donkey anti-guinea pig; Cy3-conjugated goat anti-rat; biotinylated goat anti-rat; FITC-conjugated goat anti-rat; and FITC-conjugated donkey anti-rabbit (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Biotinylated goat anti-rat was visualised using the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA, USA). For staining with ERα and ERβ, the Alexa 568 tyramide signal amplification kit (Molecular Probes, Eugene, OR, USA) was used. For nuclear staining, the sections were incubated with DAPI (Molecular Probes, Eugene, OR, USA). Images were obtained with a Nikon Eclipse E400 microscope (Nikon Instruments, Melville, New York, USA) or TissueGnostics High Throughput Imaging System (TissueGnostics, Vienna, Austria).
Measurement of apoptosis using TUNEL assay
Apoptotic cells were detected by TUNEL assay using a fluorescein in situ cell death detection kit (Roche, Indianapolis, IN, USA) 1 day after transplantation. Frozen tissue sections, 5 μm, were fixed at room temperature for 1 h in 4% (vol./vol.) PFA in PBS, pH 7.4. Samples were washed in PBS for 30 min. Following the manufacturer’s instructions, sections were permeabilised, washed, labelled, incubated and analysed. Sections were subsequently stained with guinea pig anti-human insulin as a primary antibody (1:1,000) and Cy3-conjugated donkey anti-guinea pig as a secondary antibody. Images were obtained with a Nikon Eclipse E400 microscope.
Measurement of oxygenation in transplanted islet graft
Islet oxygenation was investigated in the transplants, 1 day post-transplantation . Pimonidazole hydrochloride (hpi Hydroxyprobe-1; Omni Kit, Burlington, MA, USA) (60 mg/kg) was injected i.p. Mice were killed 3 h later, and their kidney containing the islet graft was processed for immunohistochemistry. Rabbit anti-pimonidazole antibody (hpi Hydroxyprobe-1; Omni Kit) was used to visualise pimonidazole hydrochloride accumulation in hypoxic cells of the graft.
Kidneys bearing islet grafts were sectioned in 10 μm thickness and four sections per tissue were randomly chosen for morphometric analysis. Anti-human-insulin and anti-mouse-CD31 antibodies were used to visualise beta cells and blood vessels, respectively. Morphometric analysis was conducted using the ImageJ 1.37v (rsb.info.nih.gov/ij/) program. In islet transplants, the beta cell area was calculated by dividing the insulin-positive area by the graft area. Blood vessel density was calculated by dividing the mouse-CD31-positive area by the graft area . The demarcation of an islet graft was considered to be the parenchyma of the surrounding kidney as described .
Hormone and thiobarbituric acid reactive substances assays
Human insulin and mouse glucagon was measured in serum by ELISA and RIA, respectively (Linco, St Charles, MO, USA). Systemic oxidative stress was assessed by measuring the concentration of thiobarbituric acid reactive substances (TBARS) in plasma (Cayman) and kidney homogenates (ZeptoMetrix, Franklin, MA, USA) .
Biotinylated tomato lectin (Vector Laboratories) was injected into the tail vein (200 μg) and allowed to circulate for 5 min before the mice were killed. The kidneys bearing the islet grafts were removed and prepared for histological study. Lectin staining was visualised using the Vectastain Elite ABC kit (Vector Laboratories). Functional blood vessel density was obtained by dividing the lectin-positive area by the graft area.
Data are presented as mean ± SEM unless otherwise stated. For the rodent study, data were analysed by either the unpaired Student’s t test or one-way ANOVA. A value of p < 0.05 was considered statistically significant.
Oestrogens ameliorate diabetes
Oestrogens enhance islet engraftment
We next quantified the endothelial cell area in islet grafts using the mouse endothelial cell marker CD31. At 4 weeks after PIT, mice treated with E2, 17α-E2, PPT or DPN showed an improvement in islet revascularisation compared with vehicle-treated mice (Fig. 2f, g). Conversely, the mice treated with G1 did not show any improvement compared with controls (Fig. 2f, g). To determine the functionality of the E2-induced reconstructed islet vasculature network, we applied in vivo staining for lectin—a commonly used marker of vessel functionality —to a group of mice treated with vehicle or E2. We detected increased lectin staining in E2-treated mice compared with vehicle-treated mice (Fig. 2h, i), demonstrating that E2 increased functional vessel density in the transplanted islets. While the contribution of endothelial cells from freshly isolated donor islets to the formation of functional vessels within islet grafts has been demonstrated [33, 34], cultured islets lose most of their endothelial cell populations within 3 days of culture . We thus examined whether E2 treatment could preserve the intra-islet endothelial cell population in freshly isolated mouse islets. Consistent with a previous report , we observed that the islet endothelial cell number decreased within 1 day of culture and totally disappeared after 3 days. However, we observed no beneficial effect of E2 treatment on the maintenance of intra-islet endothelial cell population (ESM Fig. 4). As E2 favours islet survival, we tested whether in vitro E2 treatment of human islets prior to PIT could improve PIT outcome using the same model system. We observed that E2 treatment of cultured human islets prior to PIT had no significant effect on subsequent glycaemic control either alone or in association with in vivo E2 treatment (ESM Fig. 5).
Improvement of islet engraftment persists in the presence of immunosuppression and after discontinuation of E2 treatment
E2 acutely improves islet-graft hypoxia
E2 acutely suppresses graft beta cell apoptosis
PPT acutely suppresses blood glucose in the absence of ERα in the islet graft
E2 acutely suppresses glucagon production
The present study shows that E2, used at physiological concentrations, improves islet engraftment in a sex-non-specific manner in an insulin-deficient nude mouse model transplanted with a marginal dose of human islets and in the absence of allorejection or autoimmunity recurrence. Several non-exclusive mechanisms could be implicated.
Suppression of hyperglucagonaemia
The acute correction of hyperglycaemia and improvement of glucose tolerance observed 1 day after transplantation and following E2 treatment occur without an increase in graft insulin secretion. In fact, E2 treatment produces a decrease in fed serum insulin concentrations, suggesting that E2 has improved insulin sensitivity. Indeed, the E2 hypoglycaemic action occurs in the face of better suppression of serum hyperglucagonaemia. This observation suggests that the E2-mediated acute suppression of blood glucose is independent of an improvement in beta cell function; rather, it is dependent on glucagon suppression. This may explain why E2-treated mice have suppressed hyperglucagonaemia and lower insulin concentrations, as a decrease in hyperglucagonaemia improves glucagon-induced hepatic insulin resistance. This observation is also consistent with the ‘glucagonocentric’ vision of diabetes pathophysiology in which glucagon excess, rather than insulin deficiency, is the sine qua non of hyperglycaemia in all forms of diabetes, at least in rodents . Indeed, total insulin deficiency by beta cell destruction in glucagon-receptor-null mice, which are unresponsive to glucagon, does not cause diabetic abnormalities . We observe that ERα and ERβ are produced in mouse and human alpha cells, further indicating that alpha cells are direct targets of E2 actions. In addition, our experiments suggest that ERα needs to be activated in alpha cells of the host pancreas as the hypoglycaemic effect of an ERα-selective agonist is lost when islets from WT mice are transplanted in Erα null mice (lacking ERα in alpha cells). Accordingly, the ERα agonist can still acutely suppress blood glucose and permanently improve diabetes when ERα-deficient islets are transplanted in WT mice. This demonstrates that ERα action in graft alpha cells is not necessary to suppress blood glucose. Recently, leptin has been shown to suppress glucagon and correct diabetes in mice, in the absence of insulin . Our results suggest that E2 requires the presence of insulin—or at least functional beta cells—to suppress glucagon as ER agonists have no hypoglycaemic effect in diabetic mice in the absence of transplanted islets. This observation is consistent with the concept that insulin suppresses glucagon secretion from alpha cells via paracrine mechanisms  and that insulin signalling in alpha cells is required for this process . Thus, in the alpha cells of diabetic mice E2 may act as an insulin sensitiser in suppressing glucagon production. Additional studies in mice lacking ERs in alpha cells are needed to address this issue.
Protection of functional beta cell mass
Oestrogens have acutely improved islet survival following transplantation, as demonstrated by E2-mediated acute suppression of graft apoptotic beta cells. This is the first evidence that E2, used at therapeutic doses, protects human islets from apoptosis in an in vivo diabetic environment. We and others have shown that E2 promotes islet survival in conditions of oxidative stress and pro-inflammatory cytokine injury in culture and in vivo [11, 12, 13, 17]. Higher islet functionality and decreased apoptosis are also observed after transplantation of rat islets recovered from E2-treated brain-dead donors . This anti-apoptotic protection probably accounts for the early retention of graft beta cell mass, which persists after 3 weeks of ER agonist treatment. In addition, we believe that E2 anti-apoptotic protection is also mediated via activation of ERs in recipient endothelial cells. E2 dilates the mesenteric arteries and increases mesenteric blood flow [46, 47]. E2 also improves splanchnic circulation during haemorrhagic shock, which increases oxygenation . We observed that E2 acutely and dramatically improved islet-graft hypoxia, which is associated with decreased systemic and kidney oxidative damage. This protection of islet oxygenation is probably instrumental in E2 acute anti-apoptotic protection as, following PIT, hypoxic stress is a critical factor in the early loss of islet mass [8, 9, 10]. Initially, E2-treated graft beta cells are non-functional and show no insulin-secretory response to the glucose challenge, probably as a result of hyperglycaemia-induced glucose desensitisation and/or lack of islet vascularisation. However, following 3 weeks of normoglycaemia, PIT mice treated with ER agonists show a restoration of beta cell function. Thus, E2 acute normalisation of blood glucose, via suppression of hyperglucagonaemia, has favoured the subsequent restoration of beta cell function following correction of glucose desensitisation.
Action on recipient endothelial cells
Apart from E2-mediated acute improvement of islet-graft oxygenation, the likely action of oestrogens involves the enhancement of islet revascularisation through action on recipient endothelial cells, as E2 is a potent angiogenic factor in the endometrium and following vascular injury [22, 23]. Accordingly, E2 increases the functional blood vessel density of the human islet graft. More importantly, immunosuppression with sirolimus and tacrolimus typically inhibits islet revascularisation and engraftment. Yet E2 stimulation of revascularisation persists in the presence of this immunosuppression regimen. Furthermore, after discontinuation of E2 treatment the correction of diabetes endures, demonstrating that E2 has induced a stable engraftment. E2 treatment promotes revascularisation of the transplanted human islets through ERs on the endothelial cell from the recipient kidney cortex and without the participation of endothelial cells from the human graft, as these cells had disappeared at the time of transplantation. However, G1, which exhibits potent anti-apoptotic action in human islets , improves PIT outcome without enhancing graft revascularisation, suggesting that the acute hypoglycaemic and anti-apoptotic effects of oestrogens are more important to the engraftment process than the moderate increase in revascularisation.
Our findings have direct therapeutic implications for PIT in type 1 diabetes. Although long-term E2 treatment following PIT is not suitable because of the risk of cancer, conversely, transient E2 treatment represents a safe and immediately available alternative to improve PIT in women. Indeed, fertile women with type 1 diabetes show E2 deficiency compared with healthy women . Therefore, women with type 1 diabetes undergoing islet transplantation have lost their endogenous E2 protection and could benefit from short-term oestrogen supplementation using therapeutic doses. Furthermore, 17α-E2, which is endogenous in humans, has few of the biological effects associated with the female hormone activity and may be a candidate for sex-neutral therapy in PIT.
In conclusion, E2 enhances islet engraftment after PIT. As oestrogens are approved by the Food and Drug Administration, further testing in women with the addition of oestrogens to the Edmonton protocol should be considered. This could provide an immediate therapeutic alternative to improve PIT and achieve insulin independence with fewer islets, long before other surrogate islet beta cell sources or beta cell regeneration therapy can be developed.
We are grateful to B. Hering from the University of Minnesota for helpful discussion and to M. Brissova and A. Powers from Vanderbilt University for providing protocol and advice on in vivo lectin infusion. We acknowledge the Integrated Islet Distribution Program (IIDP) funded by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) and the Juvenile Diabetes Research Foundation (JDRF) for providing human islet for research.
This work was supported by grants from the National Institutes of Health (RO1 DK074970, P50 HD044405), the JDRF (1-2006-837), the March of Dimes Birth Defects Foundation (6-FY07-312) and the American Heart Association (11IRG5570010) to F. Mauvais-Jarvis.
SL and GK performed experiments, analysed data and wrote the manuscript. MSM and GN performed experiments, analysed data and revised the manuscript; YW and JO participated in design of the study and manuscript revision. FMJ conceptualised and designed the study, analysed data and wrote the manuscript. All authors gave approval of the final version of this manuscript for publication.
Duality of interest
F. Mauvais-Jarvis has received research support from Pfizer. The remaining authors declare that there is no duality of interest associated with this manuscript.
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- 5.Weir GC, Bonner-Weir S (2004) Five stages of evolving beta-cell dysfunction during progression to diabetes. Diabetes 53(Suppl 3):S16–S21Google Scholar
- 18.Tiano JP, Mauvais-Jarvis F (2012) Importance of oestrogen receptors to preserve functional beta-cell mass in diabetes. Nat Rev Endocrinol 8:342–351Google Scholar
- 19.Shifren JL, Tseng JF, Zaloudek CJ et al (1996) Ovarian steroid regulation of vascular endothelial growth factor in the human endometrium: implications for angiogenesis during the menstrual cycle and in the pathogenesis of endometriosis. J Clin Endocrinol Metab 81:3112–3118PubMedCrossRefGoogle Scholar
- 29.Moos WH, Dykens JA, Howell N (2008) 17α-Estradiol: a less-feminizing estrogen. Drug Dev Res 69:177–184Google Scholar
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