Transplantation of pancreas or islets of Langerhans is a promising curative treatment in patients with complicated diabetes. Intraportal islet infusion in liver is widely used for clinical islet transplantation. However, transplantation in the liver is associated to a number of complications, such as loss of the functional islet mass, bleeding and thrombosis, and progressive deterioration of intrahepatic islet function.1,2 In the continued search for optimal sites for islet transplantation, several tissues and organs have been explored, including the kidney subcapsule,3 skeletal muscle,4,5 subcutaneous and intramuscular spaces,4 epididymal fat pad,6 spleen,7 thymus,8 bone marrow,9 lymph node,10 eye,11 etc. However, the ideal site of transplantation, allowing to reduce the required number of engrafted islets and prolong islet graft survival, remains to be finally established.
Humans and other mammals have 2 types of adipose tissue, brown adipose tissue (BAT) and white adipose tissue (WAT). Adipose tissue can induce angiogenesis and adipogenesis12,13 and contains adipose-derived stem cells that have the ability to differentiate into multiple lineages, a property that might be valuable for repairing or replacing several types of damaged cells.14 Additionally, human BAT contains a rich plexus of blood vessels15 and can therefore warm the blood to ensure the normal function of vital organs such as myocardium, kidney, and brain.16 In this respect, BAT is supposed to benefit the ectopic tissues by warming blood drainage in neovasculatures.
In this study, we explored the potential of BAT as a transplantation site for ectopic islets. We evaluated vitality and function of the engrafted islets, as well as glycemia, in diabetic mice in the short and in the long term.
MATERIALS AND METHODS
The wildtype (WT) C57BL/6J male mice and transgenic red fluorescent protein (RFP+) C57BL/6J male mice were obtained from Center of Experimental Animals, Southern Medical University. The RFP transgenic mice were generated according to the previously described method in our research.17 Briefly, heterozygous RFP transgenic mice were intercrossed to obtain the homozygous RFP transgenic mice as donor mice (RFP+ C57BL/6J) and nontransgenic littermate as recipient mice (WT C57BL/6J). Recipient WT male mice (10 wk old) were intraperitoneally injected with a single dose (190 mg/kg) of streptozotocin (STZ) to induce diabetes. Diabetes was confirmed (blood glucose concentration >300 mg/dL) 3 days after STZ injection using One-Touch blood glucose monitor (Johnson & Johnson, USA). Donor islets were isolated from 10-week-old male RFP+ C57BL/6J mice and transplanted into unilateral inguinal WAT or interscapular BAT tissue of age-matched STZ-induced diabetic mice. Transplantation was performed 5 days after the diabetes was confirmed. All mice were housed under pathogen-free conditions with drinking water and food ad libitum. Blood glucose and body weight (BW) of the mice were monitored for a short term (10 wks) and long term (1 y). Vitality and function of the islets were assessed by histological examination, fluorescence imaging, and intraperitoneal glucose tolerance test (GTT) at end of short-term and long-term study. Every effort was made to minimize the number of animals in the experimental design. All animal experiments were approved by the Ethical Guidelines for Animal Care set by the Animal Research Ethics Committee of Southern Medical University.
Pancreatic Islet Isolation and Transplantation
Pancreases from adult RFP+ C57BL/6J mice were perfused in situ with collagenase P (Roche) through the bile duct to digest islets according to the method described by Sutton et al.18 Then, the islets were washed in PBS and handpicked in room temperature. The islets with a diameter of 150–250 μm were mixed with 10 μL of Matrigel and slowly injected into interscapular BAT or unilateral inguinal WAT using BD Micro Fine Plus 1 mL Insulin Syringes (30 G). In the preexperiment, we found that the minimum number of islets in normal C57 mice was about 120. And transplanting 100 and 120 islets into the WAT of diabetic mice could reverse the diabetes (data not shown). However, due to dense architecture of BAT, >20 islets with Matrigel were left in the syringe when injection of Matrigel containing 100 islets for transplant. In this study, we used marginal number grafted islets (80 for BAT, 100 for WAT) for transplantation. All transplantations were performed under sodium pentobarbital anesthesia (60 mg/kg, intraperitoneal injection).
Whole-body and Organ Fluorescence Imaging
To detect donor RFP-positive islets, the Xenogen IVIS Lumina II Imaging System was used, following the manufacturer’s recommendations. For the exvivo fluorescence imaging of adipose tissue, tissues were dissected into ice-cold PBS, and then RFP fluorescence was visualized using Xenogen IVIS Lumina II Imaging System or stereo fluorescence microscope (Nikon, AZ100), as described previously.19
Graft Function Analysis and Histological Examination
The nonfasting blood glucose concentration was monitored using One-Touch blood glucose monitor. The grafted islet function was defined successful when mice showed 2 consecutive nonfasting blood glucose concentrations <200 mg/dL in 2 days. The intraperitoneal glucose GTTs (2 g/kg BW) were performed to evaluate the glucose tolerance, and serum insulin levels were measured by ELISA (rat/mouse insulin ELISA kit, Millipore). Serial sections of the islet-bearing adipose tissues were obtained to evaluate the morphology by staining with hematoxylin-eosin (HE) and immunohistochemistry using glucagon antibody or insulin antibody (Cell Signal Technology). The protocols for GTT, ELISA, and immunohistochemistry were previously well described.19
All data were expressed as the mean ± SD. All statistical analysis was performed with the statistical package for social sciences 22.0 software. Shapiro-Wilks test was used for normality, and all variables showed normally distributed. The 2-tailed Student’s t test was used for comparing the means of the 2 groups, and 1-way ANOVA with Bonferroni correction was applied for multiple post hoc comparisons. P value under 0.05 was considered statistically significant.
Ectopic Islets in the Adipose Tissues After Islet Transplantation
The interscapular BAT and inguinal WAT were easily accessible to observe the isolated RFP-expressing islets (Figure S1, SDC, http://links.lww.com/TP/B947) in the adipose tissues in vivo. Both in vivo and in vitro imaging showed the RFP signal (Figure 1A), which indicated that RFP+ islets survived after transplantation. As a consequence of the islet survival in adipose tissue, the blood glucose of diabetic mice (>450 mg/dL) significantly continued to decrease after 3 days after grafts and recovered to normal (<200 mg/dL) approximately 7 weeks after transplantation (Figure 1B). All mice were rescued from lethal hyperglycemia and gained body weight (Figure 1C). No mouse received a second islet transplant. The diabetic mice without islet transplantation were euthanized 3 weeks after the experiment start due to severe hyperglycemia and poor conditions. We did not observe significant differences in serum lipid levels, including total cholesterol, triglycerides, and free fatty acids, between the engrafted mice and nondiabetic age-matched mice (Figure 1D). Together, these data indicated that islets survived in the adipose tissue and had no apparent negative effects on serum lipid levels.
BAT Provided the Remarkable Site for Restoring the Function of Ectopic Islets in Short Term
To examine the characteristics of ectopic islets, we detected the expression of insulin and glucagon (Figure 2A). Interestingly, we noticed that Matrigel around the transplanted islets in BAT had been mostly absorbed by week 10 after islet transplantation. Furthermore, numerous adipocytes and endothelial-like cells infiltrated in the remnant Matrigel, suggesting that extensive vascular remodeling happened during this period. Correspondingly, abundant extra- and intraislet vasculatures were observed in BAT; the islets also showed normal architecture and morphology (Figure 2A). However, in the WAT transplantation group, the ectopic islets were still surrounded by a mass of Matrigel with congestion, although scattered neovasculatures were visible in the Matrigel (Figure 2A).
Next, we investigated whether ectopic islets could release insulin and be induced by glucose normally. After verifying that the mice remained euglycemic for 3–4 weeks, intraperitoneal GTT was performed 10 weeks after transplantation. GTT tests showed that ectopic islets both in WAT and BAT responded to the glucose and decreased its blood levels to the normal concentration at 120 minutes (Figure 2B). Instead, the blood glucose levels at 15, 30, and 90 minutes were significantly lower in BAT group than in WAT group (P < 0.05). The area under the curve for GTT was also significantly lower in the BAT group than in the WAT group (P = 0.005) (Figure 2C). However, the serum insulin levels of mice with grafted islets in WAT or BAT were similar (Figure 2D). These results could also due to the abundant extra- and intraislet vessels (shown in Figure 2A), which may facilitate ectopic islet response to glucose and instant insulin release in BAT. All diabetic mice displayed extreme high blood glucose (>600 mg/dL) 3 days after removal of islet-engrafted adipose tissue.
Islets Grafted in BAT Successfully Maintained Normoglycemia in Mice for 1 Year
Remarkably, all engrafted mice maintained normoglycemia (range 126–161 mg/dL) during the 1-year study period (Figure 3). As with islets transplanted in BAT for 10 weeks, the islets still retained normal morphology and structure, and insulin and glucagon expressions 1 year after transplantation. In WAT, the Matrigel completely disappeared, and the islets also displayed a more natural morphology, with a large number of extra- and intraislet vessels (Figure 3A). As a result of the long-term euglycemia (Figure 3B), these mice showed gradual increases in body weight with age (Figure 3C) and normal GTT results (Figure 3D, E) in WAT and BAT, respectively. After removal of islet-engrafted adipose tissue, the average blood glucose levels of diabetic mice were above the 500 mg/dL immediately, within 1 day.
BAT has loose, fatty, and areolar composition, and a rich plexus of blood vessels, which could provide a suitable matrix for sustaining the transplanted islets. In our study, we demonstrate that BAT enables a small number of ectopic islets to restore normal architecture and function in the short term and sustain the euglycemia for 1 year in diabetic mice.
The optimal site for islet implantation should improve ectopic grafted islets and enable long-term graft function. Studies in rat and mouse models indicate that the number of islets required is dependent upon the transplantation site.20 Previous researches that demonstrated successful transplantation in diabetic rats and mice generally used large numbers of purified islets (300–2000).21 In a mouse model, Nakano et al22 reported a marginal number of donor islets (250) transplanted into the liver; however, only 2 out of 14 diabetic mice receiving 250 islets became normoglycemic by day 90 after transplantation. These transplantation sites were discouraging due to lack of early neovascularization even when postoperative hyperbaric oxygen therapy was applied.4 Some studies suggested that the adipose tissue might be more suitable as transplantation site than subcutaneous and intramuscular space. Yasunami et al23 reported that transplanted islets engrafted in the inguinal subcutaneous WAT could reverse STZ-induced diabetes in mice and that allograft rejection was preventable by blockade of costimulatory signals. Moreover, adipose tissue samples from visceral and subcutaneous sites layered on chick chorioallantoic membrane express angiogenic factors and stimulate angiogenesis, and the angiogenic potency of adipose tissue is not related to its localization.12 Sarkanen et al13 also proved that adipose tissue extract induced angiogenesis and adipogenesis in vitro. It is well-known that the revascularization of grafted islets in a new-host organ determines their survival and function.24 In our study, we used marginal number grafted islets (80 for BAT, 100 for WAT) and found that islet transplantation did not fail in either group, which could be associated with the abundant neovasculatures in the adipose tissue.
Although the engrafted islets function both in BAT and in WAT, the BAT could be a more suitable site to protect and sustain the activity and function of ectopic islets. In the short term after transplantation in BAT, but not in WAT, the Matrigel around the transplanted islets was mostly absorbed, and was infiltrated by adipocytes and endothelial-like cells, suggesting that extensive vascular remodeling had taken place during the engraftment, and that the islets had restored almost normal architecture and morphology. BAT is a densely vascularized tissue with abundant oxygen, blood supply, and mitochondria,25 which is supposed to promote the survival and function of islet graft. Instead, WAT is composed of unilocular lipid droplets and has relatively few capillaries and mitochondria, which may cause the slow absorption of Matrigel and formation of fewer vessels. Concomitantly, we found that the glucose tolerance of the BAT transplantation group at 10 weeks was significantly improved than that of the WAT group. The abundant extra- and intraislet vasculatures also allowed the ectopic islets in BAT to release insulin into the blood instantly and efficiently respond to the blood glucose change. We also noticed that the engrafted mice had no abnormal serum lipid and the islet-engrafted adipose tissue showed no change in adipocyte morphology. Lipolysis can lead to the recruitment and activation of immune cells in tissues.26 In liver, a main reason for graft loss is the nonspecific inflammatory reaction, in which liver macrophages, the Kupffer cells, attack and injury allotransplanted islets.27 Instead, the BAT can release insulin like growth factor-I (IGF-I), which negatively correlates with inflammatory cytokines.28 Gunawardana et al28 demonstrated that BAT transplants completely reversed the diabetes in nonobese diabetic mice with increased plasma IGF-I level. Between human and mouse, the overall distribution of BAT is similarity. In humans, subcutaneous BAT under the clavicles, in the axilla, or inguinal fossa, could be explored as islet transplantation site, however, supraclavicular BAT might be riskier for exploring due to its anatomical relations to the brachial plexus, carotid sheath, and subclavian vessels.29 It is also known that human BAT distribution decreases in variable amounts with increasing age, and few individuals have BAT at any site by 80 years old.29 Meantime, some recent studies have focused on the strategies to increase BAT mass and the application of transplantation BAT in the human,30 which could provide more conditions for the islet transplantation or the combination transplantation of BAT and islet in clinics.
In summary, we report for the first time that BAT provides the site for restoring architecture and function of ectopic islets both in the short term and in the long term. The anatomical location of BAT lends itself to easy observation, removal, retransplantation, and potential reisolation of the transplanted islets. The valid, simple, and reproducible islet transplantation in BAT makes it a potential desirable site for transplantation in basic and clinical research.
We thank K.T.Y. and Q.C.L. for research support and assistance with histology and serum test.
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