Key words : Diabetes, Insulin secretion, Islet viability, Oxidative stress

Introduction

Transplant of pancreatic islets is considered as an alternative treatment for type 1 diabetes because it allows insulin independence to be induced by replacing isolated insulin-producing islets.1 Extracellular matrix (ECM) is undoubtedly the most important part of the islet environment,2 and the relationship of islets with their environmental compartments is necessary for their survival and function. It has been demonstrated that destruction of the ECM and devascularization during islet isolation induce apoptosis, thus decreasing islet survival and performance, leading to reduced long-term islet graft efficacy.3-5 Treatment of isolated islets with ECM proteins and growth factors can reestablish their interactions with elements in the microenvironment and enhance islet quality and transplant outcomes. According to this strategy, many interventions are being tested to improve islet survival by using materials such as ECM proteins like laminin, collagen IV, fibronectin,6,7 matrigel,8 and small intestinal submucosa (SIS) as acellular collagen-based scaffold9 and mesenchymal stem cells.9,10

In this study, we investigated the effects of pancreatic islet homogenate (PIH) on islet quality and transplant outcomes for several reasons. First, islets in the natural environment of the pancreas are enclosed with ECM, and it has been shown that islet-ECM crosstalk activates several signaling pathways that are important for islet survival and function.2 The incomplete capsule, the basement membrane, and the intra-islet microvasculature contain a range of proteins such as fibronectin, collagen types I, III, and IV, and laminin. There is a reciprocal interaction between beta cells and intra-islet vascular endothelial cells. Beta cells secrete vascular endothelial growth factor A (VEGF-A), insulin, and angiotensin 1, which have roles in enhancement of vascular development. The islet microvasculature releases hepatocyte growth factor (HGF) and thrombospondin 1, which increase islet blood flow, regulate beta cell insulin gene expression, enhance insulin secretion, and maintain beta cell mass.11,12

Excessive reactive oxygen species production caused by chronic hyperglycemia is one of the major markers of the onset and progression of diabetes and its secondary complications. Reduction of oxidative stress may be effective in treatment of diabetes and its complications. Proteins of the ECM like collagen and laminin can modulate oxidative stress in both in vivo13 and in vitro conditions.14

Thus, we hypothesized that supplementation of cultured islets with PIH, which contains bioactive proteins (fibronectin, collagen types I, III, and IV, and laminin) and growth factors (VEGF-A, HGF), may be effective in improving ECM-islet interactions, islet survival, and long-term transplant outcomes. To address these points, we performed in vitro and in vivo experiments to evaluate the effects of PIH on islet survival and the efficacy of islet transplant. In this study, isolated islets that were not suitable for transplant were homogenated and used as a supplementary biomaterial in transplantation. The use of PIH to rebuild the environment of isolated islets is safe because PIH is more adaptable in texture, immunity, and cellular-molecular mechanisms than other synthetic materials.

Materials and Methods

Ethical statement
Animal handling and experimental procedures were carried out in accordance with the standard principles of laboratory animal care to allow the humane treatment of all experimental animals. This study was approved by the Animal Care and Use Committee of Shiraz University of Medical Sciences (IR.SUMS.REC. 1396.S1010).

Study design and animals
We performed in vitro and in vivo evaluations of the effects of PIH on survival, function, and efficacy of islet transplant. Male Sprague Dawley rats between 11 and 12 weeks of age (250-280 g) were obtained from the stock of rats bred in the animal house of the Research Institute of Shiraz University of Medical Sciences (Shiraz, Iran); these animals were used as donors or as diabetic transplant recipients.15 Animals were housed in groups of 2 per cage under standard conditions (temperature 24 ± 3 °C, relative humidity 20 ± 3%, 12:12-h light-dark cycle) with free access to food and water.

For in vitro experiments, isolated islets were cultured (10 islets/well; n = 8) for 24 hours15-17; control islets were those without PIH. Insulin release, insulin content, and viability of isolated islets were evaluated after culture of islets in RPMI 1640 medium (GIBCO, catalog no. 31800) containing 5 mM D-glucose without (control islets) or with PIH.

For in vivo experiments, diabetic rats were randomized into 2 groups9 of 8 per group: the DIT group included diabetic rats transplanted with 450 islets only under the left kidney capsule), and the DIT + PIH group included diabetic rats transplanted with 450 islets and PIH under the left kidney capsule.

After islet transplant, all animals were weighed every 15 days for 60 days, and water intake was measured weekly. Weight gain ([final weight - initial weight/initial weight] ×100) was calculated for each group. Tail blood sampling was done on day 0 (before transplant) and every 15 days after transplant to day 60 for assessment of plasma glucose and insulin concentrations. Intraperitoneal glucose tolerance test (IPGTT) was performed on day 57 after transplant.15

Diabetic induction
Diabetes mellitus was induced in male rats by single intraperitoneal injection of streptozotocin (65 mg/kg; Sigma). Ten days after injection, blood glucose was measured by using a glucometer (Accutrend Plus; Roche) from the tail vein sampling. Rats were considered as having diabetes when they exhibited hyperglycemia (blood glucose levels >300 mg/dL); these animals were then used for transplant studies.

Preparation of pancreatic islet homogenate
Pancreatic islets were isolated by using the modified collagenase method from adult male Sprague Dawley rats after overnight fasting as previously reported.18 The isolated islets of 100 to 200 μm in size (islet equivalent = 150 μm) were used for transplant, and those that were out of this range and not suitable for transplant were used as PIH. For preparation of PIH, isolated islets were mixed with lysis buffer and homogenated with ultrasonic homogenizer (Bandelin prob MS72 HD-3100) and centrifuged for 40 minutes at 12 000 revolutions/min at 4 °C. The supernatant was separated, and protein concentration was assayed with the commercial Thermo Scientific Pierce BCA protein assay kit. Pancreatic islet homogenate was then aliquoted and frozen at -20 °C for later use.

Preparation and culture of isolated islets
Islets were isolated according to the method described above and cultured in 1 mL RPMI 1640 medium (RPMI 1640 containing 5 mM D-glucose, 10% fetal bovine serum, 0.5% bovine serum albumin [Sigma-Aldrich], and 1% pen strep), which was supplemented without or with PIH (900 μL RPMI + 100 μL PIH, protein: 100 μg) and incubated at 37 °C in 5% CO2 and 95% air for 24 hours.

In vitro assessment of insulin secretion and content and islet viability
After static exposure of islets to 5 mM D-glucose in 1 mL of culture medium with or without PIH, insulin concentration was assayed by rat insulin enzyme-linked immunosorbent assay (ELISA) method.

Insulin content assay was done with the use of the acid ethanol extraction protocol.15 In brief, after 24 hours of incubation of 10 isolated islets in the culture medium, islets were centrifuged, placed into 1 mL of acid-ethanol solution (0.15 M HCl in 75% [vol/vol] ethanol in water) and stored overnight at 4 °C. After centrifugation at 613g for 10 minutes, the supernatants containing insulin extracted from the islets were collected and assayed by the ELISA method, with results expressed in milligram protein of islets.

The viability of islet cells was assessed after 24 hours of incubation of 10 isolated islet groups in medium without or with 10% PIH by annexin V (Life Technologies Japan) and propidium iodide (Sigma-Aldrich) staining. Viable islet cells were stained green by annexin V, and dead cells were stained red by propidium iodide. The average viability was calculated at each time point as follows: = % viability rate number of viable cells (green)/total number of viable and dead cells (green + red) × 100.7

Islet transplant
Animals were anesthetized with ketamine and xylazine; a small incision was made in the flank about 0.5 inches to the left of the midline. The left kidney was exposed. A small hole was made at one pole of the kidney capsule with fine-tipped forceps. Aliquots of 450 isolated islet equivalents cultured for 24 hours were aspirated into polyethylene tubes; we then inserted tubes into the space under the capsule, and islets were slowly expelled as tubes were withdrawn. The kidney was replaced in the abdominal cavity, and the body wall and outer skin were sutured with 4-0 nylon. The animals were kept warm until recovery from anesthesia.15

Intraperitoneal glucose tolerance test
Intraperitoneal glucose tolerance test was conducted for both experimental groups at day 57 posttransplant after an overnight fast by intraperitoneal injection of 2 g/kg glucose.19 Blood samples were collected at various time points (0-120 min) to determine the plasma glucose and insulin concentrations.20 Plasma levels of glucose and insulin concentrations were assessed with the glucose oxidase method (Pars Azmoon) and the ultrasensitive rat insulin ELISA method, respectively. Both insulin and glucose measurements were repeated 2 times. Plasma fasting glucose and insulin concentrations were measured for calculation of homeostasis model assessment of insulin resistance index (HOMA-IR), using the following formula21: HOMA-IR = fasting glucose (mmol/L) × fasting insulin (μU/mL)/22.5.

Evaluation of oxidative stress markers
On day 60 after transplant, before animals were killed, blood samples were obtained and the serum was separated and stored at -20 °C until use to assay lipid peroxidation and antioxidant enzyme activity. The malondialdehyde (MDA) level, as the lipid peroxidation index, was assessed by the thiobarbituric acid reactive substance method, and superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities were quantified by commercially available assay kits (ZellBio) using the colorimetric method.15

Statistical analyses
Statistical analysis was performed using GraphPad Prism software, version 6.0, and results are presented as means ± SE. Repeated measures 2-way ANOVA (post hoc Bonferroni) was used to analyze body weight, water intake, and plasma glucose and insulin concentrations during IPGTT. For paired comparisons, independent sample t tests (parametric test) or
Mann-Whitney U tests (nonparametric test) were used. P < .05 was considered statistically significant.

Results

Islet quality assessment
The effects of PIH on islet quality were determined by evaluating the viability, insulin release, and content of pancreatic isolated islets after 24 hours of culture with and without PIH. To determine the viability, isolated islets were stained with annexin V/propidium iodide (viable cells were green and dead cells were red) (Figure 1A). Our results showed that the percentage of viability was significantly higher in the PIH islet group (95.22%) than in the control islet group (91.42%; P < .05) (Figure 1B).

After 24-hour incubation of islets with 5 M glucose, insulin secretion was measured. As shown in Figure 1C, insulin release was significantly higher in the PIH-treated islets than in the control islets (P < .05).

The insulin content of the islets was evaluated by acid ethanol extraction method. We found that PIH-treated islets showed increased insulin content compared with the untreated (control) islets, but the difference was not significant (Figure 1D).

Assessment of body weight and water intake
Mean body weight and percent weight gain of animals in the 2 experimental groups were measured. All experimental animals exhibited significant increases in the body weight from day 0 (day of transplant) to day 60. Although there were no significant differences in the initial and final body weight of animals between the 2 groups, percent weight gain in the DIT + PIH group was significantly higher than that in the DIT group (Figure 2A).

Water intake was measured weekly from day 0 to day 60 after transplant. As shown in Figure 2B, significant differences in water intake were observed between the 2 groups from day 14 to day 60 after transplant. The area under curve (AUC) for the DIT + PIH group was significantly lower than for the DIT group.

Fasting plasma glucose and insulin concentrations and homeostasis model assessment of insulin resistance index
At the end of the experiment, fasting plasma glucose and insulin concentrations were measured in both animal groups. The DIT + PIH group showed significantly lower fasting plasma glucose and significantly higher fasting plasma insulin levels than the DIT group. There were no significant differences between the 2 experimental groups with regard to HOMA-IR index (Figure 3).

Plasma glucose and insulin concentrations after islet transplant
Plasma glucose and insulin concentrations in the DIT group and the DIT + PIH group were measured on day 0 (before islet transplant) and on days 15, 30, 45, and 60 after islet transplant (Figure 4). There were no differences between the DIT and DIT + PIH groups in plasma glucose (22.06 ± 0.82 and 20.13 ± 1.50 mmol/L, respectively) and insulin levels (4.02 ± 0.3 and 4.18 ± 0.13 μU/L, respectively) before islet transplant (day 0).

As shown in Figure 4A, plasma glucose and insulin levels had improved after transplant of islets in the 2 diabetic rat groups. Levels of plasma glucose (Figure 4A) at all time points (15, 30, 45, and 60 days) after islet transplant were decreased, and plasma insulin levels (Figure 4B) were increased compared with that shown at day 0.

On day 30 and day 60 after islet transplant, plasma glucose and insulin levels in the DIT + PIH group were, respectively, significantly lower and higher than those in the DIT group (Figure 4).

Intraperitoneal glucose tolerance tests
As shown in Figure 5, the mean blood glucose levels significantly decreased in the DIT + PIH group compared with the DIT group during IPGTT (Figure 5A). Glucose AUC in the DIT + PIH group was significantly lower than shown in the DIT group (P < .001; Figure 5B).

As shown in Figure 5, during IPGTT, mean plasma concentrations of insulin at 30 and 60 minutes after glucose injection (Figure 5C) in the DIT + PIH group were significantly higher than those in the DIT group. The plasma insulin AUC for this group was significantly higher than for the DIT group (Figure 5D).

Oxidative markers
Serum MDA concentrations and GPx and SOD activities are shown in Table 1. The serum MDA level in the DIT group was significantly higher than that in the DIT + PIH group. Compared with the DIT group, PIH treatment led to a significant increase in the serum SOD antioxidant activity in the DIT + PIH group. There was no statistically significant difference between the DIT and DIT + PIH groups in the serum GPx antioxidant activity.

Discussion

Intact ECM and ECM-islet interactions are the most important factors in normal islet function.2 During islet isolation, destruction of the islet environment and impairment of ECM-islet crosstalk can reduce survival and function of islets, which then limits islet graft efficacy in patients with diabetes.3-5 It seems that remodeling these interactions by adding ECM proteins and growth factors to the culture media of isolated islets is a good solution for enhancing islet survival and function. According to this strategy, we consider PIH as a safe biomaterial that has various ECM proteins and growth factors and investigated its effects on islet function and survival and its effect on transplant outcomes.

We found that supplementation of the culture media with PIH enhances islet quality. Our finding demonstrated that insulin secretion, insulin content, and survival of PIH-treated islets were higher and longer than in islets that were not treated. In addition, PIH had a positive impact on the function of transplanted islets in a diabetic animal model.The plasma levels of glucose and insulin in rats transplanted with PIH-treated islets were lower and higher, respectively, than those in rats transplant with islets only; in addition, water intake was decreased and glucose tolerance was improved.

To the best of our knowledge, there is no report on the improving effects of PIH administration on quality of the islets before and after transplant. It seems that our study is the first report of the beneficial effects of PIH (endocrine part of pancreas) on islet survival and function and outcomes after islet transplant. As mentioned above, PIH is a combination of ECM proteins and growth factors, including collagen types I, III, and IV, laminin, fibronectin, VEGF-A, and HGF. The effects of some of these ECM proteins and growth factors on survival and secretory function of islets have been previously evaluated. Investigations showed that ECM-beta cell interactions induce intracellular signaling, which leads to regulation of islet proliferation, survival, insulin secretion, and islet engraftment.22 Other studies have shown that ECM proteins like collagen type IV,6,7 laminin, and fibronectin6 and growth factors like VEGF and HGF23,24 have important roles in improvement of islet survival and engraftment, angiogenesis, pancreatic beta cell proliferation, impaired insulin, and glucose homeostasis in diabetic animals. Research indicated that MIN6 beta cells cultured in an environment with ECM proteins resulted in enhanced cell survival and decreased apoptosis.10 Other studies have shown that culturing islets with ECM proteins such as collagen IV2 collagen I, laminin, fibronectin, and small intestinal submucosa25 and encapsulation of islets with collagen IV10 improved islet survival and function. In contrast, Kaido and colleagues reported that culturing islets on collagen IV-coated tissues caused marked suppression of insulin gene transcription and insulin secretion.26 Islets embedded in collagen matrix, especially collagen type IV,6,7,27,28 fibronectin, laminin,6 and SIS scaffold9,16  inhibited islets apoptosis, increased islet cell proliferation and revascularization, and improved glycemia and graft survival and function in diabetic animals.8

Angiogenesis, which is regulated by both proan-giogenic and antiangiogenic factors, is necessary for islet engraftment and survival.29 One of the potent direct agonists of angiogenesis is VEGF, which activates both endothelial cell proliferation and migration.30 Brissova and colleagues reported that mice with reduced VEGF-A expression in beta cells showed abnormalities in the islet vasculature and impaired glucose stimulated insulin secretion.31 Lai and colleagues demonstrated that VEGF supply to islet transplants enhanced vascular density and blood flow to the graft and improved the blood supply that is associated with increased beta cell proliferation and beta cell mass in islet transplants.32 Reduced VEGF production is returned by pancreatic islet transplant with adenoviral delivery of human VEGF under the kidney capsule of streptozotocin-induced diabetic mice through increased revas-cularization in the transplanted islets and improved glycemic control in diabetic mice.33 Vasir and colleagues showed that delayed expression of VEGF receptors in cultured and transplanted islets could impair angiogenesis and graft transplant outcomes in recipients with diabetes.34

Proangiogenic growth factors like HGF have a significant role in graft survival and function.35,36 The positive effects of HGF on islet mass, islet function, and transplant outcomes were reported in transgenic mice.24,37 Administration of HGF increased beta cell regeneration and proliferation and survival in mice with reduced pancreatic beta cell mass38 and decreased blood glucose in streptozotocin-induced diabetic mice.39 Golocheikine and colleagues found that subcutaneous transplant of islets with matrigel supplemented with VEGF and HGF increased the number, vasculature, and expression of vascular and intercellular adhesion molecules of islets in immune-deficient diabetic mice.40 The improving effects of PIH on islet viability, insulin secretion, and hyperglycemia in streptozotocin-induced diabetic rats may be attributed to its constituents.

In islets treated with PIH, oxidative stress in DIT + PIH animals was significantly decreased compared with the DIT group. We found that MDA level, as an oxidative damage index, was diminished and antioxidant enzyme activity, including SOD, was enhanced following PIH treatment. Oxidative stress modulation might be a secondary effect to the effects of improved survival and function in treated islets. In addition, the antioxidant effect of PIH may be related to ECM bioactive proteins or growth factors that are present in PIH. There are few studies on the improved effects of ECM and growth factors on oxidative stress. In support of our results, Wu and colleagues showed that ECM inhalation reduced hyperoxia-induced apoptosis and oxidative damage in the lung and improved alveolar cell survival and morphology.41 In addition, some studies have indicated the protective effects of HGF against oxidative stress.42,43

Despite several strengths of our present study, such as being the first study to evaluate the effects of PIH on islet quality and islet transplant outcomes, this study had some limitations, including that the presence and concentration of the ECM proteins and growth factors were not determined. Measuring the concentration of bioactive proteins of homogenized islets would make the results more accurate, but this was not possible to perform.

Conclusions

Islet treatment with PIH successfully increased islet viability, function, and transplant outcomes in diabetic rats. These improving effects may have had secondary beneficial effects on the oxidative stress modulation. Reestablishment of the islet local microenvironment and islets-ECM crosstalk is likely to have a role in the beneficial effects of islets treated with PIH. Our findings could be a valuable reference for future studies related to this issue. the exact mechanisms of the positive effects described in this study will be necessary, in addition to an appraisal of its clinical application.

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