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Keywords:

  • Autophagy;
  • islet transplantation;
  • LC3;
  • rapamycin;
  • transgenic mice

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

Autophagy is a lysosomal degradation process of redundant or faulty cell components in normal cells. However, certain diseases are associated with dysfunctional autophagy. Rapamycin, a major immunosuppressant used in islet transplantation, is an inhibitor of mammalian target of rapamycin and is known to cause induction of autophagy. The objective of this study was to evaluate the in vitro and in vivo effects of rapamycin on pancreatic β cells. Rapamycin induced upregulation of autophagy in both cultured isolated islets and pancreatic β cells of green fluorescent protein–microtubule-associated protein 1 light chain 3 transgenic mice. Rapamycin reduced the viability of isolated β cells and down-regulated their insulin function, both in vitro and in vivo. In addition, rapamycin increased the percentages of apoptotic β cells and dead cells in both isolated and in vivo intact islets. Treatment with 3-methyladenine, an inhibitor of autophagy, abrogated the effects of rapamycin and restored β-cell function in both in vitro experiments and animal experiments. We conclude that rapamycin-induced islet dysfunction is mediated through upregulation of autophagy, with associated downregulation of insulin production and apoptosis of β cells. The results also showed that the use of an autophagy inhibitor abrogated these effects and promoted islet function and survival. The study findings suggest that targeting the autophagy pathway could be beneficial in promoting islet graft survival after transplantation.


Abbreviations: 
Atg gene

autophagy related gene

BSA

bovine serum albumin

FKBP-12

12-kDa FK506-binding protein

GFP

green fluorescent protein

GAPDH

glyceraldehyde-3-phosphate

LC3

microtubule-associated protein 1 light chain 3

mTOR

mammalian target of rapamycin

PBS

phosphate-buffered saline

TUNEL

terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling

SD

standard deviation

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

Autophagy, i.e. “self-eating”, is an intracellular degradation system designed for degradation of cytoplasmic proteins and dysfunctional organelles after their sequestration in the autophagosome. To date, only microtubule-associated protein1 light chain 3 (LC3), a mammalian homolog of yeast autophagy related gene8 (Atg8), is known to exist in the autophagosomes, and therefore this protein serves as a marker for autophagosomes (1–3). The process is tightly regulated and plays an important role in cell growth, development and homeostasis, where it helps to maintain a balance between the synthesis, degradation and subsequent recycling of cellular components (1–3). Autophagy is induced dynamically by nutrient depletion to provide necessary amino acids within cells, thus helping them adapt to starvation (4). The physiological role of autophagy has been studied in various organisms and current knowledge indicates that autophagy is involved not only in adaptation to starvation but also in the quality control of intracellular proteins and organelles, to maintain cell functions, development, growth, clearance of intracellular microbes, antigen presentation and protection against disease (5–11). Thus, autophagy functions as a cell-protective mechanism and is up-regulated when cells are preparing to rid themselves of damaging cytoplasmic components, for example, during infection or protein aggregate accumulation (12).

Rapamycin is a macrolide fungicide with immunosuppressant properties that bear molecular structural similarities to the calcineurin inhibitor, tacrolimus (13). However, the mechanism of action of rapamycin is distinct from that of calcineurin inhibitor, such as cyclosporine and tacrolimus. Rapamycin binds to its intracellular receptor, the immunophilin 12-kDa FK506-binding protein (FKBP-12) and the rapamycin-FKBP-12 complex binds to and inhibits the mammalian target of rapamycin (mTOR; Ref. 14). Inhibition of mTOR leads to arrest of the cell cycle at the G1 to S phase and thus, blockade of growth-factor-driven proliferation of not only activated T cells, which constitute the basis of its immunosuppressive action, but also other hematopoietic and nonhematopoietic cells (14,15). The mTOR is ubiquitously expressed in various cell types and functionally is a serine/threonine protein kinase that regulates important cellular process, including growth, proliferation, motility, survival, protein synthesis and transcription (16). Furthermore, activation of mTOR leads to inhibition of autophagy in cells ranging from yeast to human (17). Based on the above background, it is conceivable that the inhibitory action of rapamycin on mTOR activity induces autophagy in pancreatic islets.

Islet transplantation was recently advanced by the publication of the results of the Edmonton Protocol of immunosuppressive regimen, leading to insulin independence at 1 year in 90% of patients treated with type 1 diabetes (18–21). Accordingly, rapamycin has become a part of the standard treatment in islet transplantation. Its effectiveness in preventing allorejection and autoimmunity and promoting the survival of regulatory T lymphocytes has contributed to widespread use (22–24). However, recent reports described gradual deterioration of the metabolic profile and the need for reintroduction of exogenous insulin; only 10% of islet recipients maintained insulin independence at 5 years (21,25,26). Although the cause of the decline in insulin independence rates after islet transplantation remains obscure, the decline may reflect toxicity associated with long-term use of immunosuppressive drugs on islet β cells.

The effects of calcineurin inhibitors on islet function and proliferation have been recognized (27,28), although increasing data suggest that rapamycin alone or in combination with tacrolimus could impair islet cell function and survival (29–31). In addition, the antiangiogenic and antiproliferative properties of rapamycin could also prevent vascularization of transplanted islets, with a resultant reduction of posttransplantation engrafted and surviving islet mass (32–34).

Although it has been reported that β cells of ZDF rats (a rodent model of type 2 diabetes) contain a significant number of autophagic vacuoles (35), there is little information on the physiopathological roles of autophagy in the islets, and no causal link has been reported between autophagy and pathogenesis of diabetes. The aims of this study were to evaluate the in vitro and in vivo effects of rapamycin on pancreatic β cells, including induction of autophagy, cell viability and insulin secretory function, because these factors may contribute to progressive dysfunction of islet grafts in recipients.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

In vitro autophagy induction assay and islet viability assay

Thirty cells from fresh mice islets, obtained from either C57BL/6 mice or green fluorescent protein (GFP)–LC3 transgenic mice were seeded in a 96-well culture plate and cultured for either 24 or 48 h with complete culture medium containing 1 or 10 ng/mL of rapamycin. In the first step, treated islets that were isolated from transgenic mice were observed by fluorescence microscopy to detect GFP signals, which is an accurate marker of induction of autophagy (36). Subsequently, islet viability was evaluated after 24 h treatment by monitoring metabolic activity with the colorimetric methyl tetrazolium salt (MTS) assay using the Cell Titer 96 Aqueous One reagent (Promega, Madison, WI, USA; Ref. 37). The colorimetric reagent was added to each well of the plate and incubated for 2 h, and the absorbance values read at 490 nm.

To further determine the change in islet viability before/after rapamycin treatment, fluorescence labeling was performed using tetramethyl rhodamine ethyl ester (TMRE; Molecular Probes, Eugene, OR, USA) and 7-amino actinomycin D (7-AAD; Molecular Probes; Refs. 38,39). Islets treated or untreated with rapamycin were dissociated into single cell suspensions, using Accutase (Innovative Cell Technologies Inc, San Diego, CA, USA). The dispersed cell suspensions were stained with Newport Green PDX acetoxymethylether (Molecular Probes), for identification of β cells (38). The single islet cell suspensions were incubated with 100 ng/mL TMRE for 30 min at 37°C in phosphate-buffered saline (PBS) without Ca2+ and Mg2+. This dye selectively binds to the mitochondrial membrane allowing the assessment of cells with functional mitochondria, and therefore is a good marker for cell viability. Furthermore, cells were stained with 7-AAD that binds to DNA when cell membrane permeability is altered after cell death. Stained cells were analyzed by FACSCalibur flow cytometer (BD Immunocytometry, San Jose, CA, USA). In addition, improvement in islet viability was assessed by either MTS assay, TMRE or 7-AAD staining based on the results of autophagic signal blocking. Islet viability assays were performed with the addition of 10 mM of 3-methyladenine (3-MA).

Glucose-stimulated insulin release and stimulation index (SI)

To determine the changes in the endocrinological potency of rapamycin-treated islets, static glucose challenge was performed with or without 1 or 10 ng/mL of rapamycin. After overnight culture with or without rapamycin, 100 IEQ of treated islets were incubated with either 2.8 or 20 mM of glucose in culture medium for 2 h at 37°C to stimulate insulin release. The supernatants were collected and stored at −80°C for insulin assessment by enzyme-linked immunosorbent assay (ELISA; Mercodia Inc., Uppsala, Sweden; Refs. 38–40). Glucose-stimulated insulin release was expressed as the SI, calculated as the ratio of insulin released during exposure to high glucose (20 mM) over that released during low glucose incubation (2.8 mM). To determine the in vitro islet potency with regard to autophagic signal blocking, static incubation was also performed with the addition of 10 mM of 3-MA.

In vivo studies using GFP-LC3 transgenic mice

To study the effects of starvation, transgenic mice were provided with drinking water ad libitum, but were deprived of food for 24 h (10 a.m.–10 a.m.; Ref. 36). The starved transgenic mice were sacrificed and the pancreas, brain and muscle tissues were recovered. This was followed by preparation of the tissues for fluorescence microscopy. Furthermore, to demonstrate in vivo 3-MA-induced blocking of autophagy, mice were injected intraperitoneally (i.p.) with 10 mM of 3-MA for 2 weeks, followed by starvation for 24 h.

To assess the in vivo effects of rapamycin on islets from GFP-LC3 transgenic mice, mice were randomly separated into three experimental groups, no treatment group (i.e. control group; n = 25), rapamycin-treated group (n = 25) and the combination treatment group (n = 25) treated with both rapamycin and 3-MA. Rapamycin treatment consisted of daily i.p. injection of 0.2 mg/kg rapamycin and combined treatment consisted of daily i.p. injection of 0.2 mg/kg rapamycin combined with 10 mM of 3-MA. These treatments continued for 1, 2, 3, 4 or 5 weeks (n = 5 mice, each). The rapamycin-treated transgenic mice were sacrificed, the pancreas was removed and processed for fluorescence and immunofluorescence microscopy. During rapamycin treatment, nonfasting blood glucose level was monitored daily using samples obtained from the tail vein. To further determine the effects of rapamycin on glucose tolerance, intraperitoneal glucose tolerance test (IPGTT) was conducted before and at days 14 and 28 after treatment (41). In this test, mice were fasted for 6 h and then injected intraperitoneally with 2 g glucose in saline/kg body weight. Blood glucose levels were measured for 2 h at 30 min intervals. Moreover, to detect the change in insulin secretion after in vivo rapamycin treatment, plasma insulin levels were also measured by ELISA before and after the treatment (at days 0, 7, 14, 21, 28 and 35, n = 5 mice, each group).

Fluorescence microscopy and immunofluorescence microscopy

Pancreatic tissue samples for GFP examination were prepared as follows. To prevent artificial induction of autophagy during sample preparation, mice were anesthetized by diethyl ether and immediately fixed by transcardial perfusion through the left ventricle with 4% paraformaldehyde dissolved in 0.1 M Na-phosphate buffer (pH 7.4). Subsequently, the pancreas was removed and further fixed with the same fixative for another 4 h at room temperature, followed by treatment with 5% sucrose in PBS for 2 h and then with 15% sucrose solution for 4 h, finally with 30% sucrose solution overnight. Pancreatic tissue samples were embedded in Tissue-Tek OCT compound (Sakura Finetechnical Co., Tokyo, Japan) and stored at −80°C. The tissue samples were sectioned at 7 μm thickness with a cryostat, air-dried for 30 min at room temperature and then stored at −80°C until use. Fluorescence signals were analyzed by Biozero fluorescence microscopy (Keyence, Osaka, Japan) by measuring green fluorescence (excitation, 488 nm; emission, 530 nm).

For general histological examination, cryosections were stained with hematoxylin and eosin. Furthermore, for immunofluorescence microscopy, cryosections were prepared as described earlier. After rinsing with water for 5 min, the sections were blocked with 4% bovine serum albumin (BSA)–PBS for 10 min at room temperature. Subsequently, these sections were incubated with rabbit polyclonal anti-mouse insulin Ab (SC-9168; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C diluted in 1% BSA-TBS-Tween-20 (0.05% w/v), followed by incubation with Alexa fluor555 goat anti-mouse IgG (H+L) Ab (A21429; 1:1000 dilution; Invitrogen, Carlsbad, CA, USA) for 30 min at room temperature. Fluorescence signals were observed by Biozero fluorescence microscopy (Keyence). The fluorescence intensities of insulin and GFP-LC3 in treated islets were quantified using Fluor-Chem image analyzer (Bio-Rad Laboratories Inc., Hercules, CA, USA) and expressed in arbitrary units. The mean fluorescence intensities of insulin and GFP, expressed as mean ± standard deviation (SD), were determined in islets of five rapamycin-treated mice. To identify apoptotic β cells in the pancreas of mice, islet sections were stained with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) using Tumor TACS™ In Situ Apoptosis Detection Kit (catalog# 4815–30-K, Trevigen, Gaithersburg, MD, USA) following the instructions provided by the manufacturer.

Statistical analysis

Data were expressed as mean ± SD and analyzed using Excel for Windows software. Two samples were compared with the Student's t-test. The p values <0.05 denoted the presence of statistical significance.

Details of the mice used in these experiments, islet isolation to assess the effects of rapamycin treatment in vitro and western blot analysis are presented in the Supplementary Materials and Methods in the on line version of the journal.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

In vitrooverinduction of autophagy in pancreatic islets by rapamycin

Control islets, including untreated islets and 3-MA-treated islets, showed similar levels of endogenous expression of LC3-II protein (Figure 1). Islets treated with 1 or 10 ng/mL of rapamycin showed the highest expression of LC3-II protein. The conversion of LC3-I (cytosolic form) to LC3-II (membrane-bound lapidated form) was detected by immunoblotting. The amounts of LC3-II protein were three- to fivefold higher in 1 and 10 ng/mL rapamycin-treated islets, respectively, as assessed by the LC3-II/LC3-I ratio (Figure 1). The rapamycin-induced increase in LC3-II level suggests increased autophagy flux. Quantification of LC3-II band intensities showed that blockade of autophagy by 3-MA prevented the accumulation of LC3-II protein in islets treated with 1 or 10 ng/mL of rapamycin. With regard to the conversion of LC3-I to LC3-II, the LC3-II/LC3-I ratio was significantly reduced in islets treated with rapamycin-plus-10 mM 3-MA compared with that of islets treated with rapamycin alone (Figure 1).

image

Figure 1. Changes in LC3-I and LC3-II protein expression levels in rapamycin-treated islets. LC3-I and LC3-II protein expression level were examined by western blot analysis. Protein samples extracted from either untreated islets, rapamycin-treated or rapamycin + 3-MA-treated islets were subjected to 15% SDS/PAGE and transferred onto PVDF membrane. Representative photographs are shown, together with mouse GAPDH levels as an internal control. Quantification of the intensity of the immunoreactive bands of both LC3-I and LC3-II, expressed in arbitrary units, was carried out using NIH Image J software. Results of densitometric analysis of immunoblots of LC3 in islets were expressed as the ratio of LC3-II to LC3-I. Data are mean ± SD of three independent experiments. *p < 0.05, versus control islet; +p < 0.05, versus rapamycin-treated islets.

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As shown in the top panels of Figure 2, a diffuse GFP-LC3 signal was detected in the control islets, with few GFP puncture dots. After 24 h incubation with 1 or 10 ng/mL of rapamycin, the number of GFP-LC3 dots was markedly increased; most dots were detected as cup- or ring-shaped structures (left middle panels, Figure 2). These findings indicate overinduction of autophagy in rapamycin-treated islets. In contrast, the fluorescence level of GFP-LC3 signal in rapamycin-treated islets in the presence of 10 mM 3-MA was diffuse and returned to the basal level of autophagy in control islets (left bottom panels, Figure 2). After 48 h incubation with rapamycin, many large ring- or cup-shaped structures were identified by fluorescence microscopy (right middle panels, Figure 2). Furthermore, the fluorescence signals of GFP-LC3 in rapamycin-plus-3-MA-treated islets continued to show diffuse distribution and persisted at the basal level of autophagy seen in the control islets (right bottom panels, Figure 2). Taken together, the results indicate that the blocking effects of 3-MA were persistent rather than transient.

image

Figure 2. In vitro overinduction of autophagy in response to rapamycin treatment. Fresh islets samples were prepared from GFP-LC3 transgenic mice, and then incubated for either 24 or 48 h in the absence or presence of rapamycin. In 3-MA blocking, fresh islets were incubated in the presence of both rapamycin and 3-MA. After treatment, the GFP signal was detected by fluorescence microscopy. Bars = 100 μm.

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Rapamycin-related overinduction of autophagy in islet cells reduces islet viability

To examine the effect of overinduction of autophagy by rapamycin on islet viability, we performed MTS assay (Figure 3) and fluorescence labeling with TMRE and 7-AAD (Figure 4). Viability under treatment with 3-MA alone was similar to the control islets (Figure 3). Treatment with 1 and 10 ng/mL rapamycin resulted in approximately 43% and 51% reduction of viability, respectively (Figure 3). In contrast, 3-MA ameliorated the effect of rapamycin on islet viability (Figure 3).

image

Figure 3. In vitro viability assessments of rapamycin-treated islet by MTS assay. Control islets, rapamycin-treated and rapamycin-3-MA-treated islets were assessed for islet viability. Data are mean ± SD of five independent islets preparations. The% absorbance of treated islets was expressed relative to absorbance of control islets, which was set at 100%. *p < 0.05, versus control islets; +p < 0.05, versus rapamycin-treated islets.

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image

Figure 4. In vitro analysis of the cytotoxic effect of rapamycin and upregulation of autophagy. Islets were incubated with either 1 or 10 ng/mL of rapamycin for 24 h to overinduce autophagy. In blocking assay, islets were cultured with rapamycin in the presence of 10 mM 3-MA. After dispersion of mice islets into single cell suspensions, cells were stained with TMRE or 7-AAD. (A) Pancreatic β cells were analyzed for the relative percentage of apoptotic or nonapoptotic cells by TMRE. (B) Dead cells represented 7-AAD-positive cells. Data are representative of five independent experiments using different mice islets reparations.

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To further determine the effect of rapamycin on islet viability, islet cells were stained with TMRE or 7-AAD and assessed by FACS analysis. 3-MA had no significant effect on islet viability (control, 80.5 ± 4.5%; 3-MA, 80.4 ± 5.5%) and the percentages of dead cells (i.e. 7-AAD-positive cells; control, 4.7 ± 3.5%; 3-MA, 3.9 ± 4.3%). Rapamycin significantly decreased the proportion of TMRE-positive cells (1 ng/mL of rapamycin, 62.4 ± 6.7%; 10 ng/mL of rapamycin, 52.1 ± 6.1%; compared with the control, p < 0.05), and significantly increased the percentage of 7-AAD-positive cells (1 ng/mL of rapamycin, 17.7 ± 7.6%; 10 ng/mL of rapamycin, 18.7 ± 6.7%; compared with the control islets, p < 0.05). The addition of 3-MA to rapamycin-treated islets ameliorated the effects of the latter on the percentages of both viable and dead cells (Figures 4A and B). Taken together, these data suggest that rapamycin-induced overinduction of autophagy negatively affects islet viability and mitochondrial integrity, and that these effects are blocked by 3-MA.

Rapamycin reduces islet insulin production

Islet insulin potency was assessed by static glucose challenge in vitro. In control islets, insulin was secreted at 4.4–4.5 μg/L under high glucose medium (Figure 5A). In contrast, insulin secretion under high glucose medium was significantly inhibited in rapamycin-treated islets and treatment with rapamycin elicited approximately 45–53% reduction in insulin concentration (Figure 5A). We also analyzed islets’ insulin production using the SI. The SI of untreated control islets was 1.57 ± 0.13 (Figure 5B). 3-MA did not have a significant effect on insulin production compared with the control islets. However, rapamycin significantly reduced the SI (1 ng/mL of rapamycin, 1.20 ± 0.1; 10 ng/mL of rapamycin, 1.11 ± 0.12; p < 0.05, each, compared with the control islets). The addition of 3-MA to rapamycin-treated islets markedly improved both insulin production and SI. Especially, insulin production showed complete recovery in islet treated with 1 ng/mL of rapamycin and 10 mM of 3-MA (Figures 5A and B). These results indicate that rapamycin elicits not only overinduction of autophagy but also reduction of both islet viability and in vitro insulin function.

image

Figure 5. In vitro assessment of the effect of rapamycin on insulin production from islets. Production of insulin was assessed by static glucose challenge and the results expressed as both (A) blood insulin concentration and (B) stimulation index (SI). Data are mean ± SD of five independent islet preparations. *p < 0.05, compared with the control; +p < 0.05, compared with rapamycin alone.

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Effect of nutrient starvation on autophagy in GFP-LC3 transgenic mice

To confirm the beneficial effects of 3-MA on induction of autophagy in the intact animal, we used GFP-LC3 transgenic mice and examined autophagy in 3-MA-treated transgenic mice under starvation. In the control GFP-LC3 transgenic mice, few GFP-LC3 dots were observed in pancreatic acinar cells and such dots were relatively small. In the pancreatic islets, no GFP dots were detected and these islets were clearly stained for insulin (top panels, Figure 6). The GFP-LC3 structures appeared 24 h after starvation as large cup-shaped structures in both islet and acinar cells. To validate these findings, we examined both muscle (as an example of nonessential tissue) and brain (as an essential tissue) tissues by fluorescence microscopy. As shown in Figure 7(A), no GFP dots were observed in the extensor digitorum longus muscles before starvation, however, GFP-LC3 dots appeared after 24 h starvation in muscle tissues (Figure 7B). On the other hand, in brain samples, including the cerebral cortex and medulla oblongata, no GFP-LC3 structures could be detected in spite of 24 h starvation (Figures 7D and F). In addition, islets starved for 24 h stained faintly for insulin (middle panels, Figure 6). The mean insulin staining intensity of starved islets was markedly reduced compared with that of untreated control islets, although the difference in insulin intensity was not significant (control, 61 514 ± 4364; starvation, 44 154 ± 19 925). The mean fluorescence intensity of GFP-dots was significantly higher in starved islets than untreated control islets (control, 2901 ± 576; starvation, 8306 ± 807; p < 0.01). The merged images of GFP signals of LC3 dots and the adjacent islets stained for insulin are shown in Figure 6. The merged microphotographs also showed weaker insulin intensity in starved islets compared with the control islets. The use of 3-MA during 24 h starvation ameliorated the effect of 24 h starvation as evident by the appearance—diffuse and few fluorescence signals of GFP-LC3 dots—and by the return of GFP fluorescence intensity in islets. Furthermore, the recovered islets stained positive for insulin and the intensity of such staining was similar to the control islets, as judged by both the mean staining intensity and the merged microphotographs (lower panels, Figure 6).

image

Figure 6. Starvation induced autophagy in pancreatic islets and acinar cells of GFP-LC3 transgenic mice. Representative images of islets stained with H&E and for insulin after 24 h starvation. Representative GFP images of pancreatic islets, acinar cells and merged microphotographs of GFP images and insulin staining. Numbers in the right upper corner of the photographs represent the mean ± SD intensity of GFP and insulin staining, expressed in arbitrary units, of five different islets. **p < 0.01, compared with the control; ++p < 0.01, compared with 24 h starvation. Bars = 100 μm.

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image

Figure 7. Starvation induced autophagy in muscle tissues, but not in brain. GFP images of transverse sections of extensor digitorum longus muscle (A) before starvation and (B) after starvation. GFP images of the cerebral cortex (C) before starvation and (D) after starvation. GFP images of medulla oblongata (E) before starvation and (F) after starvation. Bars = 10 μm.

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Effect of rapamycin on autophagy in GFP-LC3 transgenic mice

Finally, we assessed the effects of rapamycin on autophagy and insulin production in transgenic mice in vivo. For this purpose, the mice were treated with 0.2 mg/kg of rapamycin intraperitoneally daily for 1, 2, 3, 4 or 5 weeks. After 1 week of such treatment, small but few dots appeared in both islets and acinar cells, however, no significant difference was observed in the pancreas of rapamycin-treated mice and rapamycin-plus-3-MA (10 mM)-treated mice (top panels, Figures 8A and B). After 2, 3, 4 and 5 weeks of rapamycin treatment, a marked increase in the density of GFP-LC3 dots was observed and these dots appeared as ring- or cup-shaped structures in both islets and acinar cells (Figures 8A and B). The GFP fluorescence intensity was higher in 1-week treated islets than in untreated control islets, although no significant large GFP dots were observed (Figures 6 and 8A). After 2, 3, 4 and 5 weeks of treatment, the GFP fluorescence intensity in the treated islets was significantly up-regulated compared with those in control and 1-week treated islets. In spite of overinduction of autophagy in rapamycin-treated islets, the mean intensities of insulin in 2-, 3-, 4- and 5-week rapamycin-treated islets were significantly lower than the control untreated islets (Figure 8A). Interestingly, 3-MA ameliorated the changes in immunofluorescence, including GFP-LC3 dots and insulin staining intensity, which reflects rapamycin-induced overinduction of autophagy (Figure 8B). The merged microphotographs also demonstrated reduced insulin intensity in rapamycin-treated islets and that the degenerative change showed significant recovery in islets of the rapamycin-plus-3-MA group. TUNEL-positive cells were detected in 2-, 3-, 4- and 5-week rapamycin treated islets. In contrast, no such cells were observed in islets treated with rapamycin-plus-3-MA. Taken together, these in vivo findings correlated well with the in vitro data, including islet insulin potency and TMRE viability assay.

imageimage

Figure 8. Upregulation of autophagy in rapamycin-treated GFP-LC3 transgenic mice. Representative images of islets stained with H&E, GFP images of pancreatic islets and acinar cells, images of islets stained for insulin and images of TUNEL staining after the indicated period of treatment. Also shown are the merged microphotographs of GFP images and insulin staining. Numbers in the right upper corner of the photographs represent the mean ± SD intensity of GFP and insulin staining, expressed in arbitrary units, of five different islets. *p < 0.05, compared with the control; **p < 0.01, compared with the control; ++p < 0.01, compared with rapamycin alone. Images were obtained from GFP-LC3 transgenic mice treated with (A) 0.2 mg/kg/i.p. rapamycin alone and (B) 0.2 mg/kg/i.p. rapamycin-plus-10 mM 3-MA. Bars = 100 μm.

To further determine the effects of rapamycin on islet function in mice, we measured nonfasting blood glucose and plasma insulin concentrations. Rapamycin had no significant effect on nonfasting blood glucose levels, and near-normoglycemia was noted in mice treated with rapamycin alone and in those treated with rapamycin-plus-3-MA (Figures 9A and B). At days 14, 21, 28 and 35 after treatment, plasma insulin levels were higher in rapamycin-plus-3-MA-treated mice than in rapamycin-treated mice. Especially, plasma insulin concentration at day 14 in rapamycin-treated mice was significantly lower than in rapamycin-plus-3-MA-treated mice (Figures 9C and D). All other differences in insulin concentration were not significant between the two groups. Interestingly, in IPGTT performed at day 14, the blood glucose level of mice treated with rapamycin alone was significantly higher than in those treated with rapamycin-plus-3-MA at 15, 30, 60, 90 and 120 min after injection of glucose. Thus, rapamycin elicited a diabetic glucose pattern in mice (Figure 9E). In contrast, in the same test performed at day 28, the blood glucose levels of rapamycin-treated mice were similar to those of mice treated with rapamycin-plus-3-MA, and the pattern of blood glucose after injection was also similar between the two groups (Figure 9F). Taken together, rapamycin treatment resulted in impairment of in vivo glucose tolerance until 2 weeks after treatment and this abnormality was reversed by co-administration of 3-MA. It is possible that this abnormality of glucose tolerance represents physiological adjustment, such as reduction of insulin resistance at day 28. Further analysis of this phenomenon requires in vivo experiments of long-term rapamycin treatment.

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Figure 9. Nonfasting blood glucose and plasma insulin concentrations after treatment and glucose tolerance test at days 14 and 28. (A) Blood glucose level of GFP-LC3 transgenic mice treated with 0.2 mg/kg/i.p. rapamycin alone. (B) Blood glucose level of GFP-LC3 transgenic mice treated with 0.2 mg/kg/i.p. rapamycin-plus-10 mM 3-MA. (C) Plasma insulin concentration of mice treated with 0.2 mg/kg/i.p. rapamycin alone. (D) Plasma insulin concentration of mice treated with 0.2 mg/kg/i.p. rapamycin-plus-10 mM 3-MA. (E) Glucose tolerance test after treatment at day 14. (F) Glucose tolerance test after treatment at day 28. Closed circles: data of mice treated with 0.2 mg/kg/i.p. rapamycin alone; open circles: data of mice treated with 0.2 mg/kg/i.p. rapamycin-plus-10 mM 3-MA. Data are mean ± SD of five mice in each treatment group.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

Rapamycin has deleterious effects on islet β cell based on the blockade of VEGF-mediated survival pathways and inhibition of β-cell proliferation and by induction of apoptosis (20,34,42). Accordingly, we raised the question of whether rapamycin in islet transplantation is a friend or a foe. In this study, we focused on the effect of rapamycin on autophagy and evaluated the direct effect of rapamycin on islet β cells. Using various techniques, the results demonstrated for the first time that rapamycin at therapeutically used concentrations, overinduced autophagy both in vitro and in vivo and that this effect on islet β cells impaired both islet viability and insulin potency.

Autophagy is the degradation of redundant or faulty cell components (1–4,12). Recent studies have described a link between diabetes and autophagy (43,44). Two groups independently reported the findings of increased apoptosis and reduced proliferation of β cells with resultant reduction in β-cell mass in β-cell-specific autophagy-deficient mice (Atg7f/f: RIP-Cre mice; Refs. 43,44). These studies indicated that basal autophagy is indispensable for the maintenance of normal islet architecture, such as mitochondria and function of β cells (43,44).

As shown in Figure 1 and immunoblot analyses reported by others (36,44–46), endogenous LC3-II expression was detected in cell lysates from pancreatic islets and a low level of constitutive autophagy (here referred to as “basal autophagy”) was present in normal control islets. Our results also showed that rapamycin resulted in overinduction of autophagy in islets with consequent impairment of insulin function, both in vitro and in vivo. These results suggest that overinduction of autophagy by rapamycin in islet β cells negatively affects insulin function by modulating cell death through accelerated self-digestion and degradation of essential cellular components. Based on the effect of rapamycin on islet β cells, it is possible that excessive digestion of various types of cellular structures, including insulin granules, mitochondria and endoplasmic reticulum membranes takes place in autophagic vacuoles, because this structure lacks stringent substrate specificity, which is different from that used by the ubiquitin–proteasome system (47); in other words, any structure in the cytosol could become a substrate for autophagy (43,44). This may explain the significantly low insulin production capacity of in vitro rapamycin-treated islet β cells, the significant reduction of insulin staining intensity in islets of rapamycin-treated mice and the marked impairment of glucose tolerance assessed by IPGTT (Figures 5, 8A and 9E). Based on these results, we speculate that the main etiology of progressive dysfunction of transplanted islets is reduced insulin production related to rapamycin treatment and the related overinduction of autophagy.

Although the protective role of basal autophagy on pancreatic β-cell function has been proposed in loss-of-function studies on Atg/genes (43,44,48), accelerated autophagy seems to be involved in certain types of cell death (2,49–51). For this reason, we evaluated β-cell apoptosis by TMRE staining and TUNEL, and dead cells by 7-AAD. Figures 4A and B showed that rapamycin increased the percentages of apoptotic β cells and 7-AAD-positive dead islet cells. Furthermore, the TUNEL-positive apoptotic cells were observed in islets of rapamycin-treated mice and these apoptotic cells disappeared after the administration of 3-MA. These findings seem to indicate the existence of crosstalk between autophagy and apoptosis and various links between autophagy and cell death, which may occur in a hierarchial or independent fashion (52–55). In this regard, Masini et al. (48) reported that exposure of nondiabetic islets to high concentrations of free fatty acid resulted in accumulation of autophagic vacuoles. Together with enhanced β-cell death, which was associated with decreased LAMP2 expression. These results suggest that accelerated autophagy may contribute to β-cell death under special conditions, such as rapamycin treatment.

The upregulation of autophagy after rapamycin treatment resulted in a significant impairment of β-cell insulin function, and this effect may contribute to islet graft dysfunction observed in islet recipients. We also demonstrated that 3-MA ameliorated rapamycin-related β-cell dysfunction both in vitro and in vivo. Thus, this new modulator of autophagy, such as 3-MA, should be tested further clinically, and better therapeutic agents with specific autophagic activity need to be developed for the prevention and treatment of islet graft dysfunction.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

The authors thank Dr. F.G. Issa (http://www.word-medex.com.au) for the careful reading and editing of the manuscript. We also thank Dr. N. Kawaguchi and Prof. N. Matsuura (Osaka University) for their help in the execution of this project and the valuable suggestions.

Funding source: This work was supported by a grant from the Ministry of Education, Sports and Culture of Japan to M. T. (No. 22591520) and by Kobayashi Foundation for Cancer Research to M. T.

Disclosure

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

Supplementary Materials and Methods

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AJT_3771_sm_SuppMat.doc31KSupporting info item

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