Alberto Bartolomé,1,2,3 Maki Kimura-Koyanagi,4 Shun-Ichiro Asahara,4 Carlos Guillén,2,3

Hiroyuki Inoue,1 Kyoko Teruyama,1 Shinobu Shimizu,1 Ayumi Kanno,4 Ana García-Aguilar,2,3

Masato Koike,5 Yasuo Uchiyama,5 Manuel Benito,2,3 Tetsuo Noda,6 and Yoshiaki Kido1,4

Pancreatic b-Cell FailureMediated by mTORC1Hyperactivity and AutophagicImpairmentDiabetes 2014;63:2996–3008 | DOI: 10.2337/db13-0970

Hyperactivation of the mammalian target of rapamycincomplex 1 (mTORC1) in b-cells is usually found as a con-sequence of increased metabolic load. Although it playsan essential role in b-cell compensatory mechanisms,mTORC1 negatively regulates autophagy. Using amousemodel with b-cell–specific deletion of Tsc2 (bTsc22/2)and, consequently, mTORC1 hyperactivation, we focusedon the role that chronic mTORC1 hyperactivation mighthave on b-cell failure. mTORC1 hyperactivation drove anearly increase in b-cell mass that later declined, trigger-ing hyperglycemia. Apoptosis and endoplasmic reticulumstress markers were found in islets of older bTsc22/2

mice as well as accumulation of p62/SQSTM1 and animpaired autophagic response. Mitochondrial mass wasincreased in b-cells of bTsc22/2 mice, but mitophagywas also impaired under these circumstances. We pro-vide evidence of b-cell autophagy impairment as a linkbetween mTORC1 hyperactivation and mitochondrialdysfunction that probably contributes to b-cell failure.

Nutrient overload is one of the main causes of insulinresistance. This triggers the compensatory mechanismsleading to b-cell mass increase and hyperinsulinemia.Hyperactivation of the mammalian target of rapamycincomplex 1 (mTORC1) is elicited under nutrient overloadconditions (1–3). mTORC1 plays a positive role in b-cell

mass expansion (3–6), and rapamycin treatment impairsb-cell mass adaptation (7,8). On the other hand, mTORC1hyperactivation is also a cause of insulin resistance (1)and endoplasmic reticulum (ER) stress (9), conditionslinked with b-cell dysfunction and diabetes progression(10,11). We previously described how Tsc2 deletion inb-cells (bTsc22/2) results in chronic mTORC1 hyperacti-vation leading to a biphasic phenotype with early b-cellmass increase, hyperinsulinemia, and hypoglycemia. Thiswas followed by b-cell failure and hyperglycemia in oldermice (3). We also found how the first phase in bTsc22/2

mice is characterized by both an increase of mitochondrialmass and enhanced glucose-stimulated insulin secretion(12). Autophagy is a cytoprotective mechanism also foundessential for b-cell homeostasis (13,14). Autophagy playsa protective role under stress conditions, such as ER stress(15), and we and others have described its positive role inb-cells under these conditions (16,17). Autophagy is alsoresponsible for the turnover of mitochondria by the specificelimination of defective or damaged organelles. mTORC1 isa critical negative regulator of autophagy (18), and studieshave shown how TSC deficiency leads to impaired autoph-agy in human tumors and cell lines (19,20).

In this study, we explored the intriguing possibilitythat mTORC1 hyperactivity in b-cells, apart from beingessential for b-cell compensatory mechanisms, might

1Division of Medical Chemistry, Department of Biophysics, Kobe UniversityGraduate School of Health Sciences, Kobe, Japan2Department of Biochemistry and Molecular Biology, Faculty of Pharmacy,Complutense University of Madrid, Madrid, Spain3CIBERDEM, Instituto de Salud Carlos III, Madrid, Spain4Division of Diabetes and Endocrinology, Department of Internal Medicine, KobeUniversity Graduate School of Medicine, Kobe, Japan5Department of Cell Biology and Neurosciences, Juntendo University GraduateSchool of Medicine, Tokyo, Japan6Department of Cell Biology, Cancer Institute, Japanese Foundation of CancerResearch, Tokyo, Japan

Corresponding author: Yoshiaki Kido, kido@med.kobe-u.ac.jp.

Received 25 June 2013 and accepted 10 April 2014.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-0970/-/DC1.

© 2014 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, andthe work is not altered.

2996 Diabetes Volume 63, September 2014

ISLETSTUDIES

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trigger b-cell failure when chronically elicited. This couldbe the consequence of the dysregulation of cytoprotectivemechanisms, such as autophagy and mitochondrial turn-over (mitophagy), and the development of insulin resis-tance and ER stress.

RESEARCH DESIGN AND METHODS

AnimalsbTsc22/2 mice were obtained by crossing Tsc2flox/flox micewith those expressing Cre recombinase under rat insulin 2gene promoter, as described before (3). Tsc2flox/flox were usedas control mice. Animals were maintained as described be-fore (3,12). For rapamycin treatment, bTsc22/2 mice 20–24weeks of age were subjected to intraperitoneal injections of2 mg/kg body weight rapamycin (LC Laboratories) or vehicleevery other day. A 10 mg/mL stock solution of rapamycinwas made in 100% ethanol, stored at 20°C, diluted to 0.5mg/mL vehicle (5% Tween 80 and 5% polyethylene glycol),and used within 24 h. Only male mice were used, and allexperiments were performed following the guidelines fromthe Animal Ethics Committee of Kobe University GraduateSchool of Medicine.

Islet Isolation and ImmunostainingPancreatic islets were isolated by collagenase digestionof exocrine pancreas and Histopaque density-gradientcentrifugation as previously described (3,10,12). Isolatedislets were cultured overnight in RPMI1640 supple-mented with 10% FBS and antibiotics. For confocal imag-ing of islets, islets were fixed in paraformaldehyde 4%(weight/volume [w/v]) and processed as described before(21). Islets from at least three mice per genotype wereused for all staining experiments.

Immunoblot AnalysisIslets or cells were washed with cold PBS and lysed bysonication in a buffer containing 1% (volume/volume)Nonidet-P40, 50 mmol/L Tris-HCl, 5 mmol/L EDTA,5 mmol/L EGTA, 150 mmol/L NaCl, 20 mmol/L NaF,1 mmol/L phenylmethylsulfonyl fluoride, 10 mg/mL apro-tinin, and 2 mg/mL leupeptin, pH 7.5. Antibodies used aredescribed in Supplementary Table 1.

Confocal Microscopy ParametersFor confocal microscopy assays with whole islets, AxioObserver Z1 with LSM 700 scanning module (Zeiss) wasused. Four different solid-state lasers were used: diode405 nm, 5 mW; diode 488 nm, 10 mW; diode 555 nm, 10mW; and diode 639 nm, 5 mW. Fluorophore excitationwas generally performed at #2% laser transmission.

For whole-islet imaging, a 403 Zeiss Plan-Apochromatoil objective was used (numerical aperture = 1.3). For de-tailed imaging of cells within the islet, a 633 Zeiss Plan-Apochromat oil objective was used (numerical aperture =1.4). Fluorophores were visualized in various tracks toavoid crosstalking, DAPI was visualized with 405-nm laserexcitation and short pass (SP) 550-nm filter. Fluoresceinisothiocyanate was excited with a 488-nm laser and bandpass (BP) 420- to 528-nm filter. MitoTracker orange and

Cy3 were excited with a 555-nm laser and visualized witha 576- to 640-nm BP filter. Alexa Fluor 647 was excitedwith 639-nm laser and visualized with a long pass 640-nmfilter. Appropriate controls were used for each experimentto confirm specific signals.

All images were obtained in a 1,024 3 1,024–pixelformat, excitation speed was 0.5–5 ms/pixel, and pinholesize set to ;1 airy unit. Z-stacks were collected with 2.5-mm spacing (403 objective) or 1-mm spacing (633 objec-tive). For microscope operation and image gathering, ZEN2011 (Zeiss) software was used.

Selective Imaging of Active Mitochondrial MembranePotentialAfter overnight incubation of islets in RPMI supplementedwith 10% FBS, islets were further incubated for 30 min at37°C in serum-free medium supplemented with 500 nmol/LMitoTracker Orange CMTMRos (Life Technologies). Isletswere washed twice with cold PBS and fixed as describedbefore (21). MitoTracker CMTMRos is only incorporatedby actively respiring mitochondria and is irreversibly boundto mitochondrial proteins, allowing its use with antibodiesfor multiple staining after aldehyde fixation.

Immunohistochemistry and Morphometric AnalysisImmunofluorescence images of pancreatic sectionsstained with insulin and glucagon were gathered witha Biozero BZ-8000 microscope (Keyence) and BZ Analyzersoftware. Images of five different sections per pancreas(spaced at least 200 mm) were analyze to allow stereologicdetermination of b-cell mass. At least five mice per ageand genotype were used for immunohistochemical andb-cell mass determination assays.

Image Processing and QuantificationImage analysis was performed with NIH ImageJ software(http://rsb.info.nih.gov/ij/), ZEN 2011 software (Zeiss), andAmira (Visage Imaging). NIH ImageJ was used for densito-metric quantification of Western blot results, islet diametercalculation of isolated islets, and determination of b-cellmass. ZEN 2011 was used for analysis of confocal microscopeimages. Amira was used to construct three-dimensional mod-els from z-stacks and volumetric measurements.

Quantitative PCRRNA was isolated from islets from separate control orbTsc22/2 mice using the RNeasy kit (Qiagen). cDNA syn-thesis and PCR amplification was performed in one stepwith SYBR Green PCR Master Mix (Life Technologies) andquantified with an ABI 7900 sequencer (Life Technolo-gies) with the specific primers detailed in SupplementaryTable 2.

Cell Culture and Immunofluorescence of CulturedCellsCell culture of insulinoma-derived cell lines MIN6 andINS-1E was performed as described before (16). Reagentsused for cell culture were rapamycin (LC Laboratories), tuni-camycin, chloroquine, insulin, and IGF-I (Sigma-Aldrich).For immunofluorescence assays, cells were grown on glass

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coverslips, and fixation and antibody incubation were per-formed as previously described (16).

Lentivirus ProductionHuman embryonic kidney 293T cells were cotransfectedusing Lipofectamine 2000 (Life Technologies) with lenti-viral packaging plasmid pMD2.G (Addgene #12259) andpsPAX2 (Addgene #12260) along with lentiviral vectorpLKO.1-neo for short hairpin RNA (shRNA) production(Addgene #13425). Tsc2 and scrambled sequences (indicatedin Supplementary Table 2) were cloned according to recom-mendations from Addgene as previously described (16).

Electron Microscopy and Immunogold LabelingPancreatic tissue was obtained from two mice per age andgenotype and processed as previously described (12). Thinsections (60–70 nm) were obtained with an Ultracut E (Leica)ultramicrotome, stained with lead citrate, and examined un-der a JEM-1010 transmission electron microscope (JEOL).

Immunogold labeling of epoxy resin sections wasperformed according to standard methods. Briefly, ultra-thin sections of pancreas embedded in epoxy resin weremounted onto nickel grids. To increase resin hydrophilic-ity, sections were incubated in drops of 4% (w/v) sodiummetaperiodate and then 1% (w/v) periodic acid. Thiswas followed by incubation in PBSG (PBS containing50 mmol/L glycine), and nonspecific binding sites wereblocked by incubation of sections in PBSG containing 5%(w/v) BSA. Antibodies were diluted in PBSG containing 1%BSA, guinea pig anti-p62/SQSTM1 (1:200) or guinea piganti-insulin (1:200), the latter used as a positive controlfor the technique. Twelve-nanometer colloidal-gold donkeyanti–guinea pig secondary antibody (1:200 in PBSG contain-ing 1% BSA) was used (Jackson ImmunoResearch). Fur-ther staining and observation was performed as previouslydescribed (12).

Mitochondrial Fractionation and Protein OxidationAssaysFor detection of mitochondrial protein oxidation, 23 107

cells per condition were used. After trypsinization, centri-fugation (3 min, 110g, 4°C), and PBS washing, mitochon-dria were isolated using the Mitochondria Isolation Kit forCultured Cells (Thermo Scientific) according to manufacturerinstructions. All buffers were enriched with protease inhib-itors and 1% (volume/volume) 2-mercaptoethanol to avoidfurther oxidation of proteins after cell lysis. Mitochondrialpelleting was performed at low gravitational force to avoidperoxisome contamination of the mitochondrial fraction (15min, 3,000g, 4°C). Mitochondrial pellets were lysed in 30 mLbuffer containing 2% (w/v) CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), 10 mmol/L Tris-HCl, 150 mmol/L NaCl, and 1% (w/v) 2-mercaptoethanol,pH 7.5. Protein concentration was determined, and 10 mgwere used for either direct Western blot analysis or deriva-tization with OxyBlot kit (Merck Millipore).

Statistical AnalysisStatistically significant differences between mean valueswere determined using paired Student t test for paired

comparisons. Differences were considered statistically sig-nificant at P , 0.05.

RESULTS

Tsc2 Ablation in b-Cells Results in a BiphasicPhenotypeConsistent with our previous study (3), young bTsc22/2

mice became hypoglycemic as a consequence of increasedb-cell mass. However, after 40 weeks, b-cell mass de-clined, and hyperglycemia appeared (Fig. 1A and B). Isletmorphology analysis showed diminished b-cell composi-tion in older bTsc22/2 islets (Fig. 1C and SupplementaryFig. 1A) as well as the presence of cleaved caspase-3 inpancreatic slides and islet lysates (Fig. 1D and E). Wesought to discern the mechanisms responsible of b-cellfailure in older bTsc22/2 mice.

mTORC1 Hyperactivation in b-Cells Causes CellHypertrophy and Insulin ResistanceTsc2 ablation led to mTORC1 hyperactivation in b-cells;protein lysates from isolated bTsc22/2 islets showed in-creased phosphorylation of key proteins downstream ofmTORC1, such as S6 kinase (S6K) and the ribosomal S6protein (Fig. 2A). Volumetric analyses in 24-week-oldislets showed a fivefold increase in b-cell volume as wellas a doubled b-cell nuclei volume in bTsc22/2 islets (Fig.2B and Supplementary Movies 1 and 2). a-Cell volumeswere also slightly increased, probably as a consequenceof hyperinsulinemia or other compensatory mechanisms(22). Distribution of islet sizes is highly shifted in youngbTsc22/2 mice, with a mean diameter in control mice of201 6 10 mm and 281 6 20 mm for bTsc22/2 islets (Sup-plementary Fig. 1B–D).

Tsc2 ablation and the consequent mTORC1/S6Khyperactivation induces insulin resistance by Ser/Thrphosphorylation of insulin receptor substrates (1,2,23).MIN6 cells stably expressing Tsc2-shRNA showed impairedAkt phosphorylation in response to insulin or IGF-I, whichwas not fully recovered by rapamycin treatment (Fig. 2C),consistent with other reports (24). Insulin and IGF-I sig-naling are necessary for the correct functioning and main-taining of b-cell mass (11). FoxO1 is an important player inb-cell proliferation and stress response, being normallyexcluded from the nucleus in b-cells by autocrine/paracrineinsulin signaling. Akt directly phosphorylates FoxO1, driv-ing its nuclear exclusion (25). In bTsc22/2 islets, increasedmTORC1 activity impaired FoxO1 nuclear exclusion andpartly recovered after rapamycin treatment (Fig. 2D); theseobservations were consistent in islets from bTsc22/2 miceof 8, 24, and 40 weeks of age (data not shown). Similarobservations were performed in MIN6 Tsc2-shRNA cells.Nuclear localization and impaired phosphorylation ofFoxO1 Thr21 was observed and partly recovered afterrapamycin treatment (Supplementary Fig. 2A and B). In-sulin signaling and FoxO1 in b-cells also modulated Pdx1(25); immunofluorescence of pancreatic islets showed de-creased Pdx1-positive nuclei in bTsc22/2 throughout lifebut recovered after rapamycin treatment (Fig. 2E).

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Tsc2 Ablation in b-Cells Triggers the Unfolded ProteinResponsePrevious reports have shown how Tsc2- or Tsc1-deficientfibroblasts or human tumors display increased activationof the unfolded protein response (9). Islets from 40-week-oldbTsc22/2 mice showed increased eukaryotic initiation fac-tor 2a kinase 3 (Eif2ak3/PERK) phosphorylation as well asEif2a Ser51 phosphorylation and activating transcriptionfactor 4 (ATF4) expression (Fig. 3A). The ER chaperoneheat shock protein 5 (Hspa5/BiP) was also increasedtogether with some transcription factors associated withapoptotic outcome after ER stress, such as the DNAdamage-inducible transcript 3 (Ddit3/CHOP) or theCCAAT/enhancer-binding protein b (Cebpb) (Fig. 3A).Transcripts of Hspa5, Ddit3, and Cebpb were increasinglyexpressed in bTsc22/2 islets in an age-dependent manner(Fig. 3B). Ddit3 expression was only found in islet proteinextracts from older bTsc22/2 mice, whereas p62/SQSTM1was increased in both, as discussed in the next section(Fig. 3C). Ddit3 was found specifically in b-cell nuclei ofolder bTsc22/2 islets (Fig. 3D) as was the phosphorylatedform of Eif2a (Fig. 3E). Increased ER stress in b-cells hasbeen related to accumulation of polyubiquitinated proteins

(26). bTsc22/2 islets showed an increased profile of proteinubiquitination by Western blot or immunohistochemicalanalyses of pancreatic sections (Supplementary Fig. 3Aand B).

Impaired Autophagy in bTsc22/2 IsletsmTORC1 is a negative regulator of autophagy (18); thus,chronic upregulation of mTORC1 due to Tsc2 ablation isexpected to impair autophagy. bTsc22/2 islet proteinextracts showed no differences in basal lipidation of LC3B;however, basal levels of the adaptor protein p62/SQSTM1were markedly increased, indicating a defect in the clear-ance of autophagic substrates (Fig. 4A). Immunofluores-cence analysis of isolated islets revealed an increasednumber and diameter of p62/SQSTM1-positive punctain bTsc22/2 islets, specifically in b-cells (Fig. 4B and E).Ex vivo challenge of isolated islets to ER stress–inducedautophagy showed impaired response in bTsc22/2 islets.mTORC1 activity, seen as phosphorylation of S6, was notdownregulated after tunicamycin treatment, and lipida-tion of LC3B was not increased as observed in controlislets (Fig. 4C). Chloroquine was used as a tool to estimateautophagic flux, which was not increased in bTsc22/2 after

Figure 1—Biphasic response of b-cell mass and blood glucose levels in bTsc22/2 mice. A: Blood glucose concentration at the indicatedages. B: b-Cell mass calculated from pancreatic sections immunostained with anti-insulin antibodies. Positive insulin area divided by thetotal area of the pancreas. C: Representative images of islets from pancreatic sections stained with anti-insulin antibodies for b-cells (red),and antiglucagon for a-cells (green). Scale bars, 50 mm. D: Pancreatic sections of 35-week-old mice were subjected to immunohisto-chemistry with an antibody detecting the cleaved form of caspase-3. E: Protein extracts of isolated islets from 40-week-old animals weresubjected to Western blot analysis and incubated with specific antibodies. Densitometric quantification of cleaved caspase-3 levels isshown (n = 3). Data are mean 6 SD (n = 3–8 per age and model). *P < 0.05, **P < 0.01, ***P < 0.001. KO, knockout.

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Figure 2—mTORC1 hyperactivation in b-cells induces cell hypertrophy and insulin resistance. A: Islet protein extracts were subjected toWestern blot analysis with specific antibodies. B: Isolated islets from 24-week-old mice were incubated with specific antibodies foridentification of b-cells (insulin, red), a-cells (glucagon, green), and membranes (b-catenin, blue). Nuclei were stained with DAPI andvisualized in white. Multiple z-stack images were collected, which allowed for three-dimensional reconstruction and volume measurements,as indicated in the graphs, and expressed in cubic micrometers as mean per islet 6 SD. C: MIN6 cells stably expressing Tsc2-shRNA orcontrol shRNA (scrambled) were serum starved for 3 h, with the addition of rapamycin 20 nmol/L where indicated, and subsequentlystimulated with insulin or IGF-I for 5 min. D: Isolated islets were cultured for 24 h in RPMI containing 10% FBS and subjected to

3000 mTORC1 Hyperactivation and b-Cell Failure Diabetes Volume 63, September 2014

acute ER stress induction. Similar results were observed inthe MIN6 Tsc2-shRNA cell line (Supplementary Fig. 4A).Immunofluorescence analysis of ER stress–challenged

islets also showed impairment of LC3B puncta upregula-tion in bTsc22/2 islets (Fig. 4E). Additionally, glucosedeprivation was used as a proautophagic stimulus. In

immunofluorescence with anti-FoxO1 antibody. Representative confocal microscopy images are shown. E: Isolated islets were grownovernight in RPMI containing 10% FBS and then subjected to immunofluorescence with anti-Pdx1 antibody. Representative confocalmicroscopy images are shown. *P < 0.05, **P < 0.01, ***P < 0.001. KO, knockout; ns, not significant.

Figure 3—Marked increase in ER stress markers during aging in bTsc22/2 islets. A: Protein extracts from islets isolated in 40-week-oldmice were subjected to Western blot analysis with specific antibodies. Graphs show densitometric quantification of blots (n = 3). B:Quantitative PCR analysis of mRNA from islets isolated from mice at the indicated ages. Data are mean 6 SD (n = 3). *P < 0.05, **P <0.01, ***P < 0.001. C: Protein extracts from islets isolated in 8- and 40-week-old mice and subjected to Western blot analysis. D: Isolatedislets from 40-week-old mice were grown overnight on RPMI containing 10% FBS and then subjected to immunofluorescence with anti-Ddit3antibody. Positive nuclei are observed in bTsc22/2 islets (white arrows) but not in control islets (data not shown). E: Pancreatic sections of 35-week-old mice were subjected to immunohistochemistry with an antibody detecting the P-Eif2a Ser51. Positive cells are indicated by blackarrows. Further incubation with anti-insulin antibodies were done to confirm b-cell identity of positive stained regions, as indicated by whitearrows. KO, knockout.

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Figure 4—Autophagy is impaired in bTsc22/2 islets. A: Protein extracts from 24-week-old isolated islets were subjected to Western blotanalysis. p62/SQSTM1 levels are represented in the graph as mean 6 SD (n = 3). B: Isolated islets from 24-week-old mice were subjectedto immunostaining with anti-p62/SQSTM1 antibody. Representative confocal microscopy images are shown. Representation of the num-ber and diameter of puncta per islets is mean6 SD. C: Isolated islets were stabilized overnight in RPMI containing 10% FBS and treated exvivo with tunicamycin 2 mg/mL for 20 h in the presence or absence of chloroquine 10 mmol/L. Protein extracts were subjected to Westernblot analysis, and images from a representative experiment are shown (n = 3). D: Representative confocal microscopy image showingspecific accumulation of p62/SQSTM1 puncta in b-cells of bTsc22/2 islets. E: Representative confocal microscopy images of the same

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pancreatic islets, both energy stress and loss of autocrineinsulin signaling may trigger autophagy after glucosewithdrawal. Glucose starvation did not properly triggeran increase of LC3B-positive puncta in bTsc22/2 isletscompared with controls (Supplementary Fig. 4B). Ofnote, a-cells within bTsc22/2–starved islets showeda marked upregulation of LC3B puncta, meaning thatautophagy is only impaired in b-cells within bTsc22/2

islets (Supplementary Fig. 4C).

Increased Colocalization of p62/SQSTM1 WithMitochondria in bTsc22/2 IsletsOur previous work showed how mTORC1 hyperactivationcauses an increase in mitochondrial biogenesis in b-cellsof 10-week-old bTsc22/2 mice (12). Immunohistochemi-cal analyses showed how mitochondrial markers are also

increased in 35-week-old bTsc22/2 mice (SupplementaryFig. 5A). The finding of impaired autophagy prompted usto investigate a possible defect in autophagic clearance ofmitochondria (mitophagy). Colocalization of mitochon-drial structures with the adaptor molecule p62/SQSTM1was evident in b-cells of bTsc22/2 mice (SupplementaryFig. 5B). Tsc2 downregulation in MIN6 cells also increasedthe number of p62/SQSTM1-positive mitochondria butnot in the presence of rapamycin (Supplementary Fig. 5C).Moreover, the findings for these mitochondrial structuresin bTsc22/2 islets were negative for a mitochondrialmembrane potential (Dcm)–sensitive probe (Fig. 5A andSupplementary Movies 3 and 4). Quantification of thesemitochondrial structures (Tom20 positive) with collapsedDcm (MitoTracker negative) showed an age-dependent in-crease of p62/SQSTM1 colocalization in b-cells of bTsc22/2

experiment shown in C; islets were subjected to immunostaining with anti-LC3B antibody. Quantification of LC3B-positive puncta isshown, representing the mean number per mice 6 SD (n = 3). Scale bars, 10 mm. *P < 0.05, **P < 0.01, ***P < 0.001. KO, knockout; Tun,tunicamycin; U.A., arbitrary unit.

Figure 5—Accumulation of low Dcm and p62/SQSTM1-positive mitochondria in bTsc22/2 islets. A: Isolated islets were stabilized overnight

in RPMI containing 10% FBS and then incubated for 30 min in medium containing MitoTracker (MTR) Orange CMTMRos 500 nmol/L andsubsequently subjected to immunofluorescence assays with specific antibodies. Representative images of isolated islets from 40-week-oldmice are shown. Arrows indicate collapsed Dcm (MTR-negative) and p62/SQSTM1-positive mitochondria. B: Quantification of the numberof MTR-negative and p62/SQSTM1-positive mitochondrial structures (Tom20 positive). Number per cell is mean per islet 6 SD (severalislets from 3–5 mice per age and genotype). C: Isolated islets from mice treated with rapamycin or vehicle were subjected to staining withCox4 and p62/SQSTM1, as shown in Supplementary Fig. 5D. Bars show the average number of colocalizing spots per cell expressed asmean per islet 6 SD. *P < 0.05, **P < 0.01, ***P < 0.001. KO, knockout.

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mice (Fig. 5B), whereas the number of positive Dcm mito-chondrial structures (MitoTracker and Tom20 positive)colocalizing with p62/SQSTM1 was anecdotic and age andmodel independent (data not shown). bTsc22/2 mice thatwere subjected to rapamycin treatment showed a great re-duction of p62/SQSTM1-positive mitochondria (Fig. 5C andSupplementary Fig. 5D). p62/SQSTM1 can bind to proteinaggregates or damaged organelle, allowing recognition by theautophagic machinery (27), and its accumulation is a markerof impaired autophagy (13,14). Such differences observedbetween control and bTsc22/2 islets could only be explainedby assuming deficient mitophagy in b-cells due to mTORC1hyperactivation.

Moreover, electron micrographs from 35-week-oldmice showed mitochondrial abnormalities in b-cellsfrom bTsc22/2 mice. Mitochondrial mass was clearlyhigher in 35-week-old mice compared with controls, aspreviously reported in 10-week-old mice (12). However,mitochondria of b-cells in older bTsc22/2 mice showeda higher degree of complexity as well as highly dilatedcristae, which were frequently found with abnormal ori-entation (Fig. 6). Signs of degenerated mitochondria wereevident in electron micrographs of b-cells from 35-week-old bTsc22/2 mice and hardly found in either control or10-week-old bTsc22/2 mice (Fig. 6 and Supplementary

Fig. 6). In addition, immunogold labeling assays revealedp62/SQSTM1-positive mitochondria in b-cells of 35-week-old bTsc22/2 mice (Fig. 6F and G and SupplementaryFig. 7).

Inefficient Mitophagy Caused by Tsc2 AblationProduces the Accumulation of Oxidized MitochondrialProteinsTo assess consequences of impaired mitophagy in bTsc22/2

islets, pancreatic sections were probed with an antibodyrecognizing oxidized proteins (nitrotyrosine); bTsc22/2

islets showed increased immunoreactivity compared withreactivity in exocrine pancreas or control islets (Fig. 7A).Moreover, mitochondrial protein extracts were used for thedetection of oxidized proteins. Both MIN6 and INS-1Estably expressing Tsc2-shRNA showed increased protein ox-idation compared with controls (Fig. 7B). Mitochondrialprotein oxidation was reduced by rapamycin treatment(Fig. 7C). In addition, autophagy was found to playa role in the clearance of oxidized mitochondrial proteins.An increased pattern of oxidized proteins was onlyobserved after chemical inhibition of autophagy in con-trol MIN6 cells, not in Tsc2-shRNA cells, indicatingbasal impairment of autophagic mitochondrial clearancein Tsc2-shRNA MIN6 cells (Fig. 7D). Immunofluores-cence assays also showed increased colocalization of

Figure 6—Electron micrographs of b-cells from 35-week-old mice. A and B: Control mice, normal mitochondrial mass, complexity andmorphology. C–H: bTsc22/2 mice. C and D: Increased mitochondrial mass and complexity, degeneration of mitochondria. E: Degeneratedmitochondria, highly dilated cristae. F: Aberrant orientation of cristae in fused mitochondria, indicated by arrow. G: Immunogold labelingusing anti-p62/SQSTM1 antibody; boxed region is magnified in H, arrows indicate gold particles.

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http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-0970/-/DC1http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-0970/-/DC1http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-0970/-/DC1http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-0970/-/DC1http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-0970/-/DC1

nitrotyrosine with mitochondrial structures in MIN6Tsc2-shRNA cells (Fig. 7E).

DISCUSSION

mTORC1 signaling in b-cells is essential for the adaptivemechanisms that allow b-cell mass increase and compen-satory hyperinsulinemia. In b-cells, TSC2/mTORC1 inte-grates glucose and growth factor signaling to controlprotein synthesis and cell growth and proliferation (23).mTORC1 hyperactivation occurs as a consequence ofincreased metabolic load, being a feature of obesity-associated type 2 diabetes progression (1–3). Therefore,we aimed to study the consequences of chronic mTORC1hyperactivation in b-cells due to Tsc2 ablation.

In young bTsc22/2 mice, increased b-cell mass due tostriking hypertrophy was found. However, as previouslyreported, b-cell mass failure and hyperglycemia becameevident from 40 weeks (3). Of note, other authors usinganother strain of bTsc22/2 mice did not observe this bi-phasic behavior (4). Rapamycin treatment of bTsc22/2

mice from 20 weeks of age abolished b-cell failure (3).

On the other hand, moderate hyperactivation of mTORC1in b-cell–specific Rheb-overexpressing mice was shown toplay a protective role against diabetes development (5).Although disruption of signaling downstream of mTORC1in b-cells classically leads to diminished b-cell mass andglucose intolerance (28), increased b-cell apoptosis is alsoobserved in mice expressing a constitutively active formof S6k1 (29).

In the current study, the fact that b-cell failure andhyperglycemia are only apparent in old bTsc22/2 mice(9 months old) is consistent with other studies linkingmTORC1 hyperactivity to late-onset pathologies or defi-ciencies (30,31). Other reports described mTORC1 asa key regulator of aging (32,33); thus, increased mTORC1signaling might accelerate aging and the development ofaging-related pathologies.

The current results point to diverse deleterious con-sequences due to mTORC1 hyperactivation. First, inb-cells of bTsc22/2 mice, insulin resistance is observedthroughout life, leading to impaired FoxO1 nuclear exclu-sion and decreased Pdx1-positive nuclei. These events are

Figure 7—Tsc2 ablation in b-cells increases the level of oxidized proteins. A: Pancreatic sections of 35-week-old mice were subjected toimmunohistochemical analysis with antinitrotyrosine antibody. Islets are outlined. B: MIN6 and INS-1E cell lines expressing control (scrambled)or Tsc2-shRNA were subjected to cytosol/mitochondria fractionation. The mitochondrial fraction was derivatized using an OxyBlot kit andanalyzed by Western blot; representative images from three independent experiments are shown. C: MIN6 cells were treated for 24 h with 40nmol/L rapamycin or vehicle. Mitochondrial fractions from MIN6 Tsc2-shRNA were analyzed by direct Western blot (nitrotyrosine) or afterderivatization of oxidized proteins (OxyBlot). D: MIN6 cells were treated with bafilomycin A1 20 nmol/L for 3 h or left untreated. Mitochondrialfractions were purified and subjected to Western blot analysis. Representative blots are shown. E: Confocal microscope images of MIN6 cellsstained with antinitrotyrosine (green). Mitochondria were visualized in red by cytochrome c staining and nuclei in blue by DAPI staining. Whitearrows indicate nitrotyrosine-positive mitochondria. Scale bar: 10 mm.

diabetes.diabetesjournals.org Bartolomé and Associates 3005

linked to b-cell dysfunction (25,34) and increased b-cellsusceptibility to ER stress (35,36). Additionally, ER stressin bTsc22/2 islets becomes evident with aging. Severalreports linked the increased insulin demand in insulin-resistant states, with ER stress development resultingfrom the large load placed in the b-cell ER. This finallycauses the expression of Ddit3 and Cebpb, leading tob-cell failure (10,37).

Hyperactivation of mTORC1 in b-cells impairs basalautophagy, as p62/SQSTM1 levels were found increasedin b-cells within islets. Moreover, autophagic response toER stress challenge was also impaired in bTsc22/2 islets.Reports have indicated how autophagy is an important pro-tective mechanism in b-cells under ER stress (16,17). Auto-phagic impairment might be the underlying cause of ERstress development (17), which becomes evident with agingin bTsc22/2 mice or in other TSC-deficient models (9).

mTORC1 hyperactivation leads to enhanced mitochon-drial mass and oxidative function (12,38), but in the cur-rent study, we also found impaired mitophagy due to Tsc2ablation. Accumulation of mitochondrial structures withcollapsed mitochondrial membrane potential and positivefor the adaptor protein p62/SQSTM1 was evident, andthose structures were found in higher numbers in anage-dependent manner and decreased by rapamycin treat-ment. p62/SQSTM1 plays an important role in the rec-ognition and grouping of mitochondria before theirincorporation into the autophagic machinery (39,40).Antiautophagic activity of mTORC1 is conducted thoughinteraction with ULK1 complex (41,42), which is consis-tent with similar observations between bTsc22/2 isletsand Ulk12/2 fibroblasts (43) where increased mitochon-drial mass together with accumulation of p62/SQSTM1 isattributed to inefficient mitophagy.

Aberrant and degenerated mitochondria were found inb-cells from older bTsc22/2 mice, and atypical cristae andhighly interconnected mitochondria were frequently seen

on electron micrographs. Increased fusion of mitochon-dria has been proposed as a defense mechanism againstcellular aging (44) and deleterious conditions that mighttrigger apoptosis (45), also enabling mitochondrial func-tion under these conditions (46). Chronic mTORC1 hy-peractivity leading to impaired mitophagy is likely theunderlying cause of increased mitochondrial degenerationand elongation observed in b-cells from older bTsc22/2

mice. Although mTORC1 plays a well-described role inmitochondrial biogenesis (12,38), its particular impact onmitochondrial dynamics is understudied. Consistent withthe results shown here, another report showed howmTORC1 inhibition is also required for mitophagy (47).

Other reports have shown how impaired mitophagyleads to the accumulation of dysfunctional mitochondriaand excess production of reactive oxygen species (48,49).bTsc22/2 islets also showed increased oxidation of pro-teins, and by using a cellular model of mTORC1 hyper-activation in b-cells, we confirmed the finding of increasedoxidation of mitochondrial proteins. Mitochondrial dys-function is a characteristic feature of b-cell failure (50).Other reports have also linked insulin resistance andFoxO1 activity with mitochondrial dysfunction (51).

We cannot fully determine one specific cause responsiblefor b-cell failure under mTORC1 hyperactivity. Still, the ex-perimental evidence points to autophagic impairment as themajor cause. Rapamycin treatment prevents b-cell failure inthis model (3), and we show how it ameliorates autophagicdeficiency. However, rapamycin cannot fully restore insulinsignaling toward mTORC2/Akt, consistent with otherreports (24,52). Therefore, we believe that other deleteriousevents overshadowed by autophagic impairment are theleading causes of b-cell failure in aged bTsc22/2 mice.

In conclusion, mTORC1 hyperactivation may, in a firststage, play a positive role in b-cell compensatory mecha-nisms leading to hypertrophy, increased mitochondrialmass, and hyperinsulinemia (Fig. 8). However, we report

Figure 8—In an early stage, mTORC1 hyperactivation drives the increase of b-cell mass and function. However, when this hyperactivation becomeschronic, other deleterious effects might trigger b-cell failure.

3006 mTORC1 Hyperactivation and b-Cell Failure Diabetes Volume 63, September 2014

on how chronic activation of these compensatory mecha-nisms driven by mTORC1 may result in insulin resistancein b-cells, ER stress, autophagic impairment, and accumu-lation of dysfunctional mitochondria that could lead tob-cell failure and diabetes onset.

Acknowledgments. The authors thank Agustín Fernández and MaríaLuisa García (Centro Nacional de Microscopía Electrónica, Universidad Complu-tense de Madrid, Spain) for technical assistance and expertise in electron mi-croscopy experiments.Funding. A.B. acknowledges an FPU (Formación de personal ProfesoradoUniversitario) fellowship from the Spanish Ministry of Education, Spain. Thiswork is supported by a Grant-in-Aid for Scientific Research from MEXT (Ministryof Education, Culture, Sports, Science and Technology–Japan) to Y.K. (22590981)and grant SAF2011-22555 from the Spanish Ministry of Science and Innovation.Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. A.B. researched data and contributed to theexperimental design, discussion, and writing of the manuscript. M.K.-K.researched data and contributed to the experimental design, discussion, andreview and editing of the manuscript. S.-I.A. and Y.K. contributed to the exper-imental design, discussion, and review and editing of the manuscript. C.G.researched data and contributed to the discussion and review and editing ofthe manuscript. H.I. researched data and contributed to the discussion. K.T., A.K.,and T.N. contributed to the discussion. S.S. contributed to the experimentaldesign and discussion. A.G.-A., M.K., and Y.U. researched data. M.B. contributedto the discussion and review and editing of the manuscript. Y.K. is the guarantorof this work and, as such, had full access to all the data in the study and takesresponsibility for the integrity of the data and the accuracy of the data analysis.Prior Presentation. Parts of this work were presented at the 48th AnnualMeeting of the European Association for the Study of Diabetes, Berlin, Germany,1–5 October 2012.

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