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doi:10.1152/ajpendo.00262.2009 297:1013-1022, 2009. First published Aug 18, 2009;Am J Physiol Endocrinol Metab

MacDougald, Martin G. Myers, Jr. and Kun-Liang Guan Villanueva, Miro Faouzi, Tsuneo Ikenoue, David J. Kwiatkowski, Ormond A. Hiroyuki Mori, Ken Inoki, Darren Opland, Heike Münzberg, Eneida C.

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Critical roles for the TSC-mTOR pathway in �-cell function

Hiroyuki Mori,1,2 Ken Inoki,1,2 Darren Opland,3,4 Heike Munzberg,3 Eneida C. Villanueva,2,3

Miro Faouzi,3 Tsuneo Ikenoue,1 David J. Kwiatkowski,5 Ormond A. MacDougald,2,3

Martin G. Myers, Jr.,2,3,4 and Kun-Liang Guan1,6,7,8

1Life Sciences Institute, 2Department of Molecular and Integrative Physiology, 3Department of Medicine, 4Program inNeuroscience, 6Department of Biological Chemistry, and 7Institute of Gerontology, University of Michigan, Ann Arbor,Michigan; 5Division of Translational Medicine, Department of Medicine, Brigham and Women’s Hospital, Harvard MedicalSchool, Boston, Massachusetts; and 8Department of Pharmacology and Moores Cancer Center, University of California SanDiego, La Jolla, CA

Submitted 27 April 2009; accepted in final form 15 August 2009

Mori H, Inoki K, Opland D, Munzberg H, Villanueva EC, Faouzi M,Ikenoue T, Kwiatkowski DJ, MacDougald OA, Myers MG Jr, GuanKL. Critical roles for the TSC-mTOR pathway in �-cell function.Am J Physiol Endocrinol Metab 297: E1013–E1022, 2009. Firstpublished August 18, 2009; doi:10.1152/ajpendo.00262.2009.—TSC1 is a tumor suppressor that associates with TSC2 to inactivateRheb, thereby inhibiting signaling by the mammalian target of rapa-mycin (mTOR) complex 1 (mTORC1). mTORC1 stimulates cellgrowth by promoting anabolic cellular processes, such as translation,in response to growth factors and nutrient signals. To test roles forTSC1 and mTORC1 in �-cell function, we utilized Rip2/Cre togenerate mice lacking Tsc1 in pancreatic �-cells (Rip-Tsc1cKO mice).Although obesity developed due to hypothalamic Tsc1 excision inolder Rip-Tsc1cKO animals, young animals displayed a prominentgain-of-function �-cell phenotype prior to the onset of obesity. Theyoung Rip-Tsc1cKO animals displayed improved glycemic controldue to mTOR-mediated enhancement of �-cell size, mass, and insulinproduction but not determinants of �-cell number (proliferation andapoptosis), consistent with an important anabolic role for mTOR in�-cell function. Furthermore, mTOR mediated these effects in theface of impaired Akt signaling in �-cells. Thus, mTOR promulgates adominant signal to promote �-cell/islet size and insulin production,and this pathway is crucial for �-cell function and glycemic control.

tuberous sclerosis complex; mammalian target of rapamycin; pancre-atic �-cell; conditional knockout mice; rat insulin promoter 2

TUBEROUS SCLEROSIS COMPLEX (TSC) is an autosomal dominantgenetic disorder characterized by benign tumor growth in awide range of tissues. TSC is exclusively associated withmutations in either the TSC1 or TSC2 tumor suppressor gene.TSC1 and TSC2 proteins form a tight physical complex thatpossesses GTPase-activating protein (GAP) activity towardsRheb, a small GTPase of the Ras family. In the TSC1/TSC2complex, TSC2 contains the catalytic Rheb-GAP activity,whereas TSC1 functions as a regulatory subunit that controlsthe stability and cellular localization of the complex. Active,GTP-bound Rheb directly binds to the mammalian target ofrapamycin (mTOR) complex 1 (mTORC1) to promote mTORC1kinase activity. Interference with TSC1/2 (e.g., in TSC mutantcells) promotes Rheb GTP loading and constitutive activationof mTORC1.

mTOR is an evolutionarily conserved serine/threonine ki-nase that promotes anabolic cellular processes such as protein

synthesis in response to growth factors, nutrients (amino acidsand glucose), and stress (43). mTOR exists in two distinctcomplexes, the rapamycin-sensitive TORC1 and the rapamy-cin-insensitive TORC2 (23). mTORC1 consists of mTOR,mLST8, raptor, and PRAS40. In addition to the shared mTORand mLST8, mTORC2 also contains two unique subunits,rictor and sin1. Interestingly, TSC1/TSC2 and Rheb directlyregulate TORC1 but not TORC2 (45). Furthermore, mTORC1phosphorylates S6 kinase (S6k) and eukaryotic initiation factor4E-binding protein-1 (4E-BP1), whereas mTORC2 is requiredfor phosphorylation of Akt on Ser473, serum and glucocorti-coid-inducible kinase, and conventional protein kinase C.Therefore, the two TORC complexes differ in their subunitcompositions, physiological functions, and regulations. For thepurposes of this article, mTOR refers to mTORC1.

Insulin and insulin-like growth factor I potently activatemTOR likely through the phosphatidylinositol 3-kinase (PI3K)and Akt pathways (34). Cellular energy levels also stronglyregulate mTOR activity, and the AMP-activated protein kinase(which is active in states of low cellular energy) blunts mTORsignaling (13). The mechanism of mTOR regulation by aminoacids is not completely understood, although recent studieshave implicated the Rag family of GTPases as potential me-diators in this pathway (15, 32). The TSC1/2 complex inte-grates upstream signals from growth factors, nutrients, andcellular energy levels to modulate mTOR. In response toinsulin, the PI3K-Akt pathway phosphorylates and inhibitsTSC2 to activate mTOR (4, 12, 24); the Akt-dependent phos-phorylation of PRAS40 may also promote mTOR activity (30,33, 39, 40). Thus, the TSC1/2 complex integrates multipleupstream signals to modulate mTORC1 activity (6, 11, 12, 24, 43).

Several studies using genetically engineered mouse modelshave established an important role for the insulin-insulin re-ceptor (IR)-insulin receptor substrate (IRS)-PI3K-Akt cascadenot only in glucose metabolism in peripheral tissue but also inpancreatic �-cell development and function. For instance, in-activation of mediators of insulin signaling [such as IR, IRS,phosphoinositide-dependent protein kinase-1 (PDK1), Akt,and glucose transporter-4 (GLUT4)] not only produces periph-eral insulin resistance (1, 14, 29, 37, 42) but also attenuatespancreatic �-cell function (9, 17, 18, 21). The use of rapamycinto inhibit mTOR has suggested crucial roles for mTOR in�-cell function. Not only does rapamycin inhibit �-cell prolif-eration in vitro (22), it also blocks the effects of glucose andAkt activation on �-cell mass and proliferation (20). Recentstudies have also begun to explore roles for increased mTORsignaling in �-cell function (8, 35). Here, we utilize genetic

Address for reprint requests and other correspondence: K.-L. Guan, Dept. ofPharmacology and Moores Cancer Center, Univ. of California San Diego, LaJolla, CA 92093-0815 (e-mail: [email protected]).

Am J Physiol Endocrinol Metab 297: E1013–E1022, 2009.First published August 18, 2009; doi:10.1152/ajpendo.00262.2009.

0193-1849/09 $8.00 Copyright © 2009 the American Physiological Societyhttp://www.ajpendo.org E1013


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ablation of the mTOR inhibitor TSC1 to examine mTORfunction in �-cells. Since the conventional knockout of Tsc1leads to embryonic lethality (16, 19, 27), we generated miceconditionally lacking Tsc1 in pancreatic �-cells using Rip2/Cre[Rip-Tsc1c-knockout (KO) mice]. Although older Rip-Tsc1cKO an-imals displayed hyperphagia and obesity due to the activationof mTOR in Rip/Cre-expressing hypothalamic neurons (26),we examined �-cell function in younger mice prior to the onsetof obesity and insulin resistance. These studies demonstratedthat constitutive activation of mTOR in �-cells by Tsc1KOcauses �-cell hypertrophy, increased insulin synthesis andrelease, and consequent hyperinsulinemia. These results sug-gest that the cellular TSC-mTOR pathway regulates insulinproduction as well as the function of appetite-suppressingneural circuits.

MATERIALS AND METHODS

Animals and animal care. Tsc1lox/lox mice, with exons 17 and 18 ofTsc1 flanked by loxP sites by homologous recombination, have beendescribed (25, 38). We generated �-cell and hypothalamic -specificTsc1-knockout mice (Rip-Tsc1cKO) by breeding Tsc1lox/lox mice withmice that express the Cre recombinase gene under the control of therat insulin 2 gene promoter (The Jackson Laboratory). Mice weremaintained on the mixed genetic background (C57Bl/6 � 129Sv �BALB/c). We performed experiments using Rip-Cre/Tsc1lox/lox (Rip-Tsc1cKO) mice and littermates Tsc1lox/lox as control. Mice werehoused on a 12:12-h light-dark cycle in the Unit for LaboratoryAnimal Medicine at the University of Michigan with free access towater and standard mouse chow. Animal experiments were conductedfollowing protocols approved by the University Committee on the Useand Care of Animals.

In vivo physiological studies. Blood glucose levels were deter-mined using an automated blood glucose reader (Accu-Check;Roche). Serum insulin and leptin levels were measured by ELISA(Crystal Chem, Downers Grove, IL). Glucose tolerance tests wereperformed on mice that were fasted overnight (16 h). Blood wascollected immediately before as well as 15, 30, 60, and 120 min afterthe intraperitoneal injection of glucose (2 g/kg body wt). For insulintolerance tests, mice were fasted for 3 h and injected with 0.75 U/kgbody wt of human insulin (Novolin R; Novo Nordisk). For glucose-stimulated insulin secretion experiments, mice were deprived of foodfor 16 h and then anesthetized with pentobarbital sodium. Bloodsamples were taken from tail vein at 0, 1, 2, 5, 10, and 15 min afterthe intravenous glucose injection (1 g/kg body wt). Insulin levels weremeasured as described above. Serum growth hormone levels were mea-sured by ELISA (Linco Research). Serum glucagon levels weremeasured by Hormone Assay & Analytical Services Core, MMPC,Vanderbilt University [National Institutes of Health (NIH) Grant no.DK-59637].

Rapamycin treatment studies. Rapamycin (LC Laboratories) wasinitially dissolved in 100% ethanol, stored at �20°C, and furtherdiluted in an aqueous solution of 5.2% Tween-80 and 5.2% PEG 400(final ethanol concentration 2%) immediately before use (41). Two-week-old Rip-Tsc1cKO and control mice were injected with rapamy-cin intraperitoneally (1 mg/kg body wt every other day) for �4 or 6wk. Both pancreas and brain tissues for Western blot analysis, RT-PCR, and immunohistochemistry (IHC) were harvested after 12 h ofthe last treatment of rapamycin.

Insulin content measurement. For measurement of insulin contentof whole pancreas, excised pancreata were frozen in liquid nitrogenand disrupted. Then the pancreas powder was suspended in coldacid-ethanol, and insulin was extracted overnight at 4°C. The super-natants were diluted and subjected to ELISA as described above.Pancreas insulin content was determined using a rat insulin ELISA kitfollowing acid-ethanol extraction.

Islet isolation and insulin secretion. Mice were euthanized, thecommon bile ducts cannulated, and their duodenal ends occluded byclamping. One and a half milligrams per milliliter collagenase P [1mg/ml in Hanks� balanced salt solution (HBSS); Roche Diagnostics,Indianapolis, IN] was injected into the duct to distend the pancreas.The pancreas was excised, incubated at 37°C for 20 min, and me-chanically disrupted in 10 ml of HBSS. Cellular components werecollected by centrifugation (220 g for 1 min) and resuspended in 10 mlof HBSS. Islets were handpicked under a microscope and washed inHBSS.

Freshly isolated islets were incubated for 1 h in RPMI 1640medium supplemented with 10% fetal calf serum, 10 mM HEPES, 2mM L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, 100�g/ml streptomycin, and 11.1 mM glucose at 37°C in a 5% CO2

incubator. Size-matched islets were preincubated in 12-well plates for2 h in RPMI 1640 medium at 2.8 mM glucose and then 1 h inKrebs-Ringer bicarbonate-HEPES buffer (KRBH) plus 0.5% bovineserum albumin and 2.8 mM glucose. Insulin secretion was assessedduring a subsequent 1-h incubation in KRBH and 0.5% bovine serumalbumin at 2.8 or 16.7 mM glucose at 37°C (7).

IHC. Pancreas tissue was harvested following transcardiac perfu-sion with 4% paraformaldehyde and postfixed in 4% paraformalde-hyde. Pancreatic sections were stained with antibodies against insulinand glucagon (DakoCytomation), phosphorylated S6 (p-S6; Ser235/236,IHC preferred; Cell Signaling Technology), p-S6 (Ser235/236 AlexaFluor 488 conjugate for double staining of pancreas), and �-catenin(Cell Signaling Technology). For analysis of pancreatic morphometry,we scored six animals/genotype and nine sections/animal spaced 100�m apart using NI Vision Assistant (version 7.1.0; National Instru-ments). We also used ApopTag (Millipore) for terminal deoxynucleo-tidyl transferase dUTP nick-end labeling (TUNEL) assay, Ki-67antibody (Leica), and ProLong Gold antifade with 4�,6-diamidino-2-phenylindole (invitrogen) for proliferation assay.

Brains were also harvested from 24-h-fasted mice at the beginningof light cycle. After transcardiac perfusion with 4% paraformalde-hyde, brains were postfixed in 4% paraformaldehyde and then trans-ferred to 20% sucrose solution for cryoprotection overnight. Tissueswere frozen on dry ice and cut into 25-�m coronal sections on asliding microtome, collected in five series, and stored in antifreezesolution (50% PBS, 15% ethyl glycol, and 35% glycerol) at �20°Cuntil further use. Brain sections were stained with p-S6 antibodiesusing free-floating method.

Western blot analysis. After isolation, 40 islets/individual mousewere incubated at 37°C for 12 h (RPMI 1640, 100 mg/ml glucose,1.5% FBS). Islet extracts were immunoblotted with Tsc1, Tsc2,IRS-1, IRS-2, p-S6k (Thr398), S6k, p-4E-BP1 (Thr37/46), 4E-BP1,p-S6 (Ser240/244), S6, p-Akt (Thr308), p-Akt (Ser473), Akt (Cell Sig-naling), and �-tubulin (Sigma Aldrich). p-IRS-1 (Ser307) antibodywas kindly provided by Dr. L. Rui (University of Michigan). Forquantitative phosphorylation, after immunoblotting with p-S6k, S6k,p-S6, S6, p-4E-BP1, and 4E-BP1, the signals were quantified usingImage J software.

Statistical analyses. All data are presented as means � SE and wereanalyzed by Student’s t-test or analyses of variance. The differenceswere considered to be significant if P � 0.05.

RESULTS

Tsc1 knockout by Rip-cre activates mTORC1 in islets. Toinvestigate the function of the TSC-mTOR pathway in pancre-atic �-cells, we sought to establish a mouse model withpancreatic �-cell-specific deletion of Tsc1. Mice containinghomozygous floxed Tsc1 alleles (Tsc1lox/lox) (25, 38) weremated with the cre transgenic line driven by the rat insulinpromoter 2 (Rip) (5, 17, 21, 28, 36). All subsequent experi-ments were performed using Rip-cre/Tsc1lox/lox (Rip-Tsc1cKO)

E1014 TSC-mTOR PATHWAY IN �-CELL FUNCTION

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mice and littermate Tsc1lox/lox (control) mice. Western blottingconfirmed markedly reduced Tsc1 expression in isolated isletsfrom Rip-Tsc1cKO mice (Fig. 1A). Tsc2 content was alsodecreased in Rip-Tsc1cKO mice, consistent with the stabiliza-tion of TSC2 by TSC1 (Fig. 1A). We further determined theactivation status of mTOR in islets by immunoblotting forp-S6, which serves as a convenient downstream readout formTOR, since activated mTOR stimulates the phosphorylationof S6 by S6k. Loss of Tsc1 was associated with increased S6phosphorylation (p-S6) in Tsc1cKO islets (Fig. 1A), indicatingthat mTOR was activated in the mutant islets. Immunostainingfor p-S6 showed that, within the pancreas, mTOR was acti-vated only in the �-cells of Rip-Tsc1cKO mice and not theneighboring cells (Fig. 1, B and C), consistent with a cell-autonomous effect of Tsc1 on mTOR. These data also revealedlarge islets, suggesting the potential expansion of islet mass inthe Rip-Tsc1cKO mice. Tsc1 protein levels in brain, heart,kidney, liver, and muscle were indistinguishable between Rip-Tsc1cKO and control mice (data not shown). In additionto Tsc1 deletion in islets, we demonstrated previously thatRip-Tsc1cKO mice demonstrate Tsc1 excision and subse-quently increase p-S6 immunoreactivity in the hypothalamus (Fig.1D) (26).

Normal insulin responsiveness in 4-wk-old Rip-Tsc1cKOmice. The hypothalamic deletion of Tsc1 in Rip-Tsc1cKO miceleads to obesity and insulin resistance in older animals (26).Since obesity and insulin resistance would confound our ex-amination of �-cell function in these animals, we initiallyexamined insulin levels and parameters of glycemic controlwith age in Rip-Tsc1cKO mice to determine an appropriateyoung age prior to the onset of obesity at which to examine�-cell function (Fig. 2). We focused on animals 4 wk of ageprior to the onset of obesity (Fig. 2A) and compared theirphenotype to that of older animals. Serum insulin concentra-

tions in Rip-Tsc1cKO mice were elevated relative to controls atall ages examined, although this was most dramatic in the older(8- and 24-wk-old) animals, consistent with the dramatic obe-sity and insulin resistance in these older animals (Fig. 2B).Indeed, whereas older Rip-Tsc1cKO mice displayed normal orincreased blood glucose levels despite dramatically increasedcirculating insulin levels (consistent with insulin resistance),4-wk-old Rip-Tsc1cKO mice demonstrated decreased bloodglucose relative to controls (Fig. 2C). These data are consistentwith the hypersecretion of insulin in young Rip-Tsc1cKOrelative to the wild-type mice (4-wk-old; Fig. 2D), indicating anormal insulin response in the Rip-Tsc1cKO animals. Todirectly examine insulin sensitivity in these animals, insulintolerance tests were performed. Commensurate with their obe-sity, by 8 wk of age, Rip-Tsc1cKO mice exhibited an impairedhypoglycemic response to exogenous insulin. Consistently,insulin-induced Akt phosphorylation in both liver and musclewere significantly compromised in Rip-Tsc1cKO mice at 12wk of age along with the expression of IRS-1 protein in muscle(Supplemental Fig. S1; Supplemental Material for this article isavailable at the AJP-Endocrinology and Metabolism website).The finding that pair-feeding Rip-Tsc1cKO and control micenormalized the insulin responsiveness of Rip-Tsc1cKO animals(Fig. 2E) suggests that the insulin resistance of the olderRip-Tsc1cKO mice results from their increased adiposity ratherthan some other alteration (such as hypothalamic function).

In contrast to the insulin resistance in older animals, bloodglucose levels fell at a similar rate and to a similar extent inRip-Tsc1cKO compared with control mice at 4 wk of age (Fig.2D, left). Thus, consistent with the reduction in blood glucoselevels appropriate for the hyperinsulinemia displayed by 4-wk-old Rip-Tsc1cKO mice, these young animals exhibit normalinsulin sensitivity, suggesting that the elevated insulin levels inyoung Rip-Tsc1cKO mice reflect a primary enhancement in

Fig. 1. Tuberous sclerosis complex 1 (Tsc1)knockout (KO) by Rip-Cre activates mamma-lian target of rapamycin (mTOR) complex 1in islets. A: elevated S6 phosphorylation inTsc1-knockout islets. Immunoblot analysis ofTsc1, Tsc2, phospho-S6 (p-S6), S6, and �-tu-bulin are indicated. Islets were isolated andextracts prepared from independent animals.B: S6 phosphorylation in Tsc1cKO �-cells.Immunostaining of the pancreatic sectionsfrom 4-wk-old Rip-Tsc1cKO and controlmice were performed with p-S6 (Ser235/236)antibody. The scale bar represents 500 (lowmag., low magnification) and 100 �m (highmag., high magnification). C: S6 phosphory-lation was observed in Tsc1cKO �-cells.Double labeling of the pancreatic sectionsfrom 4-wk-old Rip-Tsc1cKO and controlmice were performed with p-S6 (Ser235/236)antibody and insulin antibody. The scale barrepresents 100 �m. D: Rip-Tsc1cKO in-creases S6 phosphorylation in hypothalamus.Representative p-S6 (Ser235/236) staining of hy-pothalamus from ad libitum-fed Rip-Tsc1cKOand control mice at 4-wk ages. Ct., control;Rip-cKO, Rip-Tsc1cKO, which is Tsc1 tissue-specific knockout using Rip/Cre; IHC, immu-nohistochemistry; ME, median eminence; 3V,3rd ventricle.

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insulin production and that 4 wk represents an appropriate ageto examine islet function without confounding effect of hypo-thalamic obesity.

Increased �-cell size in 4-wk-old Rip-Tsc1cKO mice. Giventhe apparent enlargement of islets observed in the p-S6 staining(Fig. 1B), we examined the morphology of the islets in Rip-Tsc1cKO mice more carefully (Fig. 3). As shown in Fig. 3A,the average islet area was increased significantly in Rip-Tsc1cKO pancreas at 4 wk of age compared with that ofcontrol littermates. Consistently, the distribution of islet sizewas skewed toward larger islets in Rip-Tsc1cKO pancreas (Fig.3B), and protein content per islet (not normalized for size) wasincreased almost twofold in islets isolated from Rip-Tsc1cKOmice compared with those from control mice (Fig. 3C). Todetermine whether the increase in islet mass reflected increasedcell size (rather than cell number), the size of individual �-cellswas also measured. We used insulin staining to mark the�-cells and �-catenin staining to highlight cell boundaries. Wefound that �-cells in Rip-Tsc1cKO were significantly largerthan those in control mice (Fig. 3D). In contrast, Rip-Tsc1cKOhad no effect on �-cell number (Fig. 3E). Furthermore, we didnot observe significant differences in the proportion of Ki-67-positive nuclei or TUNEL-positive nuclei in the islets betweencontrol and Rip-Tsc1cKO mice at 4 wk of age (Fig. 3, F andG). Therefore, ablation of Tsc1 and subsequent mTOR activa-tion primarily promotes increased cell growth/size rather thanproliferation or number of �-cells. Consistent with this idea,there was no evidence of invasiveness of islet tissue.

Increased mTOR activity in �-cells from Rip-Tsc1cKO micepromotes insulin production. To test whether the phenotype ofthe pancreatic �-cells in Rip-Tsc1cKO animals is mediated by

mTOR activation, we treated 2-wk-old Rip-Tsc1cKO and con-trol mice with rapamycin, a specific mTOR inhibitor, at 1mg/kg every other day for 2 wk (until 4 wk of age). Rapamycintreatment abrogated the increased mTOR activity in the mutantpancreas �-cells, as determined by the p-S6 immunostaining(Fig. 4A). In contrast, treatment with this low dose of rapamy-cin failed to normalize p-S6 immunoreactivity in the hypothal-amus of Rip-Tsc1cKO animals (Fig. 4B) and did not restorefeeding regulation or adiposity in these animals (SupplementalFig. S2) (26). Thus, the effects of treating Rip-Tsc1cKOanimals with 1 mg/kg rapamycin were not attributable tonormalization of hypothalamic mTOR signaling but rather tothe inhibition of hyperactive mTOR in the �-cells.

Serum insulin levels were higher in Rip-Tsc1cKO micecompared with control mice in both fasting and fed states (Fig.4C). Rapamycin treatment significantly decreased the seruminsulin levels in Rip-Tsc1cKO mice, making them similar tocontrol mice (Fig. 4C).

We performed intraperitoneal glucose tolerance tests in the4-wk-old rapamycin-treated and control mice. As shown inFig. 4D, the injected glucose was cleared more rapidly inRip-Tsc1cKO mice compared with controls. In both genotypes,the rapamycin-treated mice showed impairments in their abilityto clear blood glucose. Insulin secretion in response to glucosewas also dramatically increased in Rip-Tsc1cKO mice com-pared with that in control mice (Fig. 4E). This observation isconsistent with the higher insulin level in the Rip-Tsc1cKOmice (Figs. 2A and 4C). Rapamycin treatment diminished theinsulin secretion in response to glucose injection in bothgenotypes (Fig. 4E). A sharp increase of the serum insulinlevels by glucose injection in Rip-Tsc1cKO mice suggests that

Fig. 2. Normal insulin responsiveness in 4 wk-old Rip-Tsc1cKO mice A: body weight in male littermates fed on regular chow diet at 4 wk of age (n 22).B: age-dependent increase of insulin levels in Rip-cKO mice. Serum insulin levels were determined in overnight-fasted or random-fed mice of each genotype(4 and 8 wk, n 6–11; 24 wk, n 12–15). C: blood glucose levels in random-fed Rip-Tsc1cKO and control male mice were determined (n 16–17).D: impaired insulin tolerance in Rip-cKO mice. Insulin tolerance tests in 3-h-fasted mice at the age of 4 (left; n 9–10), 8 (middle; n 9–12), and 20–24 wk(right; n 6–10). E: insulin tolerance tests in 3-h-fasted 8-wk-old mice. Rip-cKO and control mice were pair-fed from weeks 4 to 8 before the insulin tolerancetest (n 5–8). Values are means � SE. *P � 0.05; **P � 0.01.




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glucose-sensing and insulin secretion mechanisms in mutant�-cells are preserved.

Normoglycemia in Rip-Tsc1cKO mice at 4 wk of age. Sur-prisingly, despite a large increase in insulin levels in Rip-Tsc1cKO mice, the blood glucose levels were only modestlydecreased relative to controls (Figs. 2B and 5A). Since insulinsensitivity in 4-wk-old animals was similar between genotypes,we examined circulating levels of the counterregulatory hor-mones glucagon and growth hormone (Fig. 5, B and C).Interestingly, both glucagon and growth hormone were in-creased significantly in Rip-Tsc1cKO mice compared withthose of control mice, indicating that to maintain euglycemia,Rip-Tsc1cKO mice produce more glucagon and growth hor-mone to counterbalance the glucose-lowering effect of theelevated insulin. Consistent with this idea, rapamycin treatmentnormalized both glucagon and growth hormone levels concom-itant with the reduction in insulin levels. These results suggestthat Tsc1 deletion mediated by Rip/Cre results in increased�-cell size and islet mass, and higher insulin content andsecretion by �-cells due to activation of mTOR signaling.

Increased insulin production in Rip-Tsc1cKO mice. Next,insulin content in pancreas was measured to determine theability of Tsc1cKO �-cells to synthesize insulin. We found thatTsc1cKO pancreas had approximately twice as much insulin ascontrols (Fig. 6A). Rapamycin treatment significantly de-creased the insulin content in Rip-Tsc1cKO mice and had asimilar, albeit milder, effect in control mice. Importantly, both

Rip-Tsc1cKO and control mice showed similar pancreaticinsulin content under rapamycin treatment. These results dem-onstrate that mTORC1 plays an important role in normalinsulin synthesis in �-cells, and the constitutive mTORC1activation in Rip-Tsc1cKO mice is responsible for the highinsulin levels in both the Tsc1KO �-cells and serum.

We also studied insulin release from size-matched isolatedislets from Rip-Tsc1cKO and control mice (Fig. 6B). In re-sponse to high glucose, there was no significant difference ininsulin secretion in Rip-Tsc1cKO islets compared with that ofcontrol islets. Given the overall increase in �-cell and islet size,these data suggest a primary effect of mTOR activation on�-cell mass and insulin content, rather than insulin secretion, tomediate increased circulating insulin in the Rip-Tsc1cKO ani-mals.

Immunoblotting for crucial signaling mediators of mTORand �-cell function from control and Rip-Tsc1cKO micerevealed that phosphorylation of 4E-BP1, S6k, and S6 wasincreased in Rip-Tsc1cKO islets (Fig. 6C). As expected, thephosphorylation of S6k in both Rip-Tsc1cKO and controlislets was inhibited by rapamycin (data not shown). We alsoobserved that Akt phosphorylation (Ser473 and Thr308) wasdecreased dramatically in the Rip-Tsc1cKO islets, consistentwith the negative feedback effect of high S6k in the Tsc1-knockout cells (Fig. 6D). Indeed, although we detected noincrease in the phosphorylation of IRS-1 on Ser307 ordecrease in IRS-1 or IRS-2 expression, the electrophoretic

Fig. 3. Increased �-cell size in 4-wk-old Rip-cKO mice. A: islet size increases in Rip-cKO. Immunostaining was performed with antibodies against insulin(brown) and glucagon (red). Scale bar represents 500 and 100 �m. Total insulin- and glucagon-positive areas were expressed as %total pancreas area. We scored6 animals/genotype and 9 sections/animal spaced 100 �m apart. B: Size distribution of islets expressed as %total islets. Values are means � SE (n 6). C: isletprotein content increase in Rip-cKO. Lysates from 40 islets randomly picked per individual mouse were measured for protein content using the bicinchoninicacid-containing protein assay (n 5 each group). D: �-cell size increases in Rip-cKO. Pancreatic sections were stained with antibodies against insulin (red) and�-catenin (brown) to determine the size of individual �-cells. The scale bar represents 100 �m. The graph shows an average size of �250 �-cells from eachof 4 mice of each genotype. E: �-cell numbers/islet in Rip-cKO mice. The islet area averaged from 6 animals, with 9 sections from each animal divided by �-cellsize. F: �-cell proliferation in Rip-cKO mice. Pancreatic sections were stained with the anti-insulin Ki-67 antibody and 4�,6-diamidino-2-phenylindole. The graphshows % Ki-67-positive nuclei in insulin-stained sections from control and Rip-cKO mice. G: �-cell apoptosis in Rip-cKO mice. Numbers of all nuclei and ofTUNEL-positive nuclei in the islets of control and Rip-cKO mice were counted, and then the proportions were calculated. Values are means � SE. *P � 0.05;**P � 0.01. ns, Not significant.




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mobility of IRS-2 was reduced in Rip-Tsc1cKO samples,consistent with increased serine phosphorylation (2), andthis change was reversed by rapamycin treatment. Theincreased serine/threonine phosphorylation of IRS-2 in Rip-Tsc1cKO islets may blunt IRS-2-dependent signaling to Aktand other downstream effectors. Akt signaling, which de-pends upon its phosphorylation, promotes �-cell size as wellas proliferation and survival. Thus, the mTOR activation

following Tsc1 ablation increases �-cell mass and insulinproduction to augment glycemic control even in the face ofblunted Akt signaling.

DISCUSSION

We have generated Rip-Tsc1cKO mice, in which Rip2/Credeletes the Tsc1 gene in both pancreatic �-cells and the

Fig. 4. Tsc1 deletion improves �-cell function. A: p-S6 (Ser235/236) staining of pancreatic sections from Rip-cKO with or without rapamycin treatment. Scalebar, 100 �m. B: Rip-cKO increases S6 phosphorylation in hypothalamus. Hypothalamic sections from ad libitum-fed Rip-Tsc1cKO and control mice at 4-wkages were stained with p-S6 (Ser235/236) antibody. For A and B, tissue was harvested 12 h after the last treatment of rapamycin. C: serum insulin levels of 4-wk-oldmale mice under 16 h of fasting or in a fed state (no treatment: n 12–18; rapamycin treatment: n 9–13). Similar effects of Tsc1 deletion on serum insulinlevel were also observed in female mice (data not shown). Fa, fasted. D: blood glucose levels after intraperitoneal (ip) injection of glucose (2 g/kg body wt) in4-wk-old mice with or without rapamycin treatment (n 12–19). E: serum insulin levels after an intravenous (iv) glucose injection. To evaluate acute-phaseinsulin secretion, 4-wk-old mice were injected with glucose (1 g/kg body wt); no treatment: n 7–10; rapamycin treatment: n 4. Rapamycin treatment (Rap):2-wk-old Rip-cKO and control mice were injected with rapamycin (1 mg/kg body wt) every other day for �4 wk. Graphs show means � SE. *P � 0.05; **P �0.01; ***P � 0.001.




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hypothalamus, and studied islet and �-cell function in theseanimals. Although it is well known that mutation of Tsc1causes tumors in multiple organs, there was no evidence oftumors in the pancreas or brain during the observation periodsof this study (6 mo). Although we noted the development ofprolactin-producing pituitary tumors in Rip-Tsc1cKO mice atadvanced ages, this is unrelated to the phenotypes described inthis report because the tumor occurred in only a few animalsand at a later stage, and the prolactin levels were normal inyoung Rip-Tsc1cKO animals (data not shown).

Previous studies have established important roles of insulinsignaling in maintaining �-cell size, cell number, and insulinsecretion (5, 9, 17, 18, 21). Mutations of several genes in theinsulin pathway, such as Irs (5, 17, 21), Pten (28, 36), and Pdk1(9), affect multiple pathways, including mTOR. In contrast,Tsc1 mutation specifically activates mTORC1. The Irs2 Rip-Cre (Rip-Irs2cKO) mice showed insulin deficiency with afailure of �-cell growth (5, 17, 21), whereas Pten Rip-Cre(Rip-PtencKO) mice displayed increased islet mass and �-cellnumbers. The use of rapamycin to inhibit mTOR has suggestedcrucial roles for mTOR in �-cell function. Not only doesrapamycin inhibit �-cell proliferation in vitro, it also blocks theeffects of Akt activation on �-cell mass and proliferation (2,20, 22). Here, we utilize genetic ablation of the mTOR inhib-itor Tsc1 to examine mTOR function in �-cells.

We observed significant increases in islet mass, �-cell size,and insulin content in Rip-Tsc1cKO mice; these effects werecounteracted by rapamycin treatment, indicating the criticalrole of mTORC1 activation in �-cell mass. Although both isletand �-cell size were increased in the Rip-Tsc1cKO mice, thenumber of �-cells per islet was not increased, suggesting thatmTOR activation increases cell growth but not �-cell number.Indeed, the similar number of Ki-67- and TUNEL-reactivecells in islets from Rip-Tsc1cKO and control mice is consistentwith unaltered proliferation and apoptosis with mTOR activa-tion in Rip-Tsc1cKO islets. By contrast, others have shown thatinhibition of mTOR decreases �-cell proliferation and number,including in response to Akt activation (2, 3, 22, 46). Thus,although mTOR signaling appears to be required for cellproliferation, it is not sufficient; this finding is consistent withthe results of others and with the low oncogenic potential of thetumors in TSC (10, 35). Importantly, however, our data revealthat increased mTOR signaling suffices to promote increased

�-cell size and pancreatic islet mass even in the face of reducedbaseline Akt signaling, consistent with the previous findings ofothers suggesting that mTOR mediates important aspects of theresponse to the insulin-like signaling pathway and Akt.

The enlarged �-cell size and increased pancreatic insulincontent in Rip-Tsc1cKO mice likely results from increasedoverall protein synthesis, including insulin synthesis, that isdriven by high mTOR activity. Although our data do not revealan increase in per-islet insulin secretion in Rip-Tsc1cKO isletsthat are size-matched to controls (suggesting that mTOR mayminimally effect the insulin secretory machinery), the overallincreased pancreatic islet mass and insulin content result in thegreater release of insulin in Rip-Tsc1cKO mice compared withcontrols. Although Rip-Cre has been reported to blunt insulinsecretion even in the absence of conditional alleles, this isunlikely to be a factor in the Rip-Tsc1cKO mice because theyexhibit increased first-phase insulin secretion in vivo. Also,although rapamycin treatment may promote the increased sen-sitivity on target tissues to insulin, the decreased glucosetolerance of rapamycin-treated animals, coupled with the de-creased secretion of insulin, suggests a primary effect on �-cellfunction in this case. Thus, although the TSC-mTOR pathwaydoes not mediate �-cell proliferation as does the PI3K path-way, mTOR plays a crucial role in the regulation of �-cellmass and islet function by controlling �-cell size and insulinproduction.

Our observations of �-cell hypertrophy and hyperinsulin-emia are consistent with Tsc2 deletion studies in pancreatic�-cells (31, 35). However, Tsc2 knockout by Rip/Cre did notaffect body weight, and the knockout mice maintain normalinsulin sensitivity at advanced age. In contrast, we found thatRip-Tsc1cKO mice show obesity and insulin resistance. Fur-thermore, we observed that Tsc1 knockout increased only�-cell size and not cell proliferation/apoptosis/numbers, where-as Rachdi et al. (31) reported that Tsc2 knockout also increased �-cellproliferation. These differences could be due to a functional differ-ence between TSC1 and TSC2, although it is generally believed thatTSC1 and TSC2 function together as a complex. Alternatively, theRip/Cre expression, especially in the hypothalamus, may be influ-enced by genetic background. Also, if the Tsc2cKO animals dis-played some mild, undetected increase in adiposity in the olderanimals studied, it is possible that the stimulus of mild insulinresistance may have promoted some increase in �-cell proliferation.

Fig. 5. Normoglycemia in Rip-cKO mice at 4-wk of age A: blood glucose levels. Overnight fasting (16 h) or ad libitum-fed blood glucose levels were measuredin 4-wk-old male mice. Control or Rip-cKO mice were treated with or without rapamycin as indicated (1 mg/kg body wt; n 16–21). B: serum glucagon levelsof fed-state 4-wk-old mice of each group with or without rapamycin (n 5–8). C: serum growth hormone levels of fed-state 4-wk-old mice of each group withor without rapamycin (n 11–16). Graphs show means � SE. *P � 0.05; **P � 0.01.




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Shegeyama et al. (35) reported that Tsc2 knockout by Rip/Creresulted in mice that had epileptic seizures and died around 3 wk ofage. It is not clear why the Tsc2 Rip/Cre knockout by two groupsproduced vastly different phenotypes. Our Tsc1-knockout mice byRip/Cre have normal viability up to 6 mo of age. Consistent with ourobservations, Tsc2 knockout increased �-cell size but not cell num-bers at young ages (31, 35). Furthermore, transgenic mice thatexpress Rheb under the control of the rat insulin promoter werereported to have a similar phenotype with increased �-cell mass butwithout increased �-cell proliferation (8). Therefore, our data with

Tsc1 and the majority of other observations with Tsc2 suggest thatmTOR plays a major role in �-cell size but not proliferation.

In the prediabetic stage, before �-cell failure and progres-sion to type 2 diabetes, islet �-cells normally produce moreinsulin to compensate for the insulin resistance in the periph-eral tissues and maintain euglycemia. Indeed, mTOR plays anessential role in promoting pancreatic �-cell function in re-sponse to growth factors and nutrients in vitro (44), and theprocess of prediabetic compensation is similar to that observedhere in Rip-Tsc1cKO mice with constitutive activation of

Fig. 6. Increased insulin production and secretion in Rip-cKO mice. A: insulin contents of the pancreas from mice of each genotype with or without rapamycintreatment at 4 wk of age (n 5–7). Two-week-old Rip-cKO and control mice were injected with rapamycin (1 mg/kg body wt) every other day for �4 wk.B: in vitro insulin release studies. Ten size-matched pancreatic islets prepared from Rip-cKO and control mice were incubated for 1 h in Krebs solution containingthe indicated glucose concentration (n 6). B, left: the amount of insulin in medium during 1-h incubation is indicated. B, right: the fold increase of the insulinresponse at 16.7 mM glucose vs. 2.8 mM glucose was calculated. C: elevated S6 kinase (S6k), S6, and eukaryotic initiation factor 4E-binding protein-1 (4E-BP1)phosphorylation in Tsc1-knockout islets. C, top: immunoblot analysis of Tsc1, Tsc2, p-S6K, S6k, p-S6, S6, p-4E-BP1, and 4E-BP1 is indicated. Islets wereisolated and extracts prepared from independent animals. C, bottom; quantitative phosphorylation (p-S6K/S6K, p-S6/S6, p-4E-BP1/4E-BP1) was measured inislets from both genotypes (n 5 each group). D: negative feedback by increased mTOR/S6K in Tsc1-knockout islets. Immunoblot analysis of Tsc1, p-Akt(Ser473 and Thr308), p-Irs1 (Ser307), Irs1, and Irs2 are indicated. Islets were isolated and extracts prepared from independent animals. Rapamycin, 50 nMrapamycin. Treatment was done during incubation (12 h). For A–C, graphs show means � SE. *P � 0.05; **P � 0.01.




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mTOR. Together, these observations indicate that cellularmTOR signaling promotes �-cell growth and function andlikely plays an important role in the compensatory response toinsulin resistance.

ACKNOWLEDGMENTS

We thank Marta Dzaman, Wanda Snead, Chris Edwards, Naoko Wanibu-chi, Dr. Zhaoyang Feng, Michael Reid, and Sam Langberg for technical adviceand assistance and Dr. Ryoko Takahashi and other members of the Guan,MacDougald, and Myers Laboratory for helpful discussions and assistance.

Present address of H. Munzberg: Pennington Biological Research Center,Baton Rouge, LA.

GRANTS

This work was supported by grants from the NIH and US Department ofDefense (K. -L. Guan), DK-57768 and DK-56731 (M. G. Myers, Jr.), DK-51563 and DK-62876 (O. A. MacDougald), NIH/NCI 1 P01 CA120964 (D. J.Kwiatkowski), and an American Heart Association Scientist DevelopmentGrant to H. Munzberg. H. Mori was supported by the Uehara MemorialFoundation and a mentor-based postdoctoral fellowship from the AmericanDiabetes Association.

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