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doi: 10.1152/ajpendo.00100.2010298:E1261-E1273, 2010. First published 30 March 2010;Am J Physiol Endocrinol Metab

Isabelle Leclerc and Guy A. RutterGao Sun, Andrei I. Tarasov, James A. McGinty, Paul M. French, Angela McDonald,secretion in vivo

-cell morphology and enhances insulinpancreatic transgene modifiesRIP2.CreLKB1 deletion with the

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LKB1 deletion with the RIP2.Cre transgene modifies pancreatic -cellmorphology and enhances insulin secretion in vivo

Gao Sun,1 Andrei I. Tarasov,1 James A. McGinty,2 Paul M. French,2 Angela McDonald,1 Isabelle Leclerc,1and Guy A. Rutter11Section of Cell Biology, Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, and 2PhotonicsGroup, Department of Physics, Imperial College London, London, United KingdomSubmitted 11 February 2010; accepted in final form 29 March 2010

Sun G, Tarasov AI, McGinty JA, French PM, McDonald A,Leclerc I, Rutter GA. LKB1 deletion with the RIP2.Cre transgenemodifies pancreatic -cell morphology and enhances insulin secretionin vivo. Am J Physiol Endocrinol Metab 298: E1261E1273, 2010.First published March 30, 2010; doi:10.1152/ajpendo.00100.2010.The tumor suppressor liver kinase B1 (LKB1), also called STK11, isa protein kinase mutated in Peutz-Jeghers syndrome. LKB1 phosphor-ylates AMP-activated protein kinase (AMPK) and several relatedprotein kinases. Whereas deletion of both catalytic isoforms of AMPKfrom the pancreatic -cell and hypothalamic neurons using the ratinsulin promoter (RIP2).Cre transgene (AMPKdKO) diminishesinsulin secretion in vivo, deletion of LKB1 in the -cell with aninducible Pdx-1.CreER transgene enhances insulin secretion in mice.To determine whether the differences between these models reflectgenuinely distinct roles for the two kinases in the -cell or simplydifferences in the timing and site(s) of deletion, we have thereforecreated mice deleted for LKB1 with the RIP2.Cre transgene. Inmarked contrast to AMPKdKO mice, LKB1KO mice showeddiminished food intake and weight gain, enhanced insulin secretion,unchanged insulin sensitivity, and improved glucose tolerance. In linewith the phenotype of Pdx1-CreER mice, total -cell mass and thesize of individual islets and -cells were increased and islet architec-ture was markedly altered in LKB1KO islets. Signaling by mam-malian target of rapamycin (mTOR) to eIF4-binding protein-1 andribosomal S6 kinase was also enhanced. In contrast to Pdx1-CreER-mediated deletion, the expression of Glut2, glucose-induced changesin membrane potential and intracellular Ca2 were sharply reduced inLKB1KO mouse islets and the stimulation of insulin secretion wasmodestly inhibited. We conclude that LKB1 and AMPK play distinctroles in the control of insulin secretion and that the timing of LKB1deletion, and/or its loss from extrapancreatic sites, influences the finalimpact on -cell function.

AMP-activated protein kinase; -cell; insulin secretion; food intake;liver kinase B1; pancreas

LIVER KINASE B1 (LKB1, also called STK11) is a potent tumorsuppressor whose inactivation in Peutz-Jeghers syndrome(PJS) (21) is characterised by melanotic macules, hamartoma-tous polyps in the gastrointestinal tract, and increased risk ofall cancers (7). LKB1 is a partial mammalian homolog of theSaccharomyces cerevisae kinases Elm1, Pak1, and Tos3,which phosphorylate yeast snf1 (47), the yeast homolog ofmammalian AMP-activated protein kinase (AMPK) (47). Ac-tivation of AMPK, a target of several glucose-lowering agentsused in diabetes treatment, including metformin and the thia-zolidenediones (31), stimulates insulin action in peripheral

tissues, acting to phosphorylate and stimulate the TSC1:TSC2complex, which subsequently inactivates mammalian target ofrapamycin/regulatory associated protein of mTOR (mTOR/Raptor) (3). AMPK is also implicated in the control of -cellsurvival (27, 39, 40) and insulin secretion (11, 12, 42). In themediobasal hypothalamus, changes in AMPK activity in pro-opiomelanocortin (POMC)-, agouti-related peptide (AgRP)-,and neuropeptide Y (NPY)-expressing neurons are also impli-cated in the control of feeding and body weight (9, 33). Recentdata (4) have suggested that calmodulin-dependent proteinkinase kinase- (CaMKK), another upstream kinase forAMPK (20), is involved in these cells.

Compelling evidence for a conserved role for an LKB1-AMPK signaling cassette comes from studies in Caenorhab-ditis. elegans, where inactivation of the corresponding ho-mologs leads to reversal of the dauer phenotype (36), charac-terized by metabolic inhibition and growth arrest. LKB1 andAMPK are also implicated in the control of cell polarity. Thus,deletion of the LKB1 homologues in Drosophila melanogaster(dLKB1) and C. elegans (par4) disrupts epithelial cell polarity(26, 32), and in Drosophila this change is rescued by trans-genic overexpression of AMPK (48). Although forced over-expression of LKB1 induces cell polarization in intestinalepithelial cancer cell lines (5), the requirement for LKB1 inmaintaining the polarity of mammalian cells is less clear (44).However, there is also growing evidence that the effects ofLKB1 in mammalian cells may be, at least in part, independentof AMPK, since LKB1 phosphorylates 11 further kinases ofthe AMPK subfamily in vitro (30, 41).

We (46) have recently demonstrated that deletion of bothcatalytic isoforms of AMPK from the pancreatic -cell by use ofthe rat insulin promoter (RIP2).Cre transgene (AMPKdKOmouse) leads to impaired glucose tolerance and defective insulinsecretion in vivo but enhanced glucose-stimulated insulin secre-tion from isolated islets. However, and in marked contrast, Fu etal. (16) and Granot et al. (18) have demonstrated that deletion ofLKB1 from the pancreatic -cell (and probably intestinal incretin-producing cells) from adult mice by means of an induciblePdx1-CreER transgene leads to increased insulin production andenhanced -cell size. To determine whether these markedly di-vergent phenotypes may reflect true biological differences in theroles of LKB1 and AMPK in the pancreatic -cell rather thanbeing the result of differences in the timing and sites of deletion,using the RIP2.Cre transgene, we have therefore generated micein which LKB1 is deleted in the -cell.

Inactivation of LKB1 in -cells with this strategy led tomarked increases in -cell size and insulin production in vivo,consistent with the findings using Pdx1-CreER-mediated dele-tion (16, 18). However, and in contrast to the latter model,

Address for reprint requests and other correspondence: G. A. Rutter, Sectionof Cell Biology, Division of Diabetes, Endocrinology and Metabolism, Dept.of Medicine, Imperial College London, London, UK (e-mail: [email protected]).

Am J Physiol Endocrinol Metab 298: E1261E1273, 2010.First published March 30, 2010; doi:10.1152/ajpendo.00100.2010.

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-cell glucose signaling and insulin secretion were inhibited invitro. Importantly, the above alterations in LKB1 mice con-trast sharply with the effects of deleting both AMPK subunitsusing the same RIP2.Cre transgene (46), wherein unaltered-cell mass and a decrease in mean -cell size are observed. Inaddition, we observed a decrease in body weight and improvedglycemia but unaltered insulin sensitivity, suggestive of a rolefor LKB1 in RIP2.Cre neurons to control satiety distinct fromthat of AMPK. Overall, the present findings indicate that LKB1and AMPK control distinct signaling pathways in the -cell toregulate insulin production. The results also support the viewthat inhibition of LKB1, or its downstream targets, may be auseful approach to increase -cell mass in some forms ofinsulin-secretory insufficiency, including type 2 diabetes, andthat these changes are likely to be mediated by member(s) ofthe AMPK superfamily distinct from AMPK.

METHODS

Generation of Mutant Mice Selectively Lacking LKB1 in Pancreatic-Cells and RIP2.Cre Neurons

Mice homozygous for floxd alleles of the lkb1/stk11 gene (MouseModels of Human Cancer Consortium, http://mouse.ncifcrf.gov/)were first crossed with heterozygous RIP2.Cre-expressing transgenicmice (expressing Cre recombinase under the rat insulin 2 promoter;Jackson Laboratory). The resulting double heterozygous LKB1fl/,Crewere intercrossed with LKB1fl/ mice to generate LKB1KO (LKB1fl/fl,Cre), LKB1het (LKB1fl/,Cre), and LKB1Wt (LKB/,Cre). LKB1KO, LKB1het, and LKB1Wt mice were born atthe expected Mendelian ratios and kept on a mixed FVB/129S6 andC57BL/6 background.

Mouse Maintenance and Diet

Mice were housed with 25 animals per cage in a pathogen-freefacility on a 12:12-h light-dark cycle. Mice were fed ad libitum witha standard mouse chow diet or a high-fat diet [60% (wt/wt) fatcontent; Research Diet, New Brunswick, NJ]. Where indicated, 4-wk-old mice were transferred onto high-fat diet for a period of 6 wk. Allin vivo procedures stated were performed in the Imperial CollegeCentral Biomedical Service (CBS) and approved by the UK HomeOffice according to the Animals Scientific Procedures, Act of 1986.

Body Weight and Food Intake

Fed or 15-h overnight-fasted mice were weighed at the age of 68wk. Food intake was measured daily at fed status either for 3consecutive days or at 30 min and 1 h after refeeding the mice fastedfor 15 h using a metabolic cage.

In Vivo Physiological Studies

Intraperitoneal glucose tolerance test. Mice fasted for 15 h (waterallowed) were intraperitoneally injected with 1 g glucose/kg mousewt. Blood from the tail vein was obtained at 0, 15, 30, 60, 90, and 120min after injection. Blood glucose levels were measured with anautomatic glucometer (Accuchek; Roche, Burgess Hill, UK).

Plasma insulin measurement. Mice fasted for 15 h were intraperi-toneally injected with 3 g glucose/kg mouse wt. Blood from micestail veins was collected into a heparin-coated tube (Sarstedt, Beau-mont Leys, UK) at 0, 15, and 30 min after injection. Plasma wasseparated by centrifuging the blood at 2,000 g for 5 min. Plasmainsulin levels were measured using an ultrasensitive mouse insulinELISA kit (Mercodia, Uppsala, Sweden). Normal fed plasma insulinlevels were measured from blood collected from 6- to 8-wk-oldmices tail veins between 10:00 and 11:00 AM.

Insulin tolerance tests. Bovine insulin (Sigma, Dorset, UK; 0.75U/kg) was intraperitoneally injected into fed mice. Blood glucoselevels were measured at 0, 15, 30, and 60 min after injection as above.Islet Isolation and In Vitro Insulin Secretion Measurement

Islet isolation by in situ collagenase digestion, and in vitro insulinsecretion measurement, were performed as previously described (28).

RNA Extraction and RT-PCR

Total cellular RNA from mouse islets or other tissues was obtainedusing TRIzol reagent (Invitrogen, Paisley, UK), and RNA was furtherpurified against DNA contamination with a DNA-free kit (Appliedbiosystems, Warrington, UK). Total RNA (1.52 g) was then re-verse transcribed into cDNA with a high-capacity reverse transcrip-tion kit (Applied Biosystems) according to the manufacturers instruc-tions. To detect deletion of LKB1 exons 3 to 6, two pairs of primerswithin exon 1 (LKB fwd: AGGTGAAGGAGGTGCTGG) and 8(LKB rev: TCTGGGCTTGGTGGGATA) were designed (Fig. 1A).The PCR reaction was preheated at 95C for 2 min, and amplificationwas performed for 30 cycles under the following conditions: 94C for30 s, 57C for 30 s, and 72C for 1 min. At the end of the last cycle,a prolonged extension step was carried out at 72C for 10 min.

qRT-PCR

The expression levels of lkb1/stk11, kir6.2, sur1, glut2, ki67,insulin (ins1 and ins2), glucagon, gck, NPY, POMC, and AgRPgenes were quantitated by real-time PCR using the SYBR Greenmethod (11).

Immunoprecipitation, AMPK Activity Measurement, and Western(Immuno-) Blot Analysis

To determine total AMPK activity in islet extracts, 10 g of totalprotein from 150200 islets isolated from fed mice was incubated inRPMI supplemented with 11 mM glucose for 35 days prior to AMPK assaywith synthetic SAMS peptide (HMRSAMSGLHLVKRR) as substrate(28). To determine AMPK1 and -2 activities in the hypothalamus,dissected tissue was placed in liquid nitrogen immediately afterextraction and lysed in 800 l of ice-cold lysis buffer [in mM: 50TrisHCl (pH 7.4, 4C), 250 sucrose, 50 NaF, 1 Na pyrophosphate, 1EDTA, 1 EGTA, 1 DTT, 0.1 benzamidine, and 0.1 PMSF, 5 g/mlsoybean trypsin inhibitor, and 1% (vol/vol) Triton X-100] [with 10%(wt/vol) sucrose in lysis buffer for hypothalamus]. Total extracts (100g of protein) were used for immunoprecipitating with anti-AMPK1/2 antibodies (Upstate, Dundee) conjugated to proteinG-Sepharose and were subjected to AMPK activity measurement asabove. For Western blot analysis, 50 g of protein from 300400islets was used.

Immunohistochemistry and Analysis of Islet ArchitectureIsolated pancreata were fixed in 10% (vol/vol) formalin and em-

bedded in paraffin wax within 24 h of removal. Head-to-tail sections(5 m lengthwise) were cut and incubated at 37C overnight onsuperfrost slides. Slides were submerged sequentially in 100% (vol/vol) xylene followed by decreasing concentrations of industrial meth-ylated spirits for removal of paraffin wax. Antigen epitopes were thenretrieved (de-cross-linked) in Tris-EDTA-0.05% (vol/vol) Tweenbuffer (pH 9.0). Slides were subsequently blocked in 5% (vol/vol)goat serum in Tris-bbuffered saline with 0.05% (vol/vol) Tween(TBS-T) for 20 min at room temperature and then incubated in amixture of primary antibodies at the concentrations indicated at 4Covernight, After being washed in TBS-T for three times of 5 min eachtime, slices blotted with primary antibodies were visualized withAlexa fluor 568- or 488-conjugated IgG (1:500; Invitrogen, Paisley,UK) under fluorescent microscopy using a Zeiss Axiovert-200 con-

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focal microscope with an Improvision/Nokigawa spinning disc andrunning Volocity 5.0 (Improvision, Coventry, UK) software (10).

To quantify the number of rosette-like structures (i.e., 810 cellsarranged concentrically around an identifiable central hub; seeRESULTS) in islets, we used E-cadherin and DAPI staining of pancre-atic sections. Structures were included where the void at the centerwas negative for DAPI. Ten islets from three pairs of mice pergenotype were assessed.

Antibodies

Antibodies used in Western (immuno-)blot analysis and immuno-histochemistry were the following: rabbit anti-LKB1 (Millipore,Watford, UK), rabbit anti-E-cadherin, anti-phospho-S6 ribosomalprotein (Ser235/236), anti-VEGFR2, rabbit anti-phospho-4E-BP1 (NewEngland Biolabs, Hitchin, UK), mouse anti-actin (C2) (Santa CruzBiotechnology, Heidelburg, Germany), rabbit anti-mouse GLUT2(kind gift from Dr. Bernard Thorens, Lausanne), guinea pig anti-insulin, rabbit anti-glucagon (Dako, Ely, UK), rabbit monoclonalanti-Ki67 (Epitomics, Burlingame, USA), mouse anti--tubulin, anti--tubulin (Sigma Aldrich, Dorset, UK), and rat anti-ZO1 (kind giftfrom Dr. Paolo Meda, Geneva).

Optical Projection Tomography (OPT) and Determination ofRelative -Cell Mass, Single -Cell Size, and Proliferation

Whole pancreatic optical projection tomography, to 19 m reso-lution, was performed as described (2). Briefly, whole pancreata fixedin 4% (wt/vol) paraformaldehyde for 23 h at 4C were dehydratedand subjected to five cycles of freezing and thawing (80C to roomtemperature). Pancreata were then blocked overnight in Tris-bufferedsaline, pH7.5, containing 0.05% (vol/vol) Tween 20, 0.01% (wt/vol)sodium azide and 10% (vol/vol) goat serum, (Dako) before incubationwith guinea pig anti-swine insulin antibody (Dako, 1:1,000) dissolvedin the above buffer further supplemented with 5% (vol/vol) dimeth-ylsulfoxide overnight at 4C. To visualize insulin-positive staining,Alexa 594 goat anti-guinea pig antibody (Invitrogen, 1:1,000) wasapplied. The sample was then embedded in 1% (wt/vol) low meltingtemperature agarose, dehydrated in methanol, and cleared in benzylalcohol-benzyl benzoate (1:2) for optical tomographical scanning.

All specimens were scanned using two fluorescent channels (exci-tation: 580 15 nm and emission: 650 15 nm for insulin-positivestaining; excitation: 480 9 nm and emission: 650 25 nm forautofluorescence). The raw data for these two channels were recon-structed in a pair of 3-D voxel data sets (voxel to m 1:19.5) usingMatlab software, and cell volumes (m3) from each channel were

Fig. 1. Generation of LKB1KO mice. A: schematicrepresentation of deletion of floxd lkb1 exons (exons36) driven by RIP2.Cre expression. Location of primersused for PCR as indicated. Black arrows, flox sites; graybar, LKB1 exons. B: RT-PCR analysis of effects onLKB1 transcript levels of deleting exons 36 in pancre-atic islets and hypothalamus (Hypo). Product sizes were864 and 300 bp for floxd and null alleles, respectively.NS, nonspecific. C: qRT-PCR (C) and Western blotanalysis (D) of LKB1 mRNA and protein levels inpancreatic islets of LKB1KO mice and their heterozy-gous (het) and wild-type (Wt) controls. Total AMPKactivity in islets (E) and hypothalamus (F) of LKB1KOand LKB1het mice. Data are expressed as means SE;n 45 mice per genotype. *P 0.05, **P 0.01,***P 0.001.

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measured using Volocity 5.0 (Improvision). Data from Volocity 5.0were further analyzed in Microsoft Excel to provide size frequencyhistograms and total volume graphs. Relative -cell mass was calcu-lated by dividing the volume of insulin-positive area (total -cellmass) by that of the autofluorescence area (total pancreatic mass).

To calculate single -cell size, the area of 100 single -cells fromfive islets of each genotype from E-cadherin- and insulin-costainedpancreatic sections were measured using Image J (http://rsbweb.nih.gov/ij/). To measure relative number of proliferating -cells, thenumber of Ki67-positive -cells per islet was divided by total -cellnumber; 1520 islets per genotype were analyzed.

Electrophysiological Measurements and Ca2 Imaging

The plasma membrane potential of -cells was recorded in perfo-rated-patch whole cell configuration using an EPC9 patch clampamplifier controlled by Pulse acquisition software (HEKA Elektronik,Lambrecht/Pfalz, Germany). The pipette tip was dipped into pipettesolution (see below),and then back-filled with the same solutioncontaining 0.24 mg/ml amphotericin B. Recordings were initiatedafter 30-min exposure to substrate-free solutions at 37C, and theduration of exposure to each concentration of effector(s) was 2 min.Cells that were not responsive to tolbutamide were excluded fromanalysis.

Series resistance and cell capacitance were compensated for auto-matically by the acquisition software. Experiments were carried outby periodically switching from current-clamp to voltage-clamp mode,thus obtaining pseudo-simultaneous recordings of cell membranepotential (Vm) and KATP conductance (GKATP). This controlled for theleaks of the patch and verified that the depolarization (hyperpolariza-tion) of the membrane was linked to KATP channel closure (opening).The current-clamp protocol involved continuous recording withoutelectrical stimulation. In the voltage clamp, the membrane potentialwas held at 70 mV, and whole cell currents were evoked by10-mV 0.5-Hz pulses. Data were filtered at 0.2 kHz and digitized at0.5 kHz.

The pipette solution contained (in mmol/l): 76 K2SO4, 10 NaCl, 10KCl, 1 MgCl2, and 5 HEPES (pH7.35 with KOH). The bath solution

contained (in mmol/l): 137 NaCl, 5.6 KCl, 10 HEPES (pH 7.4 withNaOH), 2.6 CaCl2, and 1.1 MgCl2. All experiments were conductedat 3337C, and the bath solution was perifused continuously.

For Ca2 imaging, dispersed islets were incubated for 30 min inKrebs-Ringer Buffer [KRB; in mmol/l: 125 NaCl, 3.5 KCl, 1.5 CaCl2,0.5 NaH2PO4, 0.5 MgSO4, 3 glucose, 10 HEPES, and 2 NaHCO3, pH7.4, and equilibrated with O2-CO2 (95:5) and supplemented with 0.1%(wt/vol) BSA] containing 3 mmol/l glucose and 200 nM FURA-REDAM (Invitrogen UK). Cells were stimulated using the conditionsindicated and excited at 480/440 nm using an Olympus IX-81 micro-scope coupled to an F-view camera and captured using Cell^Rsoftware (Olympus UK) on a 40 oil objective. Data were expressedat the ratio of the fluorescence emission at 440/480 nm.

Statistical Analysis

Data are expressed as means SE. Significance was tested byStudents two-samples unpaired or paired Students t-tests usingExcel, or ANOVA test using Graphpad 4.0. P 0.05 was consideredsignificant.

RESULTS

LKB1KO Mice Are Lean and Hypophagic and DisplayImproved Glucose Tolerance

To generate mice lacking LKB1 selectively in the pancreatic-cell from midgestation (E911.5) and in a subset of hypo-thalamic neurons (RIP2.Cre neurons) (17), we crossed ani-mals bearing floxd Lkb1/Stk11 alleles with RIP2.Cre mice(Fig. 1A). Demonstrating efficient deletion from -cells, levelsof endogenous LKB1 mRNA were markedly reduced in isletsfrom homo- (fl/fl) vs. heterozygous (/fl) mouse islets in thepresence of the Cre transgene, with the consequent increase inthe level mRNA encoded by the null allele (Fig. 1B). Similarly,the null transcript was also present in hypothalamic extracts ofhetero- and homozygous mice (Fig. 1B). Levels of LKB1

Fig. 2. Food intake and glucose homeostasis in LKB1KO mice. AC: body weight (A), and food intake of LKB1KO mice fed (B) or refed after fasting for15 h (C). DF: blood glucose (D and E) and plasma insulin (F) of LKB1KO mice fed or fasted for 15 h. Male mice from 68 wk old were used. Data areexpressed as means SE; *P 0.05, **P 0.01; n 710 mice per genotype.




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mRNA (Fig. 1C) and immunoreactivity (Fig. 1D) in islets frommice of each genotype were compared next. Each parameter wasdecreased by 7080% in islets from LKB1KO (fl/fl,Cre) vs.wild-type (/,Cre) mice, consistent with deletion from theislet -cell compartment. Levels of LKB1 mRNA and immu-noreactivity in LKB1het mouse islets were intermediate be-tween those in LKB1Wt and LKB1KO mouse islets (Fig. 1,C and D).

Since LKB1 is likely to be the major upstream kinase forLKB1 in islets (I. Leclerc and G. A. Rutter, unpublishedresults), AMPK activities were also measured as a furtherreadout of LKB1 activity. Total cellular AMPK activity inisolated islets was reduced by 75% in LKB1KO vs.LKB1het (Fig. 1E) (comparison with levels of AMPK activ-ity in wild-type islets was not performed). AMPK1/2 activ-ities were reduced by 25% in hypothalamic extracts fromLKB1KO vs. LKB1het mice (Fig. 1F), consistent withdeletion from a subset of RIP2.Cre neurons within this tissueand/or regulation of AMPK in this tissue by distinct upstreamkinases (e.g., CaMKK) (20).

Examined at 68 wk, male (Fig. 2) and female (not shown)LKB1KO mice displayed decreased fed body weight (Fig.2A) and daily food intake (Fig. 2B) compared with LKB1Wtor LKB1het mice. In addition, after 15 h of starvation,LKB1KO mice tended to eat less in the first 30 min afterrefeeding (Fig. 2C). Reduced hypothalamic expression of theorexigenic peptide NPY mRNA and the anorexigenic peptidePOMC were also observed (Suppl. Fig. 1A; supplementarymaterials are found in the online version of this paper at theJounal website), although in each case the differences wereonly apparent between wild-type and heterozygous mice, sug-gesting they are unlikely to drive the marked differences infeeding between knockout and heterozygous animals (Fig. 1).Interestingly, we have observed in preliminary experimentsincreases in the immunoreactivity of POMC in hypothalamicextracts of LKB1KO mice (data not shown), suggesting thatLKB1-dependent posttranscriptional mechanisms may modifythe levels of this factor. Further studies will be required toexplore this possibility in detail.

Fasting and fed glycemia were significantly decreased inLKB1KO mice (Fig. 2, D and E), a change accompanied bysubstantial increases in plasma insulin levels in the fed state(Fig. 2F). Correspondingly, LKB1KO mice displayed dra-matically improved glucose tolerance (Fig. 3A) and insulinrelease (Fig. 3B) in vivo compared with LKB1Wt orLKB1het mice but unchanged insulin sensitivity (Fig. 3C).

LKB1 Controls -Cell Size, Morphology, and IsletArchitecture

To examine in detail the possible mechanisms behind thehyperinsulinism in LKB1KO mice, we used OPT (2). Thistechnique, which we have previously validated by comparisonwith conventional measurements of -cell mass based onhistochemistry (46), allowed us to determine the mean andtotal volume, size, distribution, and number of islets within theentire pancreas (Fig. 4, AD, Suppl. Fig. 2, and Supplementarymovies: betaLKB1wt, betaLKB1Het, betaLKB1KO). Total-cell mass was markedly increased with respect to LKB1wtmice, and the mean volume of individual islets was signifi-cantly elevated by 30% in LKB1KO vs. LKB1het and

20% in LKB1KO vs. LKB1wt pancreata. The mostmarked increase was apparent in the number of the largestislets present (107 m3; 10,000 cells) and certain smallislets. Although there were no evident changes in the ratio of- to -cells within islets (Fig. 4E), we observed a significant40% increase in the surface area of individual -cells, asidentified by staining with E-cadherin antibodies (see Methods;Fig. 4F). Demonstrating an increase in -cell proliferation,Ki67 staining was markedly increased in LKB1KO islets(Fig. 4G).

Since mTOR signaling (3) is implicated in controlling cellgrowth and protein synthesis, phosphorylation of ribosomal pro-tein subunit S6 (rpS6) and eIF4-binding protein-1 (4E-BP1)within islets was measured. As expected, increased phospho-rpS6and decreased phospho-4E-BP1 were detected in LKB1KOmice islets compared with LKB1Wt or LKB1het mouse islets(Fig. 4H).

Fig. 3. Glucose and insulin tolerance of LKB1KO mice. Glucose tolerance(A), and plasma insulin response (B) after intraperitoneal (ip) glucose injection,and whole body insulin sensitivity (C) monitored after ip insulin injection ofLKB1KO or control mice. Male 6- to 8-wk-old mice old were used. Data areexpressed as means SE. For A and B, *P 0.05, **P 0.01 for LKB1KOvs. LKB1Wt, P 0.05 for LKB1KO vs. LKB1het; in C, *P 0.05 withrespect to time 0 for all genotypes; n 710 mice per genotype.




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To determine whether LKB1 may be critical for the estab-lishment of basolateral-apical polarity of -cells, we examinedthe distribution of the adherens junction marker E-cadherin, aswell as actin, tubulin, and the tight junction protein zonaoccludins 1 (ZO1). Unexpectedly, polarization was not com-promised in LKB1-deficient -cells (Fig. 5). Instead, mutantislets displayed a significant increase in the number of rosette-like structures or islet acini (15) identified by E-cadherin andZO1 staining in which 810 cells were arranged around acentral void (6) (Fig. 5, A, B, E, and G). Strikingly, nuclei werepositioned in the cells comprising these rosettes away from theapical pole, while actin and tubulin staining were concentratedat the junctional zones (Fig. 5, C and D). Whereas the apicalvoids were devoid of staining for VEGFR, this was enrichedat the basolateral surface, indicating the presence of capillariesat the latter site (Fig. 5F).LKB1 Deletion Diminishes -Cell Responses to GlucoseEx Vivo

To determine whether the dramatic improvements in glucosetolerance in vivo might also in part reflect enhanced -cellglucose sensitivity, we isolated islets and single cells fromLKB1KO and LKB1Wt or LKB1het mice. Glucose-stim-ulated insulin secretion (GSIS) under static incubation wasdiminished in LKB1KO mouse islets by 30% comparedwith LKB1Wt mouse islets (Fig. 6A), although the absoluteinsulin output, as assessed from six size-matched islets, wasnot significantly different between the genotypes (Fig. 6A). Bycontrast, the insulin content of similarly sized islets fromLKB1KO mice was substantially increased (2.5-fold vs.LKB1Wt and 1.5-fold vs. LKB1Het; Fig. 6A). Glucose-stimulated changes in the conductance of ATP-sensitive Kchannels (GKATP), membrane depolarization (Vm), and in-creases in intracellular free Ca2 concentration were all mark-edly attenuated in -cells from LKB1KO islets (Fig. 6, B andC). In addition, membrane electrical activity was elevated atbasal and 3 mM glucose levels in LKB1KO islet cells,suggesting blunted glucose sensing in these cells (Fig. 6B).

Providing a possible contribution to the impaired GSIS fromLKB1KO islets, we observed a substantial decrease in thelevel of GLUT2 (slc2A2) immunoreactivity at the plasmamembrane of LKB1-deleted cells (Fig. 6D) and a correspond-ingly large (90%) reduction in GLUT2 mRNA in isolatedislets (Suppl. Fig. 1B). Likewise, the levels of mRNAs encod-ing the ATP-sensitive KATP channel subunit kir6.2 (Suppl. Fig.1B) were substantially (80%) decreased in LKB1KO vs.LKB1wt islets. By contrast, we observed no significant de-creases in the expression of other genes involved in maintaining

the differentiated function of cells, including Pdx1, nor in SUR1(ABCC8) or glucokinase (Gck; Suppl. Fig. 1), nor in neuroD1 orMafA (LKB1fl/fl.RIP2.Cre vs. LKB1fl/.RIP2.Cre, not shown)suggesting that a generalized decrease in -cell differentiation didnot occur.

Effect of Metabolic Stress on LKB1KO vs. Wild-Type MiceSince LKB1 and AMPK are implicated in the cellular stress

responses, we determined how LKB1 deficiency in -cells andhypothalamus may impact on the diabetogenic effects of ahigh-fat diet (1). After 6 wk on a high-fat diet, differences inglucose tolerance (Fig. 7A) or insulin secretion (Fig. 7B) wereno longer apparent between genotypes, consistent with agreater susceptibility of LKB1KO mice to the effects of thismetabolic stress.

DISCUSSION

LKB1 Regulates -Cell Size and Islet Architecture

The principal aim of the present study was to determinewhether, when compared using a near-identical strategy fordeletion from -cells with that used previously for AMPKsubunits (46), the loss of LKB1 may lead to similar changes ininsulin secretion and glucose homeostasis in vivo and in vitro.This has seemed important given the dramatically differentphenotypes of mice deleted for LKB1 in adults using a Pdx-1-CreER transgene (16, 18) and those deleted for AMPK usingthe RIP2.Cre transgene (46), with the latter strategy resultingin earlier (midgestational) deletion and some loss from hypo-thalamic nuclei. Although the genetic background of theLKB1-deleted mice generated here (FVB/129S6/C57BL6) andin earlier studies (16, 18) differs slightly from that of thosedeleted for AMPK (C57BL6) (46), it seems unlikely to us thatthis difference could explain the markedly different phenotypesof mice deleted for LKB1 vs. AMPK in the -cell.

Our findings confirm the view that LKB1 is a criticalregulator of -cell development and function. First, we dem-onstrated that the size of individual -cells was considerablyincreased, as was that of individual islets, as assessed by OPT.Interestingly, and as shown in Fig. 4D, the mean islet volume,as determined by OPT, was increased40% in LKB1 KO vs.wild-type islets. This compares with an increase in average(single) -cell area of 60% (Fig. 4F), equivalent to anincrease in volume of more than two fold. The differencebetween these values seems likely to be due to the contributionof an unchanged volume of other cell types within the islets,and/or a lowered number of -cells per islet.

Fig. 4. Altered islet morphology in LKB1KO mice. A: representative optical projection tomographic (OPT) images of whole pancreas. OPT was performed asdescribed under METHODS. Red staining indicates insulin-positive structures (islets), while the outline of the whole pancreas was apparent as autofluorescence andis presented as white/gray shading. Images shown correspond to 3-D projections; (see also Suppl. Fig. 2 and Suppl. Movies betaLKB1wt, betaLKB1het, andbetaLKB1KO.avi.) Note presence of a particularly large islet in the LKB1KO pancreas (right image, arrow). B: distribution of islet volumes with marked (dottedlines) section magnified. C: relative -cell mass; D: mean islet volume. Data are from 56 mice per genotype. Scale bar, 500 m. E: hematoxylin-eosin (H&E)and immunofluorescent staining of pancreatic sections using guinea pig anti-insulin (1:200; green) and rabbit anti-glucagon (1:100; red) antibodies. Nuclei areshown with DAPI (blue) staining. Scale bar, 75 m. F: representative E-cadherin staining of pancreatic sections and quanification of single islet -cell sizes.Average area of 100 single -cells from 5 islets of each genotype, costained in pancreatic sections for E-cadherin and insulin, were analyzed. Scale bar, 12 m.G: staining for proliferation marker Ki67 and quantification, based on 1520 islets per pancreas; n 3 mice per genotype. H: Western blot analysis of mTORsignaling pathway markers phospho-ribosomal protein (rp)S6 and phospho-4E-BP1 of pancreatic islet extracts from LKB1KO and control mice. Islets extractedfrom fed mice were incubated in RPMI supplemented with 11 mM glucose for 16 h and lysed for analysis. Data are expressed as means SE. *P 0.05,**P 0.01. In B, *P 0.05, for LKB1KO vs. LKB1Wt, $P 0.05 for LKB1KO vs. LKB1het.




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Fig. 5. -Cell polarity is enhanced in LKB1KO mice withincreased rosette-like (islet acini) structures and accumu-lation of cell junction proteins. AE: immunofluorescencestaining of pancreatic sections for the adherens junctionmarker (A and B) E-cadherin (1:100, green), (C) actinfilament (1:50, red; note imaged areas were confirmed aslying over an islet by inspection of corresponding brightfield images), (D) -tubulin (1:100, green), (E) tight junc-tion marker zona occludins 1 (ZO1, whole serum; green) orcapillary marker (F) VEGFR2 (1:100, green). White square,voids at center of rosettes. G: quantification of no. ofrosette-like structures per islet in LKB1KO mice based onE-cadherin and DAPI staining. Such structure in islet iscounted as 1 by E-cadherin staining where the voids at thecenter of the rosette is absent of DAPI staining. Ten isletsfrom 3 pairs of mice per genotype were assessed. Data areexpressed as means SE. **P 0.01, ***P 0.001. Scalebars, 50 m (A), 12 m (B), 10 m (C), and 25 m (DF).




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As also recently reported (2), the distribution of islets inwild-type mouse pancreata was highly nonuniform, with thelargest islets representing as much as 50% of the total -cellmass. Using the same approach in LKB1KO islets, we dem-onstrated that the abundance of the largest islets was signifi-cantly increased. This shift seems likely to be the result, at leastin large part, of an increase in the average volume of individual-cells (60%), as well as increases in -cell proliferation.TSC1/2 deletion in -cells, using the same RIP2.Cre mice,leads to increased -cell mass (35), a phenotype similar towhat we describe here in LKB1KO mice. Increased phosphor-ylation of rpS6 and decreased phosphorylation of 4E-BP1 inLKB1KO mice islets suggested that the increased -cell sizeand islet volume are likely to be due to elevated mTORsignaling.

The present results also confirm the view (16, 18) that LKB1is involved in the establishment of cell polarity in mammalianepithelial cells, consistent with findings from Shorning et al.(44) in intestinal paneth and goblet cells, as well as the findingsin -cells (16, 18). Thus, we found that rosette-like arrange-ments of -cells (6), containing strikingly polarized -cells,were twice as frequently observed in LKB1KO islets. Weconsidered the possibility that this may be due simply to anincrease in the size of individual cells. Arguing against thispossibility, Hamada et al. (19) demonstrated an increase in-cell volume of 60% after transgenic overexpression ofmTORC1 in -cells, similar to the increase observed after lkb1knockout. However, no obvious increase in the number ofrosettes was apparent in the case of mTORC1-deleted -cellsin contrast to the findings described here, nor after induction of-cell hypertrophy by dexamethasone (38). Similarly, suchstructures did not appear to be more abundant in the islets oftransgenic mice expressing the cell cycle regulator CDK4/R24C under the rat insulin promoter, in which a massiveincrease in -cell mass (to almost 20% of pancreatic volume)was observed, in this case due to enhanced proliferation (34).

We have considered the possibility that the effects of LKB1deletion reflect the requirement for this enzyme to activateAMPK. However, deletion of the AMPK1 and AMPK2subunits in -cells by use of the RIP2.Cre transgene leads todefective insulin release in vivo, with none of the changes in-cell mass or cellular architecture observed here inLKB1KO islets (46). Instead, and as previously proposed (16,18) the increased number of islet acini most likely involvesalternative kinases such as MARKS13. In this context, Par-1b(MARK2) was recently shown to promote cell-cell adhesionand association of E-cadherin with the actin cytoskeleton (13).A loss of this interaction may be involved in the relocalizationof the latter to the apical pole as observed in LKB1KO-cells. Par1b/MARK2 has also recently been shown to regu-late glucose metabolism by adipose tissue in vivo (25), possi-bly by interacting with syntaxin-4 (14) to control vesiculartrafficking of GLUT4 to the plasma membrane. As syntaxin-4is implicated in the second phase of GSIS (45), it is possiblethat abnormal phosphorylation of Par1b/MARK2 in the ab-sence of LKB1 may also influence the trafficking of -cellsecretory granules to the cell surface. Most importantly, -cellsfrom mice deleted globally for Par1b/MARK2/ display asimilar redistribution of nuclei to that described here (18).

The results of the two very recent studies mentioned above(16, 18), contrast with our own findings in several important

respects. Notably, LKB1-deficient -cells were found in thepresent studies to display abnormalities in the expression of theglucose transporter GLUT2, and the KATP channel subunitKir6.2 (but not of several other genes important in maintainingthe glucose responsiveness of -cells; see RESULTS), with cor-responding alterations in basal and glucose-induced changes inmembrane potential and intracellular free Ca2. Althoughneither of the earlier studies examined the levels of expressionof -cell-specific genes in detail or involved studies of glucosesensing at the single -cell level, the subcellular distribution ofGLUT2 immunoreactivity within individual -cells was re-portedly altered by LKB1 deficiency (18). We suspect that therelatively well-preserved GSIS from isolated islets may in bothcases therefore represent alterations in -cell--cell (or -cell--cell contact) or between -cells and the capillary network.Moreover, the well-preserved stimulation of insulin secretionby glucose observed here in LKB1-deleted islets (Fig. 6A),despite changes in KATP channel conductance, resting mem-brane potential, and a marked (50%) decrease in the rise inintracellular free Ca2 in response to the sugar (Fig. 6, BD),may suggest a compensatory increase in KATP-independent(22) mechanisms of activation, serving to enhance the sensi-tivity of the secretory machinery to Ca2 changes. A moredetailed assessment of this possibility will require furtheranalysis, including a comparison of the transcriptome of isletsfrom LKBKO and wild-type islets. It should also be stressed,however, that the substantial increase in -cell size and mass(4-fold; Fig. 2, C and D) seems likely to predominate overthe relatively more minor changes in -cell glucose sensing inLKB1-deleted -cells and to underlie the substantial increasein vivo in insulin release under glucose challenge.

Does LKB1 Control Neuronal Function to Regulate FoodIntake and Body Weight?

A striking finding in the present study was that deletion ofLKB1 using the RIP2.Cre transgene led to a decrease in bodyweight and feeding. We have considered the possibility thatthese changes may simply reflect the increased levels of insu-lin, with the latter serving as a satiety factor (37). However, wesuspect that the metabolic and cellular changes resulting fromLKB1 deficiency in the brain and in the pancreas are, at leastin large part, independent. Thus, mice inactivated for LKB1 byPdx1.CreER-mediated excision, which leads to deletion in allpancreatic lineages but not in the hypothalamus, show similarincreases in circulating insulin levels and marked improve-ments in glucose tolerance (23) without a change in bodyweight.

Importantly, we show that changes in food intake and bodymass in LKB1KO mice were accompanied by significantdecreases in the hypothalamic expression of the orexigenicpeptide NPY and of the anorexigenic peptide POMC at themRNA level, suggesting that RIP2.Cre neurons may controlthe activity of neighboring cells in the melanocortin circuitry(43). On the other hand, in preliminary experiments (notshown) we have observed by Western blotting a small butdetectable increase in POMC immunoreactivity in hypotha-lamic extracts from LKB1KO mice vs. wild-type controls,suggesting possible posttranslational modifications throughwhich LKB1 may control the levels of this peptide in feedingcenters. Further studies will be necessary to explore this




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possibility and the mechanisms through which deletion ofLKB1 in RIP2.Cre neurons, not usually thought to expresseither NPY or POMC (24), may influence the levels of thesepeptides in the ventromedial hypothalamus. Interestingly, de-letion of insulin receptor substrate-2 (IRS-2), using an identicalstrategy to that used here, has previously been shown to causehyperphagia, diminished -cell mass, and glucose intolerancein mice (29). Conversely, potentation of insulin signaling bydeletion in RIP2.Cre cells of the inositol phospholipid 5=-phosphatase PTEN (8) led to diminished growth and bodyweight. LKB1 deletion in the same nuclei thus leads to astrikingly similar phenotype to the latter model. This similaritysuggests that LKB1 may also oppose insulin, or possibly leptin,signaling in RIP2.Cre neurons to enhance appetite and bodyweight. Surprisingly, however, deletion of TSC1, also expectedto enhance mTOR signaling in the same neuronal population,led to hyperphagia and obesity (35), suggesting that LKB1 mayengage other signaling pathways to influence the activity ofthese and associated neurons in the melanocortin network.

Interestingly, although LKB1-null mice displayed cleardecreases in hypothalamic and -cell AMPK activity (Fig. 1),mice deleted for both AMPK catalytic subunits in the samecells do not display a feeding or body weight phenotype (46),indicating that the hypothalamic effects of LKB1 may be

mediated by alternative downstream kinases (30) (Table 1) orother effectors. In any case, we stress that further studies willbe required in the future, perhaps involving the selectivedeletion of LKB1 in the hypothalamus by the injection of Crerecombinase into this brain region in LKB1 floxd mice, toformally confirm or refute the possibility of distinct roles forcentral and pancreatic islet LKB1 in the control of glucosehomeostasis and food intake.

Conclusion

Using an identical strategy for tissue-selective deletion inmice, we have shown that LKB1 plays a distinct role(s) fromthat of AMPK in controlling -cell mass and insulin secretionin vivo and in vitro. Further dissection of the signaling path-way(s) through which LKB1 acts at each site may provideexciting new modalities for the treatment of metabolic diseaseincluding type 2 diabetes.

ACKNOWLEDGMENTSWe thank Dr. Blerina Kola (Queen Mary, University of London) for useful

discussion and Lorraine Lawrence for the preparation of pancreatic slices.

GRANTSThis work was supported by grants to G. A. Rutter from the Wellcome Trust

(Programme Grant 081958/2/07/Z), The European Union (FP6 Save Beta),

Fig. 6. LKB1KO -cells display abnormal electrical, Ca2, and secretory responses and decreased GLUT2 immunoreactivity at the plasma membrane.A: glucose-stimulated insulin secretion and total insulin content of 6 size-matched islets statically incubated with indicated glucose for 0.5 h from LKB1KO,heterozygous, and WT mice; n 3 mice per genotype. B: representative traces of whole cell KATP channel conductance (GKATP) and plasma membrane potential(Vm) from perforated patch clamp measurements. Three to six -cells from 3 pairs of mice of each genotype were recorded. C: representative traces andquantification of free [Ca2] with fura 2-AM in dissociated -cells in LKB1KO mice. Cells (3860) from 3 pairs of mice of each genotype were analyzed.D: immunofluorescence staining and quantification of Glut2 expression at the plasma membrane of pancreatic islet -cells. Fifteen islets from 2 pairs of miceper genotype were examined. Scale bar, 75 m. Data are expressed as means SE. *P 0.05, **P 0.01, ***P 0.001.

Fig. 7. In vivo metabolic advantages of LKB1 deletion from insulin-expressingcells are lost on a high-fat diet. Glucose tolerance (A) and plasma insulinchanges (B) after ip glucose injection of LKB1KO mice on a high-fat diet for6 wk (see METHODS).

Table 1. Relative abundance of LKB1 target kinases inpancreatic islets

Protein KinasemRNA Level

(Relative Intensity)LKB1/STK11 404CaMKK2 152AMPK1 (PRKAA1) 416AMPK 2 (PRKAA2) 104Snf-related kinase (SNRK) 396Nuak1/Snf-1 line kinase-1/ARK5 314Nuak2/SNARK 172Salt-inducible kinase 1 (SIK1/MSK/SNF1LK1) 35Salt-inducible kinase (SIK2/QIK/ SNF1LK2) 54MARK1 214MARK2 (PAR1B) 422MARK3 (PAR1A/TAK1/MAP3K7) 568MARK4 ABRSK1 (SAD-A) ABRSK2 (SAD-B) A

Pancreatic islets isolated from 12-wk-old wild-type male C57Bl/6 mice bycollagenase digestion and histopaque gradient purification as described (27)were incubated in RPMI supplemented with 11 mM glucose for 16 h. Totalcellular RNA (400-600 ng) extracted from islets by use of RNAeasy kit(Qiagen UK) was used for microarray analysis. Four independent hybridiza-tions were performed on Mouse 430 2.0 chip (Affymetrix, Santa Clara, CA).Data were exported to GeneSpringX to perform quality control and subsequentdownstream analysis. Robust multichip average (RMA) summary and quantilenormalization was employed to produce expression values for the probe sets.Data are expressed as means of observations on islets from 4 separate mice,which agreed within 10%. A, absent.




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the Medical Research Council (G0401641) and National Institutes of Health(RO1 DK-071962-01), and a JDRFI PostDoctoral Fellowship to A. I. Tarasov.

DISCLOSURESNo conflicts of interest were reported by the authors.

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