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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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