human islets were obtained from donors of both sexes , of ages ranging from 5 to 70 years and with a majority being in the 2050 year range , and with purity ranging from 5095% , through the islet cell resource centers ( icrs ) and juvenile diabetes research foundation basic science islet distribution programs .
human kidney cell lines , hkc8 , and 293 with or without human pth1r cdna were used as controls for pth1r expression .
adenovirus ( ad ) constructs with cdnas were generated ( 8) including those for -galactosidase ( lacz ) , cre recombinase , and green fluorescent protein , which were used as controls . for cell - cycle analysis
, human islets transduced with ad constructs for 72 h were dispersed into single cells and 10,000 cells were analyzed on a bd lsr ii flow cytometer ( becton dickinson biosciences , san jose , ca ) .
data analysis was performed using modfit lt 3.0 software ( verity software house , topsham , me ) .
-cell proliferation was analyzed on either ad - transduced or peptide - treated dispersed human islet cultures stained for dapi , bromodeoxyuridine ( brdu ) , and insulin using olympus fluoview confocal or olympus provis fluorescence microscopes ( 8) .
insulin release from uninfected , pthrp(1 - 36 ) treated , and ad - transduced islets was measured in triplicate from human islets either transduced with ad constructs for 24 h or treated with 100 nmol / l of pthrp(1 - 36 ) for 24 h for insulin content or 30 min for gsis ( 8) .
briefly , uninfected or islets transduced with ad for 24 h were preincubated in krebs - ringer bicarbonate buffer supplemented with 10 mmol / l hepes , 1% bsa , and 5 mmol / l glucose for 1 h at 37c in a 5% co2 incubator . after washing the islets once with the same solution
, groups of 15 islet equivalents ( ieqs ) for each condition were incubated in 1 ml fresh krebs - ringer bicarbonate buffer plus 1% bsa and 5.5 or 22 mmol / l glucose .
pthrp peptide or vehicle was added to the islets at the same time as the glucose and incubated for 30 min , after which buffer was removed and frozen at 20c until insulin measurement by radioimmunoissay ( ria ) ( linco research , st .
islets were then digested overnight in naoh at 37c , and protein was measured by the bradford method after neutralization with hcl .
results are expressed as a percentage of insulin concentration obtained with uninfected islets incubated at 5.5 mmol / l glucose .
insulin content was measured by radioimmunoassay on acid ethanol extracts of human islets either transduced with ad - control or ad - pthrp or treated with vehicle or 100 nmol / l of pthrp(1 - 36 ) peptide for 24h ( 8,17 ) .
gene expression in whole islets was analyzed by real - time pcr performed on an abi 7300 system and western blot analysis on 2050 g extracts using the image j program ( nih ) for quantitative densitometry ( 8) .
pthrp(1 - 36 ) was quantitated using an immunoradiometric assay or an enzyme immunoassay ( peninsula laboratories , san carlos , ca ) .
statistical significance considered at p < 0.05 was determined by an unpaired two - tailed student 's t test or a one - way anova with tukey 's honestly significant difference post hoc test . additional details regarding research design and methods are available in an online appendix ( http://diabetes.diabetesjournals.org/cgi/content/full/db09-1796/dc1 ) .
human islets were obtained from donors of both sexes , of ages ranging from 5 to 70 years and with a majority being in the 2050 year range , and with purity ranging from 5095% , through the islet cell resource centers ( icrs ) and juvenile diabetes research foundation basic science islet distribution programs .
human kidney cell lines , hkc8 , and 293 with or without human pth1r cdna were used as controls for pth1r expression .
adenovirus ( ad ) constructs with cdnas were generated ( 8) including those for -galactosidase ( lacz ) , cre recombinase , and green fluorescent protein , which were used as controls .
for cell - cycle analysis , human islets transduced with ad constructs for 72 h were dispersed into single cells and 10,000 cells were analyzed on a bd lsr ii flow cytometer ( becton dickinson biosciences , san jose , ca ) .
data analysis was performed using modfit lt 3.0 software ( verity software house , topsham , me ) .
-cell proliferation was analyzed on either ad - transduced or peptide - treated dispersed human islet cultures stained for dapi , bromodeoxyuridine ( brdu ) , and insulin using olympus fluoview confocal or olympus provis fluorescence microscopes ( 8) .
insulin release from uninfected , pthrp(1 - 36 ) treated , and ad - transduced islets was measured in triplicate from human islets either transduced with ad constructs for 24 h or treated with 100 nmol / l of pthrp(1 - 36 ) for 24 h for insulin content or 30 min for gsis ( 8) .
briefly , uninfected or islets transduced with ad for 24 h were preincubated in krebs - ringer bicarbonate buffer supplemented with 10 mmol / l hepes , 1% bsa , and 5 mmol / l glucose for 1 h at 37c in a 5% co2 incubator .
after washing the islets once with the same solution , groups of 15 islet equivalents ( ieqs ) for each condition were incubated in 1 ml fresh krebs - ringer bicarbonate buffer plus 1% bsa and 5.5 or 22 mmol / l glucose .
pthrp peptide or vehicle was added to the islets at the same time as the glucose and incubated for 30 min , after which buffer was removed and frozen at 20c until insulin measurement by radioimmunoissay ( ria ) ( linco research , st .
islets were then digested overnight in naoh at 37c , and protein was measured by the bradford method after neutralization with hcl .
results are expressed as a percentage of insulin concentration obtained with uninfected islets incubated at 5.5 mmol / l glucose .
insulin content was measured by radioimmunoassay on acid ethanol extracts of human islets either transduced with ad - control or ad - pthrp or treated with vehicle or 100 nmol / l of pthrp(1 - 36 ) peptide for 24h ( 8,17 ) .
gene expression in whole islets was analyzed by real - time pcr performed on an abi 7300 system and western blot analysis on 2050 g extracts using the image j program ( nih ) for quantitative densitometry ( 8) .
pthrp(1 - 36 ) was quantitated using an immunoradiometric assay or an enzyme immunoassay ( peninsula laboratories , san carlos , ca ) .
< 0.05 was determined by an unpaired two - tailed student 's t test or a one - way anova with tukey 's honestly significant difference post hoc test .
additional details regarding research design and methods are available in an online appendix ( http://diabetes.diabetesjournals.org/cgi/content/full/db09-1796/dc1 ) .
pthrp mrna and protein are expressed in human islets and insulinomas ( 5,13,14 ) . however , whether the receptor for pthrp , pth1r , is expressed in human islets is unknown .
we analyzed the expression of pth1r mrna by real - time pcr in human islets using hkc8 , a human kidney cell line with high pth1r expression , as a positive control and 293 cells that lack pth1r expression as a negative control .
importantly , pth1r is expressed in human -cells , demonstrated by costaining of human islet cell cultures for pth1r and insulin ( fig .
pth1r and pthrp expression in human islets . a : pth1r mrna expression measured by real - time pcr in three human islet preps ( h1h3 ) ; hkc8 , a human kidney proximal tubule cell line , used as positive control ; and the human embryonic kidney cell line 293 , used as negative control .
b : western blot analysis of pth1r and actin expression in a positive ( + ) control line ( 293 cells stably transfected with human pth1r cdna ) and two human islet preps ( h1 and h2 ) uninfected ( u ) or transduced with ad - lacz ( l ) or ad - pthrp ( p ) . the line divides samples run on two different gels .
c : photomicrograph of human islet cell cultures costained for nuclear dapi ( blue ) , pth1r ( green ) , and insulin ( red ) .
d : representative western blot analysis of pthrp and actin in human islets either uninfected ( u ) or transduced with ad - lacz ( l ) , ad - pthrp ( p ) , or ad-sp mutant ( s ) . the line divides samples from different regions run on the same gel .
e : quantitation of pthrp(1 - 36 ) by immunoradiometric assay in medium collected after 48 h of transduction of human islets with adenoviral constructs containing lacz , pthrp , or sp sequences ( n = 2 islet preparations ) .
f : representative images of human islet cell cultures transduced with ad - lacz , ad - pthrp , or ad-sp and costained for dapi ( blue ) , insulin ( green ) , and ha ( red ) . (
a high - quality digital representation of this figure is available in the online issue . ) to overexpress pthrp in human islets , an ad construct containing hemagglutinin ( ha)-tagged human pthrp cdna was transduced into islets .
ad - pthrp  transduced human islets display increased pthrp expression relative to uninfected and ad - lacz  transduced islets as demonstrated by western blot using pthrp antibody ( fig .
furthermore , pthrp overexpression did not result in downregulation of pth1r expression in human islets ( fig .
1b ) . since pthrp is a secreted protein , we measured its concentration in medium from human islets transduced with ad - pthrp or ad - lacz .
pmol / l , in medium from ad - pthrp  infected islets versus ad - lacz  infected islets ( 4 pmol / l ) ( fig .
finally , pthrp is expressed in -cells of ad - pthrp  transduced human islet cell cultures demonstrated by coimmunostaining with ha and insulin antibodies ( fig .
we examined the effect of pthrp overexpression on human -cell proliferation by costaining human islet cell cultures transduced with ad - pthrp or ad - control with brdu and insulin antibodies ( fig .
there was a significant , approximately threefold , increase in human -cell proliferation induced by pthrp compared with ad - control  transduced or uninfected human islet cell cultures ( fig .
a : representative confocal images of human islet cell cultures transduced with ad - control ( i ) or ad - pthrp ( ii  iv ) and costained for insulin ( green , panel b ) , brdu ( red , panel c ) , and dapi ( blue , panel d ) .
the merged images of all three stainings are shown in panel a , merge for dapi and brdu in panel e , and merge for insulin and brdu in panel f. b : quantitation of the percentage of brdu - positive -cells from human islet cell cultures uninfected ( ui ) or transduced with the adenovirus ( adv ) constructs control ( ctrl ) , pthrp , or sp for 72 h or treated with 100 nmol / l of either 1 - 36 or 38 - 94 pthrp peptides for 24 h. there was a significant two- to threefold increase in -cell proliferation in pthrp - transduced and 1 - 36treated vs. ad - control  transduced or uninfected control islet cells
. basal rate of -cell proliferation in uninfected controls was 0.08  0.03% ( n = 411 individual human islet preps at least in duplicate ) .
* p < 0.05 vs. uninfected or ad - control by student 's t test ; # p < 0.001 vs. ad - control or ad-sp and & p < 0.01 vs. ui or 38 - 94 by one - way anova .
c : expression of differentiation markers by real - time pcr from human islets treated for 24 h with vehicle control ( ctrl ) or pthrp(1 - 36 ) peptide ( gray bars ) .
pcr cycles for each gene were compared , with actin used as an internal control .
the graph is depicted as fold over control , with values from vehicle - treated islets taken as 1 ( n = 47 human islet preparations in duplicate ) .
d : insulin content per ieq in extracts of human islets treated with vehicle ( veh ) or 100 nmol / l pthrp(1 - 36 ) peptide or transduced with ad - control ( ctrl ) or ad - pthrp for 24 h ( n = 5 human islet preparations in triplicate ) .
e and f : insulin secretion measured at 5.5 and 22 mmol / l glucose from human islets transduced with ad - control or ad - pthrp for 24 h ( e ) and treated with vehicle or 100 nmol / l pthrp(1 - 36 ) peptide for 30 min ( f ) .
insulin secretion is depicted as percentage of vehicle - treated control at 5.5 mmol / l glucose , which was 455.4  109.3 pg/g protein in 30 min .
n = 78 human islet preparations in triplicate . * p < 0.05 vs. insulin secretion at 5.5 mmol / l glucose with the same treatment ; # p < 0.05 vs. insulin secretion at equivalent glucose concentrations of control .
( a high - quality digital representation of this figure is available in the online issue . ) based on studies in rodent -cells , we evaluated whether secreted pthrp is required for human -cell proliferation by transducing human islets with ad-sp , a ha - tagged nonsecretory pthrp mutant that lacks the sp .
as expected , secreted pthrp levels in the medium were much lower in ad-sp ( 22 pmol / l ) versus ad - pthrp  transduced ( 600 pmol / l ) human islets ( fig .
however , pthrp levels in islet extracts from ad-sp were at least comparable with ad - pthrp  transduced human islets ( fig .
moreover , pthrp was expressed in the -cells of ad-sp  transduced human islet cultures as shown by ha and insulin costaining ( fig .
despite abundant intracellular pthrp , ad-sp did not stimulate proliferation of human -cells relative to uninfected and ad - control  infected islet cells ( fig .
2b ) , demonstrating that secreted pthrp is important for mediating human -cell proliferation . to determine which specific secretory form of pthrp induces human -cell proliferation ,
human islet cultures were treated for 24 h with 100 nmol / l of either amino - terminal pthrp(1 - 36 ) or mid - region pthrp(38 - 94 ) peptide .
there was a significant twofold increase in human -cell proliferation only with pthrp(1 - 36 ) and not with pthrp ( 3894 ) ( fig .
the concentration of pthrp(1 - 36 ) remaining in the culture medium of ad - transduced and peptide - treated human islet cell cultures was measured at the end of the experiments .
cells treated with vehicle or ad - control had undetectable pthrp(1 - 36 ) , whereas ad - pthrp  transduced cells had 469.8  57.4 pg / ml ( 117.5
pmol / l ) and pthrp(1 - 36)treated cells had 245.1  20.6 ng / ml ( 61.25
nmol / l ) of pthrp(1 - 36 ) in the culture media ( n = 45 human islet cultures ) .
since induction of proliferation of human -cells can lead to their dedifferentiation ( 17 ) , we examined mrna transcripts encoding differentiation markers in human islets treated with vehicle or pthrp(1 - 36 ) .
there was no change in transcripts encoding islet hormones ( insulin , glucagon , and somatostatin ) , glucose sensors ( glut-2 and glucokinase ) , or transcription factors important for -cell differentiation and function ( pdx1 , mafa , mafb , nkx6.1 , nkx2.2 , neurod , and isl1 ) with pthrp(1 - 36 ) treatment ( fig .
either transduction with ad - pthrp or treatment with pthrp(1 - 36 ) for 24 h did not affect the insulin content in islets ( fig .
whether pthrp affects human -cell function was evaluated by measuring insulin secretion from human islets transduced for 24 h with ad - pthrp or treated for 30 min with pthrp(1 - 36 ) peptide .
there was no change in basal insulin secretion at 5.5 mmol / l glucose , and there was a small ( 27% ) but not significant increase in insulin secretion at 22 mmol / l glucose from ad - pthrp  versus ad - control  transduced islets ( fig .
2e ) , suggesting that ad - pthrp does not negatively impact human -cell function . on the other hand , pthrp(1 - 36 ) peptide caused a significant increase in insulin secretion at 5.5 mmol / l glucose ( 36% ) , with a further enhancement at 22 mmol
thus , pthrp(1 - 36 ) enhances insulin secretion in human islets at both physiological and pathophysiological glucose concentrations . to begin to assess the molecular mechanisms of pthrp - induced human -cell proliferation , we measured expression of the g1/s cell cycle regulators .
there was no significant change in the level of mrna expression of any of the cyclins ( cyclin d13 , e12 , and a12 ) , cyclin - dependent kinases ( cdks ) ( 1,2,4,6 ) , cyclin inhibitory proteins / kinase inhibitory proteins ( cip / kip ) ( p21 , p27 , and p57 ) or the inhibitor of kinase ( ink ) ( p16 , p18 , and p19 ) family of inhibitors examined with pthrp(1 - 36 ) treatment ( fig .
pthrp increases expression of cyclin ( cyc ) e and cdk2 proteins but not mrna in human islets .
a : expression of g1/s cell cycle regulators by real - time pcr from human islets treated for 24 h with vehicle control ( ctrl ) or pthrp(1 - 36 ) peptide ( gray bars ) .
pcr cycles for each gene were compared , with actin used as an internal control .
the graph is depicted as fold over control , with values from vehicle - treated islets taken as 1 ( n = 47 human islet preparations in duplicate ) .
b : representative western blot analysis of the g1/s cell cycle activators and inhibitors from human islets treated for 24 h with vehicle ( veh ) or pthrp(1 - 36 ) using actin or tubulin as the internal housekeeping ( hk ) gene control .
quantitation of the ratio of cyclin e / hk ( c ) and cdk2/hk protein ( d ) shows a significant increase in both these proteins in pthrp(1 - 36)treated islets vs. control ( veh ) , depicted as 100% .
however , given that cell cycle molecules could be regulated posttranscriptionally , we examined the effect of pthrp(1 - 36 ) on the expression of g1/s cell - cycle proteins by western blot .
there was no change in expression of the early g1/s cell - cycle activators cyclins d1/3 or cdk4/6 or in the ink and cip / kip inhibitors p16 , p18 , p21 , p27 , and p57 ( fig .
, the late g1/s cell cycle activator proteins cyclin e ( fig . 3b and c ) and cdk2 ( fig . 3b and d ) were significantly increased , by twofold , in pthrp - treated human islets . to ascertain whether expression of cyclin e and/or cdk2 per se can drive human -cell proliferation
, these proteins were overexpressed in human islets by transduction with ad - cdk2/ad - cyclin e , relative to uninfected and ad - control  transduced islets ( fig .
4b ) revealed a significant 2.5-fold increase in the percentage of cells in the s - phase of the cell cycle in ad - cdk2/ad  cyclin e 
transduced whole human islets compared with uninfected or ad - control  transduced human islets ( fig .
a : western blot analysis of human islets uninfected ( ui ) or transduced with ad - control ( ad - ctrl ) or a combination of ad - cdk2/ad - cyclin e for expression of cdk2 , cyclin e , and actin as an internal control .
representative cell cycle phase distribution profiles ( b ) and percentage of cells in s - phase ( c ) of the cell cycle of uninfected , ad - control , or ad - cdk2/ad - cyclin e  transduced human islet cells analyzed using flow cytometry .
the percentage of cells in the g1-phase was reduced , although not significantly , in ad - cdk2/ad - cyclin e  transduced ( g1 : 91.1  1.0% , g2/m : 5.4  1.2% ) vs. uninfected ( g1 : 93.0  1.3% , g2/m : 5.4  1.2% ) and ad - control  transduced ( g1 : 94.0  1.0% , g2/m : 4.6  0.9% ) human islets , whereas the percentage of cells in s - phase of the cell cycle in ad - cdk2/ad - cyclin e  transduced human islets was significantly greater compared with uninfected or ad - control  transduced human islets ( n = 10 human islet preparations ) .
d : representative images of human islet cell cultures transduced with 100 moi of ad - control , 50 moi of ad - control plus 50 moi of ad - cdk2 , or 50 moi of ad - cyclin e and 50 moi each of ad - cdk2 and ad - cyclin e and costained for dapi ( blue ) , insulin ( green ) , and brdu ( red ) .
e : quantitation of the percentage of brdu - positive -cells from human islet cell cultures transduced with the ad constructs described for c. there was a significant 5-fold and 16-fold increase in -cell proliferation in cyclin e and cdk2/cyclin e vs. ad - control  transduced islet cells , respectively
. basal rate of -cell proliferation in controls was 0.17  0.06% , taken as 100% .
n = 9 ; 3 human islet preparations in triplicate . * p < 0.01 vs. control by student 's t test ; # p < 0.001 vs. the other three groups by one - way anova .
( a high - quality digital representation of this figure is available in the online issue . ) to independently confirm these observations and to demonstrate whether human -cell proliferation was induced by cyclin e and/or cdk2 , staining for insulin , brdu , and dapi was performed on human islet cultures ( fig .
however , expression of cyclin e alone caused a significant fivefold increase in human -cell proliferation compared with control cells .
cyclin e and cdk2 together caused a substantive synergistic 16-fold increase in human -cell proliferation over cells transduced with equivalent multiplicity of infection ( moi ) of control virus and also a significant increase over cells transduced with ad - cdk2 or ad - cyclin e alone ( fig .
pthrp mrna and protein are expressed in human islets and insulinomas ( 5,13,14 ) . however , whether the receptor for pthrp , pth1r , is expressed in human islets is unknown .
we analyzed the expression of pth1r mrna by real - time pcr in human islets using hkc8 , a human kidney cell line with high pth1r expression , as a positive control and 293 cells that lack pth1r expression as a negative control .
importantly , pth1r is expressed in human -cells , demonstrated by costaining of human islet cell cultures for pth1r and insulin ( fig .
pth1r and pthrp expression in human islets . a : pth1r mrna expression measured by real - time pcr in three human islet preps ( h1h3 ) ; hkc8 , a human kidney proximal tubule cell line , used as positive control ; and the human embryonic kidney cell line 293 , used as negative control .
b : western blot analysis of pth1r and actin expression in a positive ( + ) control line ( 293 cells stably transfected with human pth1r cdna ) and two human islet preps ( h1 and h2 ) uninfected ( u ) or transduced with ad - lacz ( l ) or ad - pthrp ( p ) . the line divides samples run on two different gels .
c : photomicrograph of human islet cell cultures costained for nuclear dapi ( blue ) , pth1r ( green ) , and insulin ( red ) .
d : representative western blot analysis of pthrp and actin in human islets either uninfected ( u ) or transduced with ad - lacz ( l ) , ad - pthrp ( p ) , or ad-sp mutant ( s ) . the line divides samples from different regions run on the same gel .
e : quantitation of pthrp(1 - 36 ) by immunoradiometric assay in medium collected after 48 h of transduction of human islets with adenoviral constructs containing lacz , pthrp , or sp sequences ( n = 2 islet preparations ) .
f : representative images of human islet cell cultures transduced with ad - lacz , ad - pthrp , or ad-sp and costained for dapi ( blue ) , insulin ( green ) , and ha ( red ) . (
a high - quality digital representation of this figure is available in the online issue . ) to overexpress pthrp in human islets , an ad construct containing hemagglutinin ( ha)-tagged human pthrp cdna was transduced into islets .
ad - pthrp  transduced human islets display increased pthrp expression relative to uninfected and ad - lacz  transduced islets as demonstrated by western blot using pthrp antibody ( fig .
furthermore , pthrp overexpression did not result in downregulation of pth1r expression in human islets ( fig .
1b ) . since pthrp is a secreted protein , we measured its concentration in medium from human islets transduced with ad - pthrp or ad - lacz .
pmol / l , in medium from ad - pthrp  infected islets versus ad - lacz  infected islets ( 4 pmol / l ) ( fig .
finally , pthrp is expressed in -cells of ad - pthrp  transduced human islet cell cultures demonstrated by coimmunostaining with ha and insulin antibodies ( fig .
we examined the effect of pthrp overexpression on human -cell proliferation by costaining human islet cell cultures transduced with ad - pthrp or ad - control with brdu and insulin antibodies ( fig .
2a ) . there was a significant , approximately threefold , increase in human -cell proliferation induced by pthrp compared with ad - control  transduced or uninfected human islet cell cultures ( fig .
pthrp increases human -cell proliferation and function without causing dedifferentiation . a : representative confocal images of human islet cell cultures transduced with ad - control ( i ) or ad - pthrp ( ii  iv ) and costained for insulin ( green , panel b ) , brdu ( red , panel c ) , and dapi ( blue , panel d ) .
the merged images of all three stainings are shown in panel a , merge for dapi and brdu in panel e , and merge for insulin and brdu in panel f. b : quantitation of the percentage of brdu - positive -cells from human islet cell cultures uninfected ( ui ) or transduced with the adenovirus ( adv ) constructs control ( ctrl ) , pthrp , or sp for 72 h or treated with 100 nmol / l of either 1 - 36 or 38 - 94 pthrp peptides for 24 h. there was a significant two- to threefold increase in -cell proliferation in pthrp - transduced and 1 - 36treated vs. ad - control  transduced or uninfected control islet cells . basal rate of -cell proliferation in uninfected controls was 0.08  0.03% ( n = 411 individual human islet preps at least in duplicate ) . * p < 0.05 vs. uninfected or ad - control by student 's t test ; # p < 0.001 vs. ad - control or ad-sp and & p < 0.01 vs. ui or 38 - 94 by one - way anova .
c : expression of differentiation markers by real - time pcr from human islets treated for 24 h with vehicle control ( ctrl ) or pthrp(1 - 36 ) peptide ( gray bars ) .
pcr cycles for each gene were compared , with actin used as an internal control .
the graph is depicted as fold over control , with values from vehicle - treated islets taken as 1 ( n = 47 human islet preparations in duplicate ) .
d : insulin content per ieq in extracts of human islets treated with vehicle ( veh ) or 100 nmol / l pthrp(1 - 36 ) peptide or transduced with ad - control ( ctrl ) or ad - pthrp for 24 h ( n = 5 human islet preparations in triplicate ) .
e and f : insulin secretion measured at 5.5 and 22 mmol / l glucose from human islets transduced with ad - control or ad - pthrp for 24 h ( e ) and treated with vehicle or 100 nmol / l pthrp(1 - 36 ) peptide for 30 min ( f ) .
insulin secretion is depicted as percentage of vehicle - treated control at 5.5 mmol / l glucose , which was 455.4  109.3 pg/g protein in 30 min .
n = 78 human islet preparations in triplicate . * p < 0.05 vs. insulin secretion at 5.5 mmol / l glucose with the same treatment ; # p < 0.05 vs. insulin secretion at equivalent glucose concentrations of control .
( a high - quality digital representation of this figure is available in the online issue . )
based on studies in rodent -cells , we evaluated whether secreted pthrp is required for human -cell proliferation by transducing human islets with ad-sp , a ha - tagged nonsecretory pthrp mutant that lacks the sp .
as expected , secreted pthrp levels in the medium were much lower in ad-sp ( 22 pmol / l ) versus ad - pthrp  transduced ( 600 pmol / l ) human islets ( fig .
however , pthrp levels in islet extracts from ad-sp were at least comparable with ad - pthrp  transduced human islets ( fig .
moreover , pthrp was expressed in the -cells of ad-sp  transduced human islet cultures as shown by ha and insulin costaining ( fig .
despite abundant intracellular pthrp , ad-sp did not stimulate proliferation of human -cells relative to uninfected and ad - control  infected islet cells ( fig .
2b ) , demonstrating that secreted pthrp is important for mediating human -cell proliferation . to determine which specific secretory form of pthrp induces human -cell proliferation ,
human islet cultures were treated for 24 h with 100 nmol / l of either amino - terminal pthrp(1 - 36 ) or mid - region pthrp(38 - 94 ) peptide .
there was a significant twofold increase in human -cell proliferation only with pthrp(1 - 36 ) and not with pthrp ( 3894 ) ( fig .
the concentration of pthrp(1 - 36 ) remaining in the culture medium of ad - transduced and peptide - treated human islet cell cultures was measured at the end of the experiments .
cells treated with vehicle or ad - control had undetectable pthrp(1 - 36 ) , whereas ad - pthrp  transduced cells had 469.8  57.4 pg / ml ( 117.5
pmol / l ) and pthrp(1 - 36)treated cells had 245.1  20.6 ng / ml ( 61.25
nmol / l ) of pthrp(1 - 36 ) in the culture media ( n = 45 human islet cultures ) .
since induction of proliferation of human -cells can lead to their dedifferentiation ( 17 ) , we examined mrna transcripts encoding differentiation markers in human islets treated with vehicle or pthrp(1 - 36 ) .
there was no change in transcripts encoding islet hormones ( insulin , glucagon , and somatostatin ) , glucose sensors ( glut-2 and glucokinase ) , or transcription factors important for -cell differentiation and function ( pdx1 , mafa , mafb , nkx6.1 , nkx2.2 , neurod , and isl1 ) with pthrp(1 - 36 ) treatment ( fig .
either transduction with ad - pthrp or treatment with pthrp(1 - 36 ) for 24 h did not affect the insulin content in islets ( fig .
whether pthrp affects human -cell function was evaluated by measuring insulin secretion from human islets transduced for 24 h with ad - pthrp or treated for 30 min with pthrp(1 - 36 ) peptide .
there was no change in basal insulin secretion at 5.5 mmol / l glucose , and there was a small ( 27% ) but not significant increase in insulin secretion at 22 mmol / l glucose from ad - pthrp  versus ad - control  transduced islets ( fig .
2e ) , suggesting that ad - pthrp does not negatively impact human -cell function . on the other hand , pthrp(1 - 36 ) peptide caused a significant increase in insulin secretion at 5.5 mmol / l glucose ( 36% ) , with a further enhancement at 22 mmol
thus , pthrp(1 - 36 ) enhances insulin secretion in human islets at both physiological and pathophysiological glucose concentrations .
to begin to assess the molecular mechanisms of pthrp - induced human -cell proliferation , we measured expression of the g1/s cell cycle regulators
. there was no significant change in the level of mrna expression of any of the cyclins ( cyclin d13 , e12 , and a12 ) , cyclin - dependent kinases ( cdks ) ( 1,2,4,6 ) , cyclin inhibitory proteins / kinase inhibitory proteins ( cip / kip ) ( p21 , p27 , and p57 ) or the inhibitor of kinase ( ink ) ( p16 , p18 , and p19 ) family of inhibitors examined with pthrp(1 - 36 ) treatment ( fig . 3a ) .
pthrp increases expression of cyclin ( cyc ) e and cdk2 proteins but not mrna in human islets .
a : expression of g1/s cell cycle regulators by real - time pcr from human islets treated for 24 h with vehicle control ( ctrl ) or pthrp(1 - 36 ) peptide ( gray bars ) .
pcr cycles for each gene were compared , with actin used as an internal control .
the graph is depicted as fold over control , with values from vehicle - treated islets taken as 1 ( n = 47 human islet preparations in duplicate ) .
b : representative western blot analysis of the g1/s cell cycle activators and inhibitors from human islets treated for 24 h with vehicle ( veh ) or pthrp(1 - 36 ) using actin or tubulin as the internal housekeeping ( hk ) gene control .
quantitation of the ratio of cyclin e / hk ( c ) and cdk2/hk protein ( d ) shows a significant increase in both these proteins in pthrp(1 - 36)treated islets vs. control ( veh ) , depicted as 100% . * p < 0.05 ( n = 49 human islet preparations ) . however , given that cell cycle molecules could be regulated posttranscriptionally , we examined the effect of pthrp(1 - 36 ) on the expression of g1/s cell - cycle proteins by western blot .
there was no change in expression of the early g1/s cell - cycle activators cyclins d1/3 or cdk4/6 or in the ink and cip / kip inhibitors p16 , p18 , p21 , p27 , and p57 ( fig .
, the late g1/s cell cycle activator proteins cyclin e ( fig . 3b and c ) and cdk2 ( fig . 3b and d ) were significantly increased , by twofold , in pthrp - treated human islets .
to ascertain whether expression of cyclin e and/or cdk2 per se can drive human -cell proliferation , these proteins were overexpressed in human islets by transduction with ad - cdk2/ad - cyclin e , relative to uninfected and ad - control  transduced islets ( fig .
4b ) revealed a significant 2.5-fold increase in the percentage of cells in the s - phase of the cell cycle in ad - cdk2/ad  cyclin e 
transduced whole human islets compared with uninfected or ad - control  transduced human islets ( fig .
a : western blot analysis of human islets uninfected ( ui ) or transduced with ad - control ( ad - ctrl ) or a combination of ad - cdk2/ad - cyclin e for expression of cdk2 , cyclin e , and actin as an internal control . the line divides samples from different regions on the same gel .
representative cell cycle phase distribution profiles ( b ) and percentage of cells in s - phase ( c ) of the cell cycle of uninfected , ad - control , or ad - cdk2/ad - cyclin e  transduced human islet cells analyzed using flow cytometry .
the percentage of cells in the g1-phase was reduced , although not significantly , in ad - cdk2/ad - cyclin e  transduced ( g1 : 91.1  1.0% , g2/m : 5.4  1.2% ) vs. uninfected ( g1 : 93.0  1.3% , g2/m : 5.4  1.2% ) and ad - control  transduced ( g1 : 94.0  1.0% , g2/m : 4.6  0.9% ) human islets , whereas the percentage of cells in s - phase of the cell cycle in ad - cdk2/ad - cyclin e  transduced human islets was significantly greater compared with uninfected or ad - control  transduced human islets ( n = 10 human islet preparations ) .
d : representative images of human islet cell cultures transduced with 100 moi of ad - control , 50 moi of ad - control plus 50 moi of ad - cdk2 , or 50 moi of ad - cyclin e and 50 moi each of ad - cdk2 and ad - cyclin e and costained for dapi ( blue ) , insulin ( green ) , and brdu ( red ) .
e : quantitation of the percentage of brdu - positive -cells from human islet cell cultures transduced with the ad constructs described for c. there was a significant 5-fold and 16-fold increase in -cell proliferation in cyclin e and cdk2/cyclin e vs. ad - control  transduced islet cells , respectively
. basal rate of -cell proliferation in controls was 0.17  0.06% , taken as 100% .
n = 9 ; 3 human islet preparations in triplicate . * p < 0.01 vs. control by student 's t test ; # p < 0.001 vs. the other three groups by one - way anova .
( a high - quality digital representation of this figure is available in the online issue . ) to independently confirm these observations and to demonstrate whether human -cell proliferation was induced by cyclin e and/or cdk2 , staining for insulin , brdu , and dapi was performed on human islet cultures ( fig .
however , expression of cyclin e alone caused a significant fivefold increase in human -cell proliferation compared with control cells .
cyclin e and cdk2 together caused a substantive synergistic 16-fold increase in human -cell proliferation over cells transduced with equivalent multiplicity of infection ( moi ) of control virus and also a significant increase over cells transduced with ad - cdk2 or ad - cyclin e alone ( fig .
this study documents for the first time that pthrp can enhance human -cell growth and function .
the major novel findings of this study are as follows : 1 ) the pthrp receptor is present in human -cells .
2 ) overexpression of pthrp causes a significant threefold increase in human -cell proliferation without negatively impacting function .
3 ) the secreted amino terminus 1 - 36 peptide of pthrp is sufficient to induce replication without causing significant dedifferentiation of human -cells .
4 ) pthrp(1 - 36 ) not only increases proliferation but simultaneously enhances human -cell function , as evidenced by a significant increase in insulin secretion at both physiological ( 5.5 mmol / l ) and pathophysiological ( 22
5 ) pthrp(1 - 36 ) specifically increases expression of the late g1/s cell cycle activators cyclin e and cdk2 at the posttranscriptional level in human islets .
7 ) more importantly , the combination of these two molecules has a substantive synergistic effect on human -cell proliferation .
the current study , together with previous work ( 68,1012 ) , establishes pthrp as a factor that enhances proliferation and function in primary -cells from both rodent and human islets .
whereas in some instances activation of human -cell replication may induce dedifferentiation ( 17 ) , in others  e.g . , in this study and with overexpression of cdk6/cyclin d1 or constitutively active akt ( 18,19)induction of human -cell proliferation maintains or even augments -cell function . based on studies in rodent islets ( 12 ) , it is likely that pthrp(1 - 36 ) may enhance insulin secretion in human islets through activation of the camp pathway .
this mitogenic activity is associated with changes in cyclin d1 transcription , activation of cyclin e / cdk2 kinase activity , or reduction of p57 or p27 inhibitors ( 2022 ) . in human -cells
, we found that pthrp(1 - 36 ) is sufficient to induce proliferation , causing an increase in expression at the posttrancriptional level of two activators of the late phase of the g1/s cell - cycle check point , cdk2 and cyclin e. pthrp regulates the stability of p27 in smooth - muscle cells , as well as the androgen receptor in prostate cancer cells , through the proteosomal degradation pathway ( 21,23 ) .
cyclin e protein is also tightly regulated through proteasome - mediated degradation by the e3 ubiquitin ligases ( 24 ) .
whether pthrp affects cyclin e stability through this pathway in human -cells requires further investigation .
there is ample evidence that activators of the early phase of the cell cycle , cyclin ds and cdk4/cdk6 , regulate rodent -cell proliferation and can induce human -cell proliferation ( 18,25 ) .
however , whether activators of the late phase of the cell cycle , cyclin e and cdk2 , are important for and can induce human -cell proliferation is not known .
now , we demonstrate for the first time that cyclin e , both alone and together with cdk2 , significantly enhances human -cell proliferation , suggesting that cyclin e may be one of the limiting factors in proliferation of human -cells .
understanding the regulation of , and elucidating therapeutic mechanisms to enhance , human -cell proliferation and function is critical for the long - term treatment of diabetes .
this study shows that a small amino terminus peptide of pthrp is capable of enhancing both proliferation and function in human -cells in vitro .
further , it identifies two cell - cycle activators as likely downstream targets of pthrp .
cyclin e and cdk2 together have pronounced effects on human -cell proliferation and , therefore , have the potential to be harnessed to enhance human -cell growth ex vivo .
the promise for pthrp(1 - 36 ) as a therapeutic agent for diabetes is especially attractive , given that this peptide has been shown to be both safe and effective for the treatment of osteoporosis ( 15,16 ) and that pthrp(1 - 36 ) in the 100 pmol / l range , a concentration that is likely transiently induced in humans treated with tolerable doses of pthrp(1 - 36 ) ( 15,16 ) , is sufficient to enhance human -cell proliferation in vitro .
future studies will determine whether this peptide can be used either ex vivo or in vivo to improve human islet growth and function .
 objectiveinducing human -cell growth while enhancing function is a major goal in the treatment of diabetes . 
 parathyroid hormone  related protein ( pthrp ) enhances rodent -cell growth and function through the parathyroid hormone-1 receptor ( pth1r ) . 
 based on this , we hypothesized that pth1r is expressed in human -cells and that pthrp has the potential to enhance human -cell proliferation and/or function.research design and methodspth1r expression , -cell proliferation , glucose - stimulated insulin secretion ( gsis ) , and expression of differentiation and cell - cycle genes were analyzed in human islets transduced with adenoviral pthrp constructs or treated with pthrp peptides . 
 the effect of overexpression of late g1/s cell cycle molecules was also assessed on human -cell proliferation.resultswe found that human -cells express pth1r . 
 more importantly , overexpression of pthrp causes a significant approximately threefold increase in human -cell proliferation . 
 furthermore , the amino terminus pthrp(1 - 36 ) peptide is sufficient to increase replication as well as expression of the late g1/s cell - cycle proteins cyclin e and cyclin - dependent kinase 2 ( cdk2 ) in human islets . 
 notably , pthrp(1 - 36 ) also enhances gsis . 
 finally , overexpression of cyclin e alone , but not cdk2 , augments human -cell proliferation , and when both molecules are expressed simultaneously there is a further marked synergistic increase in replication.conclusionspthrp(1-36 ) peptide enhances human -cell proliferation as well as function , with associated upregulation of two specific cell - cycle activators that together can induce human -cell proliferation several fold . 
 the future therapeutic potential of pthrp(1 - 36 ) for the treatment of diabetes is especially relevant given the complementary therapeutic efficacy of pthrp(1 - 36 ) in postmenopausal osteoporosis . 