to generate -cell  specific pkckn transgenic mouse lines , the rip - i / pkckn chimeric gene containing the k376r mutation was excised from the plasmid , purified , and microinjected into fertilized eggs as described previously ( 10,11 ) .
two transgenic mouse lines ( # 179 and # 162 ) on a c57bl/6 background were selected and analyzed .
for in vivo experiments control mice ( wild type ) were littermates of transgenic mice .
all animal experiments were done in accordance with the accepted standard of human care of animals and were approved by the local animal care and use committee .
ins-1e cells were infected with a retrovirus containing the wild - type or pkckn construct .
transfected cells were selected by geneticin ( g418 ) and subcloned by separation of single cells .
mice were fed either a standard diet or hfd and kept under a light / dark cycle of 12/12 h. hfd consisting of 45% kcal fat from lard was fed to 4-week - old mice for 8 weeks ( d12451 ; research diet , new brunswick , nj ) .
blood glucose was determined in overnight - fasted mice using a glucometer elite ( bayer , elkhart , in ) .
glucose tolerance tests ( intraperitoneal glucose tolerance tests [ ipgtts ] ) were performed in overnight - fasted mice .
animals were injected intraperitoneally with a single dose of d - glucose ( 2 g / kg body wt ) , and blood glucose concentrations were detected at the indicated times . for glucose - stimulated insulin release ,
a d - glucose dose of 3 g /kg body wt was used . to determine insulin tolerance ,
a bolus of human insulin ( 1 unit / kg body wt ) was injected intraperitoneally into fed mice and glucose concentrations were determined .
mouse islets were isolated and cultured as described previously ( 12 ) . in brief , after culture islets were preincubated for 1 h at 37c in modified krebs - ringer bicarbonate buffer containing ( in mmol / l ) 140 nacl , 5.6 kcl , 1.2 mgcl2 , 2.6 cacl2 , 10 hepes , 2.8 glucose , and 4
g / l bsa ( ffa free ; sigma , deisenhofen , germany ) , ph 7.4 .
thereafter , batches of 5 islets/0.5 ml were incubated for 30 min at 37c in the presence of test substances as indicated for each experiment .
insulin released into the supernatant and insulin content after extraction with acid ethanol ( hcl 1.5% [ vol / vol]/ethanol 75% [ vol / vol ] ) were measured by radioimmunoassay .
activation of caspase-3 was examined by immunohistochemical staining against cleaved caspase-3 in pancreatic slices of mice fed standard diet or hfd .
sections of frozen pancreatic tissue were fixed with 4% paraformaldehyde in pbs for 20 min , permeabilized with 0.2% triton x-100 for 2 min on ice , and then blocked with 10% fcs in pbs for 45 min .
primary antibody against cleaved caspase-3 ( 1:200 ; cell signaling technology , danvers , ma ) was applied overnight in pbs supplemented with 10% fcs .
after washing with pbs supplemented with 10% fcs , the slices were incubated for 1 h with an anti - rabbit secondary antibody ( 1:400 ; alexa - fluor546 igg , invitrogen , paisley , u.k . ) .
transferase - mediated dutp nick - end labeling ( tunel ) staining was performed in isolated islet cells that were prepared as described previously and cultured in rpmi-1640 supplemented with 10% fcs , 10 mmol / l hepes , 1 mmol / l na pyruvate , 2 mmol / l l - glutamine , 100 iu / ml penicillin , and 100 g / ml streptomycin ( 13 ) .
prior to the addition to the culture medium palmitate from a 200 mmol / l stock solution dissolved in dmso was coupled to fcs at a concentration of 6 mmol / l .
the palmitate - to - albumine ratio was 10:1 , with the final concentration of dmso of 0.3% .
after 3 days of culture in the presence of 0.6 mmol / l palmitate , apoptosis was quantified in islet cells by tunel staining using a commercial kit ( roche diagnostics , mannheim , germany ) .
nuclei were stained with 1 mol / l to - pro3 in pbs for 1 h ( invitrogen , karlsruhe , germany ) .
the fluorescence was examined with a confocal microscope ( leica , wetzlar , germany ) using a 40 objective and excitation wavelengths of 546 nm ( for cleaved caspase-3 staining ) , 488 nm ( for tunel staining ) , and 633 nm ( for nuclei staining ) .
mitochondrial potential (  ) was measured in isolated and cultured islet cells loaded with rhodamine-123 as described previously ( 13 ) . briefly
, cultured islet cell clusters were treated for 3 days with 0.6 mmol / l palmitate .
thereafter the cells were loaded with 10 mg / l rhodamine-123 in modified krebs - ringer bicarbonate buffer solution containing 0.5 mmol / l glucose for 10 min at 37c .
the fluorescence was measured using a device provided by till photonics ( grfelfing , germany ) .
mitochondrial hyperpolarization induced by increasing glucose concentration is expressed as percentage of maximal increase of fluorescence induced by carbonylcyanide - p - trifluoromethoxyphenylhydrazone ( 1 mol / l fccp ) .
sections of frozen pancreatic tissue , cultured isolated islet cells , and ins-1e cells , control or stably transfected with rip - i / pkc constructs , were fixed with 4% paraformaldehyde in pbs , permeabilized with pbs containing 0.2% triton x-100 , and blocked with 10% fcs in pbs for 45 min .
primary antibodies against pkc ( 1:500 ; bd transduction laboratories , heidelberg , germany ) , insulin ( 1:150 ; dako denmark , denmark ) , and forkhead box class o ( foxo)-1 ( 1:200 ; santa cruz biotechnology , santa cruz , ca ) were applied overnight in pbs containing 10% fcs .
after 30 min washing with pbs supplemented with 10% fcs , the samples were incubated for 1 h with the appropriate secondary antibodies ( 1:400 in 10% fcs - pbs ) : alexa - fluor488 anti - mouse igg ( for pkc ) , alexa - fluor546 anti - rabbit igg ( for foxo1 ) , and alexa - fluor546 anti - guinea pig igg ( for insulin ) .
mol / l to - pro3 in pbs for 1 h. for morphometric estimation of -cell mass , insulin antibody binding on pancreatic sections was visualized with a second antibody coupled to horseradish peroxidase .
the islet sizes ( insulin - stained areas ) of every 10th cryosection of a mouse pancreas were measured with the axiovision le documentation program ( axiovs40 le v 4.4.0.0 . ;
isolated islets , excised mouse tissues , and ins-1e cells were lysed in buffer containing 125 mmol / l nacl , 1% ( v / v ) triton x-100 , 0.5% sodiumdeoxycholate , 0.1% sds , 10 mmol / l edta , 25 mmol / l hepes ph 7.3 , 10 mmol / l napp , 10 mmol / l naf , 1 mmol / l na - vanadate , 10 g / ml pepstatin a , 10 g / ml aprotinin , and 0.1 mmol / l phenylmethylsulfonyl fluoride .
cytosolic and nuclear fractions of ins-1e cells were prepared using a commercial kit ( # 78833 ; pierce biotechnology , rockford , il ) .
the 10,000 g supernatant of homogenates or the cell fractions were subjected to a sds - page ( 812% ) and blotted onto nitrocellulose membranes ( schleicher and schuell , dassel , germany ) .
the membranes were incubated overnight with primary antibodies against foxo1 , pkc , pkc , pkc , histone h1 , and glyceraldehyde-3-phosphate dehydrogenase ( each 1:1,000 in tris - buffered saline [ tbs ] containing 5% milk , santa cruz biotechnology ) ; p - ser256-foxo1 and tubulin ( each 1:1,000 in tbs containing 5% bsa , cell signaling technology ) ; followed by incubation with a secondary antibody ( horseradish peroxidase  linked anti - rabbit igg ; 1:2,000 in tbs containing 5% milk ) .
insulin was extracted by acid ethanol from homogenized whole pancreata of 9-month - old mice .
data are expressed as means  sem ; p < 0.05 ( unpaired student t test ) was considered to be statistically significant .
to generate -cell  specific pkckn transgenic mouse lines , the rip - i / pkckn chimeric gene containing the k376r mutation was excised from the plasmid , purified , and microinjected into fertilized eggs as described previously ( 10,11 ) .
two transgenic mouse lines ( # 179 and # 162 ) on a c57bl/6 background were selected and analyzed .
for in vivo experiments control mice ( wild type ) were littermates of transgenic mice .
all animal experiments were done in accordance with the accepted standard of human care of animals and were approved by the local animal care and use committee .
ins-1e cells were infected with a retrovirus containing the wild - type or pkckn construct .
transfected cells were selected by geneticin ( g418 ) and subcloned by separation of single cells .
mice were fed either a standard diet or hfd and kept under a light / dark cycle of 12/12 h. hfd consisting of 45% kcal fat from lard was fed to 4-week - old mice for 8 weeks ( d12451 ; research diet , new brunswick , nj ) .
blood glucose was determined in overnight - fasted mice using a glucometer elite ( bayer , elkhart , in ) .
glucose tolerance tests ( intraperitoneal glucose tolerance tests [ ipgtts ] ) were performed in overnight - fasted mice .
animals were injected intraperitoneally with a single dose of d - glucose ( 2 g / kg body wt ) , and blood glucose concentrations were detected at the indicated times . for glucose - stimulated insulin release ,
a d - glucose dose of 3 g /kg body wt was used . to determine insulin tolerance ,
a bolus of human insulin ( 1 unit / kg body wt ) was injected intraperitoneally into fed mice and glucose concentrations were determined .
mouse islets were isolated and cultured as described previously ( 12 ) . in brief , after culture islets were preincubated for 1 h at 37c in modified krebs - ringer bicarbonate buffer containing ( in mmol / l ) 140 nacl , 5.6 kcl , 1.2 mgcl2 , 2.6 cacl2 , 10 hepes , 2.8 glucose , and 4
g / l bsa ( ffa free ; sigma , deisenhofen , germany ) , ph 7.4 .
thereafter , batches of 5 islets/0.5 ml were incubated for 30 min at 37c in the presence of test substances as indicated for each experiment .
insulin released into the supernatant and insulin content after extraction with acid ethanol ( hcl 1.5% [ vol / vol]/ethanol 75% [ vol / vol ] ) were measured by radioimmunoassay .
activation of caspase-3 was examined by immunohistochemical staining against cleaved caspase-3 in pancreatic slices of mice fed standard diet or hfd .
sections of frozen pancreatic tissue were fixed with 4% paraformaldehyde in pbs for 20 min , permeabilized with 0.2% triton x-100 for 2 min on ice , and then blocked with 10% fcs in pbs for 45 min .
primary antibody against cleaved caspase-3 ( 1:200 ; cell signaling technology , danvers , ma ) was applied overnight in pbs supplemented with 10% fcs . after washing with pbs supplemented with 10% fcs , the slices were incubated for 1 h with an anti - rabbit secondary antibody ( 1:400 ; alexa - fluor546 igg , invitrogen , paisley , u.k . ) .
transferase - mediated dutp nick - end labeling ( tunel ) staining was performed in isolated islet cells that were prepared as described previously and cultured in rpmi-1640 supplemented with 10% fcs , 10 mmol / l hepes , 1 mmol / l na pyruvate , 2 mmol / l l - glutamine , 100 iu / ml penicillin , and 100 g / ml streptomycin ( 13 ) . prior to the addition to the culture medium palmitate from a 200 mmol / l stock solution dissolved in dmso
the palmitate - to - albumine ratio was 10:1 , with the final concentration of dmso of 0.3% .
after 3 days of culture in the presence of 0.6 mmol / l palmitate , apoptosis was quantified in islet cells by tunel staining using a commercial kit ( roche diagnostics , mannheim , germany ) .
mol / l to - pro3 in pbs for 1 h ( invitrogen , karlsruhe , germany ) .
the fluorescence was examined with a confocal microscope ( leica , wetzlar , germany ) using a 40 objective and excitation wavelengths of 546 nm ( for cleaved caspase-3 staining ) , 488 nm ( for tunel staining ) , and 633 nm ( for nuclei staining ) .
mitochondrial potential (  ) was measured in isolated and cultured islet cells loaded with rhodamine-123 as described previously ( 13 ) . briefly
, cultured islet cell clusters were treated for 3 days with 0.6 mmol / l palmitate .
thereafter the cells were loaded with 10 mg / l rhodamine-123 in modified krebs - ringer bicarbonate buffer solution containing 0.5 mmol / l glucose for 10 min at 37c .
the fluorescence was measured using a device provided by till photonics ( grfelfing , germany ) .
mitochondrial hyperpolarization induced by increasing glucose concentration is expressed as percentage of maximal increase of fluorescence induced by carbonylcyanide - p - trifluoromethoxyphenylhydrazone ( 1 mol / l fccp ) .
sections of frozen pancreatic tissue , cultured isolated islet cells , and ins-1e cells , control or stably transfected with rip - i / pkc constructs , were fixed with 4% paraformaldehyde in pbs , permeabilized with pbs containing 0.2% triton x-100 , and blocked with 10% fcs in pbs for 45 min .
primary antibodies against pkc ( 1:500 ; bd transduction laboratories , heidelberg , germany ) , insulin ( 1:150 ; dako denmark , denmark ) , and forkhead box class o ( foxo)-1 ( 1:200 ; santa cruz biotechnology , santa cruz , ca ) were applied overnight in pbs containing 10% fcs .
after 30 min washing with pbs supplemented with 10% fcs , the samples were incubated for 1 h with the appropriate secondary antibodies ( 1:400 in 10% fcs - pbs ) : alexa - fluor488 anti - mouse igg ( for pkc ) , alexa - fluor546 anti - rabbit igg ( for foxo1 ) , and alexa - fluor546 anti - guinea pig igg ( for insulin ) .
nuclei were stained with 1 mol / l to - pro3 in pbs for 1 h. for morphometric estimation of -cell mass , insulin antibody binding on pancreatic sections was visualized with a second antibody coupled to horseradish peroxidase .
the islet sizes ( insulin - stained areas ) of every 10th cryosection of a mouse pancreas were measured with the axiovision le documentation program ( axiovs40 le v 4.4.0.0 . ;
isolated islets , excised mouse tissues , and ins-1e cells were lysed in buffer containing 125 mmol / l nacl , 1% ( v / v ) triton x-100 , 0.5% sodiumdeoxycholate , 0.1% sds , 10 mmol / l edta , 25 mmol / l hepes ph 7.3 , 10 mmol / l napp , 10 mmol / l naf , 1 mmol / l na - vanadate , 10 g / ml pepstatin a , 10 g / ml aprotinin , and 0.1 mmol / l phenylmethylsulfonyl fluoride .
cytosolic and nuclear fractions of ins-1e cells were prepared using a commercial kit ( # 78833 ; pierce biotechnology , rockford , il ) .
the 10,000 g supernatant of homogenates or the cell fractions were subjected to a sds - page ( 812% ) and blotted onto nitrocellulose membranes ( schleicher and schuell , dassel , germany ) .
the membranes were incubated overnight with primary antibodies against foxo1 , pkc , pkc , pkc , histone h1 , and glyceraldehyde-3-phosphate dehydrogenase ( each 1:1,000 in tris - buffered saline [ tbs ] containing 5% milk , santa cruz biotechnology ) ; p - ser256-foxo1 and tubulin ( each 1:1,000 in tbs containing 5% bsa , cell signaling technology ) ; followed by incubation with a secondary antibody ( horseradish peroxidase  linked anti - rabbit igg ; 1:2,000 in tbs containing 5% milk ) .
insulin was extracted by acid ethanol from homogenized whole pancreata of 9-month - old mice .
data are expressed as means  sem ; p < 0.05 ( unpaired student t test ) was considered to be statistically significant .
to define the physiological and molecular consequences of depleted pkc signaling in pancreatic -cells , we established two transgenic mouse lines ( # 179 and # 162 ) that overexpress pkckn in insulin - producing cells . pkckn overexpression was verified by specific immunoblotting of islet extracts containing equal amounts of total protein and by immunohistochemistry ( fig .
s1a , available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-0512/dc1 ) . the data revealed that pkckn was highly overexpressed in line # 179 compared with line # 162 , and a low endogenous expression of pkc was detected in islets of wild - type controls .
immunoblotting of other tissues revealed that the overexpression of pkc was restricted to pancreatic -cells ( fig .
s1b ) . expression of pkc in islets and other tissues of control and pkckn - overexpressing mice . a : detection of pkc by western blotting of homogenates of isolated islets from control and pkckn mice ( # 179 ) .
b : in islet cells of pancreatic slices that stain positive for insulin ( red ) , a low endogenous and a significant overexpression of pkc ( green ) is detected in control and pkckn - overexpressing mice ( # 179 ) , respectively .
c : western blot analysis of tissue homogenates ( 50 g mouse line # 179 ) of control ( wt ) and pkckn - overexpressing ( kn ) mice reveals a high expression of pkc in brain , spleen , and hypothalamus ( hypothal ) but a low expression of pkc in heart , kidney , liver , muscle , and fat . as control , homogenate of ins-1e cells transfected with pkcwt ( 15 g ) is blotted on the left .
( a high - quality color digital representation of this figure is available in the online issue . ) to study the impact of impaired pkc signaling in pancreatic -cells on glucose homeostasis in vivo , glucose tolerance tests ( ipgtt ) were performed in both lines ( fig .
2a , control and transgenic mice ( line # 179 ) displayed the same increase in blood glucose levels at 6 weeks of age ( circles ) .
with aging , 9-month - old control and pkckn mice had comparable fasting glucose levels and similar body weight ( fig .
2a , triangles ; supplemental table s1 ) but seemed to be partly protected against age - related glucose intolerance because 120 min after a glucose load , plasma glucose levels were lower in 9-month - old pkckn animals compared with control mice ; this was also true for the second transgenic line ( fig .
however , the ipgtts expressed as area under the curve ( auc glucose ) were not significantly improved .
consistently , serum insulin increased up to 1.3  0.13 ng / ml throughout the glucose tolerance test in pkckn animals , whereas aging control mice were not able to increase insulin secretion during the ipgtt ( fig .
a and b : blood glucose and serum insulin concentrations were measured during ipgtt of control (  ,  ) and pkckn (  ,  ) mice at the age of 6 weeks ( circles ) and 9 months ( triangles ) .
blood glucose concentrations were measured after injection of insulin ( 1 unit / kg body wt ) at 0 min as described in details under research design and methods . shown are means  sem of control mice ( n = 3 ;  ,  ) and pkckn mice ( # 179 , n = 3 ;  ,  ) .
d and e : blood glucose and serum insulin concentrations were measured during ipgtt in mice fed hfd for 8 weeks .
f : serum insulin concentrations measured during ipgtt in mice fed hfd after injection of 3 g glucose / kg body wt ; shown are the means  sem of n = 3 control mice (  ,  ) and n = 4 pkckn mice ( # 179 ,  ,  ) . * p < 0.05 against the value of control mice at the same time point . to examine whether pkc plays a decisive role in fatty acid  mediated dysfunction of pancreatic -cells in vivo
after hfd , body weight and insulin sensitivity were comparable between control and pkckn mice ( fig . 2c ; supplemental table s1 ) .
however , glucose levels during ipgtt , expressed as auc glucose , were greatly improved in transgenic mice ( control mice : 46.047  8.837 mg  dl  min vs. pkckn : 28.907  6.085 mg  dl  min ; n = 3 , p < 0.05 ; fig .
, basal insulin levels tend to be higher in pkckn mice , and insulin secretion rose approximately twofold in the first 30 min and sustained at a significantly higher level up to 120 min in pkckn transgenic mice , whereas insulin levels declined already after 60 min in control animals ( fig .
already after 30 min , serum insulin levels of pkckn mice remained significantly higher than in control mice after hfd feeding ( fig .
as expected , transgenic mice that express low levels of pkckn ( line # 162 ) were less protected against hfd - impaired glucose tolerance ( supplemental fig .
therefore , further experiments were performed with islets of line # 179 . in isolated islets of wild - type and pkckn mice ,
insulin secretion was stimulated to the same extent by glucose , phorbol ester , and forskolin , and pretreatment with palmitate for 3 days resulted in the same glucose - independent hypersecretion ( fig .
these results indicate that insulin secretion is not directly regulated by pkc. insulin secretion of isolated islets of control and pkckn - overexpressing mice .
islets were isolated from control ( wt ) and pkckn - overexpressing mice ( # 179 ) and treated as described in details under research design and methods .
results are presented as means  sem of n = 12 observations from three independent experiments .
a : insulin secretion expressed as perdent of content of islets of control mice (  ) and of pkckn mice ( # 179 ,  ) .
b : glucose - dependent secretion after 3 days control culture (  ) and after 3 days culture in the presence of 0.6 mmol / l palmitate (  ) .
as the improvement of insulin secretion in vivo in pkckn - overexpressing mice may result from an increased insulin disposability due to increased -cell mass , pancreatic insulin content and islet size were assessed .
indeed , in 9-month - old transgenic mice , total pancreatic insulin content was significantly higher than in control littermates ( fig .
4a ) . in addition , according to insulin staining the mean islet size was nearly doubled in old pkckn transgenic animals , and the islets of transgenic mice fed hfd were also significantly larger than that of control animals ( fig .
however , brdu and ki67 staining suggested no increase in proliferation rates ( data not shown ) . increased pancreatic insulin content and islet size and protection against hfd- and palmitate - induced cell death in pkckn - overexpressing mice . a : insulin content of whole pancreatic extracts .
b : mean islet size assessed in pancreatic slices of aged ( > 9 months ) control (  ) and pkckn - overexpressing ( # 179 ,  ) mice fed standard chow .
c : mean islet size assessed in pancreatic slices of 3-month - old control (  ) and pkckn - overexpressing ( # 179 ,  ) mice fed hfd .
results are presented as means  sem of n = 4 pancreata ( a ) and of the number of islets as indicated in each column from two mice ( b and c ) .
d : representative pictures of insulin ( red ) and cleaved caspase-3 ( green ) staining in pancreatic slices of control and pkckn mice after either chow ( cd , upper panels ) or hfd ( lower panels ) feeding .
e : means  sem of cleaved caspase-3positive cells in islets ( n = 3040 ) of control (  ) and pkckn (  ) mice .
f : isolated islet cells were cultured for 3 days under control culture conditions and in the presence of 0.6 mmol / l palmitate as indicated .
the percentage of apoptotic tunel - positive cells is expressed as means  sem of n = 3 independent experiments .
( a high - quality color digital representation of this figure is available in the online issue . ) as apoptosis modulates -cell mass , the effect of pkckn overexpression on cell death was examined next . in pancreatic slices from wild - type but not from pkckn mice ,
inhibition of apoptosis by overexpression of pkckn was further confirmed in cultured isolated islet cells pretreated with palmitate . in cells of pkckn mice ,
palmitate did not change the percentage of apoptotic cells , whereas in control cells palmitate doubled the amount of tunel - positive cells ( fig .
therefore , the inhibition of palmitate - induced cell death in pkckn - overexpressing cells suggests that pkc indeed transmits palmitate - mediated -cell dysfunction .
apoptotic activation of caspase-3 implies cytochrome c release and mitochondrial depolarisation . to examine the integrity of mitochondria , glucose - induced hyperpolarization was assessed .
the rise of glucose from 0.5 to 16.7 mmol / l hyperpolarized mitochondria in isolated islet cells from control and pkckn mice to the same extent .
after pretreatment with palmitate , the effect of glucose was reduced by 60% in islet cells of control but not of pkckn mice ( fig .
isolated islet cells were cultured for 3 days under control culture conditions and in the presence of 0.6 mmol / l palmitate as indicated .
b : the hyperpolarizing effect of 16.7 mmol / l glucose expressed as percent of the maximal increase in fluorescence measured after addition of carbonylcyanide - p - trifluoromethoxyphenylhydrazone ( 1 mol / l fccp ) is given as means  sem of n = 1830 cells of three independent experiments . *
p < 0.05 against islet cells of control mice cultured under the same condition . because it has been described that the dominant - negative transcription factor foxo1 inhibited ffa - dependent -cell death ( 14 ) ,
experiments were performed to examine whether nuclear translocation of foxo1 is altered by pkckn . in control
palmitate triggered foxo1 accumulation into the nucleus , and this effect was inhibited in cells overexpressing pkckn ( fig .
is further confirmed by experiments performed with the insulin - secreting cell line ins-1e overexpressing either wild - type pkc ( pkcwt ) or pkckn ( fig . 6b ; supplemental fig .
s2 ) , whereas other novel pkcs ( pkc and pkc ) remained undetectable ( data not shown ) . in control
ins-1e cells foxo1 accumulated in the nucleus after palmitate treatment ( fig . 6b and 7a  c ) .
overexpression of pkcwt resulted in an increased nuclear staining of foxo1 already under control conditions , whereas overexpression of pkckn inhibited nuclear accumulation of foxo1 ( fig . 6b and 7a  c ) .
because jun nh2-terminal kinase ( jnk ) inhibition by sp600125 did not affect foxo1 accumulation induced by palmitate in either control ins-1e cells or pkcwt  ins-1e cells , it is suggested that foxo1 accumulation does not depend on jnk activity ( supplemental fig .
because phosphorylation of foxo1 at ser256 stimulates its nuclear extrusion and degradation , the amount of foxo1 protein and its phosphorylation were examined in control and transfected ins-1e cells . indeed , increased phosphorylation of foxo1 and significantly lower protein levels were detected in pkckn  ins-1e cells , whereas reduced phosphorylation of foxo1 and increased foxo1 protein levels were found in pkcwt  ins-1e cells ( fig . 7d 
, these data suggest that overexpression of pkckn in -cells inhibits palmitate - mediated -cell dysfunction by protecting against mitochondrial dysfunction and apoptosis while counteracting nuclear accumulation of foxo1 .
palmitate - mediated nuclear accumulation of foxo1 was inhibited by overexpression of pkckn in insulin secreting cells .
a : islet cells from control and pkckn mice and b : control ins-1e cells and ins-1e cells overexpressing pkcwt or pkckn were cultured under control conditions or in the presence of 0.6 mmol / l palmitate for 1 day .
( a high - quality color digital representation of this figure is available in the online issue . )
overexpression of pkcwt and pkckn changes foxo1 cellular distribution , phosphorylation , and protein concentration in insulin - secreting cells .
ins-1e cells and ins-1e cells overexpressing pkcwt or pkckn were cultured under control conditions and in the presence of 0.6 mmol / l palmitate for 1 h and 1 day .
b and c : representative western blot for foxo1 detection in nuclear fractions with means  sem of n = 4 independent experiments .
d  f : representative western blot for foxo1 , p - ser256-foxo1 , and tubulin ( as loading control ) of cell homogenates with means  sem of n = 4 independent experiments of relative amounts of foxo1 and phosphorylation of foxo1 ( p - foxo1/foxo1 ) by setting the respective bands of control ins-1 cells to 100% . * significant difference to ins-1e under control condition ;  significant difference to the respective condition of ins-1e ; # significant difference to pkckn at the same condition .
to define the physiological and molecular consequences of depleted pkc signaling in pancreatic -cells , we established two transgenic mouse lines ( # 179 and # 162 ) that overexpress pkckn in insulin - producing cells . pkckn overexpression was verified by specific immunoblotting of islet extracts containing equal amounts of total protein and by immunohistochemistry ( fig .
s1a , available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-0512/dc1 ) . the data revealed that pkckn was highly overexpressed in line # 179 compared with line # 162 , and a low endogenous expression of pkc was detected in islets of wild - type controls .
immunoblotting of other tissues revealed that the overexpression of pkc was restricted to pancreatic -cells ( fig .
s1b ) . expression of pkc in islets and other tissues of control and pkckn - overexpressing mice . a : detection of pkc by western blotting of homogenates of isolated islets from control and pkckn mice ( # 179 ) .
b : in islet cells of pancreatic slices that stain positive for insulin ( red ) , a low endogenous and a significant overexpression of pkc ( green ) is detected in control and pkckn - overexpressing mice ( # 179 ) , respectively .
c : western blot analysis of tissue homogenates ( 50 g mouse line # 179 ) of control ( wt ) and pkckn - overexpressing ( kn ) mice reveals a high expression of pkc in brain , spleen , and hypothalamus ( hypothal ) but a low expression of pkc in heart , kidney , liver , muscle , and fat . as control , homogenate of ins-1e cells transfected with pkcwt ( 15 g ) is blotted on the left .
( a high - quality color digital representation of this figure is available in the online issue . ) to study the impact of impaired pkc signaling in pancreatic -cells on glucose homeostasis in vivo , glucose tolerance tests ( ipgtt ) were performed in both lines ( fig .
2a , control and transgenic mice ( line # 179 ) displayed the same increase in blood glucose levels at 6 weeks of age ( circles ) .
with aging , 9-month - old control and pkckn mice had comparable fasting glucose levels and similar body weight ( fig .
2a , triangles ; supplemental table s1 ) but seemed to be partly protected against age - related glucose intolerance because 120 min after a glucose load , plasma glucose levels were lower in 9-month - old pkckn animals compared with control mice ; this was also true for the second transgenic line ( fig .
however , the ipgtts expressed as area under the curve ( auc glucose ) were not significantly improved .
consistently , serum insulin increased up to 1.3  0.13 ng / ml throughout the glucose tolerance test in pkckn animals , whereas aging control mice were not able to increase insulin secretion during the ipgtt ( fig .
a and b : blood glucose and serum insulin concentrations were measured during ipgtt of control (  ,  ) and pkckn (  ,  ) mice at the age of 6 weeks ( circles ) and 9 months ( triangles ) .
blood glucose concentrations were measured after injection of insulin ( 1 unit / kg body wt ) at 0 min as described in details under research design and methods . shown are means  sem of control mice ( n = 3 ;  ,  ) and pkckn mice ( # 179 , n = 3 ;  ,  ) .
d and e : blood glucose and serum insulin concentrations were measured during ipgtt in mice fed hfd for 8 weeks .
f : serum insulin concentrations measured during ipgtt in mice fed hfd after injection of 3 g glucose / kg body wt ; shown are the means  sem of n = 3 control mice (  ,  ) and n = 4 pkckn mice ( # 179 ,  ,  ) . * p < 0.05 against the value of control mice at the same time point . to examine whether pkc plays a decisive role in fatty acid  mediated dysfunction of pancreatic -cells in vivo
after hfd , body weight and insulin sensitivity were comparable between control and pkckn mice ( fig . 2c ; supplemental table s1 ) .
however , glucose levels during ipgtt , expressed as auc glucose , were greatly improved in transgenic mice ( control mice : 46.047  8.837 mg  dl  min vs. pkckn : 28.907  6.085 mg  dl  min ; n = 3 , p < 0.05 ; fig .
, basal insulin levels tend to be higher in pkckn mice , and insulin secretion rose approximately twofold in the first 30 min and sustained at a significantly higher level up to 120 min in pkckn transgenic mice , whereas insulin levels declined already after 60 min in control animals ( fig .
already after 30 min , serum insulin levels of pkckn mice remained significantly higher than in control mice after hfd feeding ( fig .
as expected , transgenic mice that express low levels of pkckn ( line # 162 ) were less protected against hfd - impaired glucose tolerance ( supplemental fig .
therefore , further experiments were performed with islets of line # 179 . in isolated islets of wild - type and pkckn mice ,
insulin secretion was stimulated to the same extent by glucose , phorbol ester , and forskolin , and pretreatment with palmitate for 3 days resulted in the same glucose - independent hypersecretion ( fig .
these results indicate that insulin secretion is not directly regulated by pkc. insulin secretion of isolated islets of control and pkckn - overexpressing mice .
islets were isolated from control ( wt ) and pkckn - overexpressing mice ( # 179 ) and treated as described in details under research design and methods .
results are presented as means  sem of n = 12 observations from three independent experiments .
a : insulin secretion expressed as perdent of content of islets of control mice (  ) and of pkckn mice ( # 179 ,  ) .
b : glucose - dependent secretion after 3 days control culture (  ) and after 3 days culture in the presence of 0.6 mmol / l palmitate (  ) .
as the improvement of insulin secretion in vivo in pkckn - overexpressing mice may result from an increased insulin disposability due to increased -cell mass , pancreatic insulin content and islet size were assessed .
indeed , in 9-month - old transgenic mice , total pancreatic insulin content was significantly higher than in control littermates ( fig .
4a ) . in addition , according to insulin staining the mean islet size was nearly doubled in old pkckn transgenic animals , and the islets of transgenic mice fed hfd were also significantly larger than that of control animals ( fig .
4b and c ) . however , brdu and ki67 staining suggested no increase in proliferation rates ( data not shown ) . increased pancreatic insulin content and islet size and protection against hfd- and palmitate - induced cell death in pkckn - overexpressing mice
b : mean islet size assessed in pancreatic slices of aged ( > 9 months ) control (  ) and pkckn - overexpressing ( # 179 ,  ) mice fed standard chow .
c : mean islet size assessed in pancreatic slices of 3-month - old control (  ) and pkckn - overexpressing ( # 179 ,  ) mice fed hfd .
results are presented as means  sem of n = 4 pancreata ( a ) and of the number of islets as indicated in each column from two mice ( b and c ) .
d : representative pictures of insulin ( red ) and cleaved caspase-3 ( green ) staining in pancreatic slices of control and pkckn mice after either chow ( cd , upper panels ) or hfd ( lower panels ) feeding .
e : means  sem of cleaved caspase-3positive cells in islets ( n = 3040 ) of control (  ) and pkckn (  ) mice .
f : isolated islet cells were cultured for 3 days under control culture conditions and in the presence of 0.6 mmol
the percentage of apoptotic tunel - positive cells is expressed as means  sem of n = 3 independent experiments .
( a high - quality color digital representation of this figure is available in the online issue . ) as apoptosis modulates -cell mass , the effect of pkckn overexpression on cell death was examined next . in pancreatic slices from wild - type but not from pkckn mice ,
inhibition of apoptosis by overexpression of pkckn was further confirmed in cultured isolated islet cells pretreated with palmitate . in cells of pkckn mice ,
palmitate did not change the percentage of apoptotic cells , whereas in control cells palmitate doubled the amount of tunel - positive cells ( fig .
therefore , the inhibition of palmitate - induced cell death in pkckn - overexpressing cells suggests that pkc indeed transmits palmitate - mediated -cell dysfunction .
apoptotic activation of caspase-3 implies cytochrome c release and mitochondrial depolarisation . to examine the integrity of mitochondria , glucose - induced hyperpolarization was assessed .
the rise of glucose from 0.5 to 16.7 mmol / l hyperpolarized mitochondria in isolated islet cells from control and pkckn mice to the same extent .
after pretreatment with palmitate , the effect of glucose was reduced by 60% in islet cells of control but not of pkckn mice ( fig .
isolated islet cells were cultured for 3 days under control culture conditions and in the presence of 0.6 mmol / l palmitate as indicated .
b : the hyperpolarizing effect of 16.7 mmol / l glucose expressed as percent of the maximal increase in fluorescence measured after addition of carbonylcyanide - p - trifluoromethoxyphenylhydrazone ( 1 mol / l fccp ) is given as means  sem of n = 1830 cells of three independent experiments . *
p < 0.05 against control ;  p < 0.05 against islet cells of control mice cultured under the same condition .
because it has been described that the dominant - negative transcription factor foxo1 inhibited ffa - dependent -cell death ( 14 ) , experiments were performed to examine whether nuclear translocation of foxo1 is altered by pkckn . in control
palmitate triggered foxo1 accumulation into the nucleus , and this effect was inhibited in cells overexpressing pkckn ( fig .
is further confirmed by experiments performed with the insulin - secreting cell line ins-1e overexpressing either wild - type pkc ( pkcwt ) or pkckn ( fig . 6b ; supplemental fig .
s2 ) , whereas other novel pkcs ( pkc and pkc ) remained undetectable ( data not shown ) . in control
ins-1e cells foxo1 accumulated in the nucleus after palmitate treatment ( fig . 6b and 7a  c ) .
overexpression of pkcwt resulted in an increased nuclear staining of foxo1 already under control conditions , whereas overexpression of pkckn inhibited nuclear accumulation of foxo1 ( fig . 6b and 7a  c ) .
because jun nh2-terminal kinase ( jnk ) inhibition by sp600125 did not affect foxo1 accumulation induced by palmitate in either control ins-1e cells or pkcwt  ins-1e cells , it is suggested that foxo1 accumulation does not depend on jnk activity ( supplemental fig .
because phosphorylation of foxo1 at ser256 stimulates its nuclear extrusion and degradation , the amount of foxo1 protein and its phosphorylation were examined in control and transfected ins-1e cells . indeed , increased phosphorylation of foxo1 and significantly lower protein levels were detected in pkckn 
ins-1e cells , whereas reduced phosphorylation of foxo1 and increased foxo1 protein levels were found in pkcwt  ins-1e cells ( fig . 7d 
, these data suggest that overexpression of pkckn in -cells inhibits palmitate - mediated -cell dysfunction by protecting against mitochondrial dysfunction and apoptosis while counteracting nuclear accumulation of foxo1 .
palmitate - mediated nuclear accumulation of foxo1 was inhibited by overexpression of pkckn in insulin secreting cells .
a : islet cells from control and pkckn mice and b : control ins-1e cells and ins-1e cells overexpressing pkcwt or pkckn were cultured under control conditions or in the presence of 0.6 mmol / l palmitate for 1 day .
( a high - quality color digital representation of this figure is available in the online issue . )
overexpression of pkcwt and pkckn changes foxo1 cellular distribution , phosphorylation , and protein concentration in insulin - secreting cells .
ins-1e cells and ins-1e cells overexpressing pkcwt or pkckn were cultured under control conditions and in the presence of 0.6 mmol / l palmitate for 1 h and 1 day .
b and c : representative western blot for foxo1 detection in nuclear fractions with means  sem of n = 4 independent experiments .
d  f : representative western blot for foxo1 , p - ser256-foxo1 , and tubulin ( as loading control ) of cell homogenates with means  sem of n = 4 independent experiments of relative amounts of foxo1 and phosphorylation of foxo1 ( p - foxo1/foxo1 ) by setting the respective bands of control ins-1 cells to 100% .
* significant difference to ins-1e under control condition ;  significant difference to the respective condition of ins-1e ; # significant difference to pkckn at the same condition .
the observation that overexpression of pkckn in -cells protects against hfd - induced glucose intolerance strongly supports the idea that pkc plays a central role in lipotoxicity .
indeed , significantly lower blood glucose and increased plasma insulin concentrations during hfd were observed in pkckn - overexpressing mice , whereas animals fed standard chow displayed no significant difference .
our study , therefore , differs from observations in whole - body pkc knockout ( pkcko ) mice that displayed glucose intolerance ( 9 ) .
the directly opposed results may be explained first by the difference between whole - body knockout versus -cell  specific inhibition of pkc because , especially in brain tissue , large amounts of pkc are expressed . to exclude alterations in other tissues than  - cells , we therefore restricted expression of the transgene to insulin - secreting cells .
moreover , the most significant effect of pkckn overexpression was observed after high - fat feeding in our study , a condition that was not studied in pkcko mice .
the conclusion that pkckn protects against lipotoxicity in vivo was further substantiated by the fact that the percentage of islet cells that stained positive for cleaved caspase-3 after hfd was not increased and that palmitate - induced apoptotic cell death was inhibited in pkckn - expressing -cells .
because we were unable to detect proliferation , i.e. , brdu incorporation or ki67-positive cells in pancreatic slices of hfd - fed mice ( data not shown ) , it is anticipated that pkckn inhibits cell death in insulin - secreting cells ; this effect contributes to the increase in islet size and the increase in pancreatic insulin content .
this is in contrast to studies that demonstrated that pkc exerts stimulatory or inhibitory effects on cell proliferation .
these opposing effects of pkc may depend on the cell cycle status of the respective cell ( 15 ) . because -cell mass depends on proper insulin / igf-1 receptor
( 5 ) reported that pkc stimulation by pma inhibits protein kinase b ( pkb)/akt activation in insulin - secreting cells and described a reduced binding of insulin receptor substrate ( irs)-2 to p85 after pma stimulation of the cells .
this observation may be explained by putative pkc-mediated serine / threonine phosphorylations of irs-2 that inhibit irs-2 activation in parallel to known pkc phosphorylation sites in irs-1 ( 16 ) .
further experiments are needed to clarify whether pkc mediates changes of irs-2 activity via serine phosphorylation that reduce pkb / akt activation . in -cells ,
the mitochondrial potential plays a central role in normal glucose responsiveness of insulin secretion , whereas the collapse of mitochondrial potential is an essential step of the intrinsic pathways of apoptotic cell death ( 17,18 ) .
the protective effect of pkckn on mitochondrial potential strengthens the idea that pkc interferes with mitochondrial function . in line with this concept ,
salivary epithelial cells overexpressing pkckn were resistant to the loss of mitochondrial potential and apoptosis induced by etoposide , ultraviolet irradiation , bredfeldina , and paclitaxel ( 19 ) .
this complex transfers the endoplasmic reticulum ( er ) stress to mitochondria , which further leads to apoptotic cell death ( 20,21 ) .
although we can not exclude direct effects of pkc in mitochondria and in er , our data favor the idea that pkc leads to nuclear accumulation of foxo1 with palmitate treatment .
in fact , it has been previously demonstrated that ffa - induced er stress and apoptosis are inhibited in insulin - secreting cells expressing the dominant - negative form of foxo1 ( 14 ) .
activation of foxo1 leads to multiple cellular changes and plays a major role in stress resistance and survival of -cells ( 2227 ) .
in contrast , inhibition of foxo1 is necessary for adaptive -cell proliferation during insulin resistance ( 28,29 ) .
our study therefore provides strong evidence that ffas stimulate nuclear sequestration of foxo1 through the activation of pkc because the nuclear accumulation of foxo1 is inhibited in cells overexpressing pkckn .
moreover , phosphorylation of foxo1 at the pkb / akt phosphorylation site ser256 is reduced in cells overexpressing pkcwt but increased in cells expressing pkckn .
the signaling pathway through pkc that leads to reduced ser256 phosphorylation of foxo1 may involve activation of inhibitory pathways such as jnk and protein phosphatase 2a ( 30,31 ) .
however , jnk inhibition did not affect nuclear foxo1 accumulation induced by palmitate , suggesting a pkc-dependent but jnk - independent mechanism .
our study delivers in vivo evidence that activation of pkc by ffas mediates -cell failure .
because pkckn inhibited ffa - induced nuclear accumulation of foxo1 , mitochondrial dysfunction , and apoptosis , it is anticipated that the underlying mechanism involves pkc-dependent chronic activation of foxo1 .
in fact , a gene variation of foxo1 associates with impaired glucose tolerance and type 2 diabetes in humans ( 32 ) .
 objectivein vitro models suggest that free fatty acid  induced apoptotic -cell death is mediated through protein kinase c ( pkc). to examine the role of pkc signaling in vivo , transgenic mice overexpressing a kinase - negative pkc ( pkckn ) selectively in -cells were generated and analyzed for glucose homeostasis and -cell survival.research design and methodsmice were fed a standard or high - fat diet ( hfd ) . 
 blood glucose and insulin levels were determined after glucose loads . 
 islet size , cleaved caspase-3 , and pkc expression were estimated by immunohistochemistry . in isolated islet cells 
 apoptosis was assessed with tunel / to - pro3 dna staining and the mitochondrial potential by rhodamine-123 staining . 
 changes in phosphorylation and subcellular distribution of forkhead box class o1 ( foxo1 ) were analyzed by western blotting and immunohistochemistry.resultspkckn mice were protected from hfd - induced glucose intolerance . 
 this was accompanied by increased insulin levels in vivo , by an increased islet size , and by a reduced staining of -cells for cleaved caspase-3 compared with wild - type littermates . in accordance , long - term treatment with palmitate increased apoptotic cell death of isolated islet cells from wild - type but not from pkckn mice . 
 pkckn overexpression protected islet cells from palmitate - induced mitochondrial dysfunction and inhibited nuclear accumulation of foxo1 in mouse islet and ins-1e cells . 
 the inhibition of nuclear accumulation of foxo1 by pkckn was accompanied by an increased phosphorylation of foxo1 at ser256 and a significant reduction of foxo1 protein.conclusionsoverexpression of pkckn in -cells protects from hfd - induced -cell failure in vivo by a mechanism that involves inhibition of fatty acid  mediated apoptosis , inhibition of mitochondrial dysfunction , and inhibition of foxo1 activation . 
RESEARCH DESIGN AND METHODS
Generation of PKCKN transgenic mice and stably transfected INS-1E cells.
Glucose and insulin tolerance test and insulin secretion in vivo.
In vitro insulin secretion.
Apoptosis.
Mitochondrial potential.
Immunohistochemistry.
Western blotting.
Pancreatic insulin content.
Statistics.
RESULTS
Overexpression of PKCKN in mice protects against HFD-induced glucose intolerance.
PKCKN overexpression augments -cell mass and protects against apoptotic cell death induced by HFD and palmitate.
PKCKN overexpression interferes with FOXO1 regulation.
DISCUSSION
Supplementary Material