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 ) .