a total of 54 sprague - dawley rats ( wt ; n = 25 ) and rats expressing human iapp ( hip rats ; n = 29 ) were used . the generation of the human iapp transgenic rats has been described in detail previously ( 16 ) . rats were bred and housed individually throughout the study at the university of california , los angeles ( ucla ) animal housing facility and subjected to a standard 12-h light - dark cycle . the ucla institutional animal care and use committee approved all surgical and experimental procedures . at 2 months of age , wt and hip rats were randomly assigned to either an hfd ( 60% fat , 20% protein , and 20% carbohydrates ; research diets , new brunswick , nj ) or a control regular chow diet ( 10% fat , 20% protein , and 70% carbohydrates ; research diets ) and fed ad libitum for 10 weeks . after 10 weeks of either diet , treatment animals were anesthetized with isoflurane ( 2.5% ) by inhalation until effect , and indwelling catheters were then inserted into the right internal jugular vein and left carotid artery for subsequent in vivo metabolic studies as previously described ( 26 ) . all catheters were filled with 100 u / ml heparin / saline solution , exteriorized to the back of the neck , and encased in the infusion harness ( instech , plymouth meeting , pa ) . after surgery , all rats maintained preoperative body weight and had normal food intake and mean hematocrit ( > 40% ) . to assess glucose and arginine - stimulated insulin secretion , hip ( n = 10 ) , hip + hfd ( n = 9 ) , wt ( n = 6 ) , and wt + hfd ( n = 12 ) rats underwent a hyperglycemic clamp followed by an arginine bolus injection as previously described ( 18 ) . in brief , after a 30-min equilibration period , plasma samples were taken for measurements of fasting glucose , insulin , c - peptide , and free fatty acids . thereafter , animals received an intravenous glucose bolus ( 375 mg / kg ) followed by a variable 50% ( wt / vol ) glucose infusion to clamp arterial glucose at 250 mg / dl ( 070 min ) . at 60 min , rats received a bolus injection of l - arginine solution ( 1 mmol / kg ; sigma , st . arterial blood samples ( 50 l ) were taken at baseline ( 30 and 0 min ) , at 1 and 5 min , and every 15 min thereafter during the clamp for immediate determination of plasma glucose and subsequent analysis for insulin and c - peptide . to assess insulin sensitivity and glucose turnover , hip ( n = 7 ) , hip + hfd ( n = 7 ) , wt ( n = 6 ) , and wt + hfd ( n = 7 ) rats underwent a hyperinsulinemic - euglycemic clamp with concomitant infusion of [ 3-h]glucose to assess glucose turnover as previously described ( 18 ) . rats received primed ( 3 ci ) continuous ( 0.05 ci / min ) infusion of [ 3-h]glucose ( perkin elmer , boston , ma ) for a 90-min basal period increased to 0.2 ci / min for 120 min throughout the hyperinsulinemic - euglycemic clamp . plasma glucose was determined every 10 min , and additional blood samples ( 150 l ) for determination of tracer - specific activity at fasting were drawn from 40 to 0 min and during insulin infusion from 120 to 150 min . at the end of the hyperinsulinemic - euglycemic clamp , rats were euthanized and the pancreas was then rapidly removed and fixed in 4% paraformaldehyde overnight at 4c . paraffin - embedded pancreatic sections were stained first for hematoxylin and eosin , insulin ( guinea pig anti - insulin , 1:100 ; zymed , carlsbad , ca ) . the -cell mass was measured by quantifying the pancreatic cross - sectional area positive for insulin and multiplying this by the pancreatic weight . -cell size was determined as a mean distance between neighboring insulin - positive nuclei in 10 representative islets per animal . -cell nuclear diameter was determined as a mean ( of 180 measurements per nucleus ) nuclear diameter in insulin - positive nuclei in 10 representative islets per animal . sections were also costained by immunofluorescence for insulin ( guinea pig anti - insulin , 1:100 ; zymed ) and terminal deoxynucleotidyl transferase biotin - dutp nick - end labeling ( tunel method ; roche diagnostics , mannheim , germany ) for quantification of -cell apoptosis and insulin ( guinea pig anti - insulin , 1:100 ; zymed ) and ki-67 ki-67 , 1:50 ; dako , carpinteria , ca ) for determination of -cell replication . additional sections were costained by immunofluorescence for insulin ( guinea pig anti - insulin , 1:100 ; zymed ) and anti - c / ebp homologous protein ( chop ) ( rabbit ant - chop ; santa cruz biotechnology ) as previously described in detail ( 19,20 ) . all islets per pancreatic section ( 100 islets ) were examined in detail at 200 magnification ( 20 objective , 10 ocular ) for the total number of tunel , ki-67 , and chop - positive -cells . the frequency of tunel , ki-67 , and chop expression in each animal was presented as total number of stained -cells per total number of islets . fluorescent slides were analyzed and imaged using a leica dm600 microscope ( leica microsystems , wetzlar , germany ) , and images were acquired using openlab software ( improvision ) and analyzed using imagepro plus software . to obtain rat islets , hip ( n = 4 ) , hip + hfd ( n = 4 ) , wt ( n = 3 ) , and wt + hfd ( n = 3 ) rats were euthanized , and islets were isolated as previously described ( 19 ) . for western blotting experiments , islets were washed with ice - cold pbs and lysed in 2 laemmli sample buffer immediately following the isolation procedure . proteins ( 50 g / lane ) were separated on a 4% to 12% bis - tris nupage gel and transferred to polyvinylidine fluoride membranes ( bio - rad ) . membranes were blocked with 5% nonfat dry milk in tbs/0.1% tween-20 and incubated overnight at 4c with chop ( 1:500 ; santa cruz biotechnology ) , anti - p - perk ( 1:2,000 ; cell signaling technology , beverly , ma ) , anti - grp78 ( 1:10,000 ; stressgen , victoria , bc , canada ) , anti - hiapp ( 1:6,000 , 2537 aa ; peninsula laboratories , san carlos , ca ) , anti - insulin ( 1:100 ; zymed ) , and anti - gapdh ( 1:8,000 ; cell signaling technologies ) . membranes were washed with tbs/0.1% tween-20 and incubated with horseradish peroxidase  conjugated secondary antibodies ( 1:3,000 ; jackson laboratories , bar harbor , me ) for 1 h. after washes , proteins were visualized using enhanced chemiluminescence ( bio - rad ) and protein expression levels were quantified using un - scan - it software ( silk scientific , orem , ut ) . plasma glucose concentrations were measured by the glucose oxidase method ( beckman glucose analyzer 2 ; beckman , fullerton , ca ) . plasma insulin and c - peptide levels were measured using elisa assay ( alpco diagnostics , salem , nh ) . plasma free fatty acid levels were measured using in vitro standard enzymatic colorimetric methods ( wako chemicals , richmond , va ) . plasma glucose specific activity , hepatic glucose production , and glucose disposal were calculated as previously described in detail ( 18 ) . regression analysis was performed using statistica , version 6 ( statsoft , tulsa , ok ) . a total of 54 sprague - dawley rats ( wt ; n = 25 ) and rats expressing human iapp ( hip rats ; n = 29 ) were used . the generation of the human iapp transgenic rats has been described in detail previously ( 16 ) . rats were bred and housed individually throughout the study at the university of california , los angeles ( ucla ) animal housing facility and subjected to a standard 12-h light - dark cycle . the ucla institutional animal care and use committee approved all surgical and experimental procedures . at 2 months of age , wt and hip rats were randomly assigned to either an hfd ( 60% fat , 20% protein , and 20% carbohydrates ; research diets , new brunswick , nj ) or a control regular chow diet ( 10% fat , 20% protein , and 70% carbohydrates ; research diets ) and fed ad libitum for 10 weeks . after 10 weeks of either diet , treatment animals were anesthetized with isoflurane ( 2.5% ) by inhalation until effect , and indwelling catheters were then inserted into the right internal jugular vein and left carotid artery for subsequent in vivo metabolic studies as previously described ( 26 ) . all catheters were filled with 100 u / ml heparin / saline solution , exteriorized to the back of the neck , and encased in the infusion harness ( instech , plymouth meeting , pa ) . after surgery , all rats maintained preoperative body weight and had normal food intake and mean hematocrit ( > 40% ) . to assess glucose and arginine - stimulated insulin secretion , hip ( n = 10 ) , hip + hfd ( n = 9 ) , wt ( n = 6 ) , and wt + hfd ( n = 12 ) rats underwent a hyperglycemic clamp followed by an arginine bolus injection as previously described ( 18 ) . in brief , after a 30-min equilibration period , plasma samples were taken for measurements of fasting glucose , insulin , c - peptide , and free fatty acids . thereafter , animals received an intravenous glucose bolus ( 375 mg / kg ) followed by a variable 50% ( wt / vol ) glucose infusion to clamp arterial glucose at 250 mg / dl ( 070 min ) . at 60 min , rats received a bolus injection of l - arginine solution ( 1 mmol / kg ; sigma , st . arterial blood samples ( 50 l ) were taken at baseline ( 30 and 0 min ) , at 1 and 5 min , and every 15 min thereafter during the clamp for immediate determination of plasma glucose and subsequent analysis for insulin and c - peptide . to assess insulin sensitivity and glucose turnover , hip ( n = 7 ) , hip + hfd ( n = 7 ) , wt ( n = 6 ) , and wt + hfd ( n = 7 ) rats underwent a hyperinsulinemic - euglycemic clamp with concomitant infusion of [ 3-h]glucose to assess glucose turnover as previously described ( 18 ) . rats received primed ( 3 ci ) continuous ( 0.05 ci / min ) infusion of [ 3-h]glucose ( perkin elmer , boston , ma ) for a 90-min basal period increased to 0.2 ci / min for 120 min throughout the hyperinsulinemic - euglycemic clamp . plasma glucose was determined every 10 min , and additional blood samples ( 150 l ) for determination of tracer - specific activity at fasting were drawn from 40 to 0 min and during insulin infusion from 120 to 150 min . at the end of the hyperinsulinemic - euglycemic clamp , rats were euthanized and the pancreas was then rapidly removed and fixed in 4% paraformaldehyde overnight at 4c . paraffin - embedded pancreatic sections were stained first for hematoxylin and eosin , insulin ( guinea pig anti - insulin , 1:100 ; zymed , carlsbad , ca ) . the -cell mass was measured by quantifying the pancreatic cross - sectional area positive for insulin and multiplying this by the pancreatic weight . -cell size was determined as a mean distance between neighboring insulin - positive nuclei in 10 representative islets per animal . -cell nuclear diameter was determined as a mean ( of 180 measurements per nucleus ) nuclear diameter in insulin - positive nuclei in 10 representative islets per animal . sections were also costained by immunofluorescence for insulin ( guinea pig anti - insulin , 1:100 ; zymed ) and terminal deoxynucleotidyl transferase biotin - dutp nick - end labeling ( tunel method ; roche diagnostics , mannheim , germany ) for quantification of -cell apoptosis and insulin ( guinea pig anti - insulin , 1:100 ; zymed ) and ki-67 ( mouse anti ki-67 , 1:50 ; dako , carpinteria , ca ) for determination of -cell replication . additional sections were costained by immunofluorescence for insulin ( guinea pig anti - insulin , 1:100 ; zymed ) and anti - c / ebp homologous protein ( chop ) ( rabbit ant - chop ; santa cruz biotechnology ) as previously described in detail ( 19,20 ) . all islets per pancreatic section ( 100 islets ) were examined in detail at 200 magnification ( 20 objective , 10 ocular ) for the total number of tunel , ki-67 , and chop - positive -cells . the frequency of tunel , ki-67 , and chop expression in each animal was presented as total number of stained -cells per total number of islets . fluorescent slides were analyzed and imaged using a leica dm600 microscope ( leica microsystems , wetzlar , germany ) , and images were acquired using openlab software ( improvision ) and analyzed using imagepro plus software . to obtain rat islets , hip ( n = 4 ) , hip + hfd ( n = 4 ) , wt ( n = 3 ) , and wt + hfd ( n = 3 ) rats were euthanized , and islets were isolated as previously described ( 19 ) . for western blotting experiments , islets were washed with ice - cold pbs and lysed in 2 laemmli sample buffer immediately following the isolation procedure . proteins ( 50 g / lane ) were separated on a 4% to 12% bis - tris nupage gel and transferred to polyvinylidine fluoride membranes ( bio - rad ) . membranes were blocked with 5% nonfat dry milk in tbs/0.1% tween-20 and incubated overnight at 4c with chop ( 1:500 ; santa cruz biotechnology ) , anti - p - perk ( 1:2,000 ; cell signaling technology , beverly , ma ) , anti - grp78 ( 1:10,000 ; stressgen , victoria , bc , canada ) , anti - hiapp ( 1:6,000 , 2537 aa ; peninsula laboratories , san carlos , ca ) , anti - insulin ( 1:100 ; zymed ) , and anti - gapdh ( 1:8,000 ; cell signaling technologies ) . membranes were washed with tbs/0.1% tween-20 and incubated with horseradish peroxidase  conjugated secondary antibodies ( 1:3,000 ; jackson laboratories , bar harbor , me ) for 1 h. after washes , proteins were visualized using enhanced chemiluminescence ( bio - rad ) and protein expression levels were quantified using un - scan - it software ( silk scientific , orem , ut ) . plasma glucose concentrations were measured by the glucose oxidase method ( beckman glucose analyzer 2 ; beckman , fullerton , ca ) . plasma insulin and c - peptide levels plasma free fatty acid levels were measured using in vitro standard enzymatic colorimetric methods ( wako chemicals , richmond , va ) . plasma glucose specific activity , hepatic glucose production , and glucose disposal were calculated as previously described in detail ( 18 ) . regression analysis was performed using statistica , version 6 ( statsoft , tulsa , ok ) . food intake , body weight , and plasma free fatty acid levels increased comparably in wt and hip rats on an hfd ( fig . 1b and c ; the fasting plasma glucose concentrations in hip and wt rats were comparable at the beginning of the study ( p = 0.85 between groups ) . on a regular chow diet , fasting plasma glucose values remained unchanged in the wt rats but increased to the impaired fasting glucose concentration range in the hip rats , consistent with prior studies over the same age range ( 16,18 ) . on the hfd , there was a modest increase in blood glucose in wt rats ( 110  3 versus 98  2 mg / dl ; p = nonsignificant for wt + hfd versus wt ; fig . 1a ) , but in contrast , hip + hfd rats developed diabetes ( 178  20 mg / dl ; p < 0.0001 versus all other groups ; fig . although there was no relation between change in either body weight or food intake and the fasting blood glucose concentration in wt rats , both food intake ( r = 0.9 , p < 0.01 ) and increase in body weight ( r = 0.9 , p < 0.01 ) during the 10-week study predicted the subsequent fasting blood glucose concentrations in hip rats ( fig . fasting plasma glucose ( a ) , body weight ( b ) , and mean daily food intake ( c ) during 10-week treatment with 60% high fat ( hfd ) or regular chow diet in wt ( n = 19 ) and hip rats ( n = 21 ) . the relationship between the change in body weight ( d ) and daily caloric food intake ( e ) versus fasting glucose levels in hip rats ( solid line ) and wt rats ( broken line ) during 10-week treatment with 60% hfd or regular chow diet . f : the relation between the mean daily caloric food intake and change in body weight in individual wt rats ( broken line ) and hip rats ( solid line ) . * p < 0.001 for hip + hfd versus all groups ; p < 0.001 for regular chow versus hfd . , wt ;  , wt + hfd ;  , hip ;  , hip + hfd . metabolic characteristics of hip and wt rats after 10 weeks on an hfd or regular chow diet data are expressed as means  sem . * p < 0.05 for hfd versus regular chow . p < 0.05 for hip + hfd versus all other groups . p < 0.05 for wt + hfd versus all groups . the hfd provoked a 46% increase in -cell mass in wt rats ( 13  2 versus 19  2 mg , wt versus wt + hfd ; p < 0.05 ) , but no change of -cell mass in hip rats ( 12  1 versus 13  2 mg , hip versus hip+hfd ; p = nonsignificant ; fig . the increase in -cell mass in wt rats on an hfd was the result of -cell hyperplasia rather than hypertrophy because -cell size and nuclear diameter were unchanged ( fig . -cell apoptosis was increased almost 13-fold ( 0.04  0.006 versus 0.003  0.001 cells / islet ; fig . 3a and c , p < 0.001 ) in hip versus wt rats on a regular diet , but this was partially offset by a sixfold increase in -cell replication ( 0.018  0.003 versus 0.003  0.003 cells / islet ; fig . although there was no increase ( p = 0.22 ) in -cell apoptosis in wt rats on an hfd , -cell apoptosis increased still further in hip rats on the hfd ( fig . 3a and c , p < 0.0001 , 34-fold versus wt rats ) . in contrast , there was no further increase in -cell replication in hip rats on the hfd ( fig . compensatory increase in -cell mass in response to an hfd is absent in hip rats . a : typical islets from wild - type ( wt ) and hip rats stained for insulin and hematoxylin after 10 weeks of 60% high - fat ( hfd ) or regular chow diet . quantification of -cell mass ( b ) , mean islet number per section ( c ) , mean -cell size ( d ) , and -cell nuclear diameter ( e ) after 10 weeks of 60% hfd or regular chow diet in wt ( n = 19 ) and hip rats ( n = 21 ) . data are means  se . * p < 0.05 ; * * p < 0.01 . ( a high - quality digital representation of this figure is available in the online issue . ) hfd leads to increased -cell apoptosis in hip rats and increased -cell replication in wild - type rats . a and b : examples of islets stained for insulin ( green ) and apoptosis marker tunel ( red ) ( a ) and islets stained for insulin and replication marker ki-67 ( red ) and nuclear stain dapi ( blue ) ( b ) imaged at 200. c and d : quantified frequency of -cell apoptosis and replication after 10 weeks of 60% hfd or regular chow diet in wt ( n = 19 ) and hip rats ( n = 21 ) . data are means  se . * p < 0.05 ; * * p < 0.01 . note that the frequency of tunel- and ki67-positive cells in islets shown in a and b are higher than the mean to reveal the fidelity of the immunostaining rather than representative of the mean . ( a high - quality digital representation of this figure is available in the online issue . ) to further elucidate the underlying mechanism by which an hfd led to increased -cell apoptosis in the hip rats but not the wt rats , we next investigated the unfolded protein response ( p - perk and grp-78 ) and er stress by examining the protein expression and nuclear colocalization of the er stress marker chop . in wt rats , the hfd increased both perk phosphorylation and grp78 expression in relation to the primary er client protein insulin ( fig . were already increased on a regular chow diet in comparison to wt rats in relation to insulin expression . in hip rats , however , neither p - perk nor grp78 expression were further increased in response to the hfd ( fig . 4b and d ) despite the further increased expression of both insulin and iapp , so the protective unfolded protein response was not further increased in the hfd - fed rats despite this marked increased er load . nuclear chop expression was 13-fold increased in hip versus wt rats on a regular diet ( 0.049  0.011 versus 0.0037  0.002 -cells / islet , p < 0.01 ; fig . e ) and was increased further in hip , but not wt , rats with an hfd ( 0.049  0.011 versus 0.09  0.01 -cells / islet , p < 0.01 ; fig . there was a positive correlation between the frequency of -cell nuclear chop and -cell apoptosis ( r = 0.6 , p < 0.001 ; fig . western blot analysis of protein lysates from isolated islets confirmed the immunohistochemically detected increased chop protein expression in hip rats on a regular diet increased further on the hfd ( fig . induced apoptosis in the hip rat , but did not induce er stress in wt rats , implying that the propensity of human iapp to form toxic oligomers is an important link between an hfd and er stress  induced -cell apoptosis . . a : immunoblot analysis of p - perk , grp-78 , insulin , iapp , and chop in protein lysates from islets isolated after 10 weeks of a 60% hfd or regular chow diet in wt ( n = 6 ) and hip rats ( n = 8) . b and c : quantification of p - perk and grp-78 expression normalized to insulin expression . d : representative examples of islets stained by immunofluorescence for insulin ( green ) , er stress marker chop ( red ) , and nuclear stain dapi ( blue ) imaged at 200. e and f : quantified frequency of nuclear chop expression per islet and ( e ) a relation between the frequency of -cell chop versus tunel expression after 10 weeks of 60% hfd or regular chow diet ( f ) in wt ( n = 19 ) and hip rats ( n = 21 ) rats . note that the frequency of chop - positive cells in islets shown in d are higher than the mean to reveal the fidelity of the immunostaining rather than representative of the mean . ( a high - quality digital representation of this figure is available in the online issue . ) in the fasting state , insulin secretion was impaired in hip rats on an hfd , as illustrated by failure to increase c - peptide concentrations despite hyperglycemia ( fig . in contrast , c - peptide concentrations increased by 1.6-fold in wt rats on an hfd ( 1.9  0.2 versus 3.1  0.5 ng / ml , p < 0.05 ; fig . in contrast , although insulin secretion was impaired in hip rats in the fasting state ( evaluated by c - peptide concentrations ) , systemic insulin concentrations were comparable to those in wt rats ( table 1 ) , indicating that hepatic insulin clearance of endogenously secreted insulin was decreased in hip rats on a chow diet or hfd . to evaluate glucose - mediated insulin secretion under comparable conditions , we performed hyperglycemic clamp studies in which , by design , plasma glucose levels were maintained at 265  10 mg / dl ( supplemental fig . 1 , available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db08-1464 ) . in response to hfd , in wt rats , the first - phase insulin response to glucose was increased by 1.7-fold ( 966  90 versus 1,677  108 pmol / l , p < 0.01 , wt versus wt + hfd ; fig . 5b , e , and h ) and the second phase by twofold ( 705  33 versus 1,462  116 1 , available in the online appendix ) . in contrast , in hip rats , there was a markedly blunted first- and second - phase response to hyperglycemia and no increment with an hfd ( fig . likewise , although there was a 1.5-fold increase in arginine - stimulated insulin release in wt rats in response to an hfd , there was no increase in arginine - induced insulin secretion at the same glucose concentration in the hip rat on a regular diet or hfd ( fig . consistent with these findings , we observed a positive correlation between -cell mass and fasting c - peptide ( r = 0.5 , p < 0.01 ; fig . 5d and g ) , first - phase insulin response to glucose ( r = 0.4 , p < 0.01 ; fig . 5e and h ) , and first - phase insulin response to arginine ( r = 0.5 , p < 0.01 ; fig . hip rats fail to adaptively increase fasting , glucose - stimulated , and arginine - stimulated insulin secretion in response to an hfd . mean fasting c - peptide levels ( a ) , first - phase glucose ( b ) , and arginine - stimulated insulin secretion ( c ) . d  i : regression analysis of relations between changes in -cell mass and fasting , glucose - stimulated , and arginine - stimulated insulin secretion in wt ( n = 18 ) and hip rats ( n = 19 ) after 10-week treatment with 60% hfd or regular chow diet . * p < 0.05 ; * * p < 0.01 . to further elucidate the relation between hepatic and extrahepatic insulin sensitivity and -cell failure in hip rats exposed to an hfd , we undertook hyperinsulinemic - euglycemic clamps while measuring glucose turnover . by design , plasma glucose and insulin concentrations were comparable during the hyperinsulinemic - euglycemic clamps in hip and wt rats on a regular chow diet or hfd ( fig . whole - body insulin sensitivity , assessed by the mean glucose infusion rates during the hyperinsulinemic clamp , was comparably decreased by hfd in hip and wt rats ( fig . 6b ) . the isotope dilution technique revealed that fasting hyperglycemia in the hip + hfd group was attributed to a twofold increase in fasting hepatic glucose production ( fig . 6c , p < 0.05 versus all other groups ) . in hip rats , there was hepatic insulin resistance ( impaired suppression of hepatic glucose release during the clamp ) ( fig . isotopically measured insulin - stimulated glucose uptake was comparably decreased by hfd in both hip and wt rats ( fig . an hfd had its primary effect on insulin sensitivity by inducing extrahepatic insulin resistance , whereas the hip rat has a selective defect in hepatic insulin sensitivity independent of the hfd . a : fasting ( 30 and 0 min ) and hyperinsulinemic - euglycemic clamp glucose concentrations . c and d : mean rates of hepatic glucose production ( c ) and glucose disposal ( d ) at fasting and during the final 30 min of the hyperinsulinemic - euglycemic clamp in wt ( n = 13 ) and hip rats ( n = 14 ) after 10-week treatment with 60% hfd or regular chow . data are means  se . * p < 0.05 for hip versus wt ; * * p < 0.05 for hip + hfd versus all groups ; p < 0.05 for hfd versus chow . in summary , wt and hip rats provided either a regular chow diet or hfd for 10 weeks had an average daily food intake that varied from 100 to 200 kcal / day . weight gain and subsequent insulin resistance was proportionate to average food intake , which was comparable in wt and hip rats . in wt rats , the adaptive increase in -cell mass was accomplished by increased -cell replication in the absence of any increase in -cell apoptosis ( fig . in contrast , in hip rats , neither -cell function nor mass adaptively increased to the increased demand imposed by an hfd ( fig . 7e ) . in hip rats , increasing insulin resistance provoked increased -cell apoptosis ( fig . hfd - induced insulin resistance correlates with the increase in -cell apoptosis in hip rats and increase in -cell replication in wt rats . regression analysis of relations among the -cell apoptosis , -cell replication , and -cell mass versus hyperinsulinemic - euglycemic clamp determined insulin sensitivity in wt rats ( n = 13 ) ( b , d , and f ) and hip rats ( n = 14 ) ( a , c , and e ) after 10-week treatment with 60% hfd or regular chow . food intake , body weight , and plasma free fatty acid levels increased comparably in wt and hip rats on an hfd ( fig . 1b and c ; the fasting plasma glucose concentrations in hip and wt rats were comparable at the beginning of the study ( p = 0.85 between groups ) . on a regular chow diet , fasting plasma glucose values remained unchanged in the wt rats but increased to the impaired fasting glucose concentration range in the hip rats , consistent with prior studies over the same age range ( 16,18 ) . on the hfd , there was a modest increase in blood glucose in wt rats ( 110  3 versus 98  2 mg / dl ; p = nonsignificant for wt + hfd versus wt ; fig . 1a ) , but in contrast , hip + hfd rats developed diabetes ( 178  20 mg / dl ; p < 0.0001 versus all other groups ; fig . although there was no relation between change in either body weight or food intake and the fasting blood glucose concentration in wt rats , both food intake ( r = 0.9 , p < 0.01 ) and increase in body weight ( r = 0.9 , p < 0.01 ) during the 10-week study predicted the subsequent fasting blood glucose concentrations in hip rats ( fig . fasting plasma glucose ( a ) , body weight ( b ) , and mean daily food intake ( c ) during 10-week treatment with 60% high fat ( hfd ) or regular chow diet in wt ( n = 19 ) and hip rats ( n = 21 ) . the relationship between the change in body weight ( d ) and daily caloric food intake ( e ) versus fasting glucose levels in hip rats ( solid line ) and wt rats ( broken line ) during 10-week treatment with 60% hfd or regular chow diet . f : the relation between the mean daily caloric food intake and change in body weight in individual wt rats ( broken line ) and hip rats ( solid line ) . * p < 0.001 for hip + hfd versus all groups ; p < 0.001 for regular chow versus hfd . , wt ;  , wt + hfd ;  , hip ;  , hip + hfd . metabolic characteristics of hip and wt rats after 10 weeks on an hfd or regular chow diet data are expressed as means  sem . * p < 0.05 for hfd versus regular chow . p < 0.05 for hip + hfd versus all other groups . the hfd provoked a 46% increase in -cell mass in wt rats ( 13  2 versus 19  2 mg , wt versus wt + hfd ; p < 0.05 ) , but no change of -cell mass in hip rats ( 12  1 versus 13  2 mg , hip versus hip+hfd ; p = nonsignificant ; fig . the increase in -cell mass in wt rats on an hfd was the result of -cell hyperplasia rather than hypertrophy because -cell size and nuclear diameter were unchanged ( fig . 2d and e ) . -cell apoptosis was increased almost 13-fold ( 0.04  0.006 versus 0.003  0.001 cells / islet ; fig . 3a and c , p < 0.001 ) in hip versus wt rats on a regular diet , but this was partially offset by a sixfold increase in -cell replication ( 0.018  0.003 versus 0.003  0.003 cells / islet ; fig . although there was no increase ( p = 0.22 ) in -cell apoptosis in wt rats on an hfd , -cell apoptosis increased still further in hip rats on the hfd ( fig . in contrast , there was no further increase in -cell replication in hip rats on the hfd ( fig . compensatory increase in -cell mass in response to an hfd is absent in hip rats . a : typical islets from wild - type ( wt ) and hip rats stained for insulin and hematoxylin after 10 weeks of 60% high - fat ( hfd ) or regular chow diet . quantification of -cell mass ( b ) , mean islet number per section ( c ) , mean -cell size ( d ) , and -cell nuclear diameter ( e ) after 10 weeks of 60% hfd or regular chow diet in wt ( n = 19 ) and hip rats ( n = 21 ) . data are means  se . * p < 0.05 ; * * p < 0.01 . ( a high - quality digital representation of this figure is available in the online issue . ) hfd leads to increased -cell apoptosis in hip rats and increased -cell replication in wild - type rats . a and b : examples of islets stained for insulin ( green ) and apoptosis marker tunel ( red ) ( a ) and islets stained for insulin and replication marker ki-67 ( red ) and nuclear stain dapi ( blue ) ( b ) imaged at 200. c and d : quantified frequency of -cell apoptosis and replication after 10 weeks of 60% hfd or regular chow diet in wt ( n = 19 ) and hip rats ( n = 21 ) . note that the frequency of tunel- and ki67-positive cells in islets shown in a and b are higher than the mean to reveal the fidelity of the immunostaining rather than representative of the mean . ( a high - quality digital representation of this figure is available in the online issue . ) to further elucidate the underlying mechanism by which an hfd led to increased -cell apoptosis in the hip rats but not the wt rats , we next investigated the unfolded protein response ( p - perk and grp-78 ) and er stress by examining the protein expression and nuclear colocalization of the er stress marker chop . in wt rats , the hfd increased both perk phosphorylation and grp78 expression in relation to the primary er client protein insulin ( fig . were already increased on a regular chow diet in comparison to wt rats in relation to insulin expression . in hip rats , however , neither p - perk nor grp78 expression were further increased in response to the hfd ( fig . 4b and d ) despite the further increased expression of both insulin and iapp , so the protective unfolded protein response was not further increased in the hfd - fed rats despite this marked increased er load . nuclear chop expression was 13-fold increased in hip versus wt rats on a regular diet ( 0.049  0.011 versus 0.0037  0.002 -cells / islet , p < 0.01 ; fig . 4d e ) and was increased further in hip , but not wt , rats with an hfd ( 0.049  0.011 versus 0.09  0.01 -cells / islet , p < 0.01 ; fig . there was a positive correlation between the frequency of -cell nuclear chop and -cell apoptosis ( r = 0.6 , p < 0.001 ; fig . western blot analysis of protein lysates from isolated islets confirmed the immunohistochemically detected increased chop protein expression in hip rats on a regular diet increased further on the hfd ( fig . these data reveal that an hfd exacerbated er stress  induced apoptosis in the hip rat , but did not induce er stress in wt rats , implying that the propensity of human iapp to form toxic oligomers is an important link between an hfd and er stress  induced -cell apoptosis . hfd leads to increased er stress in hip rats . a : immunoblot analysis of p - perk , grp-78 , insulin , iapp , and chop in protein lysates from islets isolated after 10 weeks of a 60% hfd or regular chow diet in wt ( n = 6 ) and hip rats ( n = 8) . b and c : quantification of p - perk and grp-78 expression normalized to insulin expression . d : representative examples of islets stained by immunofluorescence for insulin ( green ) , er stress marker chop ( red ) , and nuclear stain dapi ( blue ) imaged at 200. e and f : quantified frequency of nuclear chop expression per islet and ( e ) a relation between the frequency of -cell chop versus tunel expression after 10 weeks of 60% hfd or regular chow diet ( f ) in wt ( n = 19 ) and hip rats ( n = 21 ) rats . data are means  se . * p < 0.05 ; * * p < 0.05 . note that the frequency of chop - positive cells in islets shown in d are higher than the mean to reveal the fidelity of the immunostaining rather than representative of the mean . ( a high - quality digital representation of this figure is available in the online issue . ) in the fasting state , insulin secretion was impaired in hip rats on an hfd , as illustrated by failure to increase c - peptide concentrations despite hyperglycemia ( fig . in contrast , c - peptide concentrations increased by 1.6-fold in wt rats on an hfd ( 1.9  0.2 versus 3.1  0.5 ng / ml , p < 0.05 ; fig . in contrast , although insulin secretion was impaired in hip rats in the fasting state ( evaluated by c - peptide concentrations ) , systemic insulin concentrations were comparable to those in wt rats ( table 1 ) , indicating that hepatic insulin clearance of endogenously secreted insulin was decreased in hip rats on a chow diet or hfd . to evaluate glucose - mediated insulin secretion under comparable conditions , we performed hyperglycemic clamp studies in which , by design , plasma glucose levels were maintained at 265  10 mg / dl ( supplemental fig . 1 , available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db08-1464 ) . in response to hfd , in wt rats , the first - phase insulin response to glucose was increased by 1.7-fold ( 966  90 versus 1,677  108 pmol / l , p < 0.01 , wt versus wt + hfd ; fig . 5b , e , and h ) and the second phase by twofold ( 705  33 versus 1,462  116 pmol / l , p < 0.01 , wt versus wt+hfd ; supplemental fig . 1 , available in the online appendix ) . in contrast , in hip rats , there was a markedly blunted first- and second - phase response to hyperglycemia and no increment with an hfd ( fig . likewise , although there was a 1.5-fold increase in arginine - stimulated insulin release in wt rats in response to an hfd , there was no increase in arginine - induced insulin secretion at the same glucose concentration in the hip rat on a regular diet or hfd ( fig . consistent with these findings , we observed a positive correlation between -cell mass and fasting c - peptide ( r = 0.5 , p < 0.01 ; fig . 5d and g ) , first - phase insulin response to glucose ( r = 0.4 , p < 0.01 ; fig . 5e and h ) , and first - phase insulin response to arginine ( r = 0.5 , p < 0.01 ; fig . hip rats fail to adaptively increase fasting , glucose - stimulated , and arginine - stimulated insulin secretion in response to an hfd . mean fasting c - peptide levels ( a ) , first - phase glucose ( b ) , and arginine - stimulated insulin secretion ( c ) . d  i : regression analysis of relations between changes in -cell mass and fasting , glucose - stimulated , and arginine - stimulated insulin secretion in wt ( n = 18 ) and hip rats ( n = 19 ) after 10-week treatment with 60% hfd or regular chow diet . to further elucidate the relation between hepatic and extrahepatic insulin sensitivity and -cell failure in hip rats exposed to an hfd , we undertook hyperinsulinemic - euglycemic clamps while measuring glucose turnover . by design , plasma glucose and insulin concentrations were comparable during the hyperinsulinemic - euglycemic clamps in hip and wt rats on a regular chow diet or hfd ( fig . whole - body insulin sensitivity , assessed by the mean glucose infusion rates during the hyperinsulinemic clamp , was comparably decreased by hfd in hip and wt rats ( fig . 6b ) . the isotope dilution technique revealed that fasting hyperglycemia in the hip + hfd group was attributed to a twofold increase in fasting hepatic glucose production ( fig . 6c , p < 0.05 versus all other groups ) . in hip rats , there was hepatic insulin resistance ( impaired suppression of hepatic glucose release during the clamp ) ( fig . isotopically measured insulin - stimulated glucose uptake was comparably decreased by hfd in both hip and wt rats ( fig . an hfd had its primary effect on insulin sensitivity by inducing extrahepatic insulin resistance , whereas the hip rat has a selective defect in hepatic insulin sensitivity independent of the hfd . hip rats on hfd exhibit increased fasting and insulin - stimulated hepatic glucose production . a : fasting ( 30 and 0 min ) and hyperinsulinemic - euglycemic clamp glucose concentrations . c and d : mean rates of hepatic glucose production ( c ) and glucose disposal ( d ) at fasting and during the final 30 min of the hyperinsulinemic - euglycemic clamp in wt ( n = 13 ) and hip rats ( n = 14 ) after 10-week treatment with 60% hfd or regular chow . p < 0.05 for hip versus wt ; * * p < 0.05 for hip + hfd versus all groups ; p < 0.05 for hfd versus chow . in summary , wt and hip rats provided either a regular chow diet or hfd for 10 weeks had an average daily food intake that varied from 100 to 200 kcal / day . weight gain and subsequent insulin resistance was proportionate to average food intake , which was comparable in wt and hip rats . in wt rats , the adaptive increase in -cell mass was accomplished by increased -cell replication in the absence of any increase in -cell apoptosis ( fig . in contrast , in hip rats , neither -cell function nor mass adaptively increased to the increased demand imposed by an hfd ( fig . 7e ) . in hip rats , increasing insulin resistance provoked increased -cell apoptosis ( fig . hfd - induced insulin resistance correlates with the increase in -cell apoptosis in hip rats and increase in -cell replication in wt rats . regression analysis of relations among the -cell apoptosis , -cell replication , and -cell mass versus hyperinsulinemic - euglycemic clamp determined insulin sensitivity in wt rats ( n = 13 ) ( b , d , and f ) and hip rats ( n = 14 ) ( a , c , and e ) after 10-week treatment with 60% hfd or regular chow . an hfd leads to obesity and insulin resistance ( 1 ) that in most individuals is compensated by increased -cell mass and insulin secretion ( 6,12 ) . in those vulnerable to type 2 diabetes , hyperglycemia occurs as a consequence of impaired insulin secretion in response to insulin resistance ( 27,28 ) with characteristic islet pathology including a deficit in -cell mass ( 12,13 ) , islet amyloid derived from iapp , and er stress  induced -cell apoptosis ( 20,29 ) . we sought to gain insights into the mechanisms underlying the appropriate adaptive versus maladaptive -cell response to hfd - induced insulin resistance . in humans , prospective measurements of the adaptive changes in -cell mass and corresponding changes in insulin secretion and insulin resistance in response to obesity are unavailable because longitudinal studies of pancreatic morphology can not be performed . cross - sectional studies of islet morphology in humans report a 50% increase in -cell mass in obese individuals compared with their lean counterparts ( 12 ) . similarly , cross - sectional studies that examined changes in insulin secretion and insulin sensitivity in obesity report an 1.5- to 3-fold increase in fasting and meal - stimulated insulin secretion ( 6,7,28 ) and an 50% decrease in insulin sensitivity ( 28,30 ) . we report that 10 weeks of high - fat feeding initiated at 2 months of age in the sprague - dawley rat induced a 46% increase in -cell mass and a 1.5- to 2-fold increase in fasting and glucose - stimulated insulin secretion in response to a 50% decline in whole body insulin sensitivity . the adaptive changes in the wt rat on an hfd therefore recapitulate those observed in nondiabetic obese humans . we report for the first time in the same animals that the adaptive changes in insulin secretion in wt + hfd animals is closely related to the adaptive increase in -cell mass . in contrast to wt rats , hip rats failed to adaptively increase -cell mass and insulin secretion in response to hfd - induced insulin resistance . -cell replication was increased in hip rats compared with wt rats at baseline , but this increased -cell replication was offset by increased -cell apoptosis so that -cell mass was comparable in wt and hip rats . -cell replication failed to increase further in hip rats on an hfd despite a marked further increase in -cell apoptosis , consistent with prior studies showing that newly forming -cells have increased vulnerability to iapp - induced apoptosis ( 31 ) . a primary goal of the present studies was to gain insights into the successful versus failed adaptation to high fat having noted that failure to expand -cell mass in hip rats fed an hfd was the result of er stress  induced -cell apoptosis , we turned our attention to the role of the er in the successful versus failed adaptation to an hfd . the er in the -cell is well developed to permit synthesis , folding , processing , and export of the primary client secretory proteins , insulin , and iapp . the unfolded protein response ( upr ) is an important adaptive regulatory mechanism that seeks to balance the rate of delivery of nascent proteins into the er with the capacity of the er to fold and export these proteins ( 32 ) . the upr accomplishes this both by constraining the rate of protein synthesis as well as enhancing the capacity of this system , for example , by increasing the er chaperone protein bip that binds to all nascent er proteins as they are inserted into the er ( 33 ) . evidence that the upr was intact in wt rats is provided by increased perk phosphorylation and grp78 expression in relation to the primary er client proteins insulin and iapp . in contrast , although perk phosphorylation and grp78 expression were increased in hip rats on a regular chow diet in comparison to wt rats , in relation to insulin expression , the upr was not further increased in response to the hfd . it is therefore plausible that high expression rates of oligomeric proteins ( such as human iapp ) may lead to er stress , in part attributable to the failure of an adequate unfolded protein response . induced apoptosis , we observed a similar twofold increase in -cell apoptosis in response to an hfd in hip , but not in wt , rats . there was a positive correlation between nuclear chop and -cell apoptosis ( r = 0.6 , p = 0.0001 ; fig . induced apoptosis is related to the extent of insulin resistance in those genetically vulnerable , the frequency of -cell apoptosis was positively related to the degree of insulin resistance in hip rats ( p < 0.05 ) , but not wt rats ( p = 0.6 ) . the close parallels in the islet pathology between the hfd - fed hip rat and obese humans with type 2 diabetes ( -cell deficit resulting from er stress  induced -cell apoptosis ) are consistent with the postulate that humans genetically at risk of type 2 diabetes , like the hip rat , have a marginal capacity for er to traffic iapp that is readily overcome by induction of insulin resistance . in contrast , humans who are not genetically vulnerable to type 2 diabetes are capable of adapting to insulin resistance and so presumably have a much higher threshold for expression and trafficking of iapp . we note that both wt and hip rats developed comparable insulin resistance in extrahepatic tissues on an hfd . hepatic insulin resistance in response to high - fat feeding was more prominent in the hip rats . we previously reported that the loss of -cell mass in the hip rat leads to hepatic insulin resistance ( 18 ) . one possible explanation for this finding is that the impaired pulsatile delivery of insulin to the liver leads to hepatic insulin resistance . in health , insulin is secreted in discrete insulin secretory bursts that present the liver with an insulin concentration profile that oscillates with an amplitude of 1,000 pmol / l in the fasting state and up to 5,000 pmol / l after meal ingestion ( 34,35 ) , an observation recently confirmed in vivo in rats ( 26 ) . the magnitude of these bursts is substantially decreased in patients with type 2 diabetes ( 36 ) and in animal models of reduced -cell mass ( 37,38 ) , including the hip rat ( a.v.p . and p.c.b . , unpublished observations ) . it has been proposed that pulsatile mode of insulin delivery to liver increases hepatic insulin sensitivity ( 39,40 ) , possibly by avoiding insulin receptor downregulation and/or sustained activation of akt with attendant feedback inhibition of irs-2 ( 41 ) . we postulate that the more prominent hepatic insulin resistance in the hip rat on an hfd ( and in obese patients with type 2 diabetes ) may relate at least in part to the decline in -cell mass leading to an abnormal pattern of insulin delivery to the liver . we also document a decrease in hepatic insulin clearance in hip rats fed an hfd . although fasting insulin levels in hip rats increased almost threefold in response to an hfd , c - peptide levels remained relatively unchanged ( table 1 ) . thus , in the hip rat , the c - peptide  to  insulin molar ratio , a measure of insulin clearance , was reduced by 40% in high fat  fed hip rats . we have previously reported that the amplitude of insulin pulses directed to the liver is directly related to hepatic insulin clearance in humans ( 42 ) . consistent with this hypothesis , hepatic insulin clearance is decreased in animal models of reduced -cell mass / insulin pulse mass as well as in humans with diabetes ( 37,38,43,44 ) . because hepatic insulin extraction depends on the amplitude of insulin pulses presented to the liver ( 42 ) , deficits in insulin secretion , at least in the fasting state , would be largely offset in the systemic circulation by decreased hepatic insulin clearance . in summary , an hfd induced a comparable increase in expression of iapp and induction of upr in -cells irrespective of amyloidogenic potential of expressed iapp . however , despite the induction of the upr , an hfd still led to -cell er stress  mediated apoptosis in the hip but not wt rats with islet pathology that recapitulates that in humans with type 2 diabetes . specifically , the -cell mass was deficient in the high fat  fed hip rats , because the increased -cell apoptosis prevented an appropriate adaptive increase in -cell mass . the consequence of -cell er stress and a deficit in -cell mass in high fat  fed hip rats were impaired insulin secretion and hyperglycemia . this -cell failure led to a further exacerbation of the hfd - induced insulin resistance in the hip rat .
objectiveobesity is a known risk factor for type 2 diabetes . however , most obese individuals do not develop diabetes because they adapt to insulin resistance by increasing -cell mass and insulin secretion . islet pathology in type 2 diabetes 
 is characterized by -cell loss , islet amyloid derived from islet amyloid polypeptide ( iapp ) , and increased -cell apoptosis characterized by endoplasmic reticulum ( er ) stress . 
 we hypothesized that iapp - induced er stress distinguishes successful versus unsuccessful islet adaptation to insulin resistance.research design and methodsto address this , we fed wild - type ( wt ) and human iapp transgenic ( hip ) rats either 10 weeks of regular chow or a high - fat diet and prospectively examined the relations among -cell mass and turnover , -cell er stress , insulin secretion , and insulin sensitivity.resultsa high - fat diet led to comparable insulin resistance in wt and hip rats . 
 wt rats compensated with increased insulin secretion and -cell mass . in hip rats , in contrast , neither -cell function nor mass compensated for the increased insulin demand , leading to diabetes . 
 the failure to increase -cell mass in hip rats was the result of er stress  induced -cell apoptosis that increased in proportion to diet - induced insulin resistance.conclusionsiapp-induced er stress distinguishes the successful versus unsuccessful islet adaptation to a high - fat diet in rats 
 . these studies are consistent with the hypothesis that iapp oligomers contribute to increased -cell apoptosis and -cell failure in humans with type 2 diabetes .