we studied 16 probands of french families with clinically defined mody based on two criteria : diabetes diagnosed before age 25 years ( range of age at diagnosis : 1523 ) without requirement of exogenous insulin in the first 2 years , and an autosomal dominant inheritance of type 2 diabetes ( 16 ) .
/ mody-3 mutation , and 8 were negative for mutations in hnf4a / mody-1 , pdx1/mody-4 , and neurod1/mody-6 , which were very rarely found in french mody patients ( 16 ) .
all patients with unexplained mody were also negative for serologic markers of type 1 diabetes .
ninety - five patients diagnosed with nonautoimmune diabetes before age 35 years , and presenting with at least one affected first - degree relative ( all are caucasian and of french origin ) , were included in the study for mutation identification .
in addition , we report functional studies on a r6h mutation that was identified among 48 danish patients with early - onset diabetes and known vertical transmission of the disease ( all tested negative for a hnf4a / mody-1 , gck / mody-2 , or hnf1a / mody-3 mutation ) .
the three exons of the ins gene were screened for mutations from genomic dna of the patients by direct sequencing , as previously described ( 10 ) .
additional members in three families ( two french and one danish ) , for which an ins mutation was identified in the proband , were also screened for the mutation ( see results ) .
normoglycemic control subjects of french origin ( n = 292 ) were also sequenced and all were found negative for the identified mutations .
the clinical features of the patients carrying an ins mutation , including age at onset and presentation of diabetes , and information on past and current treatments for diabetes , have been reviewed . in the family fr
- am , two subjects ( the diabetic proband and his unaffected mother ) have undergone a measurement of body composition by dual energy x - ray absorptiometry , and on a separate occasion , a euglycemic hyperinsulinemic clamp for the measurement of glucose uptake and a graded glucose infusion for measurement of insulin secretory response .
the proband 's mother , who never developed diabetes and was normoglycemic at last examination ( after an oral glucose tolerance test [ ogtt ] at age 68 years ) , also underwent an intravenous bolus of arginine at the end of the graded glucose infusion to estimate her maximal insulin secretory capacity .
details of metabolic studies are given in supplementary data , available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-1091/dc1 .
human preproinsulin cdna cloned in ptarget vector containing enhanced green fluorescent protein inserted in the c - peptide was kindly provided by dr .
the single mutations ( r6h , r6c , l30 m , l30p , and r55c ) were inserted using a quikchange ii xl site - directed mutagenesis kit ( stratagene , agilent technologies , richardson , tx ) .
hek293 cells ( 5  10/well ) seeded on six - well plates were transfected in triplicate with wild - type or mutant ins constructs ( 4 g / well ) using lipofectamine2000 ( invitrogen , carlsbad , ca ) . after 48 h ,
normal growth media were replaced by 1 ml / well of reduced serum media ( dulbecco 's modified eagle 's medium , 2% fbs ) . after 20 h , human proinsulin was measured in the supernatant by radioimmunoassay ( human proinsulin ria ; millipore hpi-15k ) .
samples for mrna or protein expression were collected after 48 h. total rna was extracted using trizol reagent ( invitrogen ) and treated with dna - free reagent ( abi biosystems , foster city , ca ) to remove any dna contamination .
rna ( 2 g ) was reverse - transcribed using high - capacity cdna reverse transcription kit ( abi biosystems ) .
real - time pcr was carried out with cdna ( equivalent of 20 ng input rna ) using power sybr green master mix ( abi biosystems ) in a 7500 fast real - time pcr system ( abi biosystems ) .
all real - time primers ( human cyclophilin , insulin , and c / ebp homologous protein [ chop]/gadd153 ) were generated using abi primer express 3.0 .
pcr amplification of human xbp1 ( x - box binding protein 1 ) was carried out with the cdna equivalent of 100 ng input rna ( see supplementary table 1 for primer sequences ) .
for protein extraction , cells were washed twice in ice - cold pbs and lysed in 1% triton x-100 in pbs containing complete protease inhibitor cocktail ( roche diagnostics ) .
blots were blocked with 5% nonfat milk in tris - buffered saline for 1 h and , subsequently , incubated 1 h for each , first with primary antibody ( monoclonal anti - gfp , 1:1,000 [ sigma clone gsn24 ] , monoclonal anti--tubulin , 1:10,000 [ sigma clone b-512 ] ) followed by horseradish peroxidase  linked secondary antibody ( 1:10,000 ; ge healthcare ) , with 3 10-min washes with tris - buffered saline0.1% tween 20 between incubations .
blots were developed using ecl western blot detection reagent ( ge healthcare life sciences ) and exposing to hyperfilm ecl ( ge healthcare life sciences ) .
min6 mouse pancreatic -cells ( 17 ) seeded on poly - l - lysine  coated coverslips were transfected with wild - type or mutant insulin constructs ( 4 g / well ) with / without plasmid encoding dsred - er ( 2 g / well ; clontech ) using lipofectamine2000 and were allowed to overexpress for 48 h. cells coexpressing insulin granule marker were infected with neuropeptide y ( npy)-venus virus ( 100 multiplicity of infection ) ( 18 ) 24 h after transfection with plasmids .
coverslips were washed twice with pbs , fixed in 4% paraformaldehyde ( 5 min ) , washed again , and mounted on slides with prolong gold ( invitrogen ) .
( carl zeiss , jena , germany ) fitted with a planapo 63 oil - immersion objective and a 1.5 optivar attached to a nokigawa spinning disc confocal head .
samples were illuminated using steady - state 488-and 560-nm laser lines and emission was collected through et535/30 and et620/60 emission filters ( chroma ) .
images were captured using a hamamatsu em ccd digital camera , model c910013 , controlled by an improvision / nokigawa spinning disc system running volocity software .
statistical significance was estimated using anova with bonferroni multiple comparison test or paired t tests .
we studied 16 probands of french families with clinically defined mody based on two criteria : diabetes diagnosed before age 25 years ( range of age at diagnosis : 1523 ) without requirement of exogenous insulin in the first 2 years , and an autosomal dominant inheritance of type 2 diabetes ( 16 ) .
/ mody-3 mutation , and 8 were negative for mutations in hnf4a / mody-1 , pdx1/mody-4 , and neurod1/mody-6 , which were very rarely found in french mody patients ( 16 ) .
all patients with unexplained mody were also negative for serologic markers of type 1 diabetes .
ninety - five patients diagnosed with nonautoimmune diabetes before age 35 years , and presenting with at least one affected first - degree relative ( all are caucasian and of french origin ) , were included in the study for mutation identification .
in addition , we report functional studies on a r6h mutation that was identified among 48 danish patients with early - onset diabetes and known vertical transmission of the disease ( all tested negative for a hnf4a / mody-1 , gck / mody-2 , or hnf1a / mody-3 mutation ) .
the three exons of the ins gene were screened for mutations from genomic dna of the patients by direct sequencing , as previously described ( 10 ) .
additional members in three families ( two french and one danish ) , for which an ins mutation was identified in the proband , were also screened for the mutation ( see results ) .
normoglycemic control subjects of french origin ( n = 292 ) were also sequenced and all were found negative for the identified mutations .
the clinical features of the patients carrying an ins mutation , including age at onset and presentation of diabetes , and information on past and current treatments for diabetes , have been reviewed . in the family fr
- am , two subjects ( the diabetic proband and his unaffected mother ) have undergone a measurement of body composition by dual energy x - ray absorptiometry , and on a separate occasion , a euglycemic hyperinsulinemic clamp for the measurement of glucose uptake and a graded glucose infusion for measurement of insulin secretory response . the proband 's mother , who never developed diabetes and was normoglycemic at last examination ( after an oral glucose tolerance test [ ogtt ] at age 68 years ) , also underwent an intravenous bolus of arginine at the end of the graded glucose infusion to estimate her maximal insulin secretory capacity .
details of metabolic studies are given in supplementary data , available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-1091/dc1 .
human preproinsulin cdna cloned in ptarget vector containing enhanced green fluorescent protein inserted in the c - peptide was kindly provided by dr .
the single mutations ( r6h , r6c , l30 m , l30p , and r55c ) were inserted using a quikchange ii xl site - directed mutagenesis kit ( stratagene , agilent technologies , richardson , tx ) .
hek293 cells ( 5  10/well ) seeded on six - well plates were transfected in triplicate with wild - type or mutant ins constructs ( 4 g / well ) using lipofectamine2000 ( invitrogen , carlsbad , ca ) . after 48 h , normal growth media were replaced by 1 ml / well of reduced serum media ( dulbecco 's modified eagle 's medium , 2% fbs ) . after 20 h , human proinsulin was measured in the supernatant by radioimmunoassay ( human proinsulin ria ; millipore hpi-15k ) .
samples for mrna or protein expression were collected after 48 h. total rna was extracted using trizol reagent ( invitrogen ) and treated with dna - free reagent ( abi biosystems , foster city , ca ) to remove any dna contamination .
rna ( 2 g ) was reverse - transcribed using high - capacity cdna reverse transcription kit ( abi biosystems ) .
real - time pcr was carried out with cdna ( equivalent of 20 ng input rna ) using power sybr green master mix ( abi biosystems ) in a 7500 fast real - time pcr system ( abi biosystems ) .
all real - time primers ( human cyclophilin , insulin , and c / ebp homologous protein [ chop]/gadd153 ) were generated using abi primer express 3.0 .
pcr amplification of human xbp1 ( x - box binding protein 1 ) was carried out with the cdna equivalent of 100 ng input rna ( see supplementary table 1 for primer sequences ) . for protein extraction
, cells were washed twice in ice - cold pbs and lysed in 1% triton x-100 in pbs containing complete protease inhibitor cocktail ( roche diagnostics ) . total protein
blots were blocked with 5% nonfat milk in tris - buffered saline for 1 h and , subsequently , incubated 1 h for each , first with primary antibody ( monoclonal anti - gfp , 1:1,000 [ sigma clone gsn24 ] , monoclonal anti--tubulin , 1:10,000 [ sigma clone b-512 ] ) followed by horseradish peroxidase  linked secondary antibody ( 1:10,000 ; ge healthcare ) , with 3 10-min washes with tris - buffered saline0.1% tween 20 between incubations .
blots were developed using ecl western blot detection reagent ( ge healthcare life sciences ) and exposing to hyperfilm ecl ( ge healthcare life sciences ) .
min6 mouse pancreatic -cells ( 17 ) seeded on poly - l - lysine  coated coverslips were transfected with wild - type or mutant insulin constructs ( 4 g / well ) with / without plasmid encoding dsred - er ( 2 g / well ; clontech ) using lipofectamine2000 and were allowed to overexpress for 48 h. cells coexpressing insulin granule marker were infected with neuropeptide y ( npy)-venus virus ( 100 multiplicity of infection ) ( 18 ) 24 h after transfection with plasmids .
coverslips were washed twice with pbs , fixed in 4% paraformaldehyde ( 5 min ) , washed again , and mounted on slides with prolong gold ( invitrogen ) .
cells were imaged using a zeiss axiovert 200 m microscope ( carl zeiss , jena , germany ) fitted with a planapo 63 oil - immersion objective and a 1.5 optivar attached to a nokigawa spinning disc confocal head .
samples were illuminated using steady - state 488-and 560-nm laser lines and emission was collected through et535/30 and et620/60 emission filters ( chroma ) .
images were captured using a hamamatsu em ccd digital camera , model c910013 , controlled by an improvision / nokigawa spinning disc system running volocity software .
statistical significance was estimated using anova with bonferroni multiple comparison test or paired t tests .
three heterozygous missense ins mutations were identified in two french and one danish patients diagnosed with nonautoimmune diabetes before age 25 years .
no further mutations were found by screening additional french probands with later onset familial diabetes .
one novel mutation , a c.88c > g - p.l30 m change , was found in a patient with diabetes diagnosed at 17 years of age of normal body weight .
1a ) revealed that this replacement in the hydrophobic core of the insulin b chain is more conservative than a previously described mutation at the same site ( l30p ) ( 12 ) but , being located at the dimer interface , may affect insulin multimerization ( fig .
impact of l30 m and l30p mutations on insulin folding ( a ) and hexamerization ( b and c ) .
( left ) wild type , green ; l30 m , red ; ( center ) wild type , green , l30p , blue ; ( right ) l30 m , red , l30p , blue .
homology models were built using modeler9v4 software ( http://www.salilab.org ) using 1mso human insulin structure as a template ( 30 ) .
b : insulin hexamer showing l30 ( l ) and h34 ( h ) residues and zn ( 30 ) .
c and d : close - up showing the impact of l30 m ( lm , center ) and l30p ( lp , right ) mutations in insulin b - chain versus wild type ( left ) showing the increased cys - cys distance , disfavoring disulphide bond formation ( c ) , and changes to the structure after disulphide bond formation ( d ) .
leucine at position 6 of the b - chain ( l30 ) interacts with cysteine at position 6 of the a chain and leucine 17 and tryptophan 16 of the neighboring b - chain ( b ) .
l30 m and l30p mutations would therefore weaken or eliminate the interactions within the monomer and may also affect hexamer formation . at diagnosis , the revealing symptom in the patient was weight loss and the fasting glycemia was 16.5
no autoimmunity against pancreas was detected , and the pancreatic morphology ( assessed by abdominal tomodensitometry ) was normal .
initially , glycemic control was satisfactory with oral hypoglycemic agents ( ohas ; glibenclamide associated to metformin ) .
subsequently , the association of various oral drugs proved insufficient , and insulin requirement occurred 9 years after diagnosis .
a progressive increase in insulin needs was observed during the past 14 years ( from 0.45 to 0.83 ui  kg  day ) , and a1cranged from 5.0 to 9.9% .
no microangiopathy or macroangiopathy was detected in this patient after 24 years of diabetes evolution .
the l30 m mutation cosegregated in five individuals from the proband 's family with early - onset diabetes ( range of age at diagnosis : 1738 years ; range of bmi at last examination : 1925.5 kg / m ; fig .
however , the proband 's mother was found to carry the mutation but was not known to be diabetic , and had a normal ogtt at her last examination at age 68 years ( fasting and 2-h post  glucose load glycemia of 5.1 and 7.6 mmol / l , respectively ) .
the symbols denote the following : solid symbols , diabetes status ; striated symbol , impaired glucose tolerance status ; empty symbols , normoglycemic subjects ; grid symbol , one normal glucose tolerant subject who is carrier of the l30 m mutation ; and gray symbols , subjects who were not available for genetic testing .
the ins mutation status is shown under each symbol : nm as heterozygote , nn as wild type , and nt as not tested .
the text below indicates the following : age at diagnosis of diabetes or age at examination in normoglycemic subjects ( in years ) , bmi ( kg / m ) , and treatment in the diabetic subjects .
we also identified another mody proband bearing a c.163c > t - p.r55c mutation at the b / c junction ( previously reported in a norwegian case subject with apparent type 1 diabetes , but negative for autoantibodies ) .
the patient was diagnosed with diabetes at age 9 years by signs of polyuria and polydipsia , which required exogenous insulin therapy at the time of diagnosis . after a follow - up of > 50 years ,
clinical records indicated no signs of late - diabetic complications ( absence of retinopathy , nephropathy , or cardiovascular disease ) and a stable , well controlled glycemic profile ( a1c ranges < 6% ) .
the mutation was found to cosegregate with diabetes in two other diabetic relatives , who were diagnosed at ages 37 ( the mother ) and 33 ( the sister ) years ( fig .
they were initially treated with oha , then with insulin therapy ( 12 and 5 years , respectively , after their initial treatment ) .
the mother of the proband died at age 87 years ( the cause of death was not directly related to diabetes ) .
a c.17g > a substitution , leading to a r6h change in the signal peptide , was identified in a danish proband ( fig .
he was treated with diet and oha since diagnosis . at age 50 years , he was treated with metformin ( 1 g  2 ) , a1c was 6.4% , fasting serum c - peptide was 419 pmol / l , and fasting plasma glucose was 6.4
2 ) , who also carries the mutation , was diagnosed with diabetes at age 51 years . at age 53 years , he was treated with diet alone and had no signs of late - diabetic complications .
2 ) , carrying the mutation , was diagnosed with impaired glucose tolerance at age 26 years and with gestational diabetes at age 27 .
as shown in table 1 , the diabetic proband ( family fr - am ) displayed a dramatic decrease in insulin secretion during graded glucose infusion , whereas insulin sensitivity measured during the euglycemic hyperinsulinemic clamp was normal ; this is consistent with -cell failure as the cause of diabetes .
his nondiabetic mother was also found to have a clear defect in insulin secretion , which was apparent in response to both oral and intravenous glucose load and also to the intravenous arginine test during hyperglycemia , raising the question as to why she did not develop diabetes .
mmol / l ) was close to the glucose intolerance threshold ( 7.8 mmol / l ) .
however , insulin sensitivity in this person must be considered high , taking into account her age and her reported high level of physical activity .
anthropometric and metabolic characteristics of two subjects ( diabetic proband and nondiabetic mother from family fr - am ) carrying the l30m - ins mutation , compared with a group of control subjects data are means  sd .
we next examined the ability of the identified mutations to affect insulin exit from the er , initially using hek293 cells ( 12,15 ) .
these cells lack a well - defined regulated secretory pathway such that normal er exit of a proinsulin - gfp chimera ( 15 ) leads to the constitutive release into the medium .
along with the three identified mutations ( r6h , l30 m , r55c ) , two other previously reported mutations , r6c ( 9 ) and l30p ( 12 ) , were introduced in the preproinsulin - gfp chimera ( denoted henceforth as ppi ) for comparative molecular studies .
although all the mutants expressed at similar levels to the wild - type at the mrna level ( fig .
3a ) , expressed protein levels in total cell extract were lower in wild - type and r6h cells , most likely reflecting the more efficient constitutive secretion of these molecules than the other mutant proteins ( fig .
all mutant proinsulin proteins migrated to same extent as wild - type proinsulin on reducing sds - page , indicating efficient cleavage of preproinsulin signal peptide .
when secreted proinsulin was measured in the culture media , the l30 m mutant displayed a decrease in proinsulin release rate , albeit less marked than that for the l30p mutant ( fig .
in contrast , secretion of r6h was not significantly different from wild type , although mutation to cysteine at the same location ( r6c ) in the signal peptide led to substantially compromised proinsulin release ( fig .
the release of the b / c junction mutant proinsulin , r55c , was also considerably reduced ( fig .
3c ) , indicative of er retention . impact of insulin mutations on er release and secretion in hek293 cells .
a : real - time pcr measurements of recombinant human ins mrna expression in transfected hek293 cells normalized to endogenous cyclophilin ( means  sem ; n  5 ) .
b : typical western blot ( n  5 ) of total protein ( 30 g / lane ) from hek293 cells transfected or not with ins constructs separated on 12% reducing sds - page and probed first with anti - gfp antibody , followed by -tubulin as loading control .
c : secreted proinsulin content in 1 ml media of transfected hek293 cells collected for a period of 20 h ( means  sem ; n  5 ) .
data were analyzed by anova with bonferroni multiple comparison test . * * * p < 0.001 .
a large percentage of l30 m ( 69  8% ) and r55c ( 42  3% ) mutant chimera - expressing hek293 cells displayed a marked difference in the distribution of fluorescence compared with wild - type insulin gfp - expressing cells : whereas fine tubular / reticular fluorescence , reminiscent of a healthy er ( 19 ) , was evident in wild - type chimera - expressing cells , a globular , perinuclear staining was usually apparent in cells expressing the l30 m or r55c mutant constructs ( supplementary figure 1 ) .
this observation is consistent with retention of the mutant chimera in an inflated and engorged er . to extend these findings to insulin - secreting cells , we next expressed wild - type or mutant chimeras in clonal min6 -cells ( 17 ) .
the fluorescence of wild - type hprocpepgfp was closely colocalized with the co - overexpressed secretory granule marker , neuropeptide y - venus ( fig .
4 ) ( 18 ) , with little or no colocalization with the overexpressed er marker , dsred - er ( fig .
the insulin - gfp fluorescence distribution profile along a straight line drawn across one of the middle sections of individual cells in fig .
5b ) , implying compacted storage of protein in dense core vesicles and a lack of significant retention in the er . by contrast , l30 m chimera fluorescence was barely detected in the npy - venus  positive compartment ( fig .
4 ) but was strongly colocalized with dsred - er , confirming large - scale retention in the latter organelle ( fig .
the distribution of l30 m was very close to the structurally most perturbing mutation , l30p ( fig .
1 ) . on the other hand , r6h or r6c mutations in the preproinsulin signal peptide appeared to exert no untoward effects on proper folding within the er ( fig .
4 ) and as confirmed by a fluorescence profile similar to the wild - type chimera ( fig .
however , the r55c mutation displayed an altered distribution pattern of insulin chimera with both dense core vesicular targeting ( fig .
the fluorescence distribution profile of l30 m and r55c indicated absence of peaks above the cutoff and a diffuse presence throughout the er ( fig .
, the total number of cells in a population of min6 -cells that produced mutant insulin - containing granules significantly decreased in the presence of l30 m and r55c mutants ( fig .
6 ) , consistent with either complete deficiency in the former or attenuated insulin secretion in the latter ( fig .
min6 -cells transfected with mutant ins - gfp constructs were subsequently ( typically after 24 h ) infected with adenoviral vector expressing npy - cherry , a dense core vesicle marker .
protein expression after 48 h was studied under 63 oil - immersion lens of confocal microscope using 488-and 568-nm laser lines .
images shown are of single cells ( n  25 ) with typical distribution of ins mutant proteins .
note that the r6h panel captured two overlapping cells expressing the r6h mutant in same focal plane , but only the top one expressed npy - cherry .
( a high - quality digital representation of this figure is available in the online issue . ) er retention caused by ins mutations and distribution of ins - gfp mutant proteins within single clonal -cells .
a : min6 -cells cotransfected with dsred - er , an er marker along with ins - gfp constructs and protein expression was studied after 48 h , as in fig .
b and c : green fluorescence was measured ( in absolute gray values ) across a line drawn through middle section of typical transfected min6 cells and the profile plotted against distance in pixels .
the gray broken line limits a cutoff fluorescence intensity to differentiate local accumulation of high concentrations of insulin in vesicles compared with diffused appearance across er .
( a high - quality digital representation of this figure is available in the online issue .
a representative sample from the population transfected min6 -cells was chosen to count the fraction of cells showing vesicular localization of ins - gfp ( as determined by gfp colocalization with red npy - cherry ) .
bars represent means  sem ; n = numbers on bars of total number of cells counted from three separate experiments .
data were analyzed by anova with bonferroni multiple comparison test . * p < 0.05 , * * p < 0.001 .
misfolded client proteins accumulate in the er to trigger an upr that initially upregulates er chaperones but , in the longer term , evokes cell death ( 20,21 ) .
the er retention of l30p and r55c mutants , a possible result of protein misfolding , prompted us to examine whether they triggered upr in hek293 cells by measuring the er stress markers , xbp1 ( 12,22 ) and chop / gadd153 ( 23 ) .
although the er localized sarco(endo)plasmic reticulum pump blocker , thapsigargin , provoked enormous er stress ( 24 ) , indicated by substantial accumulation of spliced xbp1 ( fig .
7c ) , transfection with the l30 m , l30p , and r55c mutants also caused a moderate , yet significant increase of er stress compared with wild - type insulin .
interestingly , er stress was also observed in cells expressing the r6h mutant , as revealed by enhanced expression of chop / gadd153 , if not of spliced xbp1 ( fig .
7b and c ) , and despite the fact that only r6h mutant preproinsulin , but not proinsulin or mature insulin , differed from the corresponding wild - type proteins .
despite elevated er stress marker levels , under these experimental conditions none of the three new mutants evoked any significant apoptotic cell death that could be detected with annexin v staining of hek293 cells ( data not shown ) .
hek293 cells were either transfected with ins mutants allowing 48 h for protein expression or treated with 100 nmol / l thapsigargin ( tg ) for 4 h , and total rna was extracted .
after reverse transcription , 2 g rna was amplified either by conventional pcr ( 100 ng cdna ) or by real - time pcr ( 20 ng cdna ) . a : typical agarose gel ( 2% ) separated with tris - borate - edta ( tbe ) buffer showing spliced ( s ) and unspliced ( u ) forms of pcr - amplified xbp1 cdna .
the gray values of the bands ( as in a ) of spliced and unspliced xbp1 quantified ( means  sem ; n = 3 ) .
c : real - time quantification of chop / gadd153 mrna normalized to endogenous cyclophilin ( means  sem ; n = 3 ) .
all data were analyzed by paired t tests comparing with wild - type ins ppi . *
three heterozygous missense ins mutations were identified in two french and one danish patients diagnosed with nonautoimmune diabetes before age 25 years .
no further mutations were found by screening additional french probands with later onset familial diabetes .
one novel mutation , a c.88c > g - p.l30 m change , was found in a patient with diabetes diagnosed at 17 years of age of normal body weight .
1a ) revealed that this replacement in the hydrophobic core of the insulin b chain is more conservative than a previously described mutation at the same site ( l30p ) ( 12 ) but , being located at the dimer interface , may affect insulin multimerization ( fig .
impact of l30 m and l30p mutations on insulin folding ( a ) and hexamerization ( b and c ) .
( left ) wild type , green ; l30 m , red ; ( center ) wild type , green , l30p , blue ; ( right ) l30 m , red , l30p , blue .
homology models were built using modeler9v4 software ( http://www.salilab.org ) using 1mso human insulin structure as a template ( 30 ) .
b : insulin hexamer showing l30 ( l ) and h34 ( h ) residues and zn ( 30 ) .
c and d : close - up showing the impact of l30 m ( lm , center ) and l30p ( lp , right ) mutations in insulin b - chain versus wild type ( left ) showing the increased cys - cys distance , disfavoring disulphide bond formation ( c ) , and changes to the structure after disulphide bond formation ( d ) .
leucine at position 6 of the b - chain ( l30 ) interacts with cysteine at position 6 of the a chain and leucine 17 and tryptophan 16 of the neighboring b - chain ( b ) .
l30 m and l30p mutations would therefore weaken or eliminate the interactions within the monomer and may also affect hexamer formation . at diagnosis , the revealing symptom in the patient was weight loss and the fasting glycemia was 16.5
no autoimmunity against pancreas was detected , and the pancreatic morphology ( assessed by abdominal tomodensitometry ) was normal .
initially , glycemic control was satisfactory with oral hypoglycemic agents ( ohas ; glibenclamide associated to metformin ) .
subsequently , the association of various oral drugs proved insufficient , and insulin requirement occurred 9 years after diagnosis .
a progressive increase in insulin needs was observed during the past 14 years ( from 0.45 to 0.83 ui  kg  day ) , and a1cranged from 5.0 to 9.9% .
no microangiopathy or macroangiopathy was detected in this patient after 24 years of diabetes evolution .
the l30 m mutation cosegregated in five individuals from the proband 's family with early - onset diabetes ( range of age at diagnosis : 1738 years ; range of bmi at last examination : 1925.5 kg / m ; fig .
however , the proband 's mother was found to carry the mutation but was not known to be diabetic , and had a normal ogtt at her last examination at age 68 years ( fasting and 2-h post  glucose load glycemia of 5.1 and 7.6 mmol / l , respectively ) .
the symbols denote the following : solid symbols , diabetes status ; striated symbol , impaired glucose tolerance status ; empty symbols , normoglycemic subjects ; grid symbol , one normal glucose tolerant subject who is carrier of the l30 m mutation ; and gray symbols , subjects who were not available for genetic testing .
the ins mutation status is shown under each symbol : nm as heterozygote , nn as wild type , and nt as not tested .
the text below indicates the following : age at diagnosis of diabetes or age at examination in normoglycemic subjects ( in years ) , bmi ( kg / m ) , and treatment in the diabetic subjects .
we also identified another mody proband bearing a c.163c > t - p.r55c mutation at the b / c junction ( previously reported in a norwegian case subject with apparent type 1 diabetes , but negative for autoantibodies ) .
the patient was diagnosed with diabetes at age 9 years by signs of polyuria and polydipsia , which required exogenous insulin therapy at the time of diagnosis . after a follow - up of > 50 years ,
clinical records indicated no signs of late - diabetic complications ( absence of retinopathy , nephropathy , or cardiovascular disease ) and a stable , well controlled glycemic profile ( a1c ranges < 6% ) .
the mutation was found to cosegregate with diabetes in two other diabetic relatives , who were diagnosed at ages 37 ( the mother ) and 33 ( the sister ) years ( fig .
they were initially treated with oha , then with insulin therapy ( 12 and 5 years , respectively , after their initial treatment ) .
the mother of the proband died at age 87 years ( the cause of death was not directly related to diabetes ) .
a c.17g > a substitution , leading to a r6h change in the signal peptide , was identified in a danish proband ( fig .
he was treated with diet and oha since diagnosis . at age 50 years , he was treated with metformin ( 1 g  2 ) , a1c was 6.4% , fasting serum c - peptide was 419 pmol / l , and fasting plasma glucose was 6.4
2 ) , who also carries the mutation , was diagnosed with diabetes at age 51 years . at age 53 years , he was treated with diet alone and had no signs of late - diabetic complications .
2 ) , carrying the mutation , was diagnosed with impaired glucose tolerance at age 26 years and with gestational diabetes at age 27 .
as shown in table 1 , the diabetic proband ( family fr - am ) displayed a dramatic decrease in insulin secretion during graded glucose infusion , whereas insulin sensitivity measured during the euglycemic hyperinsulinemic clamp was normal ; this is consistent with -cell failure as the cause of diabetes .
his nondiabetic mother was also found to have a clear defect in insulin secretion , which was apparent in response to both oral and intravenous glucose load and also to the intravenous arginine test during hyperglycemia , raising the question as to why she did not develop diabetes . blood glucose level at 120 min during ogtt ( 7.6
mmol / l ) was close to the glucose intolerance threshold ( 7.8 mmol / l ) .
however , insulin sensitivity in this person must be considered high , taking into account her age and her reported high level of physical activity .
anthropometric and metabolic characteristics of two subjects ( diabetic proband and nondiabetic mother from family fr - am ) carrying the l30m - ins mutation , compared with a group of control subjects data are means  sd . the m value is an estimate of insulin sensibility during the euglycemic hyperinsulinemic clamp .
we next examined the ability of the identified mutations to affect insulin exit from the er , initially using hek293 cells ( 12,15 ) . these cells
lack a well - defined regulated secretory pathway such that normal er exit of a proinsulin - gfp chimera ( 15 ) leads to the constitutive release into the medium . along with the three identified mutations ( r6h , l30 m , r55c ) , two other previously reported mutations , r6c ( 9 ) and l30p ( 12 ) , were introduced in the preproinsulin - gfp chimera ( denoted henceforth as ppi ) for comparative molecular studies .
although all the mutants expressed at similar levels to the wild - type at the mrna level ( fig .
3a ) , expressed protein levels in total cell extract were lower in wild - type and r6h cells , most likely reflecting the more efficient constitutive secretion of these molecules than the other mutant proteins ( fig .
all mutant proinsulin proteins migrated to same extent as wild - type proinsulin on reducing sds - page , indicating efficient cleavage of preproinsulin signal peptide .
when secreted proinsulin was measured in the culture media , the l30 m mutant displayed a decrease in proinsulin release rate , albeit less marked than that for the l30p mutant ( fig .
secretion of r6h was not significantly different from wild type , although mutation to cysteine at the same location ( r6c ) in the signal peptide led to substantially compromised proinsulin release ( fig .
the release of the b / c junction mutant proinsulin , r55c , was also considerably reduced ( fig .
3c ) , indicative of er retention . impact of insulin mutations on er release and secretion in hek293 cells
. a : real - time pcr measurements of recombinant human ins mrna expression in transfected hek293 cells normalized to endogenous cyclophilin ( means  sem ; n  5 ) .
b : typical western blot ( n  5 ) of total protein ( 30 g / lane ) from hek293 cells transfected or not with ins constructs separated on 12% reducing sds - page and probed first with anti - gfp antibody , followed by -tubulin as loading control .
c : secreted proinsulin content in 1 ml media of transfected hek293 cells collected for a period of 20 h ( means  sem ; n  5 ) .
data were analyzed by anova with bonferroni multiple comparison test . * * * p < 0.001 . a large percentage of l30 m ( 69  8% ) and r55c ( 42  3% ) mutant chimera - expressing hek293 cells displayed a marked difference in the distribution of fluorescence compared with wild - type insulin gfp - expressing cells : whereas fine tubular / reticular fluorescence , reminiscent of a healthy er ( 19 ) , was evident in wild - type chimera - expressing cells , a globular , perinuclear staining was usually apparent in cells expressing the l30 m or r55c mutant constructs ( supplementary figure 1 ) .
this observation is consistent with retention of the mutant chimera in an inflated and engorged er . to extend these findings to insulin - secreting cells , we next expressed wild - type or mutant chimeras in clonal min6 -cells ( 17 ) .
the fluorescence of wild - type hprocpepgfp was closely colocalized with the co - overexpressed secretory granule marker , neuropeptide y - venus ( fig .
4 ) ( 18 ) , with little or no colocalization with the overexpressed er marker , dsred - er ( fig .
the insulin - gfp fluorescence distribution profile along a straight line drawn across one of the middle sections of individual cells in fig .
5b ) , implying compacted storage of protein in dense core vesicles and a lack of significant retention in the er . by contrast , l30 m chimera fluorescence was barely detected in the npy - venus  positive compartment ( fig .
4 ) but was strongly colocalized with dsred - er , confirming large - scale retention in the latter organelle ( fig .
the distribution of l30 m was very close to the structurally most perturbing mutation , l30p ( fig .
1 ) . on the other hand , r6h or r6c mutations in the preproinsulin signal peptide appeared to exert no untoward effects on proper folding within the er ( fig .
4 ) and as confirmed by a fluorescence profile similar to the wild - type chimera ( fig .
however , the r55c mutation displayed an altered distribution pattern of insulin chimera with both dense core vesicular targeting ( fig .
the fluorescence distribution profile of l30 m and r55c indicated absence of peaks above the cutoff and a diffuse presence throughout the er ( fig .
furthermore , the total number of cells in a population of min6 -cells that produced mutant insulin - containing granules significantly decreased in the presence of l30 m and r55c mutants ( fig .
6 ) , consistent with either complete deficiency in the former or attenuated insulin secretion in the latter ( fig .
min6 -cells transfected with mutant ins - gfp constructs were subsequently ( typically after 24 h ) infected with adenoviral vector expressing npy - cherry , a dense core vesicle marker .
protein expression after 48 h was studied under 63 oil - immersion lens of confocal microscope using 488-and 568-nm laser lines .
images shown are of single cells ( n  25 ) with typical distribution of ins mutant proteins .
note that the r6h panel captured two overlapping cells expressing the r6h mutant in same focal plane , but only the top one expressed npy - cherry .
( a high - quality digital representation of this figure is available in the online issue . )
er retention caused by ins mutations and distribution of ins - gfp mutant proteins within single clonal -cells .
a : min6 -cells cotransfected with dsred - er , an er marker along with ins - gfp constructs and protein expression was studied after 48 h , as in fig .
b and c : green fluorescence was measured ( in absolute gray values ) across a line drawn through middle section of typical transfected min6 cells and the profile plotted against distance in pixels .
the gray broken line limits a cutoff fluorescence intensity to differentiate local accumulation of high concentrations of insulin in vesicles compared with diffused appearance across er .
( a high - quality digital representation of this figure is available in the online issue .
a representative sample from the population transfected min6 -cells was chosen to count the fraction of cells showing vesicular localization of ins - gfp ( as determined by gfp colocalization with red npy - cherry ) .
bars represent means  sem ; n = numbers on bars of total number of cells counted from three separate experiments .
misfolded client proteins accumulate in the er to trigger an upr that initially upregulates er chaperones but , in the longer term , evokes cell death ( 20,21 ) .
the er retention of l30p and r55c mutants , a possible result of protein misfolding , prompted us to examine whether they triggered upr in hek293 cells by measuring the er stress markers , xbp1 ( 12,22 ) and chop / gadd153 ( 23 ) .
although the er localized sarco(endo)plasmic reticulum pump blocker , thapsigargin , provoked enormous er stress ( 24 ) , indicated by substantial accumulation of spliced xbp1 ( fig .
7c ) , transfection with the l30 m , l30p , and r55c mutants also caused a moderate , yet significant increase of er stress compared with wild - type insulin .
interestingly , er stress was also observed in cells expressing the r6h mutant , as revealed by enhanced expression of chop / gadd153 , if not of spliced xbp1 ( fig .
7b and c ) , and despite the fact that only r6h mutant preproinsulin , but not proinsulin or mature insulin , differed from the corresponding wild - type proteins .
despite elevated er stress marker levels , under these experimental conditions none of the three new mutants evoked any significant apoptotic cell death that could be detected with annexin v staining of hek293 cells ( data not shown ) .
hek293 cells were either transfected with ins mutants allowing 48 h for protein expression or treated with 100 nmol
/ l thapsigargin ( tg ) for 4 h , and total rna was extracted .
after reverse transcription , 2 g rna was amplified either by conventional pcr ( 100 ng cdna ) or by real - time pcr ( 20 ng cdna ) .
a : typical agarose gel ( 2% ) separated with tris - borate - edta ( tbe ) buffer showing spliced ( s ) and unspliced ( u ) forms of pcr - amplified xbp1 cdna .
the gray values of the bands ( as in a ) of spliced and unspliced xbp1 quantified ( means  sem ; n = 3 ) .
c : real - time quantification of chop / gadd153 mrna normalized to endogenous cyclophilin ( means  sem ; n = 3 ) .
all data were analyzed by paired t tests comparing with wild - type ins ppi . *
more than 25 separate mutations in the human ins gene have been described , leading to very early - onset diabetes diagnosed in the neonatal period or early infancy in most case subjects , but also later in childhood or in young adulthood ( 25 ) .
our study from two european cohorts of diabetic patients diagnosed with mody confirms that coding ins mutations are also associated with the mody form of diabetes , albeit with a much lower prevalence compared with the mody-2/gck and mody-3/hnf1a subtypes .
moreover , our description of three families with three separate mutations highlights a great variability in clinical presentation , as well as in the severity and evolution of diabetes , even between carriers of the same mutation within a family .
several of the ins mutations leading to the most profound pathologies ( i.e. , neonatal diabetes ) involve the appearance of an insulin molecule with an unpaired cysteine residue , as observed in akita ( 1 ) and munich ( 3 ) mice , and cause a severe folding defect , upr , and -cell apoptosis .
human ins mutations at position 6 of the b - chain , l30p or l30v , have previously been found to cause early - infancy diabetes ( 12 ) .
these mutants were shown to be substantially retained in the er and lead to abnormal splicing of xbp1 , implying a folding defect and an increased er stress response . however , the behavior and trafficking of these mutants in insulin - secreting cells has not previously been described .
interestingly , the l30 m replacement , described here as a novel mutation , led to a milder clinical presentation ( diabetes onset at 1738 years ) despite clear er retention and evidence of upr ( figs .
although the presence of a methionine at this site is likely to lead to a smaller change in the structure around the hydrophobic core than proline ( fig .
1a ) , the location of l30 at the dimer - dimer interface ( fig . 1b and c ) might affect the formation of insulin multimers .
although insulin hexamerization and condensation are generally thought to occur in the trans - golgi and in immature granules , it seems conceivable that defective dimer association may occur earlier in the secretory pathway and hence influence exit from the er of the mutant .
importantly , the more substantial vesicular targeting of the l30 m than the l30p mutant in min6 cells ( fig .
6 ) suggests that er exit is more efficient for the former , even though secretion of proinsulin from hek293 cells was below the level of detection with either mutant ( fig .
nonetheless , this difference presumably permits more sustained insulin secretion in early life for l30 m mutant carriers , with less marked -cell loss and hence a later onset of diabetes than those carrying l30p , and leads to marked phenotypic differences within one family .
interestingly , the novel mutation r6h barely affected proinsulin release or er stress , as assessed by xbp1 splicing , and yet prompted the largest induction of chop mrna of all the mutants examined . because human preproinsulin contains a signal peptide with a classical tripartite domain structure , that is , a positively charged nh2-terminal ( n- ) , a hydrophobic core ( h- ) , and a cooh - terminal polar domain ( c - region ) , and because the h - and c - regions determine processing of signal peptide ( 26 ) , the r6h mutation in the n - region should not affect signal peptide cleavage . however , a shift in net positive charge at cellular ph in the n - region due to replacement of arginine ( acid dissociation constant , 12.5 ) by histidine ( acid dissociation constant , 66.5 ) may influence both the level of protein translation and efficiency of export ( 27 ) . this in turn may cause mild er stress sufficient to prompt chop activation ( 28 ) , perhaps by activating perk , although ire1 activity and thus xbp1 splicing are unaffected .
whatever the underlying mechanisms are , these data indicate that this mutation in the preproinsulin signal peptide leads to a distinct form of upr / er stress response compared with mutants in the mature insulin molecule , which seems likely to impact on -cell survival in vivo , as suggested by the clinical presentation of diabetes in the danish family .
the r6h mutant thus acts somewhat differently to the previously described r6c mutation ( 9 ) ( fig .
although we can not exclude the possibility that the behavior of the proinsulin mutants in the cell culture systems used here may differ from that of the mutants in human -cells in vivo , earlier observations also support the above view that the molecular mechanisms underlying diabetes in carriers of different ins mutations may differ .
thus , molven et al . ( 11 ) reported abundant c - peptide in two carriers of the r55c mutation , implying a substantially retained -cell mass and normal insulin release , despite hyperglycemia and ketoacidosis at presentation .
however , split proinsulin may also have contributed to the apparent c - peptide signal in this earlier study .
because the proinsulin mutant is unlikely to be fully processed by carboxypeptidase e , the presence of an unpaired cysteine may prompt a upr . of note ,
the previously identified r89c mutation at the c - a junction led to a dramatic decrease in c - peptide levels in two patients with neonatal diabetes , suggesting massive -cell loss ( 12 ) . in summary , we show that mutations in the human preproinsulin gene are a cause of nonautoimmune diabetes with variable clinical features at presentation in early life or in young adulthood , and also in the long - term evolution .
our findings suggest that the degree of er retention , and the nature of the upr , can differ widely between mutants , generating a range of underlying -cell defects .
 objectiveheterozygous mutations in the human preproinsulin ( ins ) gene are a cause of nonsyndromic neonatal or early - infancy diabetes . here 
 , we sought to identify ins mutations associated with maturity - onset diabetes of the young ( mody ) or nonautoimmune diabetes in mid - adult life , and to explore the molecular mechanisms involved.research design and methodsthe ins gene was sequenced in 16 french probands with unexplained mody , 95 patients with nonautoimmune early - onset diabetes ( diagnosed at < 35 years ) and 292 normoglycemic control subjects of french origin . 
 three identified insulin mutants were generated by site - directed mutagenesis of cdna encoding a preproinsulin  green fluorescent protein ( gfp ) ( c - peptide ) chimera . 
 intracellular targeting was assessed in clonal -cells by immunocytochemistry and proinsulin secretion , by radioimmunoassay . spliced xbp1 and c / ebp homologous protein were quantitated by real - time pcr.resultsa novel coding mutation , l30 m , potentially affecting insulin multimerization , 
 was identified in five diabetic individuals ( diabetes onset 1736 years ) in a single family . 
 l30 m preproinsulin - gfp fluorescence largely associated with the endoplasmic reticulum ( er ) in min6 -cells , and er exit was inhibited by 50% . 
 two additional mutants , r55c ( at the b / c junction ) and r6h ( in the signal peptide ) , were normally targeted to secretory granules , but nonetheless caused substantial er stress.conclusionswe describe three ins mutations cosegregating with early - onset diabetes whose clinical presentation is compatible with mody . 
 these led to the production of ( pre)proinsulin molecules with markedly different trafficking properties and effects on er stress , demonstrating a range of molecular defects in the -cell . 
RESEARCH DESIGN AND METHODS
Subjects and mutation identification.
Assessment of clinical data.
Proinsulin construct and generation of mutants.
Proinsulin secretion assay.
Quantification of transient expression of mRNA and protein.
Immunocytochemistry.
Data analysis and statistics.
RESULTS
Identification of 
In vivo metabolic studies in two carriers of the L30M mutation.
Impact of insulin mutations on intracellular trafficking ex vivo.
Impact of insulin mutations on ER stress.
DISCUSSION
Supplementary Material