the study population consisted of 85 caucasian , northern spanish girls representing a continuum of female subjects with hyperinsulinemic androgen excess that commences in prepuberty in those girls with a combined history of lbw and precocious pubarche , is followed by a preclinical phase starting around menarche of biochemical ovarian hyperandrogenism and yields to an overt clinical period of ovarian hyperandrogenism 23 years after menarche .
all subjects were included in similar clinical trials investigating the safety and efficacy of metformin treatment on their hyperinsulinemic androgen excess .
the prepubertal girls ( n = 18 ; tanner stage i ; aged 7.9  0.2 years ; bmi 19.3  0.6 kg / m ) had a combined a history of lbw ( 2.3  0.1 kg at term birth ) and precocious pubarche ( appearance of pubic hair before age 8 years ) attributable to an exaggerated adrenarche , based on high serum levels of dehydroepiandrosterone sulfate or androstenedione at diagnosis of precocious pubarche ( 8) . the girls were , in addition , hyperinsulinemic , as judged by the insulin response after an oral glucose tolerance test ( 16 ) .
all of these subjects participated in a controlled long - term study exploring the effects of early metformin treatment ( isrctn84749320 ) ; more specifically , they form together the study subgroup that was initially randomly assigned to receive metformin for at least 1 year ( 425 mg , daily at dinner time ) ( 9 ) .
the early postmenarcheal girls ( n = 31 ; aged 12.2  0.1 years ; bmi 21.5  0.5 kg / m ) manifested biochemical androgen excess characterized as the ensemble of 1 ) absence of clinical signs or symptoms of androgen excess ( 17 ) and 2 ) hyperinsulinemia ( oral glucose tolerance test ) and 3 ) ovarian androgen excess suggested by increased testosterone levels and confirmed by 17-hydroxyprogesterone hyperresponse to gonadotropin - releasing hormone agonist ( 17,18 ) .
the girls in the present analysis were enrolled in clinical studies that were initiated before 2003 , thus before compulsory trial registration ; as described , all these girls had a history of lbw - precocious pubarche and were already in the early postmenarche phase ( 612 months beyond menarche ) at start of metformin intervention ( 8,19 ) . the adolescent girls ( n = 36 ; aged 15.5  0.3 years ; bmi 21.9  0.5 kg / m ) were consecutive patients with pcos who were at least 2 years beyond menarche and who were followed in the outpatient clinic according to standard procedures , including yearly blood sampling in the fasting state ( once with dna extraction ) and yearly assessment of body composition .
the inclusion criteria were the same as those for the early postmenarcheal girls , except that the presence of clinical markers of androgen excess was now required : hirsutism ( ferriman - gallwey score > 8) plus either amenorrhea ( menses absent for > 3 months ) or oligomenorrhea ( menstrual cycle > 45 days ) . in all postmenarcheal girls ,
baseline assessments were performed in the follicular phase ( day 37 ) or after 2 months of amenorrhea .
all postmenarcheal girls received metformin for at least 1 year in monotherapy ( 850 mg , daily at dinner time ) . at the start of metformin treatment ,
none of the 85 girls presented evidence for thyroid dysfunction , cushing syndrome , hyperprolactinemia , glucose intolerance , or late - onset adrenal hyperplasia ; for at least 6 months before the start of metformin start , none of the girls received an estro - progestagen or other medication known to affect gonadal function or carbohydrate metabolism .
height and weight were measured , and bmi was calculated ( weight in kilograms divided by the square of height in meters ) .
bmi standard deviation scores ( sdss ) were derived from regional normative data ( 20 ) .
fasting blood glucose , serum insulin , igf - i , sex hormone  binding globulin ( shbg ) , and testosterone were measured as described previously ( 9 ) .
genomic dna was purified from whole blood samples using commercial reagents ( gentra puragene cell kit ; qiagen iberia , madrid , spain ) .
the stk11 rs8111699 snp was genotyped by a fluorescent genotyping system ( kaspar snp genotyping system ; kbiosciences , hoddesdon , u.k . ) . in brief ,
10 pg genomic dna was incubated in a 8-l solution of a combined mix containing 100 mol / l allele specific primer 1 ( fam , x - axis , allele g ) , 5-gaaggtgaccaagttcatgctccactgactgttcaggaaagcac-3 , 100 mol / l allele - specific primer 2 ( vic , y - axis , allele c ) , 5-gaaggtcggagtcaacggattccactgactgttcaggaaagcag-3 , 100 mol / l common reverse primer 5-ggtggaagcctgactgtgagagt-3 , 10
mol / l tris - hcl , ph 8.3 , ktaq polymerase , 1.8 mmol / l mgcl2 , and rox as a passive reference .
the pcr mix was incubated at 94c for 15 min , followed by 20 cycles of 94c for 10 s , 57c for 5 s , and 72c for 10 s. thereafter , the pcr mix was incubated for 18 cycles at 94c for 10 s , 57c for 20 s , and 72c for 40 s. samples were then read in a fluorescence plate reader .
the kaspar system uses the fluors fam and vic for distinguishing among genotypes and rox as a passive reference .
the corresponding genotypes for this snp ( cc vs. cg vs. gg ) did not differ among prepuberty ( 33% vs. 44% vs. 22% ) , early postmenarche ( 26% vs. 49% vs. 26% ) , and pcos ( 25% vs. 42% vs. 33% ) subjects in our study ( p = 0.89 ) .
the distributions were also tested for hardy - weinberg equilibrium , and none deviated significantly .
body composition was assessed by dual - energy x - ray absorptiometry with a lunar prodigy and lunar software ( version 3.4/3.5 ; lunar , madison , wi ) ( 9 ) .
total irradiation dose per assessment was 0.1 msevert ; coefficients of variation for scanning precision are 2.2 and 2.6% for fat and lean mass ( 8,9,19 ) .
the study protocol was approved by the institutional review board of hospital of sant joan de du .
informed consent was obtained from the patients and/or from parents , and assent was obtained from girls , as appropriate .
the clinical , biochemical , and body composition data from prepubertal and early postmenarcheal subjects have been reported separately ( 8,9,19 ) .
statistical analyses were performed using spss ( version 12.0 ; spss , chicago , il ) .
quantitative phenotypic data were compared across genotypes by a repeated - measures general linear model , computing the genotype as the between - subjects effect and both baseline and 1-year values as the within - subjects effect .
differences in 1-year changes across genotypes were tested by the interaction term among the between- and within - subjects effects .
height and weight were measured , and bmi was calculated ( weight in kilograms divided by the square of height in meters ) .
bmi standard deviation scores ( sdss ) were derived from regional normative data ( 20 ) .
fasting blood glucose , serum insulin , igf - i , sex hormone  binding globulin ( shbg ) , and testosterone were measured as described previously ( 9 ) .
genomic dna was purified from whole blood samples using commercial reagents ( gentra puragene cell kit ; qiagen iberia , madrid , spain ) .
the stk11 rs8111699 snp was genotyped by a fluorescent genotyping system ( kaspar snp genotyping system ; kbiosciences , hoddesdon , u.k . ) . in brief , 10 pg genomic dna was incubated in a 8-l solution of a combined mix containing 100 mol / l allele specific primer 1 ( fam , x - axis , allele g ) , 5-gaaggtgaccaagttcatgctccactgactgttcaggaaagcac-3 , 100 mol / l allele - specific primer 2 ( vic , y - axis , allele c ) , 5-gaaggtcggagtcaacggattccactgactgttcaggaaagcag-3 , 100 mol / l common reverse primer 5-ggtggaagcctgactgtgagagt-3 , 10
mol / l tris - hcl , ph 8.3 , ktaq polymerase , 1.8 mmol / l mgcl2 , and rox as a passive reference .
the pcr mix was incubated at 94c for 15 min , followed by 20 cycles of 94c for 10 s , 57c for 5 s , and 72c for 10 s. thereafter , the pcr mix was incubated for 18 cycles at 94c for 10 s , 57c for 20 s , and 72c for 40 s. samples were then read in a fluorescence plate reader .
the kaspar system uses the fluors fam and vic for distinguishing among genotypes and rox as a passive reference .
the corresponding genotypes for this snp ( cc vs. cg vs. gg ) did not differ among prepuberty ( 33% vs. 44% vs. 22% ) , early postmenarche ( 26% vs. 49% vs. 26% ) , and pcos ( 25% vs. 42% vs. 33% ) subjects in our study ( p = 0.89 ) .
the distributions were also tested for hardy - weinberg equilibrium , and none deviated significantly .
body composition was assessed by dual - energy x - ray absorptiometry with a lunar prodigy and lunar software ( version 3.4/3.5 ; lunar , madison , wi ) ( 9 ) .
total irradiation dose per assessment was 0.1 msevert ; coefficients of variation for scanning precision are 2.2 and 2.6% for fat and lean mass ( 8,9,19 ) .
the study protocol was approved by the institutional review board of hospital of sant joan de du .
informed consent was obtained from the patients and/or from parents , and assent was obtained from girls , as appropriate .
the clinical , biochemical , and body composition data from prepubertal and early postmenarcheal subjects have been reported separately ( 8,9,19 ) .
statistical analyses were performed using spss ( version 12.0 ; spss , chicago , il ) .
quantitative phenotypic data were compared across genotypes by a repeated - measures general linear model , computing the genotype as the between - subjects effect and both baseline and 1-year values as the within - subjects effect .
differences in 1-year changes across genotypes were tested by the interaction term among the between- and within - subjects effects .
genotype effects were similar in prepubertal , early postmenarcheal , and pcos subjects , and therefore genotype - phenotype associations are presented for all 85 girls studied as a single group ( adjusted for group allocation ) to maximize the statistical power .
clinical , biochemical , and body composition variables before and after 1 year of metformin in the study subjects values are means  sem .
values in healthy girls matched for bmi : prepubertal ( n = 24 ; aged 8.3  0.3 years ) : insulin 6.5  0.5 miu / l , igf - i 200  15 ng / ml , shbg 3.6  0.1 g / dl , testosterone 16  2 ng / dl ; pubertal ( n = 24 ; age 15.3  0.2 years ) : insulin 8.8  0.4 miu / l , igf - i 384  26 ng / ml , shbg 1.9  0.1 g / dl , testosterone 31  3 ng / dl .
lipid profile : triglycerides 55  4 mg / dl ; ldl 83  3 mg / dl ; hdl 65  2 mg / dl ( 8,23 and ibez et al . ; jcem 2004 ; 89:4,331337 ) .
the g allele in stk11 rs8111699 was associated with a poorer metabolic profile , specifically with higher insulin and igf - i ( both p < 0.005 ) ( table 2 ) .
the presence of this allele was at baseline unrelated to age , weight , body composition parameters , and serum androgens and lipids ( table 2 ) .
clinical , biochemical , and body composition variables before and after 1 year on metformin according to stk11 rs8111699 genotype in the study subjects values are means  sem .
p values for comparisons of baseline , 1-year data , and 1 year changes across genotypes are from a repeated - measures general linear model .
results are adjusted for group allocation ( i.e. , prepubertal , early postmenarcheal , or pcos ) ; those for 1 year of metformin and for  01 year are also adjusted for baseline insulin levels .
p  0.005 is considered the acceptable threshold of significance in these data because of the multiple tests performed ; these values appear in bold in the table .
values in healthy girls matched for bmi : prepubertal ( n = 24 ; aged 8.3  0.3 years ) : insulin 6.5  0.5 miu / l ; igf - i 200  15 ng / ml ; shbg 3.6  0.1 g / dl ; testosterone 16  2 ng / dl ; pubertal ( n = 24 ; aged 15.3  0.2 years ) : insulin 8.8  0.4 miu / l ; igf - i 384  26 ng / ml ; shbg 1.9  0.1 g / dl ; testosterone 31  3 ng / dl .
lipid profile : triglycerides 55  4 mg / dl ; ldl 83  3 mg / dl ; hdl 65  2 mg / dl ( 8,23 ) . after 1 year of therapy , there were pronounced differences in the response to metformin according to the stk11 rs8111699 genotype , as reflected by a robust response in the main endocrine - metabolic and body composition parameters in gg homozygotes , with an intermediate response in cg heterozygotes and almost no response in cc homozygotes ( table 2 ) .
such differences were found for 1-year changes in body composition , circulating insulin , igf - i , free androgen index , and lipids ( all p < 0.005 ; table 2 ) .
our results indicate that the stk11 rs8111699 snp influences both insulin sensitivity and metformin efficacy in hyperinsulinemic girls with androgen excess . despite the fact that neither stk11 nor ampk is a direct target of metformin ( 14 ) , both are necessary for metformin actions ( 14 ) .
it has been proposed that stk11 is constitutively active and that metformin exerts its hypoglycemic effects through a modification in ampk , rendering it a better substrate for stk11 ( 10 ) .
although the role of the studied intronic snp in stk11 has yet to be defined , it is known that intronic snps may regulate gene expression and alternative splicing ( 21 ) .
the stk11 rs8111699 snp may also be in linkage disequilibrium with an active snp in the kinase gene region .
the stk11 rs8111699 snp was associated with baseline insulin sensitivity in girls with hyperinsulinemic androgen excess .
bearing in mind that stk11 may be constitutively active , we postulate that carrying the g allele in the stk11 rs8111699 snp can result in lower stk11 activity and therefore lower efficiency of ampk phosphorylation by stk11 .
the same g allele in the stk11 rs8111699 snp was associated with pronounced changes in endocrine - metabolic and body composition parameters upon metformin treatment , effects that were nearly absent in c homozygous subjects .
we suggest that metformin restores the efficiency of ampk phosphorylation by stk11 in carriers of the g allele and has less or no effect in the absence of such allele .
( 15 ) , who found a stepwise increase in ovulation rate from c / c to c / g to g / g genotype in women with pcos treated with metformin .
the stk11 rs8111699 snp may thus play a broader role in metabolic and cardiovascular diseases by modulating the baseline condition as well as the response to treatment with the above - mentioned pharmacological agents .
a limitation of our study is the insufficient power to analyze the effect of the stk11 snp in each of the three subgroups within the total study population .
such an analysis would have been of interest because each of the consecutive subgroups is thought to represent a developmental stage of hyperinsulinemic androgen excess .
it should be noted , however , that the results remained essentially the same with and without adjustment for subgroup allocation in the multivariate model .
the identification of genetic polymorphisms that contribute to predict the response to commonly prescribed medications should assist clinicians in their selection of available drugs to individualize treatment , thereby improving the therapeutic response , both on the efficacy and on the safety side .
 objectiveserine - threonine kinase stk11 catalyzes the amp - activated protein kinase complex . 
 we tested the hypothesis that a gene variant in stk11 contributes to variation in insulin sensitivity and metformin efficacy.research design and methodswe studied the effects of a single nucleotide polymorphism ( snp ) ( rs8111699 ) in stk11 on endocrine - metabolic and body composition indexes before and after 1 year of metformin in 85 hyperinsulinemic girls with androgen excess , representing a continuum from prepuberal girls with a combined history of low birth weight and precocious pubarche over to postmenarchial girls with hyperinsulinemic ovarian hyperandrogenism . 
 metformin was dosed at 425 mg / day in younger girls and 850 mg / day in older girls . 
 stk11 rs8111699 was genotyped . 
 endocrine - metabolic features were assessed in the fasting state ; body composition was estimated by absorptiometry.resultsgenotype effects were similar in younger and older girls . at baseline , 
 the mutated g allele in stk11 rs8111699 was associated with higher insulin and igf - i levels ( both p < 0.005 ) . the response to metformin differed by stk11 genotype : 
 gg homozygotes ( n = 24 ) had robust metabolic improvements , gc heterozygotes ( n = 38 ) had intermediate responses , and cc homozygotes ( n = 23 ) had almost no response . 
 such differences were found for 1-year changes in body composition , circulating insulin , igf - i , free androgen index , and lipids ( all p < 0.005).conclusionsin hyperinsulinemic girls with androgen excess , the stk11 rs8111699 snp influences insulin sensitivity and metformin efficacy , so that the girls with the least favorable endocrine - metabolic profile improve most with metformin therapy . 
RESEARCH DESIGN AND METHODS
Clinical, endocrine, and metabolic assessments
Genetic analysis
Body composition
Ethics and statistics
RESULTS
CONCLUSIONS