after approval by an ethics committee and informed written consent , 18 type 2 diabetic subjects underwent a euglycemic glucose clamp study after subcutaneous injection of 0.4 units / kg glargine and 1 week of daily glargine treatment , as previously reported ( 5 ) . glargine metabolism was studied in nine subjects ( supplementary table 1 ) from whom plasma was still available ( 5 ) . the 31-h blood samples ( samples after 31 h were not available ) were processed for radioimmunoassay determination of plasma concentration of insulin ( which detects endogenous insulin plus injected glargine and its metabolites ) ( 5 ) and for a liquid chromatography - tandem mass spectrometry ( lc - ms / ms ) assay specific for glargine and metabolites 1 ( m1 ) and 2 ( m2 ) ( 6 ) ( supplementary data ) . pmol / l for insulin glargine , m1 , and m2 . the maximum plasma concentration ( cmax ) , time to reach cmax , and predose plasma concentration the area under the concentration - time curve ( auc ) between 0 and 31 h ( auc031 ) was determined using the trapezoidal rule in which concentrations less than the lower limit of quantification were set at zero . percentage of glargine and m1 was calculated according to the equation : ( glargine or m1/total insulin [ glargine + m1 ] )  100 . calculations were performed using ncss 2007 ( ncss , llc , kaysville , ut , www.ncss.com ) . plasma glucose was maintained at 101 mg / dl ( 25th and 75th percentiles , 99 and 103 mg / dl , respectively ) as a result of variable glucose infusion ( 0.47 mg / kg / min [ 0.26 and 0.82 mg / kg / min ] ) for 31 h ( supplementary fig . the last measurable concentration was at 11 h ( 25th and 75th percentiles , 53 and 35 pmol / l , respectively ) , in two other subjects at 13 h ( 37 pmol / l for both ) , and in one subject at 18 h ( 41 pmol / l ) . m1 was detected in all subjects up to 31 h , whereas m2 was not detected in any subjects at any time point . m1 , but not glargine , was detected at baseline ( 44 pmol / l [ 19131 pmol / l ] ) , likely reflecting the metabolism of glargine injected 24 h before . in fact , 24-h postinjection values of m1 were similar to baseline values ( 60 pmol / l [ 52118 h / l ) , whereas the m1 pharmacokinetics - auc031 was 2,166 pmol  h / l ( 1,622 and 4,955 pmol  h / l ) ( fig . pmol / l ( 0 and 48 pmol / l ) , and m1 cmax was 129 overall , m1 represented 97% ( 89 and 100% , respectively ) of the total amount of insulin detected in blood after injection of glargine , with glargine contributing only 3% ( 1 and 8% ) . the pattern of plasma insulin concentration as measured by radioimmunoassay was similar to that of m1 as detected by lc - ms / ms ( supplementary fig . top panel : auc031h of m1 and glargine in the nine individual subjects ( m1 , open circles ; glargine , filled circles ) and median and 10th , 25th , 75th , and 90th percentiles auc031h in the group of m1 ( open box ) and glargine ( hatched box ) ( top left panel ) and cmax of m1 ( open circles ) and glargine ( filled circles ) in individual subjects and median and 10th , 25th , 75th , and 90th percentiles cmax in the group of m1 ( open box ) and glargine ( hatched box ) ( top right panel ) . bottom panel : individual values of auc031h of m1 and glargine ( lc - ms / ms ) ( bottom left panel ) and cmax of m1 and glargine ( m1 open columns ; glargine , hatched columns ) in the nine subjects . in the current study in type 2 diabetic subjects on glargine treatment for 1 week , a subcutaneous injection of a therapeutic dose of insulin glargine resulted in biotransformation to m1 , which constituted > 90% of circulating insulin , with only a minor presence of the originally injected glargine in plasma ( < 10% ) . to the best of our knowledge , this is the first study reporting the metabolism of insulin glargine in type 2 diabetic subjects using a specific lc - ms / ms assay ( 6 ) . overall , the results are in line with recent findings in type 1 diabetes ( bolli et al . ) . taken together , the results suggest that in humans , glargine is rapidly metabolized to m1 , whereas the originally injected glargine is , if anything , only minimally present in plasma . these findings in type 2 diabetic subjects are relevant for the interpretation in vivo of the in vitro data reporting greater affinity of glargine for igf-1r and greater mitogenic potency compared with hi in some malignant cell lines ( 7 ) . the present studies indicate that in vivo , glargine concentration in plasma is negligible and well below that shown in vitro to demonstrate greater affinity for igf-1r ( 3,8 ) and greater mitogenesis ( 9 ) . because glargine is rapidly and nearly totally metabolized to m1 , which has effects on mitogenesis no different than hi ( 3,7 ) , it is unlikely that there is an increased risk of cancer in humans following administration of insulin glargine , at least via igf-1r stimulation . therefore , the findings of in vitro studies on mitogenesis using glargine as such ( 10 ) are of limited informative value for interpretation of the results of glargine administration in vivo . we acknowledge that the current study has limitations ( i.e. , small sample size and a dose of insulin glargine just above the therapeutic range of the subjects studied ) . nevertheless , the results are homogeneous in all subjects , consistent with those in type 1 diabetes ( bolli et al . ) and with recent findings of no stimulation of igf-1r bioactivity by plasma of type 2 diabetic subjects who were given glargine doses greater than those described in the current study ( 11 ) . in conclusion , after subcutaneous injection of a therapeutic dose in glargine - treated type 2 diabetic subjects , glargine is only transiently , and at minimal concentration , detectable in plasma , whereas its metabolite m1 accounts for most ( > 90% ) of the plasma insulin concentration . thus , in vivo , glargine does not exert its long - acting metabolic effects directly as glargine but predominantly via its main metabolite m1 . while this article was in proof , an elegant in vitro study was published that showed that glargine displays higher potency than human insulin for stimulation of insulin / igf-1 hybrid receptors with greater proliferative / antiapoptotic effects in mcf-7 cells . pierre - eugene c , pagesy p , nguyen tt , neuill m , tschank g , tennagels n , hampe c , issad t. effect of insulin analogues on insulin / igf1 hybrid receptors : increased activation by glargine but not by its metabolites m1 and m2 .
objectiveto investigate concentration of plasma insulin glargine after its subcutaneous dosing compared with concentration of its metabolites 1 ( m1 ) and 2 ( m2 ) in subjects with type 2 diabetes.research design and methodsnine subjects underwent a 32-h euglycemic glucose clamp study ( 0.4 units / kg glargine after 1 week of daily glargine administration ) . 
 glargine , m1 , and m2 were measured by a specific liquid chromatography - tandem mass spectrometry assay.resultsglargine was detected in only five of the nine subjects , at few time points , and at negligible concentrations . 
 m1 was detected in all subjects and exhibited the same pattern as traditional radioimmunoassay - measured plasma insulin . 
 m2 was not detected at all.conclusionsafter subcutaneous injection , glargine was minimally detectable in blood , whereas its metabolite m1 accounted for most ( > 90% ) of the plasma insulin concentration and metabolic action of the injected glargine .