impaired insulin sensitivity ( is ) plays a crucial role in the development of type 2 diabetes ( t2d ) , but its relevance for the occurrence of ischemic stroke still remains unclear . in that context , some studies suggested that decreased is , for example , insulin resistance , is an established risk factor for ischemic stroke , [ 24 ] while there are studies which could not demonstrate this relationship .
moreover , it has been reported that decreased is is directly related to different subtypes of ischemic stroke in t2d patients and nondiabetics , measured by different metabolic tests , short insulin tolerance test , homeostasis model assessment for insulin resistance , and immunoreactive insulin after glucose loading in 2  h ogtt [ 2 , 3 ] .
simultaneously , it has been shown that higher levels of insulin resistance were found in nondiabetics with coexistence of intra- and extracranial atherosclerosis in contrast to those with only intra- or extracranial atherosclerosis .
also , it has been suggested that hyperinsulinemia might be a risk factor for ischemic stroke [ 7 , 8 ] .
hyperinsulinemia represents a surrogate measure for insulin resistance in nondiabetics , as well as in t2d patients with significant residual insulin secretion capacity .
simultaneously , higher levels of pai-1 have been found in blood from patients with t2d , in obese subjects , and in other conditions associated with insulin resistance [ 1113 ] .
moreover , it has been shown that hypofibrinolysis due to higher pai-1 levels has been related to the insulin resistance [ 1417 ] and might be involved together with insulin resistance , in the pathogenesis of ischemic stroke .
therefore , in this study we tried to determine the role of impaired is , together with the relevant changes in insulin and pai-1 levels in t2d patients as well as nondiabetics with ischemic stroke .
we divided 62 t2d patients into two groups , with ( n = 33 ) and without ( n = 29 ) atherothrombotic ischemic stroke ( atis ) , and 64 nondiabetics were assigned into group with atis ( n = 34 ) and healthy subjects ( n = 30 ) , paired with the t2d patients with respect to sex and age .
diagnosis of atis was done by a neurologist according to clinical signs and visualization methods , cranial computerized scan , and/or magnetic resonance imaging of the brain , repeated after the first 7 days from initial findings of ischemic stroke . only the patients with atis
were included in the study , while the exclusion criteria involved patients with previously documented lacunar , cardioembolic , hemorrhagic cerebral infarction or coronary heart disease ( history of myocardial infarction or coronary angiography ) .
t2d patients were treated only with oral antihyperglycemic agents , while we excluded patients treated with insulin therapy or with other endocrine diseases , renal or hepatic insufficiency , previous and current infections , hematological or rheumatic diseases , uncontrolled hypertension , or severe alcohol consumption , during the last 4 weeks . at the time of metabolic evaluation ,
all the patients , irrespective of occurrence of the stroke , showed a uniform level of their physical activity .
the patients were completely informed about the study , before they gave an informed consent to participate .
the study was done at the clinic for endocrinology , diabetes and metabolic diseases and at the clinic for neurology , clinical centre of serbia , faculty of medicine , university of belgrade , and was approved by the institutional ethics committee .
the obtaining of the medical history and physical examination , metabolic tests , and fibrinolytic activity evaluation were conducted in all the patients included the study within one - day visit .
body mass index was calculated as weight in kilograms divided by the square of height in meters .
arterial blood pressure was measured by sphygmomanometry and hypertension was diagnosed according to world health organisation criteria ( systolic / diastolic blood pressure 140/90  mm  hg ) or by the use of antihypertensive agents .
the metabolic test was performed after 6 months from occurrence of atis and following overnight fasting .
is was tested by using ivgtt with frequently sampled plasma glucose ( pg ) and pi levels , followed by the minimal model analysis .
the subjects were injected with glucose 0.3  g / kg body weight and the blood samples were taken immediately before intravenous glucose loading and sequentially every minute during the first 10 minutes and then 12 , 14 , 16 , 20 , 23 , 24 , 25 , 27 , 30 , 40 , 50 , 60 , 70 , 80 , 90 , 100 , 120 , 160 , and 180 minutes after intravenous glucose loading .
insulin was added during the test intravenously ( 4  mu / kg / min ) , between minutes 20 and 25 , in order to substitute potentially diminished insulin response .
the insulin sensitivity index ( si ) was obtained from the data of pg and pi levels by computerized minimal model analysis ( minmod program , kindly provided by dr .
pg was obtained by glucose oxidase method using a beckman glucose analyser ( beckman instruments ) .
the continuous variables within each group of patients were analyzed with analysis of variance ( anova ) with post hoc tamhane test .
the significance of the differences between correlation coefficients was analyzed by using fisher r - to - z transformation .
data were analyzed using the statistical package for the social sciences ( spss ) software ( advanced statistics , version 17.0 ) , chicago , il .
all three investigated groups of patients were matched according to the age , duration of t2d , and period of time from the onset of ischemic stroke .
simultaneously , all patients were overweight and t2d patients had satisfactory metabolic control expressed as almost equal hba1c values , before metabolic investigation .
the hypertension in patients with the stroke was significantly higher than in healthy controls , both in t2d patients and in nondiabetics .
we did not find a significant difference in bmi between t2d patients and atis compared to t2d without atis and nondiabetics with atis compared to healthy subjects ( table 1 ) .
when we analysed is , expressed as si index , the lowest si values were present in patients with t2d and atis and they were significantly lower in t2d patients with atis compared to t2d patients without atis ( 1.18  0.67 versus 2.82  0.61 
moreover , si values were significantly lower in nondiabetics with atis in comparison to healthy subjects ( 3.18  0.93 versus 6.13  1.69  min / mu / l  10 ; p < 0.001 ) ( figure 1 ) .
there is no difference in si between t2d patients without atis and nondiabetics with atis ( p = ns ) .
simultaneously , we found that pi levels were higher in t2d patients and atis compared to t2d patients without atis ( 19.61  4.08 versus 14.91  1.66 
in addition , pi levels were higher in nondiabetics with atis in comparison to healthy subjects ( 15.14    2.20 versus 7.58    2.05 ; p < 0.001 ) .
moreover , t2d patients with atis showed significantly higher pi levels than nondiabetics with stroke ( p < 0.001 ) , but we did not document difference in pi levels between t2d patients without atis and nondiabetics with atis ( figure 2 ) .
simultaneously , we found that pai-1 levels were significantly higher in t2d patients with atis compared to t2d patients without atis ( 7.75  1.04 versus 4.57  0.72  mu / l ; p < 0.001 ) and in nondiabetics with atis compared to healthy subjects ( 4.78  0.98 versus 3.49  1.04 
in addition , pai-1 levels were not different among t2d patients without atis and nondiabetics with atis .
moreover , we found that si levels correlated with pai-1 levels both in t2d ( r = 0.690 , p < 0.001 ) and in nondiabetic subjects ( r = 0.456 , p < 0.001 ) ( table 2 ) . by using fisher r - to - z transformation , we evaluated the difference between two correlations coefficients .
the correlations of si and pai-1 in the t2d patients were stronger than si and pai-1 correlation in nondiabetics ( p < 0.05 ) .
the use of binary logistic regression analysis has identified levels of pai-1 , insulin , and si as independent predictors for atis both in t2d patients and in nondiabetics ( tables 3 and 4 ) .
our study results revealed decreased is in patients with atis , whether they were t2d patients or nondiabetics , suggesting that insulin resistance may play a significant role in occurrence of the stroke .
simultaneously , t2d patients with atis showed the lowest level of is , together with the fasting hyperinsulinemia .
the documented hyperinsulinemia in these patients , in the settings of diminished is levels , suggests the existence of a significant residual insulin secretion capacity .
moreover , both metabolic abnormalities , decreased is or insulin resistance and fasting hyperinsulinemia in nondiabetics with atis , have confirmed the importance of decreased is level in pathogenesis of ischemic stroke , which is consistent with the previous data .
both experimental and human studies revealed the importance of insulin resistance for the occurrence of acute ischemic stroke [ 2326 ] .
however , our results signify the persistence of compromised is and increased pi levels even 6 months after acute phase in t2d patients with ischemic stroke .
we decided to evaluate is level in this study by using ivgtt with frequently sampled pg and pi levels with minimal model analysis , which correlates with hyperinsulinemic euglycemic clamp , previously implemented in the is studies [ 27 , 28 ] .
the existence of impaired is in different subtypes of ischemic stroke in patients with t2d has also been proposed .
also , the novel study confirmed the highest values of two other parameters of insulin resistance , homeostasis model assessment for insulin resistance and insulin after glucose challenge in 2  h ogtt in patients with atherothrombotic cerebral infarction without previously documented abnormal glucose tolerance .
the results from extensive atherosclerosis risk in communities ( aric ) study showed that fasting insulin levels , among the other investigated risk factors , are positively associated with occurrence of ischemic stroke in the general population , which highlights the influence of insulin resistance , consistent with results from the finnish cohort study that included elderly t2d patients and nondiabetics .
novel data pointed out the important role of augmented insulin resistance and related metabolic abnormalities in the development of intracranial stenosis from its early stages even in nondiabetics . on the other hand , lots of data suggested that pai-1 plays a significant role in occurrence of coronary artery and cerebrovascular disease in t2d .
recent studies shed new light on pai-1 as an important pathway for cardiovascular events , including ischemic stroke . despite those facts and the background of occurrence and progression of atherosclerosis , the link between insulin resistance , diminished fibrinolytic activity , and
our results showed higher pai-1 levels in t2d and atis , in parallel with the absence of difference in pai-1 levels between t2d patients without atis and nondiabetics with atis .
however , there are some conflicting results suggesting higher pai-1 levels in t2d without ischemic stroke compared to nondiabetics , implying the absence of further deterioration of fibrinolytic activity in ischemic stroke in t2d patients .
moreover , there are indications of decreased is , hyperinsulinemia , and increased pai-1 in first - degree relatives of patients with ischemic stroke which is related to ethnicity , together with the findings supporting the hypothesis that diminished fibrinolytic activity may exist prior to ischemic stroke [ 3537 ] .
a previous study demonstrated that the higher pai-1 activity in young adults with a first ischemic stroke was a consequence of acquired hypofibrinolysis , together with other investigations supporting the genetic control of decreased fibrinolysis . also , obese diabetics had higher pai-1 levels compared to nondiabetics , implying permanent impaired fibrinolytic activity , which potentiates thrombosis , based on effect of combination of hormonal ( hyperinsulinemia ) and metabolic ( hyperglycemia ) changes characteristic for t2d [ 13 , 40 ] . in general , increased pai-1 level in patients with atis or t2d or nondiabetics could be present even 4 years after acute cerebral ischemic event . therefore , we speculate that disturbances in fibrinolysis may precede the occurrence of atis , having in mind higher pai-1 level in t2d patients without atis and almost equal pai-1 levels in nondiabetics with atis [ 14 , 42 ] .
we found that si levels correlated with pai-1 levels both in t2d and nondiabetic subjects , with stronger correlation in t2d than in nondiabetics .
is has been independently related to pai-1 levels in our study and previously in patients with t2d and obese diabetic and nondiabetic subjects [ 43 , 44 ] . in bari 2d
trial it has been shown that diminishing of insulin resistance favorably changes the balance between fibrinolysis and thrombosis potentiating fibrinolysis during long term followup in t2d patients .
in addition , it was suggested that patients who experience progression of symptomatic intracranial atherosclerosis are characterized by impaired endogenous fibrinolysis .
pai-1 levels may influence stroke mechanisms in multiple ways , and they might differentiate between responders and nonresponders to reperfusion therapies , and they might represent potential target for stroke prevention . in order to minimize the previously confirmed harmful effect of  glucose toxicity  to the is level and fibrinolytic activity [ 13 , 47 ] , we selected t2d patients with or without ischemic stroke matched with respect to duration of disease , showing optimal metabolic control before the evaluation of insulin sensitivity level .
previously described association of obesity , insulin resistance , and higher pai-1 levels , hypersecreted from adipocytes , was the reason to include overweight subjects in our investigation .
since age is known to be strongly and independently correlated with the occurrence of ischemic stroke , investigated patients were younger than 65 years .
measurements of is were not made until at least 6 months after the stroke , providing enough time for the patients to approach maximum recovery , showing similar level of the physical activity . in order to diminish the heterogeneity of stroke
the binary logistic regression analysis applied to our data has demonstrated that insulin , pai-1 , and si levels are independent predictors of the ischemic stroke occurrence , in t2d patients as well as in nondiabetics .
in conclusion , the results demonstrated that decreased is levels together with fasting hyperinsulinemia are strongly associated with the onset of the atis , while this atherogenic effect might be strongly potentiated by increased level of pai-1 . in this context , insulin resistance and
 we analyzed ( a ) insulin sensitivity ( is ) , ( b ) plasma insulin ( pi ) , and ( c ) plasminogen activator inhibitor-1 ( pai-1 ) in type 2 diabetes ( t2d ) patients with ( group a ) and without ( group b ) atherothrombotic ischemic stroke ( atis ) , nondiabetics with atis ( group c ) , and healthy controls ( group d ) . 
 is was determined by minimal model ( si ) . 
 si was lower in a versus b ( 1.18  0.67 versus 2.82  0.61  min1/mu / l  104 ; p < 0.001 ) and in c versus d ( 3.18  0.93 versus 6.13  1.69  min1/mu / l  104 ; p < 0.001 ) . 
 pi and pai-1 were higher in a versus b ( pi : 19.61  4.08 versus 14.91  1.66  
 mu / l ; p < 0.001 , pai-1 : 7.75  1.04 versus 4.57  0.72  
 mu / l ; p < 0.001 ) and in c versus d ( pi : 15.14  2.20 versus 7.58  2.05  
 mu / l ; p < 0.001 , pai-1 : 4.78  0.98 versus 3.49  1.04  
 mu / l ; p < 0.001 ) . 
 si correlated with pai-1 in t2d patients and nondiabetics , albeit stronger in t2d . binary logistic regression identified insulin , pai-1 , and si as independent predictors for atis in t2d patients and nondiabetics . 
 the results imply that insulin resistance and fasting hyperinsulinemia might exert their atherogenic impact through the impaired fibrinolysis . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions