insulin resistance and abnormal glucose tolerance are frequently observed in chronic liver disease ( cld ) , but the precise mechanism leading to insulin resistance still remains unclear . in an attempt to explain the mechanism of insulin resistance in these patients , several investigators have studied insulin receptor bindings in patients with cld . however , the results are varied , and there remain controversies about the insulin receptor status in cld . some studies have shown that insulin receptor bindings were reduced in cld which suggested an insulin receptor defect as a primary cause of insulin resistance .
however , other studies reported that insulin receptor bindings were normal , or even increased in patients with cld , thus raising doubt about the above explanation .
the present study was performed to elucidate this controversy by observing the insulin receptor status of erythrocytes in patients with cld .
although it is not clear whether erythrocytes reflect the insulin receptor status in more relevant tissues such as liver and muscle , it is generally agreed that insulin bindings to erythrocytes and monocytes change in a parallel direction in many situations .
the studies were carried out in eleven patients with cld and in ten healthy subjects .
the patient group was composed of nine men and two women ( age distribution ; 2564 years of age , mean ; 45 ) .
they did not take any medications that might influence glucose intolerance , and evidence of recent gastrointestinal bleeding was absent .
control group was composed of 10 healthy men ( age distribution ; 3360 years of age , mean ; 43 ) . who showed no evidence of hepatobiliary , renal or endocrine disturbances .
intravenous glucose tolerance test ( ivgtt ) was performed after 25 g of glucose was infused into an antecubital vein .
patients with cld were divided into two groups by fasting plasma glucose levels ( fbs ) and the results of ivgtt .
group i was composed of 6 patients with fbs less than 100 mg / dl and normal glucose disappearance rate ( k - glucose ) , and group ii was 5 patients with higher fbs ( fbs>100 mg / dl ) and lower k - glucose values ( table 1 )
. glucose disappearance rate ( k - glucose ) was calculated according to the formula of amatuzio et al . as follow ; 
 c(t)=c(o)ektwhere c(t ) is the glucose excess in mg / dl at any time t , and c(o ) is the glucose excess above basal value in mg / dl at time zero .
the reticulocyte count in peripheral blood was performed in all of the samples . those with over 1% reticulocytes were discarded as reticulocytes were known to have insulin receptors of more than tenfold compared with those of more matured red cells .
insulin binding to erythrocytes was studied in duplicate according to the method of gambhir with some minor modifications as previously described .
twenty milliliters of overnight fasting blood was drawn into a heparinized syringe and the erythrocyte cell layer was separated by ficoll - hypaque gradient centrifugation method . the cell suspension , trace amounts of radioactive insulin [ i - a14-monoiodinated human insulin with specific activity of about 2000 ci / mmole ( amersham co. , england ) ] , and various concentrations ( 0.02 to 10 ng / ml ) of unlabeled insulin were incubated at 15c for 3 and 1/2 hours . the bound form of insulin was separated and the radioactivity was counted in a gamma counter .
the nonspecific binding was defined as the radioactivity in the presence of 10 ng / ml of unlabeled insulin .
the nonspecific binding was subtracted from the total binding and the results were expressed as specific i - insulin binding to erythrocytes of a concentration of 4.010 cells / ml .
the results of the binding studies were presented as ; ( 1 ) the specific binding of i - insulin in the absence of competing unlabeled insulin , % ( b / t)0 , ( 2 ) the specific cell bound fraction plotted as a function of total insulin concentrations ( competition curve ) , and ( 3 ) the ratio of the cell - bound insulin to bee insulin plotted as a function of cell - bound insulin ( scatchard plots ) . in the scatchard plots ,
the highest insulin concentrations used was 100 ng / ml . the binding data were analyzed on the assumption that the curvature of the scatchard plots reflects the presence of two populations of insulin binding sites ( high affinity receptors and low affinity receptors ) .
, and data in figures as means.e.m .. the statistical analysis of the data was performed using the unpaired t - test .
1 shows the mean competition curves for i - insulin binding to erythrocytes in three study groups .
the percent specific binding in the absence of unlabeled insulin was significantly lower in the group i patients compared with that of the normal subjects .
however , i - insulin binding to erythrocytes in group ii patients was very similar to that of the normal subjects .
the scatchard plots of the mean values at each concentration of the bound insulin in three groups are shown in fig .
apparent receptor numbers of both high and low affinity receptors tended to be lower in the group i patients than those in normal controls , but the statistical differences of these indices between two groups ( group i and normal controls ) were not significant probably due to the small size of the samples ( table 2 ) .
there seem to be at least two subgroups of patients in cld : one with normal fbs , normal k - glucose and brisk insulin response to intravenous glucose loading ( group i ) , and the other with higher fbs , lower k - glucose and blunted acute insulin response to glucose loading ( group ii)(table 1 ) .
present study clearly demonstrates that erythrocyte insulin receptor binding is also different between these two groups of patients .
insulin binding to erythrocytes was reduced in group i patients , but was not reduced in group ii patients .
this was quite surprising since if the primary cause of insulin resistance was the defect of insulin receptors in peripheral tissues , those patients with reduced insulin receptor binding would have more abnormal glucose tolerance than those with normal insulin receptor binding .
this was not true in the present study , and it was suggested that insulin resistance in cld might not be due to a primary defect of insulin receptors . reduced insulin receptor binding is frequently associated with many states characterized by insulin resistance and hyperinsulinemia , such as obesity , type ii diabetes mellitus and acromegaly .
cld is another state frequently associated with glucose intolerance , insulin resistance and hyperinsulinemia , and several investigators have reported that insulin receptor bindings to peripheral tissues are reduced in the patients with cld .
it has been believed that down - regulation of insulin receptor by hyperinsulinemia is the cause of insulin resistance in cld . however , contradictory results have also been reported .
proietto et al , have shown that insulin receptor binding was not altered in the patients with cld , okada reported that insulin binding was rather increased in the patients with cld , and that the degree of increase was more marked in the patients who also had diabetes mellitus .
the result of the present study suggests that the discrepancy might be due to the differences in the study populations . in the present study ,
fasting insulin levels in group i patients were significantly higher than those in normal subjects ( table 1 . ) , and down - regulation by hyperinsulinemia might be the cause of reduced insulin receptor binding in these patients ( table 2 . ) .
however , down - regulation of insulin receptor was not associated with glucose intolerance ( group i ) , and erythrocyte insulin receptor binding was almost normal in the patients with glucose intolerance ( group ii ) . the biological significance of this phenomenon is not clear at present , and indeed it is not clear whether the observed changes in insulin binding to erythrocytes in cld correlate with the changes in insulin binding to insulin sensitive tissues .
some investigators still believe that reduced insulin receptors in hepatic tissues may be the primary cause of insulin resistance in patients with cld .
however , no such direct evidence has been demonstrated until now , and at least erythrocyte insulin receptors are concerned , down - regulation of insulin receptors seems to be an epiphenomenon of hyperinsulinemia and might not be very important in the genesis of insulin resistance in these patients .
 125i - insulin binding to erythrocytes was measured to evaluate the role of insulin receptors in the mechanism of insulin resistance in partients with chronic liver disease ( cld ) . 
 patients with cld were divided into two groups by the fasting plasma glucose level ( fbs ) and the glucose disappearance rate ( k - glucose ) during intravenous glucose tolerance tests : one with normal fbs and normal k - glucose ( group i ) , and the other with higher fbs and lower k - glucose ( group ii ) . 
 insulin receptor bindings were reduced in group i patients compared with those of normal controls , but were not reduced in group ii patients . 
 fasting insulin levels were significantly higher in group i patients than those in normal subjects , and down - regulation by hyperinsulinemia might be the cause of reduced insulin receptor bindings in these patients 
 . however down - regulation of insulin receptors was not associated with glucose intolerance . 
 so it is suggested that insulin resistance in cld is not due to a primary defect of insulin receptors in peripheral tissues , and down - regulation of insulin receptors is an epiphenomenon of hyperinsulinemia . 
INTRODUCTION
MATERIALS AND METHODS
INSULIN BINDING STUDY
STATISTICAL ANALYSIS
RESULTS
DISCUSSION