eighteen sedentary obese subjects with newly diagnosed type 2 diabetes who were not being treated with diabetes medications were randomized to one of three treatment groups : 1 ) placebo ( saline ; four men and two women ; age : 60  3 years ; bmi : 36  1 kg / m ; hba1c : 7.8  0.3% ; fasting plasma glucose concentration : 8.0  0.4 mmol / l ) , 2 ) low - dose ( 30 mg / day ) recombinant methionyl human ( r - met hu ) leptin ( two men and four women ; age : 53  7 years ; bmi : 35  1 kg / m ; hba1c : 8.2  0.4% ; fasting plasma glucose concentration : 8.5  0.6 mmol / l ) , or 3 ) high - dose ( 80 mg / day ) r - met hu leptin ( three men and three women ; age : 54  4 years ; bmi : 36  1 kg / m ; hba1c : 7.8  0.3% ; fasting plasma glucose concentration : 9.5  0.5 mmol / l ) . none of the subjects smoked tobacco , were pregnant or lactating , had significant organ system dysfunction , or took medications known to affect metabolism .
all subjects provided written , informed consent before participating in the study , which was approved by the human studies committee of washington university school of medicine .
subjects were admitted to the clinical research unit ( cru ) in the afternoon before the clamp procedure .
total body mass , fat mass , and fat - free mass were determined by using dual - energy x - ray absorptiometry ( hologic qdr 4500 , waltham , ma ) . at 0600 h the next morning , after an overnight fast , one catheter was inserted into a forearm vein for infusion and a second catheter was inserted into a hand vein , which was heated to 55c by using a thermostatically controlled box , to obtain arterialized blood samples . at 0700 h , a primed , continuous infusion of
[ 6,6-h2]glucose was started and maintained until the end of the study , 10.5 h later . at 0900 h , a continuous infusion of [ 1-c]palmitate and
a primed , continuous infusion of [ 1,1,2,3,3-h5]glycerol were started and maintained for 5 h ( until the end of stage 1 of the clamp ) . at 1030 h , a two - stage ( 3.5 h each ) euglycemic - hyperinsulinemic clamp was started . during stage 1
( 3.5 to 7.0 h ) , insulin was infused at a rate of 20 mu / m body surface area ( bsa)/min ( initiated by 80 mu / m bsa / min for 5 min and then 40 mu / m bsa / min for 5 min ) . during stage 2
( 7.0 to 10.5 h ) , insulin was infused at a rate of 50 mu / m bsa / min ( initiated by 200 mu / m bsa / min for 5 min and then 100 mu / m bsa / min for 5 min ) .
these two insulin infusion rates were chosen to evaluate liver and adipose tissue insulin sensitivity ( low - dose insulin infusion to submaximally suppress hepatic glucose production and lipolysis of adipose tissue triglycerides ) and skeletal muscle insulin sensitivity ( high - dose insulin infusion to adequately stimulate muscle glucose uptake ) .
euglycemia was maintained at a blood glucose concentration of 5.5 mmol / l by variable rate infusion of 20% dextrose enriched to 2.5% with [ 6,6-h2]glucose .
the infusion rates of [ 6,6-h2]glucose , [ 1-c]palmitate , and [ 1,1,2,3,3-h5]glycerol were reduced by 50% during stage 1 , and the infusion rate of [ 6,6-h2]glucose was reduced to 25% during stage 2 to account for changes in endogenous glucose production and lipolysis .
blood samples to determine plasma leptin and insulin concentrations and substrate ( glucose , palmitate , and glycerol ) tracer - to - tracee ratios ( ttrs ) were obtained before beginning the tracer infusion and every 10 min during the final 30 min of the basal period and stages 1 and 2 of the clamp .
plasma insulin and leptin concentrations were measured by using radioimmunoassay ( linco research , st .
plasma glucose , palmitate , and glycerol ttrs were determined by using gas chromatography - mass spectroscopy .
substrate rate of appearance ( ra ) in plasma was calculated by dividing the substrate tracer infusion rate by the average plasma substrate ttr .
endogenous glucose production rate during the clamp procedure was calculated by subtracting the exogenous glucose infusion rate from glucose ra ; glucose rd was calculated as the sum of endogenous glucose ra and the rate of infused glucose .
hepatic insulin sensitivity was assessed by using the hepatic insulin sensitivity index , calculated as the product of basal endogenous glucose ra and plasma insulin concentration ( 13 ) .
after completion of the clamp procedure , subjects were randomized to treatment with subcutaneous low - dose r - met hu leptin ( 15-mg bid ) , high - dose r - met hu leptin ( 40-mg bid ) , or placebo ( saline , 2-ml bid ) for 14 days , given by a research nurse during twice - daily home visits . the low dose was intended to provide a modest ( threefold ) increase in plasma leptin concentrations , whereas the high dose was intended to provide a pharmacological exposure ( 200-fold above baseline ) .
subjects were instructed to maintain their usual dietary and physical activity habits and were weighed daily by the research nurse .
an increase in dietary intake was encouraged in any subject who demonstrated a trend toward a decrease in body weight . on day 14 of treatment
, subjects were readmitted to the cru , where the body composition analyses and the clamp procedure were repeated .
anova with repeated measures was used to compare between ( placebo vs. low - dose and high - dose leptin ) and within ( before vs. after treatment ) group differences .
based on our own data evaluating the reproducibility of the effect of insulin on glucose and fatty acid kinetics during a euglycemic - hyperinsulinemic clamp in obese , insulin - resistant subjects ( 14 ) , we estimated that six subjects in each group would allow us to detect a 25 , 30 , and 35% between - group difference in insulin - mediated effects of glucose ra , palmitate ra , and glucose rd , respectively , at the 0.05 -level and a -value of 0.20 ( i.e. , 80% power ) .
eighteen sedentary obese subjects with newly diagnosed type 2 diabetes who were not being treated with diabetes medications were randomized to one of three treatment groups : 1 ) placebo ( saline ; four men and two women ; age : 60  3 years ; bmi : 36  1 kg / m ; hba1c : 7.8  0.3% ; fasting plasma glucose concentration : 8.0  0.4 mmol / l ) , 2 ) low - dose ( 30 mg / day ) recombinant methionyl human ( r - met hu ) leptin ( two men and four women ; age : 53  7 years ; bmi : 35  1 kg / m ; hba1c : 8.2  0.4% ; fasting plasma glucose concentration : 8.5  0.6 mmol / l ) , or 3 ) high - dose ( 80 mg / day ) r - met hu leptin ( three men and three women ; age : 54  4 years ; bmi : 36  1 kg / m ; hba1c : 7.8  0.3% ; fasting plasma glucose concentration : 9.5  0.5 mmol / l ) . none of the subjects smoked tobacco , were pregnant or lactating , had significant organ system dysfunction , or took medications known to affect metabolism .
all subjects provided written , informed consent before participating in the study , which was approved by the human studies committee of washington university school of medicine .
subjects were admitted to the clinical research unit ( cru ) in the afternoon before the clamp procedure .
total body mass , fat mass , and fat - free mass were determined by using dual - energy x - ray absorptiometry ( hologic qdr 4500 , waltham , ma ) . at 0600 h the next morning , after an overnight fast , one catheter was inserted into a forearm vein for infusion and a second catheter was inserted into a hand vein , which was heated to 55c by using a thermostatically controlled box , to obtain arterialized blood samples . at 0700 h , a primed , continuous infusion of [ 6,6-h2]glucose was started and maintained until the end of the study , 10.5 h later . at 0900 h , a continuous infusion of [ 1-c]palmitate and a primed , continuous infusion of [ 1,1,2,3,3-h5]glycerol were started and maintained for 5 h ( until the end of stage 1 of the clamp ) . at 1030 h , a two - stage ( 3.5 h each ) euglycemic - hyperinsulinemic clamp was started . during stage 1
( 3.5 to 7.0 h ) , insulin was infused at a rate of 20 mu / m body surface area ( bsa)/min ( initiated by 80 mu / m bsa / min for 5 min and then 40 mu / m bsa / min for 5 min ) . during stage 2
( 7.0 to 10.5 h ) , insulin was infused at a rate of 50 mu / m bsa / min ( initiated by 200 mu / m bsa / min for 5 min and then 100 mu / m bsa / min for 5 min ) .
these two insulin infusion rates were chosen to evaluate liver and adipose tissue insulin sensitivity ( low - dose insulin infusion to submaximally suppress hepatic glucose production and lipolysis of adipose tissue triglycerides ) and skeletal muscle insulin sensitivity ( high - dose insulin infusion to adequately stimulate muscle glucose uptake ) .
euglycemia was maintained at a blood glucose concentration of 5.5 mmol / l by variable rate infusion of 20% dextrose enriched to 2.5% with [ 6,6-h2]glucose .
the infusion rates of [ 6,6-h2]glucose , [ 1-c]palmitate , and [ 1,1,2,3,3-h5]glycerol were reduced by 50% during stage 1 , and the infusion rate of [ 6,6-h2]glucose was reduced to 25% during stage 2 to account for changes in endogenous glucose production and lipolysis .
blood samples to determine plasma leptin and insulin concentrations and substrate ( glucose , palmitate , and glycerol ) tracer - to - tracee ratios ( ttrs ) were obtained before beginning the tracer infusion and every 10 min during the final 30 min of the basal period and stages 1 and 2 of the clamp .
plasma insulin and leptin concentrations were measured by using radioimmunoassay ( linco research , st .
plasma glucose , palmitate , and glycerol ttrs were determined by using gas chromatography - mass spectroscopy .
substrate rate of appearance ( ra ) in plasma was calculated by dividing the substrate tracer infusion rate by the average plasma substrate ttr .
endogenous glucose production rate during the clamp procedure was calculated by subtracting the exogenous glucose infusion rate from glucose ra ; glucose rd was calculated as the sum of endogenous glucose ra and the rate of infused glucose .
hepatic insulin sensitivity was assessed by using the hepatic insulin sensitivity index , calculated as the product of basal endogenous glucose ra and plasma insulin concentration ( 13 ) .
subjects were admitted to the clinical research unit ( cru ) in the afternoon before the clamp procedure .
total body mass , fat mass , and fat - free mass were determined by using dual - energy x - ray absorptiometry ( hologic qdr 4500 , waltham , ma ) . at 0600 h the next morning , after an overnight fast , one catheter was inserted into a forearm vein for infusion and a second catheter was inserted into a hand vein , which was heated to 55c by using a thermostatically controlled box , to obtain arterialized blood samples . at 0700 h , a primed , continuous infusion of [ 6,6-h2]glucose was started and maintained until the end of the study , 10.5 h later . at 0900 h , a continuous infusion of [ 1-c]palmitate and a primed , continuous infusion of [ 1,1,2,3,3-h5]glycerol were started and maintained for 5 h ( until the end of stage 1 of the clamp ) . at 1030 h , a two - stage ( 3.5 h each ) euglycemic - hyperinsulinemic clamp was started . during stage 1
( 3.5 to 7.0 h ) , insulin was infused at a rate of 20 mu / m body surface area ( bsa)/min ( initiated by 80 mu / m bsa / min for 5 min and then 40 mu / m bsa / min for 5 min ) . during stage 2
( 7.0 to 10.5 h ) , insulin was infused at a rate of 50 mu / m bsa / min ( initiated by 200 mu / m bsa / min for 5 min and then 100 mu / m bsa / min for 5 min ) .
these two insulin infusion rates were chosen to evaluate liver and adipose tissue insulin sensitivity ( low - dose insulin infusion to submaximally suppress hepatic glucose production and lipolysis of adipose tissue triglycerides ) and skeletal muscle insulin sensitivity ( high - dose insulin infusion to adequately stimulate muscle glucose uptake ) .
euglycemia was maintained at a blood glucose concentration of 5.5 mmol / l by variable rate infusion of 20% dextrose enriched to 2.5% with [ 6,6-h2]glucose .
the infusion rates of [ 6,6-h2]glucose , [ 1-c]palmitate , and [ 1,1,2,3,3-h5]glycerol were reduced by 50% during stage 1 , and the infusion rate of [ 6,6-h2]glucose was reduced to 25% during stage 2 to account for changes in endogenous glucose production and lipolysis .
blood samples to determine plasma leptin and insulin concentrations and substrate ( glucose , palmitate , and glycerol ) tracer - to - tracee ratios ( ttrs ) were obtained before beginning the tracer infusion and every 10 min during the final 30 min of the basal period and stages 1 and 2 of the clamp .
plasma insulin and leptin concentrations were measured by using radioimmunoassay ( linco research , st .
plasma glucose , palmitate , and glycerol ttrs were determined by using gas chromatography - mass spectroscopy .
substrate rate of appearance ( ra ) in plasma was calculated by dividing the substrate tracer infusion rate by the average plasma substrate ttr .
endogenous glucose production rate during the clamp procedure was calculated by subtracting the exogenous glucose infusion rate from glucose ra ; glucose rd was calculated as the sum of endogenous glucose ra and the rate of infused glucose .
hepatic insulin sensitivity was assessed by using the hepatic insulin sensitivity index , calculated as the product of basal endogenous glucose ra and plasma insulin concentration ( 13 ) .
after completion of the clamp procedure , subjects were randomized to treatment with subcutaneous low - dose r - met hu leptin ( 15-mg bid ) , high - dose r - met hu leptin ( 40-mg bid ) , or placebo ( saline , 2-ml bid ) for 14 days , given by a research nurse during twice - daily home visits .
the low dose was intended to provide a modest ( threefold ) increase in plasma leptin concentrations , whereas the high dose was intended to provide a pharmacological exposure ( 200-fold above baseline ) .
subjects were instructed to maintain their usual dietary and physical activity habits and were weighed daily by the research nurse .
an increase in dietary intake was encouraged in any subject who demonstrated a trend toward a decrease in body weight . on day 14 of treatment ,
subjects were readmitted to the cru , where the body composition analyses and the clamp procedure were repeated .
anova with repeated measures was used to compare between ( placebo vs. low - dose and high - dose leptin ) and within ( before vs. after treatment ) group differences .
a p value of  0.05 was considered statistically significant . based on our own data evaluating the reproducibility of the effect of insulin on glucose and fatty acid kinetics during a euglycemic - hyperinsulinemic clamp in obese , insulin - resistant subjects ( 14 )
, we estimated that six subjects in each group would allow us to detect a 25 , 30 , and 35% between - group difference in insulin - mediated effects of glucose ra , palmitate ra , and glucose rd , respectively , at the 0.05 -level and a -value of 0.20 ( i.e. , 80% power ) .
baseline body weight and body composition were similar between groups , and body weight and body composition did not change in any group after 2 weeks of treatment ( table 1 ) .
plasma leptin remained unchanged in the placebo group and increased by threefold and 150-fold , respectively , in the low - dose and high - dose leptin treatment groups ( table 1 ) . body composition and plasma leptin concentrations before and after placebo and leptin treatment values are means  se . * value significantly different from corresponding value before , p  0.05 .
there were no differences between groups in basal plasma insulin concentrations , the hepatic insulin sensitivity index , and basal substrate kinetics before and after placebo or leptin treatment ( table 2 ) .
plasma insulin concentrations and substrate kinetics during the clamp procedure were also not different between groups before and after placebo or either dose of leptin treatment ( table 2 ) .
plasma insulin concentration increased from 17 mu / l during basal conditions to 35 mu / l during stage 1 and to 85 mu / l during stage 2 both before and after 14 days of therapy .
insulin - mediated suppression of endogenous glucose ra ( 60 and 80% decrease during stages 1 and 2 , respectively ) and insulin - mediated increase in glucose rd ( 75% increase during stage 2 ) was not different between groups before and after placebo or either dose of leptin treatment .
palmitate and glycerol ra decreased by 50 and 35% , respectively , during stage 1 of the clamp procedure and were not different between groups before and after placebo or either dose of leptin treatment .
plasma insulin concentrations and metabolic kinetics during basal conditions and during the hyperinsulinemic - euglycemic clamp before and after placebo and leptin treatment values are means  se .
basal , stage 1 , and stage 2 refer to the hyperinsulinemic - euglycemic clamp as described in the text .
baseline body weight and body composition were similar between groups , and body weight and body composition did not change in any group after 2 weeks of treatment ( table 1 ) .
plasma leptin remained unchanged in the placebo group and increased by threefold and 150-fold , respectively , in the low - dose and high - dose leptin treatment groups ( table 1 ) . body composition and plasma leptin concentrations before and after placebo and leptin treatment values are means  se . * value significantly different from corresponding value before , p  0.05 .
there were no differences between groups in basal plasma insulin concentrations , the hepatic insulin sensitivity index , and basal substrate kinetics before and after placebo or leptin treatment ( table 2 ) .
plasma insulin concentrations and substrate kinetics during the clamp procedure were also not different between groups before and after placebo or either dose of leptin treatment ( table 2 ) .
plasma insulin concentration increased from 17 mu / l during basal conditions to 35 mu / l during stage 1 and to 85 mu / l during stage 2 both before and after 14 days of therapy .
insulin - mediated suppression of endogenous glucose ra ( 60 and 80% decrease during stages 1 and 2 , respectively ) and insulin - mediated increase in glucose rd ( 75% increase during stage 2 ) was not different between groups before and after placebo or either dose of leptin treatment .
palmitate and glycerol ra decreased by 50 and 35% , respectively , during stage 1 of the clamp procedure and were not different between groups before and after placebo or either dose of leptin treatment .
plasma insulin concentrations and metabolic kinetics during basal conditions and during the hyperinsulinemic - euglycemic clamp before and after placebo and leptin treatment values are means  se .
basal , stage 1 , and stage 2 refer to the hyperinsulinemic - euglycemic clamp as described in the text .
leptin is an important regulator of insulin action ; both leptin deficiency and leptin resistance are associated with insulin - resistant glucose metabolism ( 4,12 ) .
furthermore , leptin replacement improves insulin sensitivity in subjects with leptin deficiency ( 58 ) .
part of the beneficial effect of leptin replacement therapy could be a result of reductions in body weight in both leptin - deficient rodents and rodents with high - fat diet 
however , leptin therapy increases insulin sensitivity in the absence of a significant decrease in body weight or causes a greater reduction in blood glucose concentration after leptin - induced weight loss than after weight loss induced by pair - feeding alone in wild - type and leptin - deficient rodent models ( 1,2,16 ) and improves insulin sensitivity in leptin - deficient subjects , even in the absence of significant changes in body weight ( 7,8,17 ) .
in contrast , our data demonstrate that increasing leptin availability above normal plasma concentrations by treatment with r - met hu leptin does not have weight - loss  independent , clinically important effects on insulin sensitivity in obese people with type 2 diabetes . these results are consistent with data from previous leptin weight loss trials , which found that 812 weeks of leptin therapy and a low - calorie diet did not cause a greater change in plasma glucose or insulin concentrations than placebo therapy and a low - calorie diet ( 18,19 ) .
the reason(s ) for the discrepancy in results from our study and those conducted in leptin - deficient subjects and rodent obesity models is unclear .
we studied only subjects with newly diagnosed , and presumably more reversible , type 2 diabetes and used sensitive measures to evaluate insulin sensitivity in vivo to increase our ability to detect an effect of leptin therapy on insulin action .
furthermore , we gave low and high doses of r - met hu leptin to our subjects to ensure adequate plasma leptin concentrations were achieved and to evaluate potential dose - dependent effects .
it is unlikely that the 2-week intervention in our study was not long enough to affect insulin action , because leptin administration alters glucose metabolism within several hours in rodents ( 11 ) and the beneficial metabolic effects of leptin replacement therapy in subjects with leptin deficiency occur within 1 week of treatment ( 20 ) .
in addition , current treatment strategies in obese insulin - resistant subjects , such as weight loss and pharmacotherapy , demonstrate improved insulin sensitivity can occur within days ( 13,21,22 ) .
therefore , our data demonstrate that leptin treatment has different metabolic effects in subjects with leptin deficiency ( who have almost no body fat ) than in obese subjects ( who have high plasma leptin concentrations and large amounts of body fat ) .
however , we can not exclude the possibility that leptin treatment would affect insulin action in overweight or obese subjects who have a lower bmi than our cohort .
in addition , our results demonstrate that leptin does not worsen insulin sensitivity , which has been suggested because of data obtained from studies conducted in obese people and isolated adipocytes . at
any given bmi , increased plasma leptin concentration is associated with greater insulin resistance ( 9,11 ) , and withdrawal of chronic leptin therapy in leptin - deficient subjects improves insulin sensitivity ( 23 ) . furthermore , large doses of leptin decrease insulin signaling and metabolic actions of insulin in isolated rat adipocytes ( 24,25 ) . in our study
although our study was conducted in a small number of subjects , it is unlikely we missed an important therapeutic effect of leptin as a result of inadequate statistical power , because there was not even a trend in leptin - induced changes in substrate kinetics with either dose of leptin therapy compared with placebo .
in contrast , current treatment strategies for insulin resistance , such as weight loss and pharmacotherapy , improve insulin - mediated glucose uptake by 25% or more ( 13,26,27 ) . in summary
, we found that short - term treatment with either low - dose or high - dose r - met hu leptin did not improve liver , skeletal muscle , or adipose tissue insulin sensitivity in weight stable , obese subjects with type 2 diabetes .
the absence of a therapeutic effect of leptin in our study , within the context of the observed beneficial effects of leptin replacement therapy in subjects with leptin deficiency ( 4,7,8,12 ) , suggests that a small amount of leptin is important for normal insulin action , but increasing leptin availability above normal plasma concentrations does not have weight loss 
 objectiveleptin therapy improves insulin sensitivity in people with leptin deficiency , but it is not known whether it improves insulin action in people who are not leptin deficient . 
 the purpose of the current study was to determine whether leptin treatment has weight loss  
 independent effects on insulin action in obese subjects with type 2 diabetes.research design and methodswe conducted a randomized , placebo - controlled trial in obese subjects ( bmi : 35.4  0.6 kg / m2 ; mean  se ) with newly diagnosed type 2 diabetes . 
 subjects were randomized to treatment with placebo ( saline ) , low - dose ( 30 mg / day ) , or high - dose ( 80 mg / day ) recombinant methionyl human ( r - met hu ) leptin for 14 days . 
 multiorgan insulin sensitivity before and after treatment was evaluated by using the hyperinsulinemic - euglycemic clamp procedure in conjunction with stable isotopically labeled tracer infusions to measure glucose , glycerol , and fatty acid kinetics.resultslow-dose and high - dose leptin treatment resulted in a threefold ( p < 0.01 ) and 150-fold ( p < 0.001 ) increase in basal plasma leptin concentrations , respectively . however , neither low - dose nor high - dose therapy had an effect on insulin - mediated suppression of glucose , glycerol , or palmitate rates of appearance into plasma compared with placebo . 
 in addition , leptin treatment did not increase insulin - mediated stimulation of glucose disposal compared with placebo ( 14.3  3.1 , 18.4  3.6 , 16.7  2.4 vs. 17.5  2.5 , 20.7  3.0 , 19.1  3.3 mol / kg body wt / min before vs. after treatment in the placebo , low - dose , and high - dose leptin groups , respectively).conclusionsr - met hu leptin does not have weight loss  
 independent , clinically important effects on insulin sensitivity in obese people with type 2 diabetes . 
RESEARCH DESIGN AND METHODS
Subjects.
Experimental protocol
None
Intervention.
Statistical analyses and sample size considerations.
RESULTS
Body composition and plasma leptin.
Substrate kinetics and insulin sensitivity.
DISCUSSION