patients were recruited from the oregon health and science university ( ohsu ) outpatient clinics in portland , oregon . patients who were pregnant or had cardiovascular , cerebrovascular , kidney , or liver disease or any other uncontrolled chronic medical conditions were excluded . other exclusion criteria included oral or parenteral corticosteroid use , immunosuppressant use , visual or physical impairments that impede the use of a continuous glucose - monitoring device , insulin or glucagon allergy , hypoglycemia unawareness or hospitalization within the past 2 years for severe hypoglycemia , serum insulin antibody titer > 100 u / ml , or requirement of > 200 units insulin / day . the research protocol was approved by the ohsu institutional review board , and all subjects provided written informed consent . permission to carry out these studies was granted by the u.s . g080130 ) . a total of 22 closed - loop studies in 14 subjects were performed . age was 36.7  3.7 years , with a duration of diabetes of 14.1  3.1 years . the data from this study were excluded from the analysis , leaving 21 datasets from 13 subjects . as requested by the fda , five subjects participated in single 9-h studies with both insulin and glucagon to assess the safety and effectiveness of the study protocol . eight subjects underwent one study with insulin and placebo and one with insulin and glucagon ( see fig . , it was delivered using high - gain parameters in seven studies and using low - gain parameters in six . the treatment order of each paired study was determined by a randomization scheme . in paired studies , study diagram depicting the number of subjects studied under each condition and the study lengths . subjects wore two subcutaneous glucose sensors , either dexcom seven plus or medtronic guardian real - time glucose sensors . sensors were placed 824 h prior to beginning the study . for subjects taking long - acting insulin at night , the dose venous glucose was measured every 10 min in duplicate using a hemocue glucose 201 analyzer . glucose sensor readings were recorded from the receivers every 5 min . for the first 2 h , after the first 2 h , the sensed glucose values from the sensor with better accuracy were input into the algorithm every 5 min to determine the hormone delivery rates . if the sensor accuracy became suboptimal , defined as a median absolute relative difference ( mard ) exceeding 20% or median absolute difference ( mad ) exceeding 20 mg / dl , control was switched to the other sensor . if the accuracy of both sensors was poor , control was switched to venous glucose and the sensors were recalibrated . sensors were calibrated at a minimum of every 12 h. the fading memory proportional derivative ( fmpd ) algorithm ( 9,11 ) was used to determine the insulin and subcutaneous glucagon ( or placebo ) delivery rates . aspart insulin ( novo nordisk ) was delivered subcutaneously via an animas ir 1000 insulin pump . glucagon or saline placebo was given through a subcutaneous catheter via a medfusion 2001 syringe pump . one milligram of glucagon ( novo nordisk ) was mixed with 3 ml of sterile water . the glucagon preparation was freshly reconstituted every 8 h. a study physician was onsite at all times and had the ability to override the hormone infusion rates called for by the fmpd algorithm , which occurred only 1.7% of the time . either a registered nurse or physician was responsible for adjusting the insulin delivery rate and glucagon delivery rate every 5 min , based on the controller output . the fmpd algorithm determined the hormone delivery rates based on proportional error , defined as the difference between the current glucose level and the target level , and the derivative error , defined as the rate of change of the glucose . this weighting provides an adaptive component to the algorithm , as described previously ( 9,11 ) . in simple terms , the insulin rate was increased for high or rising glucose levels and glucagon was given for low or falling glucose levels . the basal insulin infusion rate ( in units per hour ) was given at a rate of 35% of the patient 's typical total daily insulin dose , divided by 24 . in the fmpd algorithm , the gain factors determined the degree to which proportional or derivative errors led to changes in hormone delivery rates . positive proportional errors ( glucose level above target ) and positive derivative errors ( rising glucose level ) called for an increase in the insulin delivery rate . the overall insulin delivery rate was determined by adding the rates called for by the proportional error ( iirpe ) , the derivative error ( iirde ) , and the basal insulin rate . the proportional error gain factor was 1.2  10  0.078  10 units / kg per mg / dl / h for glucagon studies and 1.3  10 units / kg per mg / dl / h for placebo studies . the derivative error gain factor was 2.0  10  0.096  10 units / kg per mg / dl for glucagon studies and was 2.0  10 units / kg per mg / dl for placebo studies . the mean blood glucose target was 110  1 mg / dl for glucagon studies and 110 mg / dl for placebo studies . there were no significant differences between any of these parameters between the groups . for subjects who underwent two closed - loop studies , insulin on board , the amount of insulin that had been delivered and was assumed to be active , was continually estimated using a model that we derived from data published by holmes et al . the insulin infusion was discontinued if the estimated insulin on board reached 15% of the subject 's estimated total daily insulin requirement . the proportional and derivative error gain factors for glucagon were negative , such that negative proportional and derivative errors called for an increase in the glucagon rate . for glucagon , the average weighted proportional error was calculated over a 15 min interval and the average weighted derivative error was calculated over a 10 min interval . four subjects completed 9-h studies and two subjects completed 28-h studies with low - gain factor settings . in these low - gain glucagon studies , the mean proportional error gain factor was 0.23  0.04 ml / kg per mg / dl / h , the mean derivative error gain factor was 0.06  0.009 ml / kg per mg / dl , and target glucose for glucagon infusion was 108  3 mg / dl . two subjects completed 9-h studies and five subjects completed 28-h studies with high - gain factor settings . for all of these high - gain glucagon studies , the proportional error gain factor was 2.70 ml / kg per mg / dl / hour , the derivative gain factor was 0.60 ml / kg per mg / dl , and the target glucose for glucagon infusion was 97  1 mg / dl . to avoid overdelivery of glucagon , when total glucagon delivery over the prior 50 min reached a ceiling of 1.0 g / kg , the algorithm initiated a refractory period for the subsequent 50 min , during which glucagon could not be delivered . thus , short pulses of glucagon delivery over 510 min were followed by the absence of glucagon delivery for 50 min . the insulin rate was reduced by 75% for 40 min after each maximal glucagon pulse . patients were given two meals during each 9-h study and four meals during each 28-h study . each meal was announced to the controller and an open loop premeal bolus was given . aspart insulin was given 010 min before meals , depending on the subject 's premeal glucose level . for low - gain glucagon studies , 53.3  7.0% of usual premeal insulin dose was given . the amount of premeal insulin was increased after the first four studies because of a pattern of postprandial hyperglycemia in those studies . for all placebo and high - gain glucagon studies , subjects were treated for hypoglycemia if the venous glucose value fell below 70 mg / dl . for glucose levels 6069 mg / dl , subjects were given 15 g oral carbohydrate , and the treatment repeated as needed every 15 min . for a glucose value < 60 mg / dl , 10 g dextrose was given intravenously . arterialized venous glucose values , not sensed glucose values , were used to compare hypoglycemia and glucose control between groups . glucose area under the curve ( auc ) was calculated as published elsewhere ( 13 ) . minutes in the hypoglycemic range , defined as glucose < 70 mg / dl , hypoglycemic events , treatments for hypoglycemia , units of insulin delivered , and micrograms of glucagon delivered were normalized to 24 h for data from both 9- and 28-h studies . sensor accuracy was calculated by comparing sensor glucose to reference glucose values ( 14 ) . in the fmpd algorithm , the gain factors determined the degree to which proportional or derivative errors led to changes in hormone delivery rates . there were separate gain factors for insulin and glucagon . positive proportional errors ( glucose level above target ) and positive derivative errors ( rising glucose level ) called for an increase in the insulin delivery rate . the overall insulin delivery rate was determined by adding the rates called for by the proportional error ( iirpe ) , the derivative error ( iirde ) , and the basal insulin rate . the proportional error gain factor was 1.2  10  0.078  10 units / kg per mg / dl / h for glucagon studies and 1.3  10 units / kg per mg / dl / h for placebo studies . the derivative error gain factor was 2.0  10  0.096  10 units / kg per mg / dl for glucagon studies and was 2.0  10 units / kg per mg / dl for placebo studies . the mean blood glucose target was 110  1 mg / dl for glucagon studies and 110 mg / dl for placebo studies . there were no significant differences between any of these parameters between the groups . for subjects who underwent two closed - loop studies , insulin on board , the amount of insulin that had been delivered and was assumed to be active , was continually estimated using a model that we derived from data published by holmes et al . the insulin infusion was discontinued if the estimated insulin on board reached 15% of the subject 's estimated total daily insulin requirement . the proportional and derivative error gain factors for glucagon were negative , such that negative proportional and derivative errors called for an increase in the glucagon rate . for glucagon , the average weighted proportional error was calculated over a 15 min interval and the average weighted derivative error was calculated over a 10 min interval . four subjects completed 9-h studies and two subjects completed 28-h studies with low - gain factor settings . in these low - gain glucagon studies , the mean proportional error gain factor was 0.23  0.04 ml / kg per mg / dl / h , the mean derivative error gain factor was 0.06  0.009 ml / kg per mg / dl , and target glucose for glucagon infusion was 108  3 mg / dl . two subjects completed 9-h studies and five subjects completed 28-h studies with high - gain factor settings . for all of these high - gain glucagon studies , the proportional error gain factor was 2.70 ml / kg per mg / dl / hour , the derivative gain factor was 0.60 ml / kg per mg / dl , and the target glucose for glucagon infusion was 97  1 mg / dl . to avoid overdelivery of glucagon , when total glucagon delivery over the prior 50 min reached a ceiling of 1.0 g / kg , the algorithm initiated a refractory period for the subsequent 50 min , during which glucagon could not be delivered . thus , short pulses of glucagon delivery over 510 min were followed by the absence of glucagon delivery for 50 min . the insulin rate was reduced by 75% for 40 min after each maximal glucagon pulse . patients were given two meals during each 9-h study and four meals during each 28-h study . each meal was announced to the controller and an open loop premeal bolus was given . aspart insulin was given 010 min before meals , depending on the subject 's premeal glucose level . for low - gain glucagon studies , 53.3  7.0% of usual premeal insulin dose was given . the amount of premeal insulin was increased after the first four studies because of a pattern of postprandial hyperglycemia in those studies . for all placebo and high - gain glucagon studies , subjects were treated for hypoglycemia if the venous glucose value fell below 70 mg / dl . for glucose levels 6069 mg / dl , subjects were given 15 g oral carbohydrate , and the treatment repeated as needed every 15 min . for a glucose value < 60 mg / dl , 10 g dextrose was given intravenously . arterialized venous glucose values , not sensed glucose values , were used to compare hypoglycemia and glucose control between groups . glucose area under the curve ( auc ) was calculated as published elsewhere ( 13 ) . minutes in the hypoglycemic range , defined as glucose < 70 mg / dl , hypoglycemic events , treatments for hypoglycemia , units of insulin delivered , and micrograms of glucagon delivered were normalized to 24 h for data from both 9- and 28-h studies . sensor accuracy was calculated by comparing sensor glucose to reference glucose values ( 14 ) . six women and seven men with type 1 diabetes participated in a total of 21 human closed - loop studies with a duration of 21.5  2.0 h. seven subjects received glucagon delivered in a brisk fashion ( high - gain ) and six subjects received glucagon delivered in a slower fashion ( low - gain ) . in both the high- and low - gain glucagon studies , glucagon was typically delivered at times of impending hypoglycemia when glucose was 90120 mg / dl , depending on the rate of glucose decline ( fig . venous blood glucose is noted by black diamonds , insulin delivery rate by a gray line , and glucagon delivery rate by rectangles . note that glucagon is delivered by algorithm in the late postprandial period at times of impending hypoglycemia . the high - gain glucagon results ( paired analysis ) , low - gain glucagon results ( unpaired analysis ) , and combined high- and low - gain glucagon results ( unpaired analysis ) are presented separately below . one subject who received high - gain glucagon but did not return for a placebo study was included in the combined results but was not included in the paired high - gain analysis . in six subjects who underwent both a high - gain glucagon study and a placebo study , there was a 56% reduction in time spent in the hypoglycemic range ( 18  11 vs. 41  13 min / day , p = 0.01 ) . the number of hypoglycemic events , with events lasting > 20 min being considered a new event , was also significantly reduced during the high - gain glucagon versus placebo studies ( 1.0  0.6 vs. 2.1  0.6 events / day , p = 0.01 ) , as was the number of oral or intravenous carbohydrate treatments for hypoglycemia ( 1.4  0.8 vs. 4.0  1.4 treatments / day , p = 0.01 ) . there was no significant difference in mean glucose between the high - gain glucagon versus placebo studies ( 138  17 vs. 131  17 mg / dl , p = ns ) , as shown in fig . the mean fasting glucose was also quite similar ( 123  14 vs. 120  15 mg / dl , p = ns ) . there was a nonsignificant trend toward a higher postprandial glucose in high - gain glucagon versus placebo studies , defined as mean value 0180 min after meals ( 157  18 vs. 144  17 mg / dl , p = ns ) . the amount of insulin delivered during the high - gain glucagon versus placebo studies was nearly identical ( 48.9  6.2 vs. 48.3  5.5 units per day , p = ns ) . summary of glucose levels ( means  se ) , insulin delivery rate , and , for glucagon studies , the glucagon delivery rate . venous blood glucose is noted by gray diamonds , insulin delivery rate by a black line , glucagon delivery rate by a light gray line , and meals by black triangles . insulin delivery and overall glycemic control were similar in both conditions . in six subjects who received low - gain glucagon compared with the eight subjects who received placebo , there was a nonsignificant reduction in time in the hypoglycemic range ( 15  8 vs. 40  10 min / day , p = ns ) . there was also a trend toward a reduction in the number of hypoglycemic events that did not reach statistical significance ( 1.4  0.7 vs. 2.3  0.5 events / day , p = ns ) . there was a reduction in the number of treatments for hypoglycemia in studies with low - gain glucagon of borderline significance ( 1.0  0.7 vs. 3.9  1.0 treatments / day , p = 0.05 ) . mean glucose was somewhat higher in low - gain glucagon versus placebo studies ( 157  24 vs. 135  16 mg / dl , p = 0.04 ) . there was also a trend toward higher fasting glucose in the low - gain glucagon versus placebo studies ( 137  20 vs. 122  13 mg / dl , p = ns ) . there was a similar trend , of borderline statistical significance , suggesting a larger elevation in postprandial glucose in the low - gain glucagon versus placebo studies ( 179  26 vs. 151  18 mg / dl , p = 0.05 ) . there was a nonsignificant difference in insulin delivered in low - gain glucagon versus placebo studies ( 60.1  14.1 vs. 46.9  5.5 units / day ) . the mean dose of glucagon delivered during the low - gain glucagon studies was higher than the high - gain glucagon studies but did not reach statistical significance ( 746  134 vs. 516  108 g / day , p = ns ) . glucagon , when given either via high- or low - gain , compared with placebo , led to a 63% reduction of time spent in the hypoglycemic range ( 15  6 vs. 40  10 min / day , p = 0.04 ) . the number of hypoglycemic events per day was not significantly different between glucagon versus placebo studies ( 1.1  0.4 vs. 2.3  0.5 events / day , p = ns ) . the number of treatments for hypoglycemia per day was considerably reduced in the glucagon versus placebo studies ( 1.1  0.5 vs. 3.9  1.0 treatments / day , p = 0.01 ) . mean glucose was somewhat higher in the glucagon studies , but this increase did not reach statistical significance ( 145  14 vs. 135  16 mg / dl , p = ns ) . other metrics of glycemic control , including percent of auc in the target ( 70180 mg / dl ) and hyperglycemic ( > 180 mg / dl ) ranges and mean amplitude of glycemic excursions were not significantly different between the groups ( data not shown ) . overall sensor accuracy was very good , with combined mard of 8.7  1.5% and mad of 13.3  1.5 mg / dl . sensors were calibrated on average every 5.7  0.5 h. in 8.6% of cases , venous blood , rather than sensed , glucose values were sent to the controller due to suboptimal sensor accuracy . only one subject developed transient nausea and vomiting after receiving 350 g glucagon over 175 min during a low - gain glucagon study . no subjects in the high - gain glucagon or placebo studies experienced any side effects . in six subjects who underwent both a high - gain glucagon study and a placebo study , there was a 56% reduction in time spent in the hypoglycemic range ( 18  11 vs. 41  13 min / day , p = 0.01 ) . the number of hypoglycemic events , with events lasting > 20 min being considered a new event , was also significantly reduced during the high - gain glucagon versus placebo studies ( 1.0  0.6 vs. 2.1  0.6 events / day , p = 0.01 ) , as was the number of oral or intravenous carbohydrate treatments for hypoglycemia ( 1.4  0.8 vs. 4.0  1.4 treatments / day , p = 0.01 ) . there was no significant difference in mean glucose between the high - gain glucagon versus placebo studies ( 138  17 vs. 131  17 mg / dl , p = ns ) , as shown in fig . the mean fasting glucose was also quite similar ( 123  14 vs. 120  15 mg / dl , p = ns ) . there was a nonsignificant trend toward a higher postprandial glucose in high - gain glucagon versus placebo studies , defined as mean value 0180 min after meals ( 157  18 vs. 144  17 mg / dl , p = ns ) . the amount of insulin delivered during the high - gain glucagon versus placebo studies was nearly identical ( 48.9  6.2 vs. 48.3  5.5 units per day , p = ns ) . summary of glucose levels ( means  se ) , insulin delivery rate , and , for glucagon studies , the glucagon delivery rate . venous blood glucose is noted by gray diamonds , insulin delivery rate by a black line , glucagon delivery rate by a light gray line , and meals by black triangles . in six subjects who received low - gain glucagon compared with the eight subjects who received placebo , there was a nonsignificant reduction in time in the hypoglycemic range ( 15  8 vs. 40  10 min / day , p = ns ) . there was also a trend toward a reduction in the number of hypoglycemic events that did not reach statistical significance ( 1.4  0.7 vs. 2.3  0.5 events / day , p = ns ) . there was a reduction in the number of treatments for hypoglycemia in studies with low - gain glucagon of borderline significance ( 1.0  0.7 vs. 3.9  1.0 treatments / day , p = 0.05 ) . mean glucose was somewhat higher in low - gain glucagon versus placebo studies ( 157  24 vs. 135  16 mg / dl , p = 0.04 ) . there was also a trend toward higher fasting glucose in the low - gain glucagon versus placebo studies ( 137  20 vs. 122  13 mg / dl , p = ns ) . there was a similar trend , of borderline statistical significance , suggesting a larger elevation in postprandial glucose in the low - gain glucagon versus placebo studies ( 179  26 vs. 151  18 mg / dl , p = 0.05 ) . there was a nonsignificant difference in insulin delivered in low - gain glucagon versus placebo studies ( 60.1  14.1 vs. 46.9  5.5 units / day ) . the mean dose of glucagon delivered during the low - gain glucagon studies was higher than the high - gain glucagon studies but did not reach statistical significance ( 746  134 vs. 516  108 g / day , p = ns ) . glucagon , when given either via high- or low - gain , compared with placebo , led to a 63% reduction of time spent in the hypoglycemic range ( 15  6 vs. 40  10 min / day , p = 0.04 ) . the number of hypoglycemic events per day was not significantly different between glucagon versus placebo studies ( 1.1  0.4 vs. 2.3  0.5 events / day , p = ns ) . the number of treatments for hypoglycemia per day was considerably reduced in the glucagon versus placebo studies ( 1.1  0.5 vs. 3.9  1.0 treatments / day , p = 0.01 ) . mean glucose was somewhat higher in the glucagon studies , but this increase did not reach statistical significance ( 145  14 vs. 135  16 mg / dl , p = ns ) . other metrics of glycemic control , including percent of auc in the target ( 70180 mg / dl ) and hyperglycemic ( > 180 mg / dl ) ranges and mean amplitude of glycemic excursions were not significantly different between the groups ( data not shown ) . overall sensor accuracy was very good , with combined mard of 8.7  1.5% and mad of 13.3  1.5 mg / dl . sensors were calibrated on average every 5.7  0.5 h. in 8.6% of cases , venous blood , rather than sensed , glucose values were sent to the controller due to suboptimal sensor accuracy . only one subject developed transient nausea and vomiting after receiving 350 g glucagon over 175 min during a low - gain glucagon study . no subjects in the high - gain glucagon or placebo studies experienced any side effects . in this automated glycemic control system , we compared the effect of subcutaneous glucagon , delivered in small doses at times of impending hypoglycemia , to saline placebo . in both conditions , the algorithm called for a significant reduction or discontinuation of insulin delivery during impending hypoglycemia . we found that compared with placebo , glucagon delivered in pulses using high - gain parameters significantly decreased the time spent in the hypoglycemic range , the number of hypoglycemic events , and the number of treatments needed for hypoglycemia . only the high - gain , not the low - gain , glucagon delivery system was superior to placebo in reducing all three of these outcomes , despite the fact that a lower amount of glucagon was delivered in the high - gain studies . the high - gain glucagon infusion consisted of a pulse of glucagon typically given over 510 min at a time of impending hypoglycemia followed by a 50-min off period . the low - gain glucagon was delivered in a slow , more prolonged manner without a mandatory off period . the high - gain glucagon infusion is arguably more physiologic , as glucagon is secreted rapidly in response to hypoglycemia in humans without diabetes ( 15 ) . minimizing glucagon delivery , as described here , is important to avoid potential side effects , such as acute hyperglycemia and nausea , and more severe effects , such as depletion of liver glycogen . notably , the mean glucose levels in the high - gain glucagon and placebo studies were very similar . however , larger and longer - term studies will be required to assess the effect of ongoing glucagon treatment on overall glycemic control in addition , the lower amount of premeal insulin in the low - gain glucagon studies compared with the placebo studies may have affected the results , in particular the differences in mean and postprandial glucose levels . in some regards , the need to announce the meal to the controller and the delivery of substantial amounts of premeal insulin might also be considered a limitation . a true closed - loop system without meal announcement using currently available insulin preparations delivered subcutaneously is unlikely to provide optimal blood glucose control . after reconstitution , glucagon forms fibrils over time ( 16,17 ) and is currently approved for use only immediately after reconstitution . despite the occurrence of fibrils and aggregates , our group ( 9 ) and el - khatib et al . ( 18 ) have shown that even when glucagon is aged for 1 week at room or body temperature , large doses retain full hyperglycemic activity in animals . it is possible that , after injection , the aggregates dissociate into monomeric form in the subcutaneous space . there is some evidence that glucagon can be cytotoxic if it is  aged  at very high concentrations ( 19 ) , but there are no reports of cytotoxicity during aging at concentrations of 1 mg / ml or lower . further studies are needed to examine the efficacy of glucagon used for several days after reconstitution and to assess potential cytotoxicity at clinically appropriate concentrations . it is possible that aggregation may be overcome using glucagon analogs ( 20 ) or novel methods of glucagon preparation ( 21 ) . in conclusion , we found that glucagon given to subjects with type 1 diabetes by algorithm during impending hypoglycemia is effective in preventing most cases of hypoglycemia . glycemic control was good in this study , in part due to open - loop insulin delivery before meals . these results suggest that an automated system of closed - loop glucagon delivery , with a hybrid pattern of insulin delivery including meal announcement , is able to control glycemia safely and effectively in people with type 1 diabetes . there is a need for further research into the issue of glucagon stability and for the development of a fully automated insulin and glucagon delivery device .
objectiveto minimize hypoglycemia in subjects with type 1 diabetes by automated glucagon delivery in a closed - loop insulin delivery system.research design and methodsadult subjects with type 1 diabetes underwent one closed - loop study with insulin plus placebo and one study with insulin plus glucagon , given at times of impending hypoglycemia . 
 seven subjects received glucagon using high - gain parameters , and six subjects received glucagon in a more prolonged manner using low - gain parameters . 
 blood glucose levels were measured every 10 min and insulin and glucagon infusions were adjusted every 5 min . 
 all subjects received a portion of their usual premeal insulin after meal announcement.resultsautomated glucagon plus insulin delivery , compared with placebo plus insulin , significantly reduced time spent in the hypoglycemic range ( 15  6 vs. 40  10 min / day , p = 0.04 ) . compared with placebo , 
 high - gain glucagon delivery reduced the frequency of hypoglycemic events ( 1.0  0.6 vs. 2.1  0.6 events / day , p = 0.01 ) and the need for carbohydrate treatment ( 1.4  0.8 vs. 4.0  1.4 treatments / day , p = 0.01 ) . 
 glucagon given with low - gain parameters did not significantly reduce hypoglycemic event frequency ( p = ns ) but did reduce frequency of carbohydrate treatment ( p = 0.05).conclusionsduring closed - loop treatment in subjects with type 1 diabetes , high - gain pulses of glucagon decreased the frequency of hypoglycemia . 
 larger and longer - term studies will be required to assess the effect of ongoing glucagon treatment on overall glycemic control .