diabetes remains the leading cause of renal failure , nontraumatic lower - limb amputations , and new cases of blindness among adults ( 1 ) . the risks of these complications can be substantially reduced with reduction of hyperglycemia , but more aggressive therapy increases the risk of hypoglycemia significantly ( 2 ) . because of the difficulty of managing diabetes , there are ongoing efforts to develop a closed - loop system ( 3 ) . closed - loop systems generally consist of a glucose sensor from which data are collected and input into an algorithm , which calculates a varying insulin rate for delivery via a pump . the difficulty of delivering insulin subcutaneously in such a fashion is related to its relatively slow onset and prolonged effect ( 4 ) . the slow onset often leads to hyperglycemia after meals , and the prolonged effect increases the risk of hypoglycemia prior to the next meal . in contrast to insulin , the action of glucagon is rapid and short - lived , even when given subcutaneously ( 5 ) . in 1963 , kadish ( 6 ) published a study introducing the concept of delivering glucagon in the context of a closed - loop system . in such a system , insulin is delivered to treat hyperglycemia and maintain euglycemia , while glucagon is delivered to prevent or treat hypoglycemia . our group ( 7,8 ) and others ( 911 ) have successfully treated patients with type 1 diabetes using a bihormonal closed - loop system . glucagon was well tolerated in these studies and significantly reduced the frequency of hypoglycemia compared with a placebo ( 7 ) . glucagon administration did not , however , completely eliminate hypoglycemic episodes , and in one study ( 12 ) failed to prevent hypoglycemia in one - third of cases , raising the possibility of hepatic glycogen depletion . even individuals without diabetes have been shown to have a brisk hyperglycemic response to a single glucagon dose of 0.5 mg , but a poor response to a second dose ( 13 ) . lower doses , such as those given via a closed - loop system , have not been studied . individuals with type 1 diabetes are likely to be at higher risk for glycogen depletion due to lower glycogen stores after an overnight fast ( 14 ) . in particular , poorly controlled diabetes is associated with reduced glycogen synthesis and glycogen breakdown , which improved with intensive diabetes control and nearly normalized after a year ( 15 ) . these studies suggest that in individuals with type 1 diabetes , except possibly in those with excellent control , impairments in hepatic glycogen synthesis and breakdown are common . there is an unmet need to better understand whether people with type 1 diabetes are capable of responding normally to repeated small doses of glucagon . if the response to glucagon is preserved and glycogen stores are not depleted , these findings would support the safety of repeated glucagon delivery in a bihormonal closed - loop system . eleven subjects with type 1 diabetes who were receiving insulin pump therapy were each admitted for one 40-h study . eight females and three males were studied , with a mean age of 40.5  5 years , a duration of diabetes of 12.5  2.2 years , an hba1c level of 7.2  0.1% , and a weight of 70.3  4.2 kg ( mean  sem ) . all subjects were required to be between 18 and 65 years of age , and to have a bmi between 18 and 35 kg / m . women of childbearing age were required to have a negative urine pregnancy test result prior to participation . patients with a history of cardiovascular , cerebrovascular , kidney , or liver disease or any other uncontrolled chronic medical conditions were excluded from the study . other exclusion criteria included adrenal insufficiency , oral or parenteral corticosteroid use , active foot ulceration , bleeding disorder , seizure disorder , history of weight loss of 5 pounds over the prior month , active alcohol or substance abuse , or any contraindication to an mri scan . the research protocol was approved by the oregon health & science university institutional review board , and this study was registered with clinicaltrials.gov ( clinical trial reg . subjects were given standardized meals that were consumed at home at 10:00 a.m. and 3:00 p.m. on the day of admission to the clinical research center . each subject s insulin pump was stopped , and each subject was treated intravenously with regular insulin throughout the remainder of the study to a target glucose concentration of 90130 mg / dl guided by an insulin infusion protocol previously described by goldberg et al . each subject received 8 doses of glucagon ( glucagen ; novo nordisk , copenhagen , denmark ) 2 g / kg , s.c . glucagon was reconstituted with sterile water to a concentration of 1 mg / ml just prior to each dose to avoid any degradation or aggregation prior to administration . the study schedule , including glucagon dosing interval and mrs scans , is presented in table 1 . study schedule with timing of mrs scans , meals , and glucagon doses venous blood glucose levels were measured every 15 min via a hemocue 201 glucose analyzer ( hemocue ab , ngelholm , sweden ) . the frequency of venous blood glucose measurement was increased to every 10 min for the 3 h after the first and last glucagon doses were administered . the rate of the intravenous administration of regular insulin was fixed over this 3-h time period . if the blood glucose level fell to < 90 mg / dl , dextrose was infused to avoid hypoglycemia . the meals for each individual on study day 1 were identical to the meals on study day 2 . insulin aspart ( novolog ; novo nordisk ) was administered subcutaneously just prior to meals at 75% of the subject s typical dose . semiquantitative measurements of in vivo hepatic glycogen were performed by c mrs at 7 t. hepatic glycogen levels were determined immediately after entry into the study at 6:00 p.m. , after fasting overnight at 9:00 a.m. , and again at 6:00 p.m. and 9:00 a.m. following an identical feeding pattern but with added administration of glucagon . the method used followed an adaptation of the methodology previously reported by shulman et al . . however , there is no spatial resolution of signal and therefore no imaging ; only the total signal in the interrogation volume is collected , and , rather than water h nuclei , the c nucleus is probed . the spectroscopic nature of this technique allows c nuclei to be differentiated on the basis of their chemical environments . the c1 carbon of glycogen is shifted far from all other in vivo carbon resonances ( 100.5 parts per million ) , and it therefore readily allows for determining changes in hepatic glycogen concentrations ( 19 ) . a complete description of the c mrs data acquisition methods and analytical methods are provided in the supplementary data . in brief , all spectra were acquired on a siemens magnetom 7 t mri system . data acquisition was performed using a home - built h / c surface dual - tune coil . the h coil was used for shimming and coil localization , and to acquire h water proton images of the liver . c mrs spectra were acquired , on resonance , for the c1 glycogen carbon ( 74.7370 mhz ) using 11,000 transients and a repetition time of 160 ms ; the total acquisition time was 25 min . each spectrum was acquired in the presence of a c - enriched acetonitrile ( sigma - aldrich , st . the intensity of the c1 glycogen signal was then related to the in vivo glycogen concentration by comparing it to a standardization curve generated on phantoms of oyster glycogen ( sigma - aldrich ) . axial and sagittal h images were used to correct for differences in the coil volume occupied by the liver and phantom . in this way , it was possible to obtain a semiquantitative measure of hepatic glycogen concentration ( in grams per liter ) . the primary end point was the comparison of estimated hepatic glycogen as determined by c mrs after fasting overnight on study day 1 versus study day 2 ( i.e. , before and after repeated doses of glucagon ) and after lunch on study day 1 versus study day 2 . this outcome was modeled as a continuous variable using generalized estimating equations to account for correlations between repeated measures on the same individuals . in this glycogen model , we included indicator variables for fasting and the administration of glucagon , as well as for their interaction . the scans were not obtained in the first subject because of an error in equipment setup . in a second subject , minimal hepatic glycogen was detected in all scans because of excessive separation of the coil from the liver by a thick subcutaneous adipose layer . the secondary end point was the rise in glucose after the first versus the last glucagon dose , as measured by the incremental area under the curve ( auc ) for glucose for the 90-min interval after glucagon administration ( 20 ) . a three - compartment glucoregulatory model was used to account for the effect of meals , intravenous dextrose administration , and insulin administration , therefore isolating the effect of glucagon . all 19 model parameters were fit for the first overnight period . based on these estimated parameters , glucose values for the entire time course of the study were estimated . any response due to glucagon was not modeled , thus differentiating the change in glucose due to glucagon administration . the modeled glucose values were subtracted from the true values to produce the response due to glucagon for each glucagon dose . subjects were evaluated within a hospital setting , during which time they were sedentary , which could have led to reduced insulin sensitivity . to evaluate the possibility that reduced insulin sensitivity affected the outcome of the study , a separate simulation was completed whereby insulin sensitivity was modeled to decline linearly from 100% down to 50% over the course of the study . to test for changes in the mean rise in glucose due to glucagon after each dose , we used a generalized estimating equation model with robust variance estimators and dose number as a categorical independent variable . wald tests were used to examine the significance of the overall model and of coefficients representing changes from the first dose at each subsequent dose . all analyses were performed with stata version 13.1 ( statacorp lp , college station , tx ) or matlab version r2014a ( mathworks , natick , ma ) . eleven subjects with type 1 diabetes who were receiving insulin pump therapy were each admitted for one 40-h study . eight females and three males were studied , with a mean age of 40.5  5 years , a duration of diabetes of 12.5  2.2 years , an hba1c level of 7.2  0.1% , and a weight of 70.3  4.2 kg ( mean  sem ) . all subjects were required to be between 18 and 65 years of age , and to have a bmi between 18 and 35 kg / m . women of childbearing age were required to have a negative urine pregnancy test result prior to participation . patients with a history of cardiovascular , cerebrovascular , kidney , or liver disease or any other uncontrolled chronic medical conditions were excluded from the study . other exclusion criteria included adrenal insufficiency , oral or parenteral corticosteroid use , active foot ulceration , bleeding disorder , seizure disorder , history of weight loss of 5 pounds over the prior month , active alcohol or substance abuse , or any contraindication to an mri scan . the research protocol was approved by the oregon health & science university institutional review board , and this study was registered with clinicaltrials.gov ( clinical trial reg . subjects were given standardized meals that were consumed at home at 10:00 a.m. and 3:00 p.m. on the day of admission to the clinical research center . each subject s insulin pump was stopped , and each subject was treated intravenously with regular insulin throughout the remainder of the study to a target glucose concentration of 90130 mg / dl guided by an insulin infusion protocol previously described by goldberg et al . each subject received 8 doses of glucagon ( glucagen ; novo nordisk , copenhagen , denmark ) 2 g / kg , s.c . glucagon was reconstituted with sterile water to a concentration of 1 mg / ml just prior to each dose to avoid any degradation or aggregation prior to administration . the study schedule , including glucagon dosing interval and mrs scans , is presented in table 1 . study schedule with timing of mrs scans , meals , and glucagon doses venous blood glucose levels were measured every 15 min via a hemocue 201 glucose analyzer ( hemocue ab , ngelholm , sweden ) . the frequency of venous blood glucose measurement was increased to every 10 min for the 3 h after the first and last glucagon doses were administered . the rate of the intravenous administration of regular insulin was fixed over this 3-h time period . if the blood glucose level fell to < 90 mg / dl , dextrose was infused to avoid hypoglycemia . the meals for each individual on study day 1 were identical to the meals on study day 2 . insulin aspart ( novolog ; novo nordisk ) was administered subcutaneously just prior to meals at 75% of the subject s typical dose . semiquantitative measurements of in vivo hepatic glycogen were performed by c mrs at 7 t. hepatic glycogen levels were determined immediately after entry into the study at 6:00 p.m. , after fasting overnight at 9:00 a.m. , and again at 6:00 p.m. and 9:00 a.m. following an identical feeding pattern but with added administration of glucagon . the method used followed an adaptation of the methodology previously reported by shulman et al . however , there is no spatial resolution of signal and therefore no imaging ; only the total signal in the interrogation volume is collected , and , rather than water h nuclei , the c nucleus is probed . the spectroscopic nature of this technique allows c nuclei to be differentiated on the basis of their chemical environments . the c1 carbon of glycogen is shifted far from all other in vivo carbon resonances ( 100.5 parts per million ) , and it therefore readily allows for determining changes in hepatic glycogen concentrations ( 19 ) . a complete description of the c mrs data acquisition methods and analytical methods are provided in the supplementary data . in brief , all spectra were acquired on a siemens magnetom 7 t mri system . data acquisition was performed using a home - built h / c surface dual - tune coil . the h coil was used for shimming and coil localization , and to acquire h water proton images of the liver . c mrs spectra were acquired , on resonance , for the c1 glycogen carbon ( 74.7370 mhz ) using 11,000 transients and a repetition time of 160 ms ; the total acquisition time was 25 min . each spectrum was acquired in the presence of a c - enriched acetonitrile ( sigma - aldrich , st . the intensity of the c1 glycogen signal was then related to the in vivo glycogen concentration by comparing it to a standardization curve generated on phantoms of oyster glycogen ( sigma - aldrich ) . axial and sagittal h images were used to correct for differences in the coil volume occupied by the liver and phantom . in this way , it was possible to obtain a semiquantitative measure of hepatic glycogen concentration ( in grams per liter ) . the primary end point was the comparison of estimated hepatic glycogen as determined by c mrs after fasting overnight on study day 1 versus study day 2 ( i.e. , before and after repeated doses of glucagon ) and after lunch on study day 1 versus study day 2 . this outcome was modeled as a continuous variable using generalized estimating equations to account for correlations between repeated measures on the same individuals . in this glycogen model , we included indicator variables for fasting and the administration of glucagon , as well as for their interaction . the scans were not obtained in the first subject because of an error in equipment setup . in a second subject , minimal hepatic glycogen was detected in all scans because of excessive separation of the coil from the liver by a thick subcutaneous adipose layer . the secondary end point was the rise in glucose after the first versus the last glucagon dose , as measured by the incremental area under the curve ( auc ) for glucose for the 90-min interval after glucagon administration ( 20 ) . a three - compartment glucoregulatory model was used to account for the effect of meals , intravenous dextrose administration , and insulin administration , therefore isolating the effect of glucagon . all 19 model parameters were fit for the first overnight period . based on these estimated parameters , glucose values for the entire time course of the study were estimated . any response due to glucagon was not modeled , thus differentiating the change in glucose due to glucagon administration . the modeled glucose values were subtracted from the true values to produce the response due to glucagon for each glucagon dose . subjects were evaluated within a hospital setting , during which time they were sedentary , which could have led to reduced insulin sensitivity . to evaluate the possibility that reduced insulin sensitivity affected the outcome of the study , a separate simulation was completed whereby insulin sensitivity was modeled to decline linearly from 100% down to 50% over the course of the study . to test for changes in the mean rise in glucose due to glucagon after each dose , we used a generalized estimating equation model with robust variance estimators and dose number as a categorical independent variable . wald tests were used to examine the significance of the overall model and of coefficients representing changes from the first dose at each subsequent dose . all analyses were performed with stata version 13.1 ( statacorp lp , college station , tx ) or matlab version r2014a ( mathworks , natick , ma ) . all subjects received eight doses of glucagon , each 2 g / kg , with a mean dose of 140.7  8.2 g and a total dose of 1,125.8  65.5 g over 16 h. median glucose values and insulin infusion rates for the entire study are shown in fig . dextrose was infused infrequently and only for the treatment of hypoglycemia with a mean dose of 253  164 ml of 10% dextrose , equivalent to 25 g of carbohydrate , over 44 h. median venous blood glucose values in milligrams per deciliter ( top graph ) and median insulin infusion rates in units per hour ( bottom graph ) . meals ( white squares ) and glucagon doses ( black triangles ) are also shown . the insulin infusion rate was fixed after the first and last glucagon dose , but was otherwise adjusted to bring the glucose concentration into the target range . at baseline , prior to the administration of glucagon , hepatic glycogen declined significantly after an overnight fast as expected ( 19 g / l , p < 0.001 ) . repeated administration of glucagon did not significantly lower glycogen stores . in the fasting state , glycogen averaged 21  3 g / l before glucagon administration and 25  4 g / l after glucagon administration ( p = ns ; see fig . the difference before and after glucagon was larger , though still not statistically significant : glycogen averaged 40  2 g / l before glucagon administration and 34  4 g / l after glucagon administration ( p = ns ) . estimated individual and group mean glycogen stores with sem before ( white squares ) and after ( black circles ) glucagon administration in subjects with type 1 diabetes , under fed and fasting conditions ( n = 8) . the rise in glucose level after the last dose of glucagon was comparable to the rise after the first dose as measured by the 90-min incremental auc for glucose ( 2,209  757 vs. 3,018  415 mg  min  dl , p = ns ; see fig . similarly , there was no significant difference in auc when the model included a 50% decline in insulin sensitivity that may have occurred due to subject inactivity during the hospital stay ( 2,209  757 vs. 2,812  410 mg  min  dl , p = ns ) . the average auc after any dose of glucagon was 2,698  248 mg  min  dl , and there were no significant differences across all eight doses . the modeled auc accounting for the effects of meals , dextrose , and insulin was subtracted from the observed auc to isolate the effect of each glucagon dose . all subjects received eight doses of glucagon , each 2 g / kg , with a mean dose of 140.7  8.2 g and a total dose of 1,125.8  65.5 g over 16 h. median glucose values and insulin infusion rates for the entire study are shown in fig . dextrose was infused infrequently and only for the treatment of hypoglycemia with a mean dose of 253  164 ml of 10% dextrose , equivalent to 25 g of carbohydrate , over 44 h. median venous blood glucose values in milligrams per deciliter ( top graph ) and median insulin infusion rates in units per hour ( bottom graph ) . meals ( white squares ) and glucagon doses ( black triangles ) are also shown . the insulin infusion rate was fixed after the first and last glucagon dose , but was otherwise adjusted to bring the glucose concentration into the target range . at baseline , prior to the administration of glucagon , hepatic glycogen declined significantly after an overnight fast as expected ( 19 g / l , p < 0.001 ) . repeated administration of glucagon did not significantly lower glycogen stores . in the fasting state , glycogen averaged 21  3 g / l before glucagon administration and 25  4 g / l after glucagon administration ( p = ns ; see fig . the difference before and after glucagon was larger , though still not statistically significant : glycogen averaged 40  2 g / l before glucagon administration and 34  4 g / l after glucagon administration ( p = ns ) . estimated individual and group mean glycogen stores with sem before ( white squares ) and after ( black circles ) glucagon administration in subjects with type 1 diabetes , under fed and fasting conditions ( n = 8) . the rise in glucose level after the last dose of glucagon was comparable to the rise after the first dose as measured by the 90-min incremental auc for glucose ( 2,209  757 vs. 3,018  415 mg  min  dl , p = ns ; see fig . similarly , there was no significant difference in auc when the model included a 50% decline in insulin sensitivity that may have occurred due to subject inactivity during the hospital stay ( 2,209  757 vs. 2,812  410 mg  min  dl , p = ns ) . the average auc after any dose of glucagon was 2,698  248 mg  min  dl , and there were no significant differences across all eight doses . the modeled auc accounting for the effects of meals , dextrose , and insulin was subtracted from the observed auc to isolate the effect of each glucagon dose . glucagon raises circulating glucose levels primarily via glycogenolysis , and to a lesser degree gluconeogenesis ( 23 ) . in this study , we assessed the effect of repeated doses of glucagon on glycogen stores and on circulating glucose levels . to rigorously assess for glycogen depletion in this study , we gave glucagon doses that were higher than the typical doses used in our previous closed - loop studies ( 1,126 g / day in this study vs. 282 g / day in the previous studies ) ( 7,8,24 ) . the doses given in this study are comparable to those given in a study by russell et al . ( 9 ) , but were significantly higher than those given by haidar et al . ( 10 ) , and higher than those given by our group ( 7,8,24 ) . despite these glucagon doses , we found that glycogen stores were not significantly diminished after repeated glucagon doses relative to baseline in both the fasting and fed states . there was , however , a trend toward a modest decline in glycogen stores in the fed state , with six of eight subjects showing a decline . this study was not powered to detect modest differences , and a much larger study would likely demonstrate a modest difference in the fed state . the data indicate that there is not a marked ( > 50% ) reduction in glycogen stores , and subjects retained their hyperglycemic response to glucagon even in the fasting state after receiving in excess of 1 mg glucagon . these findings suggest that glycogen stores can be replenished even following multiple doses of glucagon . these findings also provide reassurance that repeated glucagon dosing in the setting of a bihormonal closed - loop system is unlikely to cause marked glycogen depletion over the short term . glycogen depletion must be avoided , as it would leave a person with diabetes at higher risk for severe hypoglycemia . in light of these findings , it is improbable that glycogen depletion was responsible for glucagon failing to prevent hypoglycemia in one - third of cases in our past study ( 12 ) . it is more likely that the failure of glucagon to prevent hypoglycemia was related to a high prevailing insulin effect , which has been shown to dominate over the effect of glucagon at high doses ( 25 ) . the delay in the detection of impending hypoglycemia by the glucose sensor also was a contributor ( 12 ) . currently available sensors have significantly better accuracy in the hypoglycemic range ( 26 ) , allowing for earlier glucagon delivery . improved sensor technology and adjusting glucagon dosing based on insulin - on - board have led to significantly fewer glucagon failures ( 24 ) . it is important to note that the subjects in this study were eating on a regular basis , had well - controlled diabetes , and were studied over a short period of time . fasting for a prolonged period would induce glycogen depletion , and automated glucagon delivery would not be appropriate in such circumstances , such as during an episode of gastroenteritis . it is also unknown whether individuals with poorly controlled diabetes would respond to repeated glucagon doses in the same manner as those with well - controlled diabetes . glucagon is an unstable protein in aqueous solution , and currently available emergency kits are unsuitable for use in a portable pump . for this reason , glucagon was reconstituted immediately prior to each dose in this study . the logistics of delivering multiple hormones are also complicated , and are being addressed by companies such as tandem diabetes care , where the development of a dual - chamber pump is underway . prior to food and drug administration approval of such systems , longer and larger outpatient clinical trials will certainly be required .
objectiveto evaluate subjects with type 1 diabetes for hepatic glycogen depletion after repeated doses of glucagon , simulating delivery in a bihormonal closed - loop system.research design and methodseleven adult subjects with type 1 diabetes participated . 
 subjects underwent estimation of hepatic glycogen using 13c mrs . 
 mrs was performed at the following four time points : fasting and after a meal at baseline , and fasting and after a meal after eight doses of subcutaneously administered glucagon at a dose of 2 g / kg , for a total mean dose of 1,126 g over 16 h. the primary and secondary end points were , respectively , estimated hepatic glycogen by mrs and incremental area under the glucose curve for a 90-min interval after glucagon administration.resultsin the eight subjects with complete data sets , estimated glycogen stores were similar at baseline and after repeated glucagon doses . in the fasting state , glycogen averaged 21 
 3 g / l before glucagon administration and 25  4 g / l after glucagon administration ( mean  sem ) ( p = ns ) . in the fed state , 
 glycogen averaged 40  2 
 g / l before glucagon administration and 34  4 g / l after glucagon administration ( p = ns ) . with the use of an insulin action model , the rise in glucose after the last dose of glucagon was comparable to the rise after the first dose , as measured by the 90-min incremental area under the glucose curve.conclusionsin adult subjects with well - controlled type 1 diabetes ( mean a1c 7.2% ) , glycogen stores and the hyperglycemic response to glucagon administration are maintained even after receiving multiple doses of glucagon . 
 this finding supports the safety of repeated glucagon delivery in the setting of a bihormonal closed - loop system .