OBJECTIVEGlucagon delivery in closed-loop control of type 1 diabetes is effective in minimizing hypoglycemia. However, high insulin concentration lowers the hyperglycemic effect of glucagon, and small doses of glucagon in this setting are ineffective. There are no studies clearly defining the relationship between insulin levels, subcutaneous glucagon, and blood glucose.RESEARCH DESIGN AND METHODSUsing a euglycemic clamp technique in 11 subjects with type 1 diabetes, we examined endogenous glucose production (EGP) of glucagon (25, 75, 125, and 175 μg) at three insulin infusion rates (0.016, 0.032, and 0.05 units/kg/h) in a randomized, crossover study. Infused 6,6-dideuterated glucose was measured every 10 min, and EGP was determined using a validated glucoregulatory model. Area under the curve (AUC) for glucose production was the primary outcome, estimated over 60 min.RESULTSAt low insulin levels, EGP rose proportionately with glucagon dose, from 5 ± 68 to 112 ± 152 mg/kg (P = 0.038 linear trend), whereas at high levels, there was no increase in glucose output (19 ± 53 to 26 ± 38 mg/kg, P = NS). Peak glucagon serum levels and AUC correlated well with dose (r2 = 0.63, P < 0.001), as did insulin levels with insulin infusion rates (r2 = 0.59, P < 0.001).CONCLUSIONSEGP increases steeply with glucagon doses between 25 and 175 μg at lower insulin infusion rates. However, high insulin infusion rates prevent these doses of glucagon from significantly increasing glucose output and may reduce glucagon effectiveness in preventing hypoglycemia when used in the artificial pancreas.

Automated control of blood glucose in patients with type 1 diabetes has not yet been fully implemented. The aim of this study was to design and clinically evaluate a system that integrates a control algorithm with off-the-shelf subcutaneous sensors and pumps to automate the delivery of the hormones glucagon and insulin in response to continuous glucose sensor measurements. The automated component of the system runs an adaptive proportional derivative control algorithm which determines hormone delivery rates based on the sensed glucose measurements and the meal announcements by the patient. We provide details about the system design and the control algorithm, which incorporates both a fading memory proportional derivative controller (FMPD) and an adaptive system for estimating changing sensitivity to insulin based on a glucoregulatory model of insulin action. For an inpatient study carried out in eight subjects using Dexcom SEVEN PLUS sensors, pre-study HbA1c averaged 7.6, which translates to an estimated average glucose of 171 mg/dL. In contrast, during use of the automated system, after initial stabilization, glucose averaged 145 mg/dL and subjects were kept within the euglycemic range (between 70 and 180 mg/dL) for 73.1% of the time, indicating improved glycemic control. A further study on five additional subjects in which we used a newer and more reliable glucose sensor (Dexcom G4 PLATINUM) and made improvements to the insulin and glucagon pump communication system resulted in elimination of hypoglycemic events. For this G4 study, the system was able to maintain subjects’ glucose levels within the near-euglycemic range for 71.6% of the study duration and the mean venous glucose level was 151 mg/dL.

Since the discovery of insulin, great progress has been made to improve the accuracy and safety of automated insulin delivery systems to help patients with type 1 diabetes achieve their treatment goals without causing hypoglycemia. In recent years, bioengineering technology has greatly advanced diabetes management, with the development of blood glucose meters, continuous glucose monitors, insulin pumps, and control systems for automatic delivery of one or more hormones. New insulin analogues have improved subcutaneous absorption characteristics, but do not completely eliminate the risk of hypoglycemia. Insulin effect is counteracted by glucagon in non-diabetic individuals, while glucagon secretion in those with type 1 diabetes is impaired. The use of glucagon in the artificial pancreas is therefore a logical and feasible option for preventing and treating hypoglycemia. However, commercially available glucagon is not stable in aqueous solution for long periods, forming potentially cytotoxic fibrils that aggregate quickly. Therefore, a more stable formulation of glucagon is needed for long-term use and storage in a bi-hormonal pump. Additionally, a model of glucagon action in type 1 diabetes is lacking, further limiting the inclusion of glucagon into systems employing model-assisted control. As a result, although several investigators have been working to help develop bi-hormonal systems for patients with type 1 diabetes, most continue to utilize single hormone systems employing only insulin. This article seeks to focus on the attributes of glucagon and its use in bi-hormonal systems.

Glucagon is unstable and undergoes degradation and aggregation in aqueous solution. For this reason, its use in portable pumps for closed loop management of diabetes is limited to very short periods. In this study, we sought to identify the degradation mechanisms and the bioactivity of specific degradation products. We studied degradation in the alkaline range, a range at which aggregation is minimized. Native glucagon and analogs identical to glucagon degradation products were synthesized. To quantify biological activity in glucagon and in the degradation peptides, a protein kinase A-based bioassay was used. Aged, fresh, and modified peptides were analyzed by liquid chromatography with mass spectrometry (LCMS). Oxidation of glucagon at the Met residue was common but did not reduce bioactivity. Deamidation and isomerization were also common and were more prevalent at pH 10 than 9. The biological effects of deamidation and isomerization were unpredictable; deamidation at some sites did not reduce bioactivity. Deamidation of Gln 3, isomerization of Asp 9, and deamidation with isomerization at Asn 28 all caused marked potency loss. Studies with molecular-weight-cutoff membranes and LCMS revealed much greater fibrillation at pH 9 than 10. Further work is necessary to determine formulations of glucagon that minimize degradation and fibrillation.

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 µg/kg, for a total mean dose of 1,126 µg 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.

Background In bi-hormonal closed-loop systems for treatment of diabetes, glucagon sometimes fails to prevent hypoglycemia. We evaluated glucagon responses during several closed-loop studies to determine factors, such as gain factors, responsible for glucagon success and failure. Methods We extracted data from four closed-loop studies, examining blood glucose excursions over the 50 minutes after each glucagon dose and defining hypoglycemic failure as glucose values < 60 mg/dl. Secondly, we evaluated hyperglycemic excursions within the same period, where glucose was > 180 mg/dl. We evaluated several factors for association with rates of hypoglycemic failure or hyperglycemic excursion. These factors included age, weight, HbA1c, duration of diabetes, gender, automation of glucagon delivery, glucagon dose, proportional and derivative errors (PE and DE), insulin on board (IOB), night vs. day delivery, and point sensor accuracy. Results We analyzed a total of 251 glucagon deliveries during 59 closed-loop experiments performed on 48 subjects. Glucagon successfully maintained glucose within target (60 – 180 mg/dl) in 195 (78%) of instances with 40 (16%) hypoglycemic failures and 16 (6%) hyperglycemic excursions. A multivariate logistic regression model identified PE (p<0.001), DE (p<0.001), and IOB (p<0.001) as significant determinants of success in terms of avoiding hypoglycemia. Using a model of glucagon absorption and action, simulations suggested that the success rate for glucagon would be improved by giving an additional 0.8 mcg/kg. Conclusion We conclude that glucagon fails to prevent hypoglycemia when it is given at a low glucose threshold and when glucose is falling steeply. We also confirm that high IOB significantly increases the risk for glucagon failures. Tuning of glucagon subsystem parameters may help reduce this risk.

Background: Commercial glucagon is unstable due to aggregation and degradation. In closed-loop studies, it must be reconstituted frequently. For use in a portable pump for 3 days, a more stable preparation is required. At alkaline pH, curcumin inhibited glucagon aggregation. However, curcumin is not sufficiently stable for long-term use. Here, we evaluated ferulic acid, a stable breakdown product of curcumin, for its ability to stabilize glucagon.Methods: Ferulic acid-formulated glucagon (FAFG), composed of ferulic acid, glucagon, L-methionine, polysorbate-80, and human serum albumin in glycine buffer at pH 9, was aged for 7 days at 37°C. Glucagon aggregation was assessed by transmission electron microscopy (TEM) and degradation by high-performance liquid chromatography (HPLC). A cell-based protein kinase A (PKA) assay was used to assess in vitro bioactivity. Pharmacodynamics (PD) of unaged FAFG, 7-day aged FAFG, and unaged synthetic glucagon was determined in octreotide-treated swine. Results:No fibrils were observed in TEM images of fresh or aged FAFG. Aged FAFG was 94% intact based on HPLC analysis and there was no loss of bioactivity. In the PD swine analysis, the rise over baseline of glucose with unaged FAFG, aged FAFG, and synthetic native glucagon (unmodified human sequence) was similar.Conclusions: After 7 days of aging at 37°C, an alkaline ferulic acid formulation of glucagon exhibited significantly less aggregation and degradation than that seen with native glucagon and was bioactive in vitro and in vivo. Thus, this formulation may be stable for 3-7 days in a portable pump for bihormonal closed-loop treatment of T1D.

Based on these findings, a formulation of glucagon stabilized with curcumin, polysorbate-80, l-methionine, and albumin at alkaline pH in glycine buffer may be suitable for extended use in a portable pump in the setting of a bihormonal artificial endocrine pancreas.