Glycogen is the sole glucose reservoir in the brain and is mobilized to support cerebral energy metabolism during hypoglycemia.^1-3 Furthermore, in vivo ¹³C MRS studies in animal and human subjects indicated that its levels rebound to higher than normal after an acute hypoglycemic (AH) episode, a phenomenon termed “supercompensation”,^1,2 suggesting it may provide energy for the brain during subsequent periods of hypoglycemia. Glycogen supercompensation was suggested to contribute to the development of hypoglycemia associated autonomic failure (HAAF), also termed hypoglycemia unawareness,^1,2 a condition frequently encountered in type 1 diabetes (T1D) as a result of insulin use and recurrent episodes of hypoglycemia (RH).⁴ Hence, recurrent hypoglycemia may lead to higher than normal levels of brain glycogen, perhaps to provide sufficient fuel to maintain cerebral energy metabolism during subsequent hypoglycemic episodes. To assess the role of glycogen supercompensation in the generation of HAAF, we estimated the level of brain glycogen supercompensation following RH using ¹³C MRS and compared it to that following AH.²Five healthy male volunteers (age 41 ± 9 years, BMI 27 ± 2 kg/m²) received IV [1-¹³C]glucose over 80-83 hours after undergoing 3 hyperinsulinemic, euglycemic or 3 hyperinsulinemic, hypoglycemic clamps over 2 days (Fig. 1). This hypoglycemic preconditioning protocol reproducibly induces HAAF in healthy subjects.⁵ Euglycemic and hypoglycemic preconditioning experiments were separated by at least one month. Following preconditioning, [¹³C]glucose administration began with a 20g bolus of 50% enriched [1-¹³C]glucose followed by an infusion of 25% [1-¹³C]glucose adjusted to maintain blood glucose at euglycemia (~90mg/dl) for 80+ hours. Samples for blood glucose, insulin, and isotopic enrichment were obtained every 10-60 minutes. ¹³C glycogen levels in the occipital lobe were measured at ~8, 20, 32, 44, 56, 68 and 80h at 4T using methods described before.^2,6 Briefly, localization was achieved by 3D outer volume suppression combined with 1D ISIS. All ¹³C glycogen levels were corrected for the cerebrospinal fluid (CSF) content of the voxel as described before.⁷ To estimate glycogen concentration, data were fitted with a biophysical model that takes into account the tiered structure of the glycogen molecule.^6,8 Similarly, ¹³C-glycogen data obtained following a single hypoglycemic episode (AH) from a prior publication² were fitted with the same biophysical model to compare glycogen supercompensation after AH vs. RH.Four of the 5 subjects completed the 80h-long protocol after both eu- and hypoglycemic preconditioning. The infusion had to be stopped after 29h due to complications with the IV line in the euglycemic preconditioning study of one subject. The hypoglycemic preconditioning protocol (Fig. 1) induced HAAF in all volunteers, as evidenced by a blunted epinephrine response during the third relative to the first hypoglycemic clamp (Fig. 2). Plasma glucose and insulin levels and [1-¹³C]glucose enrichments were well matched between the eu- and hypoglycemic preconditioning studies; euglycemia and physiologic insulin levels were maintained throughout the ¹³C-glucose infusion in all studies (Fig. 3). Time dependent plasma glucose concentrations and enrichments were incorporated in the modeling of the data and resulted in good correspondence between experimental and simulated time courses for label incorporation into glycogen at single subject-level (Fig. 4). Estimated glycogen level was 6% higher following the recurrent hypoglycemia (RH) protocol than the recurrent euglycemia protocol (Fig. 4A), while it was 16% higher following a single hypoglycemic episode (AH) than a euglycemic episode (Fig. 4B).Glycogen levels increase after both single and recurrent episodes of hypoglycemia, but to a lesser extent after repeated episodes. These data show that the level of glycogen supercompensation is affected by the number of hypoglycemic episodes encountered and hence suggest that glycogen is involved in the pathophysiology of HAAF. Perhaps glycogen supercompensated following the first 1-2 episodes provides sufficient extra fuel during the third episode, reducing the need for further supercompensation (HAAF as evidenced by blunted hormonal response already occurs at episode #3). However a causal relationship between glycogen supercompensation and generation of HAAF remains to be established.