In response to hypoglycemia, healthy individuals rapidly antagonize insulin action on glucose and lipid metabolism, but the effects on protein metabolism are unclear. Because amino acids are an important substrate for gluconeogenesis and a fuel alternative to glucose for oxidation, we evaluated whether hypoglycemia antagonizes the hypoaminoacidemic and the antiproteolytic effects of insulin and changes the de novo synthesis of glutamine, a gluconeogenic amino acid. To this purpose, in 7 healthy subjects, we performed 2 studies, 3.5 h each, at similar insulin but different glucose concentrations (i.e., 4.9 +/- 0.1 mmol/l [euglycemic clamp] or 2.9 +/- 0.2 mmol/l [hypoglycemic clamp]). As expected, hypoglycemia antagonized the insulin suppression of glucose production achieved in euglycemia (from 21 +/- 15 to 116 +/- 12% of basal, P < 0.001), the stimulation of glucose uptake (from 207 +/- 28 to 103 +/- 7% of basal, P < 0.01) and the suppression of circulating free fatty acids (from 30 +/- 5 to 80 +/- 17% of basal, P < 0.001). In contrast, hypoglycemia increased the insulin suppression of circulating leucine (from 63 +/- 1 to 46 +/- 2% of basal, P < 0.001) and phenylalanine (from 79 +/- 3 to 64 +/- 3% of basal, P < 0.001) concentrations. Hypoglycemia did not change the insulin suppression of proteolysis (from 79 +/- 2 to 82 +/- 4% of basal, P < 0.001). However, hypoglycemia doubled the insulin suppression of the glutamine concentrations (from 84 +/- 3 to 63 +/- 3% of basal, P < 0.01) in the absence of significant changes in the glutamine rate of appearance, but it also caused an imbalance between glutamine uptake and release. This study demonstrates that successful counterregulation does not affect proteolysis. Moreover, it does not increase the availability of circulating amino acids by de novo synthesis. In contrast, despite the lower concentration of circulating amino acids, hypoglycemia increases the uptake of glutamine that can be used for gluconeogenesis and as a fuel alternative to glucose.