We examined whether plasma concentrations of non-glucose insulin secretagogues are associated with prehepatic insulin secretion rates (ISR) in non-diabetic, insulin resistant, human immunodeficiency virus (HIV)-infected, lipodystrophic patients (LIPO). Additionally, the negative feedback of insulin on ISR was evaluated. ISR were estimated by deconvolution of plasma C-peptide concentrations during fasting (basal) and during the last 30 min of a 120 min euglycemic insulin clamp (40 mU/m²). Eighteen normoglycemic LIPO were compared with 25 normoglycemic HIV-infected patients without lipodystrophy (controls). Thirty minutes before start of the clamp, a bolus of glucose was injected intravenously to stimulate endogenous insulin secretion. Insulin sensitivity index (Si_RD) was estimated from glucose tracer analysis. LIPO displayed increased basal ISR (40%), clamp ISR (53%), basal insulin (56%), and clamp insulin (24%), all P 0.001, whereas Si _RD was decreased (57%, P<0.001). In LIPO, ISR_basal correlated significantly with basal insulin, alanine and glucagon (all r>0.65, P<0.01), but not with glucose. In controls, ISR_basal correlated significantly with insulin, glucagon and glucose (all r>0.41, P<0.05), but not with alanine. In LIPO, ISR_clamp correlated significantly with clamp free fatty acids (FFA), alanine, triglyceride, and glucagon (all r>0.51, P<0.05). In controls, ISR_clamp correlated with triglyceride (r=0.45, P<0.05). Paradoxically, in LIPO, ISR_clamp correlated positively with clamp insulin (r=0.68, P<0.01), which suggests an absent negative feedback of insulin on ISR. Our data support that lipodystrophic, non-diabetic, HIV-infected patients exhibit increased ISR, which can be partially explained by an impaired negative feedback of insulin on -cells and an increased stimulation of ISR by FFA, alanine, triglyceride, and glucagon.