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Hepatic energy metabolism is a key element in many metabolic diseases. Hepatic anaplerosis provides carbons for gluconeogenesis (GNG) and triglyceride (TG) synthesis. We aimed to optimize a protocol that measures hepatic anaplerotic contribution for GNG, TG synthesis, and hepatic pentose phosphate pathway (PPP) activity using a single dose of oral [U⁻¹³C₃]glycerol paired with an oral sugar tolerance test (OSTT) in a population with significant insulin resistance. The OSTT (75 g glucose + 25 g fructose) was administered to eight obese adolescents with polycystic ovarian syndrome (PCOS) followed by ingestion of [U-¹³C₃]glycerol at t = 180 or t = 210 min. ¹³C-labeling patterns of serum glucose and TG-glycerol were determined by nuclear magnetic resonance. ¹³C enrichment in plasma TG-glycerol was detectable and stable from 240 to 390 min with the [U-¹³C₃]glycerol drink at t = 180 min(3.65 ± 2.3 to …