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D espite extensive recent studies, understanding of the normal postprandial processes underlying immediate storage of substrate and maintenance of glucose homeostasis in humans after a mixed meal has been incomplete. The present study applied 13C nuclear magnetic resonance spectroscopy to measure sequential changes in hepatic glycogen concentration, a novel tracer approach to measure postprandial suppression of hepatic glucose output, and acetaminophen to trace the pathways of hepatic glycogen synthesis to elucidate the homeostatic adaptation to the fed state in healthy human subjects. After the liquid mixed meal, liver glycogen concentration rose from 207 +/- 22 to 316 +/- 19 mmol/liter at an average rate of 0.34 mmol/liter per min and peaked at 318 +/- 31 min, falling rapidly thereafter (0.26 mmol/liter per min). The mean increment at peak represented net glycogen synthesis of 28.3 +/- 3.7 g (approximately 19% of meal carbohydrate content). The contribution of the direct pathway to overall glycogen synthesis was 46 +/- 5 and 68 +/- 8% between 2 and 4 and 4 and 6 h, respectively. Hepatic glucose output was completely suppressed within 30 min of the meal. It increased steadily from 60 to 255 min from 0.31 +/- 32 to 0.49 +/- 18 mg/kg per min then rapidly returned towards basal levels (1.90 +/- 0.04 mg/kg per min). This pattern of change mirrored precisely the plasma glucagon/insulin ratio. These data provide for the first time a comprehensive picture of normal carbohydrate metabolism in humans after ingestion of a mixed meal.