Differential 'fuel usage' has been proposed as a mechanism for life-span extension by caloric restriction (CR). Here, we report the effects of CR, initiated after weaning, on metabolic enzyme gene expression 0, 1.5, 5, and 12 h after feeding of 24-month-old mice. Plasma glucose and insulin were reduced by approximately 20 and 80%. Therefore, apparent insulin sensitivity, as judged by the glucose to insulin ratio, increased 3.3-fold in CR mice. Phosphoenolpyruvate carboxykinase mRNA and activity were transiently reduced 1.5 h after feeding, but were 20-100% higher in CR mice at other times. Glucose-6-phosphatase mRNA was induced in CR mice and repressed in control mice before, and for 5 h following feeding. Feeding transiently induced glucokinase mRNA fourfold in control mice, but only slightly in CR mice. Pyruvate kinase and pyruvate dehydrogenase activities were reduced approximately 50% in CR mice at most times. Feeding induced glutaminase mRNA, and carbamyl phosphate synthetase I and glutamine synthase activity (and mRNA). They were each approximately twofold or higher in CR mice. These results indicate that in mice, CR maintains higher rates of gluconeogenesis and protein catabolism, even in the hours after feeding. The data are consistent with the idea that CR continuously promotes the turnover and replacement of extrahepatic proteins.