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To determine the source(s) of blood and very low density lipoprotein (VLDL)-triglyceride glycerol during fasting, four men ingested (2)H(2)O from 14 to 20 h into a 60-h fast to achieve ~0.5% body water enrichment. At 60 h of fasting, glycerol flux was measured using [2-(14)C]glycerol. Blood was taken for measurement of (2)H enrichment at carbon 6 of glucose and at carbon 3 of free glycerol and VLDL-triglyceride glycerol. (2)H enrichment of the 2 hydrogens bound to carbon 3 of VLDL-triglyceride glycerol was 105 +/- 2% of the (2)H enrichment of the 2 hydrogens bound to carbon 6 of glucose, indicating isotopic equilibrium between hepatic glyceraldehyde 3-P and glycerol 3-P. The (2)H enrichment of the 2 hydrogens bound to carbon 3 of free glycerol was 17 +/- 3% of VLDL-triglyceride glycerol, indicating that a significant percentage of free glycerol in blood originated from the hydrolysis of circulating VLDL-triglyceride or a pool of glycerol with similar (2)H enrichment. Glycerol flux was 6.3 +/- 1.1 micromol. kg(-1). min(-1). Glycerol appearing from nonadipose tissue sources was then approximately 1.1 micromol. kg(-1). min(-1). Seven other subjects were fasted for 12, 42, and 60 h. A small percentage of glycerol in the circulation after 12 h of fasting was enriched with (2)H. The enrichment of the 2 hydrogens bound to carbon 3 of free glycerol in the longer periods of fasting was approximately 16% of the enrichment of the 2 hydrogens bound to carbon 6 of glucose. Therefore, as much as 15-20% of systemic glycerol turnover during fasting is not from lipolysis of adipose tissue triglyceride.

Impaired glucose effectiveness (i.e., a diminished ability of glucose per se to facilitate its own metabolism), increased gluconeogenesis, and endogenous glucose release are, together with insulin resistance and beta-cell abnormalities, established features of type 2 diabetes. To explore aspects of the pathophysiology behind type 2 diabetes, we assessed in a group of healthy people prone to develop type 2 diabetes (n = 23), namely first-degree relatives of type 2 diabetic patients (FDR), 1) endogenous glucose release and fasting gluconeogenesis measured using the 2H2O technique and 2) glucose effectiveness. The FDR group was insulin resistant when compared with an age-, sex-, and BMI-matched control group without a family history of type 2 diabetes (n = 14) (M value, clamp: 6.07 +/- 0.48 vs. 8.06 +/- 0.69 mg x kg(-1) lean body weight (lbw) x min(-1); P = 0.02). Fasting rates of gluconeogenesis (1.28 +/- 0.06 vs. 1.41 +/- 0.07 mg x kg(-1) lbw x min(-1); FDR vs. control subjects, P = 0.18) did not differ in the two groups and accounted for 53 +/- 2 and 60 +/- 3% of total endogenous glucose release. Glucose effectiveness was examined using a combined somatostatin and insulin infusion (0.17 vs. 0.14 mU x kg(-1) x min(-1), FDR vs. control subjects), the latter replacing serum insulin at near baseline levels. In addition, a 360-min labeled glucose infusion was given to simulate a prandial glucose profile. After glucose infusion, the integrated plasma glucose response above baseline (1,817 +/- 94 vs. 1,789 +/- 141 mmol/l per 6 h), the ability of glucose to simulate its own uptake (1.50 +/- 0.13 vs. 1.32 +/- 0.16 ml x kg(-1) lbw x min(-1)), and the ability of glucose per se to suppress endogenous glucose release did not differ between the FDR and control group. In conclusion, in contrast to overt type 2 diabetic patients, healthy people at high risk of developing type 2 diabetes are characterized by normal glucose effectiveness at near-basal insulinemia and normal fasting rates of gluconeogenesis.

Healthy subjects ingested (2)H(2)O. (2)H enriched the hydrogen bound to carbon 1 of blood glucose 1.3 to 1.8 times more than the hydrogens bound to carbon 6. Enrichment at carbon 1 was more than at carbon 5 after 14 h, but not after 42 h, of fasting. After overnight fasting, when [2,3-(3)H]succinate was infused, 34 times as much (3)H was bound to carbon 6 as to carbon 1. On [1-(2)H,1-(3)H, 1-(14)C]galactose infusion, the ratios of (2)H to (14)C and of (3)H to (14)C in blood glucose were 30% less than in the galactose. (3)H at carbon 6 was 1% of that at carbon 1 of the glucose. Thus, although the two hydrogens bound to carbon 1 and the two bound to carbon 6 of fructose 6-phosphate (p) during gluconeogenesis are equally enriched in (2)H via pyruvate's equilibration with alanine, one of each is further enriched via hydration of fumarate that is converted to glucose. That hydrogen at carbon 1 of fructose 6-phosphate (P) is also enriched in fructose 6-P's equilibration with mannose 6-P. (2)H from (2)H(2)O at carbon 1 to carbon 2 of blood glucose cannot then quantitate gluconeogenesis because of [1-(2)H]glucose formation during glycogenolysis. Triose-P cycling has a minimal effect on quantitation. (2)H recovery in glucose from [1-(2)H]galactose does not quantitate galactose conversion via UDP-glucose to glycogen.

The use of 2H2O in estimating gluconeogenesis' contribution to glucose production (%GNG) was examined during progressive fasting in three groups of healthy subjects. One group (n = 3) ingested 2H2O to a body water enrichment of approximately 0.35% 5 h into the fast. %GNG was determined at 2-h intervals from the ratio of the enrichments of the hydrogens at C-5 and C-2 of blood glucose, assayed in hexamethylenetetramine. %GNG increased from 40 +/- 8% at 10 h to 93 +/- 6% at 42 h. Another group ingested 2H2O over 2.25 h, beginning at 11 h (n = 7) and 19 h (n = 7) to achieve approximately 0.5% water enrichment. Enrichment in plasma water and at C-2 reached steady state approximately 1 h after completion of 2H2O ingestion. The C-5-to-C-2 ratio reached steady state by the completion of 2H2O ingestion. %GNG was 54 +/- 2% at 14 h and 64 +/- 2% at 22 h. A 3-h [6,6-2H2]glucose infusion was also begun to estimate glucose production from enrichments at C-6, again in hexamethylenetetramine. Glucose produced by gluconeogenesis was 0.99 +/- 0.06 mg.kg-1.min-1 at both 14 and 22 h. In a third group (n = 3) %GNG reached steady state approximately 2 h after 2H2O ingestion to only approximately 0.25% enrichment. In conclusion, %GNG by 2 h after 2H2O ingestion and glucose production using [6,6-2H2]glucose infusion, begun together, can be determined from hydrogen enrichments at blood glucose C-2, C-5, and C-6. %GNG increases gradually from the postabsorptive state to 42 h of fasting, without apparent change in the quantity of glucose produced by gluconeogenesis at 14 and 22 h.

Healthy subjects ingested 2H2O and after 14, 22, and 42 h of fasting the enrichments of deuterium in the hydrogens bound to carbons 2, 5, and 6 of blood glucose and in body water were determined. The hydrogens bound to the carbons were isolated in formaldehyde which was converted to hexamethylenetetramine for assay. Enrichment of the deuterium bound to carbon 5 of glucose to that in water or to carbon 2 directly equals the fraction of glucose formed by gluconeogenesis. The contribution of gluconeogenesis to glucose production was 47 +/- 49% after 14 h, 67 +/- 41% after 22 h, and 93 +/- 2% after 42 h of fasting. Glycerol's conversion to glucose is included in estimates using the enrichment at carbon 5, but not carbon 6. Equilibrations with water of the hydrogens bound to carbon 3 of pyruvate that become those bound to carbon 6 of glucose and of the hydrogen at carbon 2 of glucose produced via glycogenolysis are estimated from the enrichments to be approximately 80% complete. Thus, rates of gluconeogenesis can be determined without corrections required in other tracer methodologies. After an overnight fast gluconeogenesis accounts for approximately 50% and after 42 h of fasting for almost all of glucose production in healthy subjects.

A method is introduced for estimating the contribution of gluconeogenesis to glucose production. 2H2O is administered orally to achieve 0.5% deuterium enrichment in body water. Enrichments are determined in the hydrogens bound to carbons 2 and 6 of blood glucose and in urinary water. Enrichment at carbon 6 of glucose is assayed in hexamethylenetetramine, formed from formaldehyde produced by periodate oxidation of the glucose. Enrichment at carbon 2 is assayed in lactate formed by enzymatic transfer of the hydrogen from glucose via sorbitol to pyruvate. The fraction gluconeogenesis contributes to glucose production equals the ratio of the enrichment at carbon 6 to that at carbon 2 or in urinary water. Applying the method, the contribution of gluconeogenesis in healthy subjects was 23-42% after fasting 14 h, increasing to 59-84% after fasting 42 h. Enrichment at carbon 2 to that in urinary water was 1.12 +/- 0.13. Therefore, the assumption that hydrogen equilibrated during hexose-6-P isomerization was fulfilled. The 3H/14C ratio in glucose formed from [3-3H,3-14C]lactate given to healthy subjects was 0.1 to 0.2 of that in the lactate. Therefore equilibration during gluconeogenesis of the hydrogen bound to carbon 6 with that in body water was 80-90% complete, so that gluconeogenesis is underestimated by 10-20%. Glycerol's contribution to gluconeogenesis is not included in these estimates. The method is applicable to studies in humans of gluconeogenesis at safe doses of 2H2O.

The effect of fasting vs. refeeding on hepatic glycogen repletion by the direct pathway, i.e., glucose----glucose 6-phosphate (G-6-P)----glycogen, was determined. Acetaminophen was administered during an infusion of glucose labeled with [1-13C]- and [6-14C]glucose into four healthy volunteers after an overnight fast and into the same subjects 4 h after breakfast. 13C enrichments in C-1 and C-6 of glucose formed from urinary acetaminophen glucuronide compared with enrichments in C-1 and C-6 of plasma glucose provided an estimate of glycogen formation by the direct pathway. The specific activity of glucose from the glucuronide compared with the specific activity of the plasma glucose, along with the percentages of 14C in C-1 and C-6 of the glucose from the glucuronide, also provided an estimate of the amount of glycogen formed by the direct pathway. The estimates were similar. Those from [6-14C]glucose would have been higher than from [1-13C]glucose if the pentose cycle contribution to overall glucose utilization had been significant. After an overnight fast, during the last hour of infusion, 49 +/- 3% of the glycogen formed was formed via the direct pathway. After breakfast, at similar plasma glucose and insulin concentrations, the percentage increased to 69 +/- 7% (P less than 0.02). Thus the contributions of the pathways to hepatic glycogen formation depend on the dietary state of the individual. For a dietary regimen in which individuals consume multiple meals per day containing at least a moderate amount of carbohydrates most glycogen synthesis occurs by the direct pathway.