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Unlabelled: We are developing a methodology for the noninvasive imaging of glucose transport in vivo with PET and (18)F-labeled 6-fluoro-6-deoxy-d-glucose ((18)F-6FDG), a tracer that is transported but not phosphorylated. To validate the method, we evaluated the biodistribution of (18)F-6FDG to test whether it is consistent with the known properties of glucose transport, particularly with regard to insulin stimulation of glucose transport., Methods: Under glucose clamp conditions, rats were imaged at the baseline and under conditions of hyperinsulinemia., Results: The images showed that the radioactivity concentration in skeletal muscle was higher in the presence of insulin than at the baseline. We also found evidence that the metabolism of (18)F-6FDG was negligible in several tissues., Conclusion: (18)F-6FDG is a valid tracer that can be used in in vivo transport studies. PET studies performed under glucose clamp conditions demonstrated that the uptake of nonphosphorylated glucose transport tracer (18)F-6FDG is sensitive to insulin stimulation.

The diabetogenic effect of excess growth hormone (GH) such as that in acromegaly is well known. However, the contribution of the various components to hepatic glucose production (HGP) is not completely understood. In this study we evaluated insulin resistance, HGP, gluconeogenesis (GNG), and glycogenolysis (GLY) in five patients with acromegaly before and after pituitary microsurgery. Insulin resistance was estimated by the HOMA index. HGP was measured using a primed continuous (6,6- 2H2) glucose infusion, and GNG was measured from 2 H enrichment at carbons 2 and 5 of blood glucose on ingestion of 2H2O. The ratio of these enrichments equals the fractional contribution of GNG to HGP, and GLY was calculated as the difference between HGP and GNG. All measurements were performed after 12 hours of fasting. Levels of GH and IGF-I decreased, as did the HOMA index (p<0.05). HGP was reduced from 11.4 micromol/kg/min to 9.7 micromol/kg/min (p=0.032). GNG contributed most to HGP. GNG was unchanged, whereas GLY's fraction decreased 29% (p=0.056) postoperatively. This pilot study indicates that GNG is the main contributor to HGP and that GLY is more sensitive than is GNG to the insulin resistance existing in acromegaly.

Objective: The deuterated water method uses the ratio of deuterium on carbons 5 and 2 (C5/C2) or 3 and 2 (C3/C2) to estimate the fraction of glucose derived from gluconeogenesis. The current studies determined whether C3 and C5 glucose enrichment is influenced by processes other than gluconeogenesis., Research Design and Methods: Six nondiabetic subjects were infused with [3,5-(2)H(2)]glucose and insulin while glucose was clamped at approximately 5 mmol/l; the C5-to-C3 ratio was measured in the in UDP-glucose pool using nuclear magnetic resonance and the acetaminophen glucuronide method., Results: Whereas the C5-to-C3 ratio of the infusate was 1.07, the ratio in UDP-glucose was <1.0 in all subjects both before (0.75 +/- 0.07) and during (0.67 +/- 0.05) the insulin infusion., Conclusions: These data indicate that the deuterium on C5 of glucose is lost more rapidly relative to the deuterium on C3. The decrease in the C5-to-C3 ratio could result from exchange of the lower three carbons of fructose-6-phosphate with unlabeled three-carbon precursors via the transaldolase reaction and/or selective retention of the C3 deuterium at the level of triosephosphate isomerase due to a kinetic isotope effect. After ingestion of (2)H(2)O, these processes would increase the enrichment of C5 and decrease the enrichment of C3, respectively, with the former causing an overestimation of gluconeogenesis using the C2-to-C5 ratio and the latter an underestimation using the C3-to-C2 ratio. Future studies will be required to determine whether the impact of these processes on the measurement of gluconeogenesis differs among the disease states being evaluated (e.g., diabetes or obesity).

Glucose transport rates are estimated noninvasively in physiological and pathological states by kinetic imaging using PET. The glucose analog most often used is (18)F-labeled 2FDG. Compared with glucose, 2FDG is poorly transported by intestine and kidney. We examined the possible use of 6FDG as a tracer of glucose transport. Lacking a hydroxyl at its 6th position, 6FDG cannot be phosphorylated as 2FDG is. Prior studies have shown that 6FDG competes with glucose for transport in yeast and is actively transported by intestine. Its uptake by muscle has been reported to be unresponsive to insulin, but that study is suspect. We found that insulin stimulated 6FDG uptake 1.6-fold in 3T3-L1 adipocytes and azide stimulated the uptake 3.7-fold in Clone 9 cells. Stimulations of the uptake of 3OMG, commonly used in transport assays, were similar, and the uptakes were inhibited by cyclochalasin B. Glucose transport is by GLUT1 and GLUT4 transporters in 3T3-L1 adipocyte and by the GLUT1 transporter in Clone 9 cells. Cytochalasin B inhibits those transporters. Rats were also imaged in vivo by PET using 6(18)FDG. There was no excretion of (18)F into the urinary bladder unless phlorizin, an inhibitor of active renal transport, was also injected. (18)F activity in brain, liver, and heart over the time of scanning reached a constant level, in keeping with the 6FDG being distributed in body water. In contrast, (18)F from 2(18)FDG was excreted in relatively large amounts into the bladder, and (18)F activity rose with time in heart and brain in accord with accumulation of 2(18)FDG-6-P in those organs. We conclude that 6FDG is actively transported by kidney as well as intestine and is insulin responsive. In trace quantity, it appears to be distributed in body water unchanged. These results provide support for its use as a valid tracer of glucose transport.

Aims/hypothesis: Glycogen cycling, i.e. simultaneous glycogen synthesis and glycogenolysis, affects estimates of glucose fluxes using tracer techniques and may contribute to hyperglycaemia in diabetic conditions. This study presents a new method for quantifying hepatic glycogen cycling in the fed state. Glycogen is synthesised from glucose by the direct and indirect (gluconeogenic) pathways. Since glycogen is also synthesised from glycogen, i.e. glycogen-->glucose 1-phosphate-->glycogen, that synthesised through the direct and indirect pathways does not account for 100% of glycogen synthesis. The percentage contribution of glycogen cycling to glycogen synthesis then equals the difference between the sum of the percentage contributions of the direct and indirect pathways and 100., Materials and Methods: The indirect and direct pathways were measured independently in nine healthy volunteers who had fasted overnight. They ingested (2)H(2)O (5 ml/kg body water) and were infused with [5-(3)H]glucose and acetaminophen (paracetamol; 1 g) during hyperglycaemic clamps (7.8 mmol/l) lasting 8 h. The percentage contribution of the indirect pathway was calculated from the ratio of (2)H enrichments at carbon 5 to that at carbon 2, and the contribution of the direct pathway was determined from the (3)H-specific activity, relative to plasma glucose, of the urinary glucuronide excreted between 2 and 4, 4 and 6, and 6 and 8 h., Results: Glucose infusion rates increased (p<0.01) to approximately 50 mumol kg(-1) min(-1). Plasma insulin and the insulin : glucagon ratio rose approximately 3.6- and approximately 8.3-fold (p<0.001), respectively. From the difference between 100% and the sum of the direct (2-4 h, 54+/-6%; 4-6 h, 59+/-5%; 6-8 h, 63+/-4%) and indirect (32+/-3, 38+/-4, 36+/-3%) pathways, glycogen cycling was seen to be decreased (p<0.05) from 14+/-4% (2-4 h) to 4+/-3% (4-6 h) and 1+/-3% (6-8 h)., Conclusions/interpretation: This method allows measurement of hepatic glycogen cycling in the fed state and demonstrates that glycogen cycling occurs most in the early hours after glucose loading subsequent to a fast.

It is well established that subjects with liver cirrhosis are insulin resistant, but the contribution of defects in insulin secretion and/or action to glucose intolerance remains unresolved. Healthy individuals and subjects with liver cirrhosis were studied on two occasions: 1) an oral glucose tolerance test was performed, and 2) insulin secretion was inhibited and glucose was infused in a pattern and amount mimicking the systemic delivery rate of glucose after a carbohydrate meal. Insulin was concurrently infused to mimic a healthy postprandial insulin profile. Postabsorptive glucose concentrations were equal (5.36 +/- 0.12 vs. 5.40 +/- 0.25 mmol/l, P = 0.89), despite higher insulin (P < 0.01), C-peptide (P < 0.01), and free fatty acid (P = 0.05) concentrations in cirrhotic than in control subjects. Endogenous glucose release (EGR; 11.50 +/- 0.50 vs. 11.73 +/- 1.00 mumol.kg(-1).min(-1), P = 0.84) and the contribution of gluconeogenesis to EGR (6.60 +/- 0.47 vs. 6.28 +/- 0.64 mumol.kg(-1).min(-1), P = 0.70) were unaltered by cirrhosis. A minimal model recently developed for the oral glucose tolerance test demonstrated an impaired insulin sensitivity index (P < 0.05), whereas the beta-cell response to glucose was unaltered (P = 0.72). During prandial glucose and insulin infusions, the integrated glycemic response was greater in cirrhotic than in control subjects (P < 0.05). EGR decreased promptly and comparably in both groups, but glucose disappearance was insufficient at the prevailing glucose concentration (P < 0.05). Moreover, identical rates of [3-(3)H]glucose infusion produced higher tracer concentrations in cirrhotic than in control subjects (P < 0.05), implying a defect in glucose uptake. In conclusion, carbohydrate intolerance in liver cirrhosis is determined by insulin resistance and the ability of glucose to stimulate insulin secretion. During prandial glucose and insulin concentrations, EGR suppression was unaltered, but glucose uptake was impaired, which demonstrates that intolerance can be ascribed to a defect in glucose uptake, rather than abnormalities in glucose production or beta-cell function. Although insulin secretion ameliorates glucose intolerance, impaired glucose uptake during physiological glucose and insulin concentrations produces marked and sustained hyperglycemia, despite concurrent abnormalities in glucose production or insulin secretion.

Excess cortisol has been demonstrated to impair hepatic and extrahepatic insulin action. To determine whether glucose effectiveness and, in terms of endogenous glucose release (EGR), gluconeogenesis, also are altered by hypercortisolemia, eight healthy subjects were studied after overnight infusion with hydrocortisone or saline. Glucose effectiveness was assessed by a combined somatostatin and insulin infusion protocol to maintain insulin concentration at basal level in the presence of prandial glucose infusions. Despite elevated insulin concentrations (P < 0.05), hypercortisolemia resulted in higher glucose (P < 0.05) and free fatty acid concentrations (P < 0.05). Furthermore, basal insulin concentrations were higher during hydrocortisone than during saline infusion (P < 0.01), indicating the presence of steroid-induced insulin resistance. Postabsorptive glucose production (P = 0.64) and the fractional contribution of gluconeogenesis to EGR (P = 0.33) did not differ on the two study days. During the prandial glucose infusion, the integrated glycemic response above baseline was higher in the presence of hydrocortisone than during saline infusion (P < 0.05), implying a decrease in net glucose effectiveness (4.42 +/- 0.52 vs. 6.65 +/- 0.83 ml.kg-1.min-1; P < 0.05). To determine whether this defect is attributable to an impaired ability of glucose to suppress glucose production, to stimulate its own uptake, or both, glucose turnover and "hot" (labeled) indexes of glucose effectiveness (GE) were calculated. Hepatic GE was lower during cortisol than during saline infusion (2.39 +/- 0.24 vs. 3.82 +/- 0.51 ml.kg-1.min-1; P < 0.05), indicating a defect in the ability of glucose to restrain its own production. In addition, in the presence of excess cortisol, glucose disappearance was inappropriate for the prevailing glucose concentration, implying a decrease in glucose clearance (P < 0.05). The decrease in glucose clearance was confirmed by the higher increment in [3-3H]glucose during hydrocortisone than during saline infusion (P < 0.05), despite the administration of identical tracer infusion rates. In conclusion, short-term hypercortisolemia in healthy individuals with normal beta-cell function decreases insulin action but does not alter rates of EGR and gluconeogenesis. In addition, cortisol impairs the ability of glucose to suppress its own production, which due to accumulation of glucose in the glucose space results in impaired peripheral glucose clearance. These results suggest that cortisol excess impairs glucose tolerance by decreasing both insulin action and glucose effectiveness.

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.

Based on our earlier work, a 2.5-fold increase in insulin secretion should completely inhibit hepatic glucose production through the hormone's direct effect on hepatic glycogen metabolism. The aim of the present study was to test the accuracy of this prediction and to confirm that gluconeogenic flux, as measured by three independent techniques, was unaffected by the increase in insulin. A 40-min basal period was followed by a 180-min experimental period in which an increase in insulin was induced, with euglycemia maintained by peripheral glucose infusion. Arterial and hepatic sinusoidal insulin levels increased from 10 +/- 2 to 19 +/- 3 and 20 +/- 4 to 45 +/- 5 microU/ml, respectively. Net hepatic glucose output decreased rapidly from 1.90 +/- 0.13 to 0.23 +/- 0.16 mg. kg(-1). min(-1). Three methods of measuring gluconeogenesis and glycogenolysis were used: 1) the hepatic arteriovenous difference technique (n = 8), 2) the [(14)C]phosphoenolpyruvate technique (n = 4), and 3) the (2)H(2)O technique (n = 4). The net hepatic glycogenolytic rate decreased from 1.72 +/- 0.20 to -0.28 +/- 0.15 mg. kg(-1). min(-1) (P < 0.05), whereas none of the above methods showed a significant change in hepatic gluconeogenic flux (rate of conversion of phosphoenolpyruvate to glucose-6-phosphate). These results indicate that liver glycogenolysis is acutely sensitive to small changes in plasma insulin, whereas gluconeogenic flux is not.

Contributions of gluconeogenesis to glucose production were determined between 14 to 22 hours into a fast in type 2 diabetics (n = 9) and age-weight-matched controls (n = 7); ages, 60.4 +/- 2.3 versus 55.6 +/- 1.2 years and body mass indices (BMI) 28.6 +/- 2.3 versus 26.6 +/- 0.8 kg/m2. Production was measured using a primed-continuous [6,6-2H2]glucose infusion and gluconeogenesis from 2H enrichment at carbons 2 and 5 of blood glucose on 2H2O ingestion. Plasma glucose concentration declined from 9.6 +/- 0.6 at 14 hours to 7.3 +/- 0.6 at 22 hours in the diabetics (P = .001) and from 5.4 +/- 0.1 to 5.0 +/- 0.1 in the controls (P < .05). Production from the 17th to 22nd hour declined 27.1% +/- 0.6% in the diabetics versus 18.5% +/- 0.8% in the controls (P = .001); from 10.4 +/- 0.3 to 7.6 +/- 0.2 versus 10.0 +/- 0.4 to 8.2 +/- 0.4 micromol/kg/min. Percent contributions of gluconeogenesis to production measured at 1 1/2 to 2-hour intervals beginning the 15th hour were 6.8% +/- 1.0% more in the diabetics than controls. The quantity of glucose contributed by gluconeogenesis declined 19.8% +/- 3.8% (P < .001) in the diabetics and 6.9% +/- 2.3% in the controls (P = .05); 7.21 +/- 0.32 to 5.74 +/- 0.26 versus 6.20 +/- 0.28 to 5.75 +/- 0.24 micromol/kg/min. The contribution of glycogenolysis to production, estimated from the difference between production and gluconeogenesis, declined to the same extent in diabetic and control subjects, 40.7% +/- 6.6% and 37.7% +/- 4.1%; from 3.23 +/- 0.35 to 1.86 +/- 0.26 versus 3.81 +/- 0.22 to 2.42 +/- 0.28 micromol/kg/min. Thus, gluconeogenesis contributed more to glucose production in the diabetic than control subjects. Production and the contribution of gluconeogenesis declined more in the diabetic subjects during the fast. The factors regulating these changes remain uncertain.

To examine the mechanism by which metformin lowers endogenous glucose production in type 2 diabetic patients, we studied seven type 2 diabetic subjects, with fasting hyperglycemia (15.5 +/- 1.3 mmol/l), before and after 3 months of metformin treatment. Seven healthy subjects, matched for sex, age, and BMI, served as control subjects. Rates of net hepatic glycogenolysis, estimated by 13C nuclear magnetic resonance spectroscopy, were combined with estimates of contributions to glucose production of gluconeogenesis and glycogenolysis, measured by labeling of blood glucose by 2H from ingested 2H2O. Glucose production was measured using [6,6-2H2]glucose. The rate of glucose production was twice as high in the diabetic subjects as in control subjects (0.70 +/- 0.05 vs. 0.36 +/- 0.03 mmol x m(-2) min(-1), P < 0.0001). Metformin reduced that rate by 24% (to 0.53 +/- 0.03 mmol x m(-2) x min(-1), P = 0.0009) and fasting plasma glucose concentration by 30% (to 10.8 +/- 0.9 mmol/l, P = 0.0002). The rate of gluconeogenesis was three times higher in the diabetic subjects than in the control subjects (0.59 +/- 0.03 vs. 0.18 +/- 0.03 mmol x m(-2) min(-1) and metformin reduced that rate by 36% (to 0.38 +/- 0.03 mmol x m(-2) x min(-1), P = 0.01). By the 2H2O method, there was a twofold increase in rates of gluconeogenesis in diabetic subjects (0.42 +/- 0.04 mmol m(-2) x min(-1), which decreased by 33% after metformin treatment (0.28 +/- 0.03 mmol x m(-2) x min(-1), P = 0.0002). There was no glycogen cycling in the control subjects, but in the diabetic subjects, glycogen cycling contributed to 25% of glucose production and explains the differences between the two methods used. In conclusion, patients with poorly controlled type 2 diabetes have increased rates of endogenous glucose production, which can be attributed to increased rates of gluconeogenesis. Metformin lowered the rate of glucose production in these patients through a reduction in gluconeogenesis.

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.

Phenylacetate ingestion has been used to probe Krebs cycle metabolism and to augment waste nitrogen excretion in urea cycle disorders. Phenylalkanoic acids, including phenylacetate, have been proposed as potential therapeutic agents in the treatment of diabetes. They inhibit gluconeogenesis in the liver in vitro and reduce the blood glucose concentration in diabetic rats. The effect of sodium phenylacetate ingestion on blood glucose and the contribution of gluconeogenesis to glucose production have now been studied in 7 type 2 diabetic patients. The study was not designed to test whether the changes in glucose metabolism observed in the rat could be reproduced in humans. After an overnight fast, over a period of 1 hour, 4.8 g phenylacetate was ingested, which is the highest dose used to probe Krebs cycle metabolism. Glucose production was measured by tracer kinetics using [6,6-(2)H2]glucose and gluconeogenesis by the labeling of the hydrogens of blood glucose on (2)H20 ingestion. The concentration of phenylacetate in plasma peaked by 2 hours after its ingestion, and about 40% of the dose was excreted in 5 hours. The plasma glucose concentration and production, and the contribution of gluconeogenesis to glucose production, were unaffected by phenylacetate ingestion at the highest dose used to probe Krebs cycle metabolism.

Effects of free fatty acids (FFAs) on endogenous glucose production (EGP) and gluconeogenesis (GNG) were examined in healthy subjects (n = 6) during stepwise increased Intralipid/heparin infusion (plasma FFAs 0.8+/-0.1, 1.8+/-0.2, and 2.8+/-0.3 mmol/l) and during glycerol infusion (plasma FFAs approximately 0.5 mmol/l). Rates of EGP were determined with D-[6,6-2H2]glucose and contributions of GNG from 2H enrichments in carbons 2 and 5 of blood glucose after 2H2O ingestion. Plasma glucose concentrations decreased by approximately 10% (P < 0.01), whereas plasma insulin increased by approximately 47% (P = 0.02) after 9 h of lipid infusion. EGP declined from 9.3+/-0.5 (lipid) and 9.0+/-0.8 pmol x kg(-1) x min(-1) (glycerol) to 8.4+/-0.5 and 8.2+/-0.7 micromol x kg(-1) x min(-1), respectively (P < 0.01). Contribution of GNG similarly rose (P < 0.01) from 46+/-4 and 52+/-3% to 65+/-8 and 78+/-7%. To exclude interaction of FFAs with insulin secretion, the study was repeated at fasting plasma insulin (approximately 35 pmol/l) and glucagon (approximately 90 ng/ml) concentrations using somatostatin-insulin-glucagon clamps. Plasma glucose increased by approximately 50% (P < 0.005) during lipid but decreased by approximately 12% during glycerol infusion (P < 0.005). EGP remained unchanged over the 9-h period (9.9+/-1.2 vs. 9.0+/-1.1 micromol x kg(-1) x min(-1)). GNG accounted for 62+/-5 (lipid) and 60+/-6% (glycerol) of EGP at time 0 and rose to 74+/-3% during lipid infusion only (P < 0.05 vs. glycerol: 64+/-4%). In conclusion, high plasma FFA concentrations increase the percent contribution of GNG to EGP and may contribute to increased rates of GNG in patients with type 2 diabetes.

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.

Net hepatic glycogenolysis and gluconeogenesis were examined in normal (n = 4) and cirrhotic (n = 8) subjects using two independent methods [13C nuclear magnetic resonance spectroscopy (NMR) and a 2H2O method]. Rates of net hepatic glycogenolysis were calculated by the change in hepatic glycogen content before ( approximately 11:00 PM) and after ( approximately 7:00 AM) an overnight fast using 13C NMR and magnetic resonance imaging. Gluconeogenesis was calculated as the difference between the rates of glucose production determined with an infusion of [6,6-2H2]glucose and net hepatic glycogenolysis. In addition, the contribution of gluconeogenesis to glucose production was determined by the 2H enrichment in C-5/C-2 of blood glucose after intake of 2H2O (5 ml/kg body water). Plasma levels of total and free insulin-like growth factor I (IGF-I) and IGF-I binding proteins-1 and -3 were significantly decreased in the cirrhotic subjects (P < 0.01 vs. controls). Postprandial hepatic glycogen concentrations were 34% lower in the cirrhotic subjects (P = 0.007). Rates of glucose production were similar between the cirrhotic and healthy subjects [9.0 +/- 0.9 and 10.0 +/- 0.8 micromol. kg body wt-1. min-1, respectively]. Net hepatic glycogenolysis was 3.5-fold lower in the cirrhotic subjects (P = 0.01) and accounted for only 13 +/- 6% of glucose production compared with 40 +/- 10% (P = 0.03) in the control subjects. Gluconeogenesis was markedly increased in the cirrhotic subjects and accounted for 87 +/- 6% of glucose production vs. controls: 60 +/- 10% (P = 0.03). Gluconeogenesis in the cirrhotic subjects, as determined from the 2H enrichment in glucose C-5/C-2, was also increased and accounted for 68 +/- 3% of glucose production compared with 54 +/- 2% (P = 0.02) in the control subjects. In conclusion, cirrhotic subjects have increased rates of gluconeogenesis and decreased rates of net hepatic glycogenolysis compared with control subjects. These alterations are likely important contributing factors to their altered carbohydrate metabolism.

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.

In order to examine metabolic zonation in human liver, [2-14C]glycerol, which labels carbons 2 and 5 of glucose-6-P, and [1-14C]lactate, which labels carbons 3 and 4 of glucose-6-P, in the process of gluconeogenesis, were infused intravenously into healthy subjects who ingested acetaminophen and had fasted 36 h. Distributions of 14C were determined in glucose in blood and in the glucuronic acid moiety of acetaminophen glucuronide excreted in urine. Ratios of 14C in carbons 2 and 5 to 14C in carbons 3 and 4 were significantly higher in blood glucose than in glucuronide. Since glucose and glucuronic acid are formed from glucose-6-P in liver without randomization of carbon, the differences in the ratios indicate that the pool of glucose-6-P in liver is not homogeneous. The glucuronide sampled glucose-6-P with more label from lactate than glycerol compared to the glucose-6-P sampled by the glucose. The apparent explanation is the greater decrease in glycerol compared with lactate concentration as blood streams from the periportal to the perivenous zones of the liver lobule. Glucuronidation is then expressed in humans relatively more in the perivenous than periportal zones and gluconeogenesis from glycerol more in the periportal than perivenous zones.

Normal subjects, fasted 60 h, and patients with insulin-dependent diabetes mellitus (IDDM), withdrawn from insulin and fasted overnight, were given phenylacetate orally and intravenously infused with [3-14C]lactate and 13C-bicarbonate. Rates of hepatic gluconeogenesis relative to Krebs cycle rates were estimated from the 14C distribution in glutamate from urinary phenylacetylglutamine. Assuming the 13C enrichment of breath CO2 was that of the CO2 fixed by pyruvate, the enrichment to be expected in blood glucose, if all hepatic glucose production had been by gluconeogenesis, was then estimated. That estimate was compared with the actual enrichment in blood glucose, yielding the fraction of glucose production due to gluconeogenesis. Relative rates were similar in the 60-h fasted healthy subjects and the diabetic patients. Conversion of oxaloacetate to phosphoenolpyruvate was two to eight times Krebs cycle flux and decarboxylation of pyruvate to acetyl-CoA, oxidized in the cycle, was less than one-30th the fixation by pyruvate of CO2. Thus, in estimating the contribution of a gluconeogenic substrate to glucose production by measuring the incorporation of label from the labelled substrate into glucose, dilution of label at the level of oxaloacetate is relatively small. Pyruvate cycling was as much as one-half the rate of conversion of pyruvate to oxaloacetate. Glucose and glutamate carbons were derived from oxaloacetate formed by similar pathways if not from a common pool. In the 60-h fasted subjects, over 80% of glucose production was via gluconeogenesis. In the diabetic subjects the percentages averaged about 45%.

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.

Purposes of this study were 1) to estimate in humans, using 14C-labeled propionate, the rate of hepatic gluconeogenesis relative to the rate of Krebs cycle flux; 2) to compare those rates with estimates previously made using [3-14C]lactate and [2-14C]acetate; 3) to determine if the amount of ATP required for that rate of gluconeogenesis could be generated in liver, calculated from that rate of Krebs cycle flux and splanchnic balance measurements, previously made, and 4) to test whether hepatic succinyl-CoA is channeled during its metabolism through the Krebs cycle. [2-14C]propionate, [3-14C]-propionate, and [2,3-14C]succinate were given along with phenyl acetate to normal subjects, fasted 60 h. Distributions of 14C were determined in the carbons of blood glucose and of glutamate from excreted phenylacetylglutamine. Corrections to the distributions for 14CO2 fixation were made from the specific activities of urinary urea and the specific activities in glucose, glutamate, and urea previously found on administering [14C]-bicarbonate. Uncertainties in the corrections and in the contributions of pyruvate and Cori cyclings limit the quantitations. The rate of gluconeogenesis appears to be two or more times the rate of Krebs cycle flux and pyruvate's decarboxylation to acetyl-CoA, metabolized in the cycle, less than one-twenty-fifth the rate of its decarboxylation. Such estimates were previously made using [3-14C]lactate. The findings support the use of phenyl acetate to sample hepatic alpha-ketoglutarate. Ratios of specific activities of glucose to glutamate and glucose to urinary urea and expired CO2 indicate succinate's extensive metabolism when presented in trace amounts to liver. Utilizations of the labeled compounds by liver relative to other tissues were in the order succinate = lactate > propionate > acetate. ATP required for gluconeogenesis and urea formation was approximately 40% of the amount of ATP generated in liver. There was no channeling of succinyl-CoA in the Krebs cycle in the hepatic mitochondria.

Estimating the rate of hepatic gluconeogenesis in vivo from the incorporation of 14C from 14CO2 into glucose requires determination of the rates in liver of equilibration of oxaloacetate with fumarate, conversion of oxaloacetate to phosphoenolpyruvate (PEP), and conversion of PEP to pyruvate, all relative to the rate of tricarboxylic acid cycle flux. With the use of a model of mitochondrial metabolism and gluconeogenesis, expressions are derived relating specific activity of carboxyl of PEP from 14CO2 to those rates and specific activity of mitochondrial CO2. If those rates and specific activity of mitochondrial CO2 are known, specific activity of PEP, calculated using the expressions, should, on a mole basis, be one-half the specific activity of the glucose formed. At steady state, in the 60-h fasted individual, where glucose formation is solely by gluconeogenesis, twice estimated specific activity of PEP should then approximate that of blood glucose. Estimates of relative rates in 60-h fasted humans, previously made from distribution of 14C in glutamate from phenylacetylglutamine excreted when [3-14C]lactate and phenylacetate were given, were applied to the expressions. Specific activity of mitochondrial CO2 was equated to that of CO2 expired by 60-h fasted subjects given NaH14CO3 and alpha-[1-14C]ketoisocaproate. Predicted specific activities approximated actual specific activities of blood glucose when NaH14CO3 was administered. alpha-[1-14C]ketoisocaproate administrations gave underestimates. This is attributable to differences between specific activities of hepatic mitochondrial CO2 and expired CO2, which is evidenced by higher incorporations of 14C in glucose than in expired CO2 from alpha-[1-14C]ketoisocaproate than from NaH14CO3.(ABSTRACT TRUNCATED AT 250 WORDS)

A method is introduced for quantitating cycling between hepatic glycogen and glucose-1-P in humans. It depends on the administration of trace [2-3H,6-14C]galactose, a glucose load, and acetaminophen. The ratio of 3H to 14C in the glucuronide of the acetaminophen excreted in urine to that in the administered galactose provides the measure of the fraction of glycogen synthesized that is synthesized from glucose-1-P formed from glycogen. The quantity of glucose-1-P formed from glycogen that is not reconverted to glycogen is not measured. It is assumed that the glucuronide samples the UDP-glucose pool in liver from which glycogen is formed, the last glucosyl units formed from UDP-glucose in glycogen synthesis are the first broken down, and the equilibration of [2-3H]glucose-1-P with fructose-6-P is rapid relative to its conversion to UDP-glucose. During a 5-hour period, while three normal subjects and three non-insulin-dependent diabetics, who had fasted overnight, were infused with 4 mg/kg/min of glucose, the rate of glycogen breakdown, as measured using the method, was only a small percentage of the rate of glycogen synthesis.

To examine the fate of the carbons of acetate and to evaluate the usefulness of labeled acetate in assessing intrahepatic metabolic processes during gluconeogenesis, [2-14C]acetate, [2-14C]ethanol, and [1-14C]ethanol were infused into normal subjects fasted 60 h and given phenyl acetate. Distributions of 14C in the carbons of blood glucose and glutamate from urinary phenylacetylglutamine were determined. With [2-14C]acetate and [2-14C]ethanol, carbon 1 of glucose had about twice as much 14C as carbon 3. Carbon 2 of glutamate had about twice as much 14C as carbon 1 and one-half to one-third as much as carbon 4. There was only a small amount in carbon 5. These distributions are incompatible with the metabolism of [2-14C]acetate being primarily in liver. Therefore, [2-14C]acetate cannot be used to study Krebs cycle metabolism in liver and in relationship to gluconeogenesis, as has been done. The distributions can be explained by: (a) fixation of 14CO2 from [2-14C]acetate in the formation of the 14C-labeled glucose and glutamate in liver and (b) the formation of 14C-labeled glutamate in a second site, proposed to be muscle. [1,3-14C]Acetone formation from the [2-14C]acetate does not contribute to the distributions, as evidenced by the absence of 14C in carbons 2-4 of glutamate after [1-14C]ethanol administration.

To quantify intrahepatic Krebs cycle metabolism, phenyl acetate, excreted in urine as a glutamine conjugate, was given to healthy subjects infused with [3-14C]lactate. They were studied after 60 h of fasting and when given glucose after an overnight fast. Distributions of 14C in glutamate from urinary phenylacetylglutamine and blood glucose were determined. Corrections to the distributions because of the fixation of 14CO2 formed from the [3-14C]lactate were determined by administering [14C]bicarbonate. Comparisons of distributions in glucose and glutamate support the assumption that the glutamate distributions reflect those in hepatic alpha-ketoglutarate. From the distributions in glutamate, the extent of exchange of labeled with unlabeled carbons and relative flow rates in the cycle in liver were estimated. Dilution of 14C by 12C in the cycle was found in the fasted but not the fed state. In the fasted state, pyruvate carboxylation was estimated to be at least twice the rate of Krebs cycle flux and the rate of pyruvate's decarboxylation less than 1/25 the rate of its carboxylation. In the fed state, the rate of decarboxylation was estimated to be between one-sixth and one-half the rate of carboxylation. The rate of conversion of oxalacetate to fumarate in both states appeared to be greater than 6 times the rate of Krebs cycle flux.

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.

Hepatic fructose-6-phosphate (fructose-6-P) cycling and pentose cycle activity were quantified in hyperthyroid patients. A measure of the fructose-6-P cycle was the incorporation of 14C, on administering [3-3H,6-14C]galactose, into carbon 1 of blood glucose and the 3H/14C ratio in blood glucose. The measure of the pentose cycle was the randomization of 14C to carbon 1 of blood glucose on administering [2-14C]galactose. [2-3H]Galactose was also administered, so the 3H/14C ratio in blood glucose measured the extent of equilibration of glucose-6-P with fructose-6-P. Patients given [3-3H,6-14C]galactose were restudied when euthyroid. Of the 14C from [3-3H,6-14C]galactose, 7.7-9.5% was in carbon 1 of glucose in both states. 3H/14C ratios were also the same in both states. Fructose-6-P cycling was estimated to be 13 +/- 1% the rate of glucose turnover in the euthyroid and 15 +/- 1% that in the hyperthyroid state. The pentose cycle contributed about 2% to glucose utilization, similar to previous estimates in healthy humans. As in healthy individuals, about 25% of 3H was retained in the conversion of [2-3H]glucose-6-P to glucose. Thus, the fractions of glucose turnover participating in hepatic fructose-6-P and pentose cycling are similar in hyperthyroid and healthy subjects. As a result, augmented fructose-6-P cycling does not substantially contribute to increased hepatic oxygen consumption in hyperthyroidism.

Yields of (14)CO(2) from [2-(14)C]mevalonate and [5-(14)C]mevalonate have been used by others to estimate the activity of the non-sterol-forming pathway, also called the mevalonate shunt pathway, and yields of (14)C in sterols have been used to estimate the activity of the sterol-forming pathway. Both these pathways operate following the conversion of carbon 1 of mevalonate to CO(2). The estimations of the shunt pathway contribution are dependent upon the fractions of carbons 2 and 5 of mevalonate that are oxidized to CO(2) in the Krebs cycle after leaving the pathway. Unless all of carbons 2 and 5 are oxidized to CO(2), the estimates are minimal. The metabolism of mevalonate has now been examined in slices of livers and kidneys from fasted rats and rats in diabetic ketosis. Yields of (14)CO(2) from [1-(14)C]mevalonate are used as the measure of the contributions of all the pathways by which carbon 1 of mevalonate is converted to CO(2). Yields of (3)H-labeled nonsaponifiable lipids from [5-(3)H]mevalonate are used as the measure of the sterol-forming pathway. The differences in these yields are then taken as the measure of the non-sterol-forming pathway or pathways. Yields of (14)CO(2) from [1-(14)C]mevalonate markedly exceeded the sum of the yields of (14)C in CO(2) and nonsaponifiable lipids from either [2-(14)C]mevalonate or [5-(14)C]mevalonate. Therefore, in liver and kidney, under the conditions of this study, either one or more pathways other than the shunt pathway, by which mevalonate can be metabolized to other than sterols, is operative to a marked degree, or estimates of the shunt pathway's contributions as judged by yields of (14)CO(2) from [2-(14)C]mevalonate and [5-(14)C]mevalonate are significantly underestimated.-Brady, P. S., W. C. Schumann, S. Ohgaku, R. F. Scofield, and B. R. Landau. Evidence for an underestimation of the shunt pathway of mevalonate metabolism in slices of livers and kidneys from fasted rats and rats in diabetic ketosis.

The relative contributions of the direct and the indirect pathways to hepatic glycogen formation following a glucose load given to humans four hours after a substantial breakfast have been examined. Glucose loads labeled with [6-(14)C]glucose were given to six healthy volunteers along with diflunisal (1 g) or acetaminophen (1.5 g), drugs excreted in urine as glucuronides. Distribution of 14C in the glucose unit of the glucuronide was taken as a measure of the extent to which glucose was deposited directly in liver glycogen (ie, glucose----glucose-6-phosphate----glycogen) rather than indirectly (ie, glucose----C3-compound----glucose-6-phosphate----glycogen). The maximum contribution to glycogen formation by the direct pathway was estimated to be 77% +/- 4%, which is somewhat higher than previous estimates in humans fasted overnight (65% +/- 1%, P less than 0.05). Thus, the indirect pathway of liver glycogen formation following a glucose load is operative in both the overnight fasted and the fed state, although its contribution may be somewhat less in the fed state.

In estimating glucose and fructose 6-phosphate futile cycling in vivo, complete detritiation of [2-3H]glucose is assumed at the glucose 6-phosphate level, [3-3H]glucose at triose phosphate formation, and [6-3H]glucose in its conversion to glucose via pyruvate. [3-3H]glucose detritiation via the pentose cycle is assumed to be negligible. Normal and non-insulin-dependent diabetic subjects, in the basal state and infused with glucose, were given [2-3H,2-14C]galactose, and 3H-to-14C ratios in blood glucose were determined. [2-3H,2-14C]glucose was given with acetaminophen, and 3H/14C in urinary glucuronide was determined. Detritiation at glucose 6-phosphate was approximately 80%. [3-3H,1-14C]fructose was infused, and 3H/14C was determined in blood glucose and urinary glucuronide. At triose phosphate, 75-90% of the 3H was removed. The pentose cycle contribution was only a few percent. [6-3H,6-14C]glucose was infused, and 3H/14C in blood lactate was determined. [3-3H,3-14C]lactate was infused, and ratios in blood glucose were determined. Maximally, 10% of 3H from [6-3H]glucose was retained. If glucose and galactose are metabolized in the same hepatic site(s), glucose conversion to three-carbon intermediates in the indirect pathway of glycogen formation occurs in extrahepatic tissue(s). Reported estimates of futile cycling, although qualitatively correct, quantitatively require correction.

[2-14C]Acetone was infused into rats that were fed or fasted. Each was infused with either a trace quantity of acetone or a large quantity that resulted in a blood concentration of acetone of at least 4 mM. The distribution of 14C in the carbons of glucose from each rat was determined. Two of the rats were given acetone in their drinking water and one was diabetic. Whether a rat was chronically exposed to acetone, fed or fasted, normal or diabetic, if given the trace dose, over 80% of the 14C in the glucose it formed was in carbons 1, 2, 5, and 6 of the glucose. If a rat was given the large dose, about 50% was in carbons 3 and 4. Thus, the major determinant of the pathways followed by acetone when it is metabolized is its concentration and not the prior dietary state of the animal or its previous exposure to acetone. Incorporation into carbons 1, 2, 5, and 6 occurs in the conversion of the carbons of [2-14C]lactate into glucose, whereas incorporation into carbons 3 and 4 occurs in the conversion of the carbons of [1-14C]acetate into glucose. Therefore, at high acetone concentration, the pathway that has been proposed for acetone's metabolism via acetate predominates, and via acetate there can be no net synthesis of glucose from acetone. When rats were given cyanamide and then the large dose of acetone, 74% of the 14C in the glucose they formed was in carbons 3 and 4 of the glucoses. Thus, the relative contribution of the pathway to lactate, or its metabolic equivalent, that has been proposed appears to be lessened by the administration of an aldehyde dehydrogenase inhibitor.

[1-14C]Ribose and [2-14C]glucose were given to normal subjects along with glucose loads (1 g per kg of body weight) after administration of diflunisal and acetaminophen, drugs that are excreted in urine as glucuronides. Distributions of 14C were determined in the carbons of the excreted glucuronides and in the glucose from blood samples drawn from hepatic veins before and after glucagon administration. Eighty percent or more of the 14C from [1-14C]ribose incorporated into the glucuronic acid moiety of the glucuronides was in carbons 1 and 3, with less than 8% in carbon 2. In glucuronic acid from glucuronide excreted when [2-14C]glucose was given, 3.5-8.1% of the 14C was in carbon 1, 2.5-4.3% in carbon 3, and more than 70% in carbon 2. These distributions are in accord with the glucuronides sampling the glucose unit of the glucose 6-phosphate pool that is a component of the pentose pathway and is intermediate in glycogen formation. It is concluded that the glucuronic acid conjugates of the drugs can serve as a noninvasive means of sampling hepatic glucose 6-phosphate. In human liver, as in animal liver, the classical pentose pathway functions, not the L-type pathway, and only a small percentage of the glucose is metabolized via the pathway.

Distributions of 14C were different from those of 13C in glucoses formed by livers of rats in diabetic ketosis and perfused with [2-14C]acetone and [2-13C]lactate. There was 32-73% of the 14C and 8-12% of the 13C in carbons 3 and 4 of the glucoses with the remaining 14C and 13C distributed about equally in the other carbons. Incorporations of 14C from [2-14C]acetone (14-39%) also exceeded those from [2-14C]pyruvate (8-10%) into carbons 3 and 4 of glucoses formed by hepatocytes from rats fed acetone or fasted. [2-14C]Acetone and [2-14C]pyruvate were infused into rats that were fed, fasted, given acetone in their drinking water, or in diabetic ketosis. Thirty-seven to 52% of the 14C in the glucoses formed was in their carbons 3 and 4 when the acetone was infused and 8 to 14% when the pyruvate was infused. [1,3-14C]Hydroxybutyrate was formed by the rats in diabetic ketosis given [2-14C]acetone. It is concluded that acetone is metabolized in rats to a large extent by a pathway in which lactate or its metabolic equivalent is not an intermediate and that pathway is via acetyl-CoA. via acetyl-CoA.

p-Aminobenzoic acid was fed to normal and alloxan-induced diabetic rats injected with [omega-14C]labeled and [2-14C]labeled fatty acids. The p-acetamidobenzoic acid that was excreted was hydrolyzed to yield acetate which was degraded. The distribution of 14C in the acetates formed when an [omega-14C]labeled fatty acid was injected was similar to that when a [2-14C]labeled fatty acid was injected. This contrasts with the finding that in acetates from 2-acetamido-4-phenylbutyric acid excreted when 2-amino-4-phenylbutyric acid was fed, there was a difference in the distributions of 14C, a difference attributable to omega-oxidation of the fatty acid. Acetylation of p-aminobenzoic acid is then concluded to occur in a different cellular environment than that of 2-amino-4-phenylbutyric acid, one in which omega-oxidation is not functional. When 2-amino-4-phenylbutyric acid was fed and [6-14C]palmitic acid injected, rather than [16-14C]palmitic acid, the distribution of 14C in acetate was the same as when [2-14C]palmitic acid was injected. This indicates that the dicarboxylic acid formed on omega-oxidation of palmitic acid does not undergo beta-oxidation to form succinyl-CoA. Thus, glucose is not formed via omega-oxidation of long-chain fatty acid.

[2-14C]Glucose, [3,4-14C]glucose, [5-14C]glucose, [4,5,6-14C]glucose, and [1-14C]ribose were perfused through livers of rats. The rats were fed or fasted and refed. In one experiment the liver perfused was regenerating and in another phenazine methosulfate was in the perfusate. Perfusion was for 30 or 90 min. Glucose from each perfusate and liver glucose-6-P and glycogen were isolated, purified, and degraded. The distributions of 14C in the carbons of the glucoses from the glycogens are similar to the distributions from the glucose 6-phosphates. The distributions of 14C are in accord with metabolism of glucose by the classical pentose pathway and not by the L-type pathway that has been proposed to function in liver.

Diflunisal, 5-(2',4'-difluorophenyl)salicylic acid, excreted in urine as its glucuronide, was given to normal humans (n = 6) along with a glucose load specifically labeled with 14C. Glucuronide excreted by each subject was reduced to its glucoside and glucose from it degraded to yield the distribution of 14 C in its six carbons. Randomization of the 14C from the specifically labeled glucose was taken as a measure of the extent to which glucose was deposited indirectly (i.e., glucose----lactate----glucose----6-P----glycogen), rather than directly (i.e., glucose----glucose-6-P----glycogen). The maximum contribution to glycogen formation by the direct pathway was estimated to be 65 +/- 1%, on the assumption that glucuronide and glycogen are derived from the same hepatic pool of glucose-6-P in liver. Evidence that supports that assumption was obtained by comparing the randomization of 14C in the urinary glucuronide with that in glucose in blood from the hepatic vein of four of the subjects before and after they were given glucagon. Other evidence supporting the assumption was obtained by comparing in two subjects 3H/14C ratios in glucose from hepatic vein blood before and after glucagon administration with that in urinary glucuronide, having labeled the uridine diphosphate (UDP)-glucose in their livers with 14C by giving them 1-[14C]galactose and their circulating glucose with 3H by giving a 5-[3H]glucose-labeled load. It is concluded that glucuronide formation in humans can be used to trace glucose metabolism in the liver, and that in humans the indirect pathway of glucose metabolism is active.

Specifically 14C-labeled mevalonic acids were administered to rats in diabetic ketosis, and the distribution of 14C was determined in the hydroxybutyric acid each rat excreted. Also, the distributions of 14C were determined in hydroxybutyric acid formed by slices of livers and kidneys from rats in diabetic ketosis and incubated with the specifically labeled mevalonic acids. The distributions found are in accord with the conversion of mevalonate to hydroxymethylglutaryl-CoA by the shunt pathway proposed by J. Edmond and G. Popják ((1974) J. Biol. Chem. 249, 66-71). That is, carbon 5 of mevalonate was metabolized to form the carboxyl of acetyl-CoA and carbons 2 and 3 of mevalonate were converted in large measure to hydroxybutyric acid without acetyl-CoA as an intermediate, i.e. the bond between carbon 2 and 3 was not cleaved, while the bond between 1 and 2, traced with [1,2-14C]mevalonate, was cleaved. Similar distributions of 14C were found in hydroxybutyric acid excreted by rats in diabetic ketosis administered specifically 14C-labeled isovaleric acids, isovaleric acid having in its metabolism intermediates common to those in the shunt pathway.

A method has been developed for estimating in the intact cell the contribution of deacylation of acetoacetyl-CoA to the formation of acetoacetate relative to acetoacetate's formation via hydroxymethylglutaryl (HMG)-CoA. Estimates depend upon the fraction of the terminal four carbons of an even carbon-containing fatty acid that are converted to acetoacetate without prior conversion to acetyl-CoA, since in the formation of acetoacetate via HMG-CoA the omega-2 and omega-3 carbons of the fatty acid are converted to acetyl-CoA. Incorporation of 14C from [16-14C]palmitic acid into carbon 2 relative to carbon 4 of acetoacetate is used as the measure of the formation of the acetoacetate from the omega and omega-1 carbons of the fatty acid without acetyl-CoA as an intermediate. Incorporation of 14C from [13-14C]palmitic acid into carbon 1 relative to carbon 3 of acetoacetate is the measure of the formation of acetoacetate from the omega-2 and omega-3 carbons without acetyl-CoA as an intermediate. Comparison of these incorporations is made with incorporation into the carbons of acetoacetate of 14C from palmitic acid labeled with 14C in any of its first 12 carbons since such incorporation must proceed via acetyl-CoA as an intermediate. In an application of this approach, the specifically 14C-labeled palmitic acids were injected into rats in diabetic ketosis. Hydroxybutyric acid that each rat excreted was isolated and degraded. From the ratios of incorporation into the carbons of the hydroxybutyrates, as a minimum, 11% of the total quantity of hydroxybutyrate excreted by the rats was formed from acetoacetyl-CoA without HMG-CoA as an intermediate.

When [6-3H,6-14C]glucose was given in glucose loads to fasted rats, the average 3H/14C ratios in the glycogens deposited in their livers, relative to that in the glucoses administered, were 0.85 and 0.88. When [3-3H,3-14C]lactate was given in trace quantity along with unlabeled glucose loads, the average 3H/14C ratio in the glycogens deposited was 0.08. This indicates that a major fraction of the carbons of the glucose loads was converted to liver glycogen without first being converted to lactate. When [3-3H,6-14C]glucose was given in glucose loads, the 3H/14C ratios in the glycogens deposited averaged 0.44. This indicates that a significant amount of H bound to carbon 3, but not carbon 6, of glucose is removed within liver in the conversion of the carbons of the glucose to glycogen. This can occur in the pentose cycle and by cycling of glucose-6-P via triose phosphates: glucose----glucose-6-P----triose phosphates----glucose-6-P----glycogen. The contributions of these pathways were estimated by giving glucose loads labeled with [1-14C]glucose, [2-14C]glucose, [5-14C]glucose, and [6-14C]glucose and degrading the glucoses obtained by hydrolyzing the glycogens that deposited. Only a few per cent of the glucose carbons deposited in glycogen were deposited in liver via glucose-6-P conversion to triose phosphates. Between 4 and 9% of the glucose utilized by the liver was utilized in the pentose cycle. While these are relatively small percentages, since three NADP3H molecules are formed from each molecule of [3-3H]glucose-6-P utilized in the cycle, a major portion of the difference between the ratios obtained with [3-3H]glucose and with [6-3H]glucose is attributable to metabolism in the pentose cycle. Because 3H of [3-3H]glucose is extensively removed during the conversion of the glucose to glycogen within liver the extent of incorporation of the 3H into liver glycogen is not the measure of glucose's metabolism in other tissues before its carbons are deposited in liver glycogen. The distributions of 14C from the 14C-labeled glucoses into the carbons of the liver glycogens mean that at a minimum about 30% of the carbons of the glucose deposited in the glycogen were first converted to lactate or its metabolic equivalent.

To determine if ketone bodies, synthesized from fatty acids by tissues other than the liver, enter the circulation, rats in diabetic ketosis were injected with sodium [6,13-14C]palmitate. Hydroxybutyrate was isolated from the urine excreted by each rat and from an aqueous extract of its carcass. The distribution of 14C in the four carbons of hydroxybutyrate in the extract was the same as in the urine. The ratio of 14C in carbon 1 to carbon 3 of the hydroxybutyrate averaged 1.80 and averaged 1.31 in carbon 2 to carbon 4. Hydroxybutyrate when formed by perfused liver has the same carbon 1-to-carbon 3 ratio as carbon 2-to-carbon 4 ratio. The results indicate that hydroxybutyrate synthesized by tissues other than the liver mixes in the circulation with that synthesized by the liver and a portion of the mix is then excreted in the urine. The difference between the carbon 1-to-3 carbon ratio 3 and carbon 2-to-carbon 4 ratio calculates to an estimated minimum of 15% to 17% of the hydroxybutyrate in the circulation of the ketotic diabetic rat having tissues other than the liver as its source. Assuming the liver and kidneys are the sources of the ketone bodies in diabetic ketosis, the ketone bodies produced by the kidneys are not excreted into the urine without first entering the circulation.

Specifically 14C-labeled palmitic acids were perfused through livers and incubated with slices of kidneys from rats in diabetic ketosis. The distribution of 14C in the hydroxybutyric acid formed was determined. In liver, the ratio of incorporation of 14C from [13-14C]palmitic acid into carbon 1 to carbon 3 of the hydroxybutyric acid was the same as the ratio in carbon 2 to carbon 4 from [6-14C]palmitic acid. In kidney, the carbon 1-to-carbon 3 ratio was more than twice the carbon 2-to-carbon 4 ratio. In both tissues, 14C from [16-14C] palmitic acid was preferentially incorporated into carbon 4 compared to carbon 2 of the hydroxybutyric acid, but more so in liver than kidney. These results mean that in liver, the sole pathway of acetoacetate formation is via hydroxymethylglutaryl-CoA, while in kidney it is not. Rather in kidney, acetoacetyl-CoA is converted to acetoacetate to a large extent by direct deacylation, presumably via a transferase- and/or deacylase-catalyzed reaction. In liver, most of the palmitic acid utilized is converted to acetoacetate while in kidney it is not. We previously estimated that, as a minimum, 11% of the hydroxybutyric acid excreted by the rat in diabetic ketosis is formed without hydroxymethylglutaryl-CoA as an intermediate. The kidney appears to be the source of this hydroxybutyric acid if the pathways operative in these tissues in vitro are those that also operate in vivo.

Pancreatic islets from ob/ob mice incubated with 3H2O and 5.5 mM glucose formed 3H-labeled glucose, 74 picoatoms incorporated/islet/h. Sixty-three percent of the 3H was bound to carbon 2 of the glucose. The amount of glucose-6-P dephosphorylated to glucose, determined from this incorporation, was 48 pmol/islet/h. Glucose utilization, measured by the formation of 3H2O from [5-3H]glucose, was 72 pmol/islet/h. The amount of glucose dephosphorylated was then about 40% of that phosphorylated. Thus, glucose-6-P is dephosphorylated to glucose to a significant extent by intact islets in vitro and presumably by the beta cells of the islets. The extent of this glucose cycling, i.e. glucose----glucose-6-P----glucose, may play a role in determining the extent of glucose-induced insulin secretion.