Determination of the enrichment of the hydrogen bound to carbon 5 of glucose on 2H2O administration.
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Carbon↗
Biomedical subjects
W C Schumann
Publications and source records attributed to W C Schumann.
Quantitative contributions of gluconeogenesis to glucose production during fasting in type 2 diabetes mellitus.
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.
Deuterium↗
Sources of blood glycerol during fasting.
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.
Adult↗
Small increases in insulin inhibit hepatic glucose production solely caused by an effect on glycogen metabolism.
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.
Animals↗
A probing dose of phenylacetate does not affect glucose production and gluconeogenesis in humans.
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.
Aged↗
Mechanism by which metformin reduces glucose production in type 2 diabetes.
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.
Calorimetry, Indirect↗
Prandial glucose effectiveness and fasting gluconeogenesis in insulin-resistant first-degree relatives of patients with type 2 diabetes.
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.
Adult↗
Effects of free fatty acid elevation on postabsorptive endogenous glucose production and gluconeogenesis in humans.
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.
Absorption↗
Contributions of net hepatic glycogenolysis and gluconeogenesis to glucose production in cirrhosis.
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.
Adult↗
Origins of the hydrogen bound to carbon 1 of glucose in fasting: significance in gluconeogenesis quantitation.
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.
Adult↗
Quantifying gluconeogenesis during fasting.
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.
Adult↗
Contributions of gluconeogenesis to glucose production in the fasted state.
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.
Adult↗
Gluconeogenesis and glucuronidation in liver in vivo and the heterogeneity of hepatocyte function.
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.
Acetaminophen↗
Estimates of Krebs cycle activity and contributions of gluconeogenesis to hepatic glucose production in fasting healthy subjects and IDDM patients.
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%.
Adult↗
Use of 2H2O for estimating rates of gluconeogenesis. Application to the fasted state.
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.
Adult↗
14C-labeled propionate metabolism in vivo and estimates of hepatic gluconeogenesis relative to Krebs cycle flux.
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.
Adult↗
Use of 14CO2 in estimating rates of hepatic gluconeogenesis.
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)
Adult↗
Noninvasive tracing of Krebs cycle metabolism in liver.
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.
Adult↗