Application of mass isotopomer analysis for determination of pathways of glycogen synthesis

1991 ◽  
Vol 261 (3) ◽  
pp. E332-E336
Author(s):  
J. Katz ◽  
W. N. Lee
Keyword(s):  
Blood Glucose ◽  
Mass Spectroscopy ◽  
Specific Activity ◽  
Glycogen Synthesis ◽  
Natural Abundance ◽  
Hepatic Glycogen ◽  
Direct Path ◽  
Isotopomer Analysis ◽  
Mass Isotopomer

An elementary exposition of the application of mass spectroscopy to studies with substrates labeled uniformly with 13C is presented. A procedure to obtain mass isotopomer spectra, corrected for natural abundance, of products labeled with 13C in several positions is outlined. The calculation for enrichment, a term equivalent to specific activity with radioisotopes, is shown. Examples of mass isotopomer patterns of blood glucose and glycogen are presented, and calculations of the contribution of the direct path to hepatic glycogen synthesis and the dilution of glucose-derived pyruvate are shown. The analysis of mass isotopomer patterns recently offered by C. Des Rosiers, B. R. Landau, and H. Brunengraber [Am. J. Physiol. 259 (Endocrinol. Metab. 22): E757-E762, 1990] is critically examined.

Mass isotopomer study of the nonoxidative pathways of the pentose cycle with [1,2-13C2]glucose

1998 ◽  
Vol 274 (5) ◽  
pp. E843-E851 ◽  
Author(s):  
Wai-Nang Paul Lee ◽  
Laszlo G. Boros ◽  
Joaquim Puigjaner ◽  
Sara Bassilian ◽  
Shu Lim ◽  
...  
Keyword(s):  
Pentose Phosphate ◽  
Gas Chromatography Mass Spectrometry ◽  
Hep G2 ◽  
Tracer Method ◽  
Human Hepatoma Cells ◽  
Glucose Flux ◽  
Isotopomer Analysis ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Mass Isotopomer

We present a single-tracer method for the study of the pentose phosphate pathway (PPP) using [1,2-13C2]glucose and mass isotopomer analysis. The metabolism of [1,2-13C2]glucose by the glucose-6-phosphate dehydrogenase, transketolase (TK), and transaldolase (TA) reactions results in unique pentose and lactate isotopomers with either one or two13C substitutions. The distribution of these isotopomers was used to estimate parameters of the PPP using the model of Katz and Rognstad (J. Katz and R. Rognstad. Biochemistry 6: 2227–2247, 1967). Mass and position isotopomers of ribose, and lactate and palmitate (products from triose phosphate) from human hepatoma cells (Hep G2) incubated with 30% enriched [1,2-13C2]glucose were determined using gas chromatography-mass spectrometry. After 24–72 h incubation, 1.9% of lactate molecules in the medium contained one 13C substitution ( m 1) and 10% contained two 13C substitutions ( m 2). A similar m 1-to- m 2ratio was found in palmitate as expected. Pentose cycle (PC) activity determined from incubation with [1,2-13C2]glucose was 5.73 ± 0.52% of the glucose flux, which was identical to the value of PC (5.55 ± 0.73%) determined by separate incubations with [1-13C] and [6-13C]glucose.13C was found to be distributed in four ribose isotopomers ([1-13C]-, [5-13C]-, [1,2-13C2]-, and [4,5-13C2]ribose). The observed ribose isotopomer distribution was best matched with that provided from simulation by substituting 0.032 for TK and 0.85 for TA activity relative to glucose uptake into the model of Katz and Rognstad. The use of [1,2-13C2]glucose not only permits the determination of PC but also allows estimation of relative rates through the TK and TA reactions.

Gluconeogenesis and the Cori cycle in 12-, 20-, and 40-h-fasted humans

1998 ◽  
Vol 275 (3) ◽  
pp. E537-E542 ◽  
Author(s):  
Joseph Katz ◽  
John A. Tayek
Keyword(s):  
Blood Glucose ◽  
Plasma Glucose ◽  
Glucose Concentration ◽  
Glucose Production ◽  
Plasma Glucose Concentration ◽  
Prolonged Fasting ◽  
Cori Cycle ◽  
Isotopomer Analysis ◽  
Mass Isotopomer

Six subjects were infused with [U-13C]glucose (0.03–0.05 mg ⋅ kg−1 ⋅ min−1) starting 8–9 h after a meal, and the production of glucose, the recycling of glucose (the Cori cycle), the dilution of glucose by unlabeled carbon into the hepatic lactate-pyruvate pool, and gluconeogenesis were determined in these fasted volunteers by use of mass isotopomer analysis and equations previously described [J. A. Tayek and J. Katz. Am. J. Physiol.272 ( Endocrinol. Metab. 35): E476–E484, 1997]. A primed continuous 11-h infusion was started at 6:00 AM, and the above parameters were calculated after 3 h (for the 12-h fast) and at the end of the infusion (for the 20-h fast). Another group of five subjects was fasted for 40 h, and the above parameters were calculated as before. At 12, 20, and 40 h of fasting, respectively, blood glucose was 93 ± 2, 83 ± 2, and 71 ± 2 (SE) mg/dl; glucose production was 2.3, 1.8, and 1.77 mg ⋅ kg−1 ⋅ min−1; the recycling of labeled carbon was 8, 15, and 15%, and that of glucose molecules (Cori cycle) was 18, 35, and 36%; the contribution of gluconeogenesis to glucose production was 41, 71, and 92% or 0.96, 1.29, and 1.64 mg ⋅ kg−1 ⋅ min−1; and the contribution of other sources to glucose production was 1.37, 0.53, and 0.15 mg ⋅ kg−1 ⋅ min−1. The recycling of glucose is important in prolonged fasting for the maintenance of plasma glucose concentration. We demonstrate here that gluconeogenesis can be easily measured and that it accounts for ∼90% of glucose production after a 40-h fast.

Quantitative comparison of pathways of hepatic glycogen repletion in fed and fasted humans.

1990 ◽  
Vol 259 (3) ◽  
pp. E335 ◽  
Author(s):  
G I Shulman ◽  
G Cline ◽  
W C Schumann ◽  
V Chandramouli ◽  
K Kumaran ◽  
...  
Keyword(s):  
Plasma Glucose ◽  
Specific Activity ◽  
Glycogen Synthesis ◽  
Hepatic Glycogen ◽  
Moderate Amount ◽  
Dietary Regimen ◽  
Direct Pathway ◽  
Glycogen Repletion ◽  
The Individual ◽  
Glycogen Formation

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.

Use of 14CO2 in estimating rates of hepatic gluconeogenesis

1992 ◽  
Vol 263 (1) ◽  
pp. E36-E41 ◽  
Author(s):  
E. Esenmo ◽  
V. Chandramouli ◽  
W. C. Schumann ◽  
K. Kumaran ◽  
J. Wahren ◽  
...  
Keyword(s):  
Blood Glucose ◽  
Specific Activity ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Hepatic Gluconeogenesis ◽  
Acid Cycle ◽  
Specific Activities ◽  
Glucose Formation

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

scholarly journals Pyrrolidine dithiocarbamate enhances hepatic glycogen synthesis and reduces FoxO1-mediated gene transcription in type 2 diabetic rats

2012 ◽  
Vol 302 (4) ◽  
pp. E409-E416 ◽  
Author(s):  
Tienian Zhu ◽  
Ruijing Zhao ◽  
Lizhong Zhang ◽  
Michel Bernier ◽  
Jiankun Liu
Keyword(s):  
Blood Glucose ◽  
Glycogen Synthesis ◽  
Fasting Blood Glucose ◽  
Diabetic Rats ◽  
Hepatic Glycogen ◽  
Pyrrolidine Dithiocarbamate ◽  
Glycogen Deposition ◽  
Gsk 3Β ◽  
Type 2 Diabetic

The aim of the present study was to examine the effects of pyrrolidine dithiocarbamate (PDTC) on hepatic glycogen synthesis and FoxO1 transcriptional activity in type 2 diabetic rats and the mechanism underlying these effects. Fasting blood glucose and glycogen deposition, together with expressions of two key genes related to gluconeogenesis, were studied in the liver of rats fed a normal diet (NC), high-fat diet (HFD)-induced insulin-resistant rats made type 2 diabetic by a single intraperitoneal injection of streptozotocin (DM), and a DM with intervention of PDTC (DM + PDTC) for 1 wk. The phosphorylation of Akt, GSK-3β, and FoxO1 was assessed in liver extracts of fasted rats by Western blot, whereas indirect immunofluorescence staining was performed to determine the cellular distribution of FoxO1. The DM rats exhibited obvious increases in fasting blood glucose as well as decreased hepatic glycogen content compared with the NC group. Activation of the Akt/GSK-3β pathway and inactivating phosphorylation of FoxO1 were reduced greatly in DM rat livers ( P < 0.01). By contrast, PDTC treatment protected DM rats against high fasting blood glucose and hepatic glycogen deposition loss. PDTC also elicited an increase in Akt/GSK-3β signaling and subsequent inactivation and nuclear export of FoxO1 in DM rat livers, which translated into a significant reduction in the expression of two FoxO1 target genes, phospho enolpyruvate carboxykinase and glucose-6-phosphatase. This study suggests that PDTC enhances hepatic glycogen synthesis, whereas it reduces FoxO1 transcriptional activity in DM rats.

Recycling of glucose and determination of the Cori Cycle and gluconeogenesis

1999 ◽  
Vol 277 (3) ◽  
pp. E401-E407 ◽  
Author(s):  
Joseph Katz ◽  
John A. Tayek
Keyword(s):  
Glucose Metabolism ◽  
Theoretical Analysis ◽  
Mathematical Analysis ◽  
Glycogen Synthesis ◽  
Numerical Examples ◽  
Cori Cycle ◽  
Isotopomer Analysis ◽  
The Difference ◽  
Glucose Recycling

We have derived equations, by employing [U-13C]glucose and mass isotopomer analysis, to determine the pathways of glycogen synthesis (J. Katz, W. P. Lee, P. A. Wals, and E. A. Bergner. J. Biol. Chem. 264: 12994–13004, 1989). More recently, by use of these methods we have derived equations to determine the rate of glucose recycling and of gluconeogenesis [Tayek and Katz. Am. J. Physiol.270 ( Endocrinol. Metab. 33): E709–E717, 1996 and 272 ( Endocrinol. Metab. 35): E476–E484, 1997, and Katz and Tayek. Am. J. Physiol. 275 ( Endocrinol. Metab. 38): E537–E542, 1988]. The former equations have been criticized and challenged by C. Des Rosiers, B. R. Landau, and H. Brunengraber [ Am. J. Physiol. 259 ( Endocrinol. Metab. 22): E757–E762, 1990], and the latter recently by B. R. Landau, J. Wahren, S. F. Previs, G. K. Ekberg, D. Yang, and H. Brunengraber [ Am. J. Physiol. 274 ( Endocrinol. Metab. 37): E954–E961, 1998]. Landau et al. claimed that our equations were in error and “corrected” them. Their analysis, and their values for recycling and gluconeogenesis (GNG) differ markedly from ours. We show here our equations and estimates of recycling and GNG to be correct. We present here a theoretical analysis of recycling and discuss the determination of the Cori Cycle and GNG. We illustrate by numerical examples the difference in parameters of glucose metabolism calculated by the methods of Katz and Landau. J. Radziuk and W. N. P. Lee [ Am. J. Physiol. 277 ( Endocrinol Metab. 40): E199–E207, 1999] and J. K. Kelleher [ Am. J. Physiol. 277 ( Endocrinol. Metab. 40): E395–E400, 1999] present a mathematical analysis that, although differing in some respects from Landau’s, supports his equation for GNG. We show in theappendix that their derivation of the equation for GNG is incorrect.

The glucose-galactose paradox in neonatal murine hepatic glycogen synthesis

1989 ◽  
Vol 257 (5) ◽  
pp. E697-E703 ◽  
Author(s):  
C. Kunst ◽  
R. Kliegman ◽  
C. Trindade
Keyword(s):  
Blood Glucose ◽  
Glycogen Synthesis ◽  
Hepatic Glycogen ◽  
Glycemic Response ◽  
Preferential Utilization ◽  
Glucose Incorporation ◽  
Old Rats ◽  
Direct Incorporation ◽  
Glucose Recycling

In adults glucose incorporation to glycogen is indirect after recycling from lactate. In neonates galactose entry to glycogen exceeds that for glucose, but the pathway is unknown. The pathway of hexose incorporation to glycogen was studied in 5-7-day-old rats and 6-h-old rats injected intraperitoneally (IP) with either double-labeled [6-3H]glucose (nonrecycling), [U-14C]glucose (recycling), or [6-3H]glucose and [U-14C]galactose in saline. In another group of pups, 1 g/kg of glucose or galactose was administered in addition to tracers to determine glycemia and net glycogen synthesis between 15 and 180 min after injection. Blood glucose increased from 3.4 +/- 0.4 to 8.5 +/- 1.5 mM in 5-7-day-old pups in response to IP glucose; there was no glycemic response to galactose, although galactose levels increased from 0.5 to 6.3 mM at 15 min. Hepatic glycogen increased after IP glucose from 14 +/- 2 at 15 min to 30 +/- 3 at 120 min (P less than 0.01), whereas after IP galactose glycogen was 44 +/- 6 mumol/g at 120 min (P less than 0.05). After IP glucose, 3H and 14C disintegration per minute in glycogen increased slowly with 14C exceeding 3H at 120 and 180 min. In contrast IP [14C]galactose resulted in a much greater peak of 14C incorporation into glycogen. The ratio of 3H to 14C in glycogen relative to the injectate after IP glucose decreased from 0.69 +/- 0.12 to 0.36 +/- 0.03 (P less than 0.01) between 15 to 180 min, whereas the ratio after galactose was 0.20 +/- 0.007 to 0.15 +/- 0.02 at these times. The 6-h-old pups also demonstrated augmented incorporation of [14C]galactose in glycogen relative to [3H-14C]glucose. In contrast to 5-7-day-old pups there was no evidence of glucose recycling in 6-h-old pups. In conclusion galactose entry into glycogen exceeds that for glucose and is not dependent on recycling. Direct incorporation of galactose exceeds that for direct incorporation from [3H]glucose, suggesting a preferential utilization of galactose for neonatal glycogen synthesis.

scholarly journals Contribution of dietary starch to hepatic and systemic carbohydrate fluxes in European seabass (Dicentrarchus labraxL.)

2015 ◽  
Vol 113 (9) ◽  
pp. 1345-1354 ◽  
Author(s):  
Ivan Viegas ◽  
João Rito ◽  
Ivana Jarak ◽  
Sara Leston ◽  
Albert Caballero-Solares ◽  
...  
Keyword(s):  
Blood Glucose ◽  
Glycogen Synthesis ◽  
Dicentrarchus Labrax ◽  
Glucose Production ◽  
Endogenous Glucose Production ◽  
Partial Substitution ◽  
Hepatic Glycogen ◽  
Dietary Carbohydrates ◽  
Indirect Pathway ◽  
European Seabass

In the present study, the effects of partial substitution of dietary protein by digestible starch on endogenous glucose production were evaluated in European seabass (Dicentrarchus labrax). The fractional contribution of dietary carbohydratesv.gluconeogenesis to blood glucose appearance and hepatic glycogen synthesis was quantified in two groups of seabass fed with a diet containing 30 % digestible starch (DS) or without a carbohydrate supplement as the control (CTRL). Measurements were performed by transferring the fish to a tank containing water enriched with 5 %2H2O over the last six feeding days, and quantifying the incorporation of2H into blood glucose and hepatic glycogen by2H NMR. For CTRL fish, gluconeogenesis accounted for the majority of circulating glucose while for the DS fish, this contribution was significantly lower (CTRL 85 (sem4) %v.DS 54 (sem2) %;P< 0·001). Hepatic glycogen synthesis via gluconeogenesis (indirect pathway) was also significantly reduced in the DS fish, in both relative (CTRL 100 (sem1) %v.DS 72 (sem1) %;P< 0·001) and absolute terms (CTRL 28 (sem1)v.DS 17 (sem1) μmol/kg per h;P< 0·001). A major fraction of the dietary carbohydrates that contributed to blood glucose appearance (33 (sem1) % of the total 47 (sem2) %) had undergone exchange with hepatic glucose 6-phosphate. This indicated the simultaneous activity of hepatic glucokinase and glucose 6-phosphatase. In conclusion, supplementation of digestible starch resulted in a significant reduction of gluconeogenic contributions to systemic glucose appearance and hepatic glycogen synthesis.

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