No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Measurement of Hepatic Glucose Output, Krebs Cycle, and Gluconeogenic Fluxes by NMR Analysis of a Single Plasma Glucose Sample
Abstract
13C and 1H NMR spectroscopy of plasma glucose was used to resolve the isotopomer contributions from tracer levels of [1,6-13C2]glucose, a novel tracer of glucose carbon skeleton turnover, and [U-13C]propionate, a tracer of hepatic citric acid cycle metabolism. This allowed simultaneous measurements of hepatic glucose production and citric acid cycle fluxes from the NMR analysis of a single plasma glucose sample in fasted animals. Glucose carbon skeleton turnover, as reported by the dilution of [1,6-13C2]glucose, was 56 +/- 2 micromol/kg/min in the presence of labeling from [U-13C]propionate and 53 +/- 4 micromol/kg/min in its absence. Therefore, as expected, the labeling contributions from [U-13C]propionate metabolism did not have a significant effect on the measurement of glucose turnover. For the group infused with both tracers, citric acid cycle flux estimates from the analysis of glucose C2 isotopomer ratios were consistent with those from our recent experiments where only [U-13C]propionate was infused, verifying that the presence of [1,6-13C2]glucose did not interfere with these measurements. This integrated analysis of hepatic glucose output and citric acid cycle fluxes from plasma glucose isotopomers yielded a noninvasive estimate of hepatic citrate synthase flux of 74 +/- 12 micromol/kg/min for 24-h fasted rats.
... Spectra were acquired with a 90°pulse after presaturation of the residual water signal and a 15-s interpulse delay. Long-range couplings between 13 C C-2 to C-6 and H-1␣ were abolished by the application of a narrow-band WALTZ-16 13 C-decoupling pulse sequence covering the 60-to 75-ppm region of the 13 C NMR spectrum (18). Two hundred fifty-six acquisitions were collected for a total collection time of 64 min. ...
... Relative anaplerotic flux (OAA 3 PEP, where OAA is oxaloacetate), pyruvate recycling flux (PEP 3 pyruvate or equivalent pathway), and gluconeogenic flux (PEP 3 glucose) were calculated from the multiplet areas measured in the 13 C NMR spectrum of urinary glucuronate or phenylacetylglutamine (PAGN), as described previously (18,20,21). For urinary glucuronate C-5 (the C-5 resonance was analyzed), the relevant equations are ...
... These include signals from glucose isotopomers generated from the gluconeogenic metabolism of [U-13 C 3 ]propionate (D12 and D123) in addition to the tracer amount of infused [1,6-13 C 2 ]glucose (D16). As previously demonstrated (18), the fraction of [1,6-13 C 2 ]glucose remaining in plasma at any time point can be quantified by measuring the contribution of [1,6-13 C 2 ]glucose to the C-1 resonance ( 13 C spectrum) and the total 13 C enrichment as reported in the H-1␣ resonance ( 1 H spectrum). The 1 H NMR spectrum of the H-1␣ proton features wellresolved 13 C satellites with sufficient signal-to-noise ratio for reliable quantitation of the 2-3% excess 13 C enrichment levels from this experiment. ...
... Recent studies have used 2 H NMR to explore the relative contributions of gluconeonegesis and glycogenolysis to plasma glucose [196,197]. The strategy followed earlier approaches designed for detection by mass spectrometry methods which involved oral administration of 2 H 2 O to humans followed by blood sampling and analysis of plasma glucose by 2 H NMR. The ratio of deuterium incorporation to H2 glucose as compared to H5 glucose allows the determination of the relatives amounts of glucose derived from gluconeogenesis to glycogenolysis, respectively [89,90]. ...
... [89,90,196,197] Glucose H2 n.s 2 H 2 O Glucose 6-P isomerase [192,196,197] four orders of magnitude smaller pK a 19±20 than the solvent exchangeable hydrogens from hydroxyl groups pK a 16 [166]. ...
... Glucose H2 n.s 2 H 2 O Glucose 6-P isomerase [192,196,197] four orders of magnitude smaller pK a 19±20 than the solvent exchangeable hydrogens from hydroxyl groups pK a 16 [166]. ...
This work was supported by grants PB 96-0864 from the Spanish Ministry of Science and Technology (to P.B. and S.C.), grants 08.1/0023/97 and 08.1/0046/98 from the Community of Madrid (to P.B. and S.C.).
... Spectra were acquired with a 90°pulse after presaturation of the residual water signal and a 15-s interpulse delay. Long-range couplings between 13 C C-2 to C-6 and H-1␣ were abolished by the application of a narrow-band WALTZ-16 13 C-decoupling pulse sequence covering the 60-to 75-ppm region of the 13 C NMR spectrum (18). Two hundred fifty-six acquisitions were collected for a total collection time of 64 min. ...
... Relative anaplerotic flux (OAA 3 PEP, where OAA is oxaloacetate), pyruvate recycling flux (PEP 3 pyruvate or equivalent pathway), and gluconeogenic flux (PEP 3 glucose) were calculated from the multiplet areas measured in the 13 C NMR spectrum of urinary glucuronate or phenylacetylglutamine (PAGN), as described previously (18,20,21). For urinary glucuronate C-5 (the C-5 resonance was analyzed), the relevant equations are ...
... These include signals from glucose isotopomers generated from the gluconeogenic metabolism of [U-13 C 3 ]propionate (D12 and D123) in addition to the tracer amount of infused [1,6-13 C 2 ]glucose (D16). As previously demonstrated (18), the fraction of [1,6-13 C 2 ]glucose remaining in plasma at any time point can be quantified by measuring the contribution of [1,6-13 C 2 ]glucose to the C-1 resonance ( 13 C spectrum) and the total 13 C enrichment as reported in the H-1␣ resonance ( 1 H spectrum). The 1 H NMR spectrum of the H-1␣ proton features wellresolved 13 C satellites with sufficient signal-to-noise ratio for reliable quantitation of the 2-3% excess 13 C enrichment levels from this experiment. ...
Hepatic glucose synthesis from glycogen, glycerol, and the tricarboxylic acid (TCA) cycle was measured in five overnight-fasted subjects by (1)H, (2)H, and (13)C NMR analysis of blood glucose, urinary acetaminophen glucuronide, and urinary phenylacetylglutamine after administration of [1,6-(13)C(2)]glucose, (2)H(2)O, and [U-(13)C(3)]propionate. This combination of tracers allows three separate elements of hepatic glucose production (GP) to be probed simultaneously in a single study: 1) endogenous GP, 2) the contribution of glycogen, phosphoenolpyruvate (PEP), and glycerol to GP, and 3) flux through PEP carboxykinase, pyruvate recycling, and the TCA cycle. Isotope-dilution measurements of [1,6-(13)C(2)] glucose by (1)H and (13)C NMR indicated that GP in 16-h-fasted humans was 10.7 +/- 0.9 micromol.kg(-1).min(-1). (2)H NMR spectra of monoacetone glucose (derived from plasma glucose) provided the relative (2)H enrichment at glucose H-2, H-5, and H-6S, which, in turn, reflects the contribution of glycogen, PEP, and glycerol to total GP (5.5 +/- 0.7, 4.8 +/- 1.0, and 0.4 +/- 0.3 micromol.kg(-1).min(-1), respectively). Interestingly, (13)C NMR isotopomer analysis of phenylacetylglutamine and acetaminophen glucuronide reported different values for PEP carboxykinase flux (68.8 +/- 9.8 vs. 37.5 +/- 7.9 micromol.kg(-1).min(-1)), PEP recycling flux (59.1 +/- 9.8 vs. 27.8 +/- 6.8 micromol.kg(-1).min(-1)), and TCA cycle flux (10.9 +/- 1.4 vs. 5.4 +/- 1.4 micromol.kg(-1).min(-1)). These differences may reflect zonation of propionate metabolism in the liver.
... In contrast, the group from UT Southwestern has extended Landau's [ 14 C]propionate method (14,15,17,18), which to this point has not really been questioned in the literature. Conceptually, the measured endpoints and mathematical model are the same as those originally hypothesized by Landau and colleagues; the adaptation to stable isotope tracers and NMRbased detection allows for greater ease of utilization and the simultaneous administration of multiple tracers to further enhance the resolution of metabolic flux (14). ...
... The NMR method that is used by the group at UT Southwestern measures the 13 C distribution pattern of glucose; particular attention is directed toward the 13 C labeling on C1, C2, and C3 (15)(16)(17)(18). As shown in Fig. 2, the conversion of [U- 13 C]propionate to glucose leads to the generation of a distinct 13 C labeling pattern depending on the pathway flux rates, i.e., equal amounts of C2Q and C2D12 glucose ( Fig. 2A); cycling of [ 13 C]oxaloacetate via pyruvate kinase (and/or malic enzyme) leads to the same 13 C labeling pattern but with different relative abundance of the species (Fig. 2C). ...
Two groups recently used different tracer methods to quantify liver-specific flux rates, the studies had a similar goal, i.e. to characterize mitochondrial oxidative function. These efforts could have a direct impact on our ability to understand metabolic abnormalities that affect the pathophysiology of fatty liver and allow us to examine mechanisms surrounding potential therapeutic interventions. Briefly, one method couples the continuous infusion of [(13)C]acetate with direct real-time measurements of [(13)C]glutamate labeling in liver; the other method administers [(13)C]propionate, in combination with other tracers, and subsequently measures the [(13)C]labeling of plasma glucose and/or acetaminophen-glucuronide. It appears that a controversy has arisen since the respective methods yielded different estimates of the anaplerotic:TCA flux ratio (VANA:VTCA) in "control" subjects, i.e. the [(13)C]acetate- and [(13)C]propionate-derived VANA:VTCA flux ratios appear to be ~ 1.4 and ~ 5, respectively. While the deep expertise in the respective groups makes it somewhat trivial for each to perform the tracer studies, the data interpretation is inherently difficult. The current perspective was undertaken to examine potential factors that could account for or contribute to the apparent differences, attention was directed towards (i) matters of practicality, (ii) issues surrounding stoichiometry and (iii) hidden assumptions. We believe that the [(13)C]acetate method has certain weaknesses which limit its utility, in contrast, the [(13)C]propionate method likely yields a more correct answer. Hopefully our discussion will help clarify the differences in the recent reports, presumably this will be of interest to investigators who are considering tracer-based studies of liver metabolism.
Copyright © 2015, American Journal of Physiology - Endocrinology and Metabolism.
... Furthermore, in contrast to acetate or lactate, Puchowicz et al. have demonstrated very large periportal-perivenous gradients for propionate uptake across the liver 23 . To examine these issues we measured glucose turnover in a group of rats infused intravenously with low (333 μmol kg −1 ) and high (666 μmol kg −1 ) doses of [U- 13 C] proprionate that are within the range used by previous studies 2, 24,25 . We found that plasma glucose concentrations increased bỹ 25 mg dl −1 during the high dose propionate infusions compared to the low dose propionate infusions. ...
... Furthermore, this increase in plasma glucose concentration during the high dose propionate infusion was associated with an ~30% increase in rates of hepatic glucose production ( Supplementary Fig. S3), compared to the low dose infusion. This stimulation of hepatic glucose production by propionate is similar to the ~40% increase in rates of endogenous glucose production [from 7.3 to 10.0 mg (kg-min) −1 ], observed when the dose of [U-13 C] propionate given to rats was increased from 222 to 825 μmol 24,25 . In contrast, doubling the infusion rates of [1-13 C] acetate or [3-13 C] lactate had no effects on glucose production, the ratio of pyruvate carboxylase flux (V PC ) to V TCA or the ratio of pyruvate dehydrogenase flux (V PDH ) to V TCA . ...
Despite the central role of the liver in the regulation of glucose and lipid metabolism, there are currently no methods to directly assess hepatic oxidative metabolism in humans in vivo. By using a new (13)C-labeling strategy in combination with (13)C magnetic resonance spectroscopy, we show that rates of mitochondrial oxidation and anaplerosis in human liver can be directly determined noninvasively. Using this approach, we found the mean rates of hepatic tricarboxylic acid (TCA) cycle flux (VTCA) and anaplerotic flux (VANA) to be 0.43 ± 0.04 μmol g(-1) min(-1) and 0.60 ± 0.11 μmol g(-1) min(-1), respectively, in twelve healthy, lean individuals. We also found the VANA/VTCA ratio to be 1.39 ± 0.22, which is severalfold lower than recently published estimates using an indirect approach. This method will be useful for understanding the pathogenesis of nonalcoholic fatty liver disease and type 2 diabetes, as well as for assessing the effectiveness of new therapies targeting these pathways in humans.
... Glucose was extracted from the remaining plasma by ion-exchange chromatography then converted to 1,2-isopropylidene glucofuranose (monoacetone glucose [MAG]), as described elsewhere (12) NMR spectroscopy. 2 H and 13 C NMR spectra were collected using a 14.1 T Inova NMR spectrometer (Varian Instruments, Palo Alto, CA), 3-mm sample probes, and parameters previously described (7,13). Multiplet areas in the 13 C spectra were measured using the line fitting routine in the PC-based NMR program, NUTS (Acorn NMR, Fremont, CA). ...
... It is also interesting to note that glucose production from glycerol in control animals infused with the additional ϳ1 mol/min glycerol did not differ from that in control animals without glycerol co-infusion (glycerol typically contributes ϳ25% in mice without infusion of any substrates; S.B., C.R.M., A.D.S., unpublished observations). 13 C NMR spectra of the same MAG samples showed that plasma glucose from control and knockout animals became enriched with 13 C that originated in [U-13 C 3 ]propionate (although all six MAG resonances were enriched with 13 C, only the C2 resonances are shown in Fig. 2). Unlike the 2 H spectra, the various multiplets in the 13 C spectra were more strikingly different in the controls versus knockouts. ...
Liver-specific PEPCK knockout mice, which are viable despite markedly abnormal lipid metabolism, exhibit mild hyperglycemia in response to fasting. We used isotopic tracer methods, biochemical measurements, and nuclear magnetic resonance spectroscopy to show that in mice lacking hepatic PEPCK, 1) whole-body glucose turnover is only slightly decreased; 2) whole-body gluconeogenesis from phosphoenolpyruvate, but not from glycerol, is moderately decreased; 3) tricarboxylic acid cycle activity is globally increased, even though pyruvate cycling and anaplerosis are decreased; 4) the liver is unable to synthesize glucose from lactate/pyruvate and produces only a minimal amount of glucose; and 5) glycogen synthesis in both the liver and muscle is impaired. Thus, although mice without hepatic PEPCK have markedly impaired hepatic gluconeogenesis, they are able to maintain a near-normal blood glucose concentration while fasting by increasing extrahepatic gluconeogenesis coupled with diminishing whole-body glucose utilization.
... NMR spectroscopy 2 H NMR spectra of urine water and MAG were acquired at 11.75 T with a Varian Unity 500 system equipped with a 5 mm broadband 'switchable' probe with z-gradient (Varian, Palo Alto, CA, USA) as described. 17 Absolute enrichment of glucose hydrogen 2 in selected MAG samples was obtained by addition of a 2 H-formate standard of known enrichment to the NMR sample. 16 13 C NMR spectra of MAG were obtained following addition of 100 ml CD 3 CN to provide a lock signal. ...
... For some samples, and for MAG prepared from [U-13 C]glucose of the infusion solution, quantification of carbon 1 enrichment was performed by 1 H NMR. This was achieved by pre-saturation of the acetonitrile signal and applying selective 13 C-decoupling in the 65-85 ppm spectral window. The decoupling procedure improves the resolution of the 13 C-satellite signals by removing long-range 13 C-1 H couplings between hydrogen 1 and carbons 2-6 while retaining the direct coupling between 13 C and 1 H in position 1. 17 For all plasma glucose samples, MAG carbon 1 13 C-enrichment was quantified from its 13 C NMR spectrum using the approach of Brainard et al. 18 Here, the intensity of the 13 C singlet component of any given glucose resonance is assumed to represent the natural abundance 13 C-enrichment of 1.11%. The intensity of a 13 C-13 C spin-coupled multiplet signal is expressed as fractional 13 C enrichment by obtaining the ratio of the multiplet and singlet signal and multiplying by 1.11%. ...
Glucose metabolism in five healthy subjects fasted for 16 h was measured with a combination of [U-13C]glucose and 2H2O tracers. Phenylbutyric acid was also provided to sample hepatic glutamine for the presence of 13C-isotopomers derived from the incorporation of [U-13C]glucose products into the hepatic Krebs cycle. Glucose production (GP) was quantified by 13C NMR analysis of the monoacetone derivative of plasma glucose following a primed infusion of [U-13C]glucose and provided reasonable estimates (1.90 +/- 0.19 mg/kg/min with a range of 1.60-2.15 mg/kg/min). The same derivative yielded measurements of plasma glucose 2H-enrichment from 2H2O by 2H NMR from which the contribution of glycogenolytic and gluconeogenic fluxes to GP was obtained (0.87 +/- 0.14 and 1.03 +/- 0.10 mg/kg/min, respectively). Hepatic glutamine 13C-isotopomers representing multiply-enriched oxaloacetate and [U-13C]acetyl-CoA were identified as multiplets in the 13C NMR signals of the glutamine moiety of urinary phenylacetylglutamine, demonstrating entry of the [U-13C]glucose tracer into both oxidative and anaplerotic pathways of the hepatic Krebs cycle. These isotopomers contributed 0.1-0.2% excess enrichment to carbons 2 and 3 and approximately 0.05% to carbon 4 of glutamine.
... This demonstrates that propionate and 2 H 2 O delivered at these levels and by this method do not alter glucose homeostasis. In a previous study wherein both propionate and [1,6-13 C 2 ]glucose (25 and 2.75 mol/min/kg, respectively) were infused into rats for 120 min, a signiWcantly higher glucose production rate (56 § 2 mol/kg/min) was found [9]. This apparent discrepancy could reXect the greater amount of [U-13 C] propionate received in the infusion protocol (a total of 825 mol was given over the 120-min infusion period versus only 222 mol given here by bolus). ...
... glucose, OAA ! PEP, and pyruvate cycling to the TCA Xux were consistent with our previous measurement for 24-h-fasted rats using [U- 13 C]propionate [9]. Absolute Xuxes related to TCA cycle were lower than those in the previous report, largely because the earlier study assumed that all glucose was produced from PEP; i.e., neither glycogen nor glycerol contributed to glucose production. ...
A triple-tracer method was developed to provide absolute fluxes contributing to endogenous glucose production and hepatic tricarboxylic acid (TCA) cycle fluxes in 24-h-fasted rats by (2)H and (13)C nuclear magnetic resonance (NMR) analysis of a single glucose derivative. A primed, intravenous [3,4-(13)C(2)]glucose infusion was used to measure endogenous glucose production; intraperitoneal (2)H(2)O (to enrich total body water) was used to quantify sources of glucose (TCA cycle, glycerol, and glycogen), and intraperitoneal [U-(13)C(3)] propionate was used to quantify hepatic anaplerosis, pyruvate cycling, and TCA cycle flux. Plasma glucose was converted to monoacetone glucose (MAG), and a single (2)H and (13)C NMR spectrum of MAG provided the following metabolic data (all in units of micromol/kg/min; n = 6): endogenous glucose production (40.4+/-2.9), gluconeogenesis from glycerol (11.5+/-3.5), gluconeogenesis from the TCA cycle (67.3+/-5.6), glycogenolysis (1.0+/-0.8), pyruvate cycling (154.4+/-43.4), PEPCK flux (221.7+/-47.6), and TCA cycle flux (49.1+/-16.8). In a separate group of rats, glucose production was not different in the absence of (2)H(2)O and [U-(13)C]propionate, demonstrating that these tracers do not alter the measurement of glucose turnover.
... Since single 13 C-labeled substrates in Krebs-Ringer are used in perfused organ studies these assumptions often appear valid, however, can still suffer from reincorporation of 13 C-labeled carbon dioxide. There have been many MFA studies of hepatocyte cultures and liver using 13 C isotopomer analysis by mass spectrometry [216,[225][226][227][228], NMR [94,110,120,[229][230][231][232][233][234][235][236], or a mixture of analytical methods [237][238][239][240][241] for a multitude of metabolic pathways [101,103,104,106,109,110,113,[242][243][244][245][246][247][248][249][250][251][252][253][254][255][256], where the reader is directed to obtain more details, as well as comprehensive reviews on MFA [1,2,12,68,70,71,95,96,210,212,214] and MCA [77,80,213]. ...
Metabolism is an interconnecting network of metabolite consumption and creation. Metabolomics has focused on metabolite concentrations in metabolic networks. Fluxomics is also required in the study of metabolism and quantifies the flux of substrate through each reaction step or a series of reaction steps (i.e., metabolic pathway or cycle), and ultimately is required for energy balance equations of the system. The primary noninvasive method of quantifying fluxes in living systems is by in vivo ¹³C nuclear magnetic resonance (NMR) spectroscopy. The present state of noninvasive in vivo NMR technology allows for just four simultaneous flux measurements of metabolic pathways: gluconeogenesis, glycogen synthesis, glycolysis, and citric acid cycle. Since the liver is the gatekeeper and metabolic center for the animal, in vivo fluxomics of liver is extensively reviewed. Additionally, other organ systems studies are discussed demonstrating interorgan cycles, such as the Cori and Randall cycles. This review discusses the basics of in vivo fluxomics focusing on the general details of the NMR experimental protocol and required hardware/software needed to analyze the data.
... Isotopomer analysis and 13 C fractional enrichments of certain metabolites, located at specific intersections, or branch points, of metabolism have been used for in silico modeling of 2D primary rat hepatocyte cultures using metabolic control analysis (MCA) (Brand et al. 1993;Ainscow and Brand 1999a, b, c, d;Berthiaume et al. 2003), or metabolic flux analysis (MFA) (Jones et al. 1998a, b;Jones et al. 2001;Jin et al. 2004;Gille et al. 2005;Burgess et al. 2007;Yoon and Lee 2007;Srivastava and Chan 2008;Yang et al. 2009), but data from 2D primary human hepatocyte cultures has never been used. Isotopomeric analysis is a powerful method for quantifying flux (Sherry and Malloy 2002;Fan et al. 2008;Lane et al. 2008a, b). ...
The development of techniques allowing the culturing of primary mammalian hepatocytes has provided great insights into liver physiology. For most applications, it is desirable for hepatocytes in culture to mimic hepatocytes in vivo. We used stable isotope resolved metabolomics (SIRM) to assess glucose and glutamine utilization in primary rat and human hepatocytes maintained in standard culture media. Primary rat hepatocytes readily metabolized 13C-glucose and 13C-glutamine made evident by 13C incorporation into glycogen, glycolytic end products, and Krebs cycle intermediates and responded to insulin and glucagon appropriately. In contrast, no glucose or glutamine consumption was detected in primary human hepatocytes over 4h of exposure to high media concentrations of 13C-glucose or 13C-glutamine even in the presence of insulin. Nonetheless, cultured human hepatocytes were metabolically active and viable, as demonstrated by incorporation of media 13C-octanoic acid into Krebs cycle intermediates and ketone bodies. The failure to utilize glucose was not due to inhibition of glucokinase (hexokinase IV) since the human hepatocytes could readily incorporate 13C-glucose into glucuronic acid, as demonstrated by the production of 13C-glucuronide conjugates after addition of acetaminophen to the media. These novel observations support inhibition of phosphofructokinase-1, the other regulatory enzyme in glycolysis. Parts of this phenotype could be reproduced in the rat hepatocytes by replacing insulin with glucagon to the media. We conclude that under standard culture conditions human hepatocytes are in an extreme starved state. We believe this may result from prolonged fasting in the human liver donors combined with exposure to stress hormones such as, epinephrine, glucagon, and cortisol. Efforts should now be exerted to find culture conditions that will reverse this state to achieve more metabolically relevant cultures of human hepatocytes.
... from the C-2 region of the 13 C NMR spectra of MAG to calculate flux rates from the TCA cycle [primarily through phosphoenolpyruvate carboxykinase (PEPCK), v6/v7], pyruvate cycling (v5/v7) back to oxaloacetate (OAA) through either pyruvate kinase (PK) or malic enzyme (ME), and a second measure of GNG PEP (v4/v7) relative to citrate synthase (CS) flux (v7) (Fig. 4) (36). The value of GNGPEP measured by [U-13 C3]propionate was used to convert the relative fluxes to absolute flux rates as previously described (38). ...
Animals that endure prolonged periods of food deprivation preserve vital organ function by sparing protein from catabolism. Much of this protein sparing is achieved by reducing metabolic rate and suppressing gluconeogenesis while fasting. Northern elephant seals (Mirounga angustirostris) endure prolonged fasts of up to 3 mo at multiple life stages. During these fasts, elephant seals maintain high levels of activity and energy expenditure associated with breeding, reproduction, lactation, and development while maintaining rates of glucose production typical of a postabsorptive mammal. Therefore, we investigated how fasting elephant seals meet the requirements of glucose-dependent tissues while suppressing protein catabolism by measuring the contribution of glycogenolysis, glycerol, and phosphoenolpyruvate (PEP) to endogenous glucose production (EGP) during their natural 2-mo postweaning fast. Additionally, pathway flux rates associated with the tricarboxylic acid (TCA) cycle were measured specifically, flux through phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate cycling. The rate of glucose production decreased during the fast (F(1,13) = 5.7, P = 0.04) but remained similar to that of postabsorptive mammals. The fractional contributions of glycogen, glycerol, and PEP did not change with fasting; PEP was the primary gluconeogenic precursor and accounted for ∼95% of EGP. This large contribution of PEP to glucose production occurred without substantial protein loss. Fluxes through the TCA cycle, PEPCK, and pyruvate cycling were higher than reported in other species and were the most energetically costly component of hepatic carbohydrate metabolism. The active pyruvate recycling fluxes detected in elephant seals may serve to rectify gluconeogeneic PEP production during restricted anaplerotic inflow in these fasting-adapted animals.
... Proton decoupled [ 13 C]NMR spectra of extracts were obtained with a Varian Inova 14.1 T spectrometer (Palo Alto, California, USA) operating at 150.9 MHz and equipped with a 5-mm broadband probe. Spectra were acquired with 2,000 scans as described previously (21), and the free-induction decays were processed with 0.5 Hz exponential multiplication before Fourier transformation. The areas of 13 C resonances were quantified by a curve-fitting program (NUTS; Acorn NMR, Fremont, California, USA). ...
Glycogen-targeting subunits of protein phosphatase-1, such as protein targeting to glycogen (PTG), direct the phosphatase to the glycogen particle, where it stimulates glycogenesis. We have investigated the metabolic impact of overexpressing PTG in liver of normal rats. After administration of PTG cDNA in a recombinant adenovirus, animals were fasted or allowed to continue feeding for 24 hours. Liver glycogen was nearly completely depleted in fasted control animals, whereas glycogen levels in fasted or fed PTG-overexpressing animals were 70% higher than in fed controls. Nevertheless, transgenic animals regulated plasma glucose, triglycerides, FFAs, ketones, and insulin normally in the fasted and fed states. Fasted PTG-overexpressing animals receiving an oral bolus of [U-(13)C]glucose exhibited a large increase in hepatic glycogen content and a 70% increase in incorporation of [(13)C]glucose into glycogen. However, incorporation of labeled glucose accounted for only a small portion of the glycogen synthesized in PTG-overexpressing animals, consistent with our earlier finding that PTG promotes glycogen synthesis from gluconeogenic precursors. We conclude that hepatic PTG overexpression activates both direct and indirect pathways of glycogen synthesis. Because of its ability to enhance glucose storage without affecting other metabolic indicators, the glycogen-targeting subunit may prove valuable in controlling blood glucose levels in diabetes.
... Less well recognized is the application of metabolic engineering tools to problems in physiology and medicine (Koffas et al., 1999). Isotopomer analysis of heart and liver metabolism has been used to quantify metabolic fluxes in the tricarboxylic acid (TCA) cycle and gluconeogenic pathway (Jeffrey et al., 1996; Jones et al., 1998; Katz et al., 1989 Katz et al., , 1991 Large et al., 1997; Yarmush et al., 1999). More recently, our laboratory has used nonisotopic metabolic flux analysis (MFA) to quantify flux changes in perfused livers (Arai et al., 2001; Lee et al., 2000), and an MFA model for human liver has been reported by C ¸ alik and Akbay (2000). ...
Hepatic metabolism can be investigated using metabolic flux analysis (MFA), which provides a comprehensive overview of the intracellular metabolic flux distribution. The characterization of intermediary metabolism in hepatocytes is important for all biotechnological applications involving liver cells, including the development of bioartificial liver (BAL) devices. During BAL operation, hepatocytes are exposed to plasma or blood from the patient, at which time they are prone to accumulate intracellular lipids and exhibit poor liver-specific functions. In a prior study, we found that preconditioning the primary rat hepatocytes in culture medium containing physiological levels of insulin, as opposed to the typical supraphysiological levels found in standard hepatocyte culture media, reduced lipid accumulation during subsequent plasma exposure. Furthermore, supplementing the plasma with amino acids restored hepatospecific functions. In the current study, we used MFA to quantify the changes in intracellular pathway fluxes of primary rat hepatocytes in response to low-insulin preconditioning and amino acid supplementation. We found that culturing hepatocytes in medium containing lower physiological levels of insulin decreased the clearance of glucose and glycerol with a concomitant decrease in glycolysis. These findings are consistent with the general notion that low insulin, especially in the presence of high glucagon levels, downregulates glycolysis in favor of gluconeogenesis in hepatocytes. The MFA model shows that, during subsequent plasma exposure, low-insulin preconditioning upregulated gluconeogenesis, with lactate as the primary precursor in unsupplemented plasma, with a greater contribution from deaminated amino acids in amino acid-supplemented plasma. Concomitantly, low-insulin preconditioning increased fatty acid oxidation, an effect that was further enhanced by amino acid supplementation to the plasma. The increase in fatty acid oxidation reduced intracellular triglyceride accumulation. Overall, these findings are consistent with the notion that the insulin level in medium culture presets the metabolic machinery of hepatocytes such that it directly impacts on their metabolic behavior during subsequent plasma culture.
... This technique has attracted considerable interest because of the rapid increase in incidence of type II diabetes in human populations. Similarly, oral or intravenous administration of a gluconeogenic 13 C tracer, such as [U- 13 C 3 ]propionate, can provide metabolic flux information at the level of the hepatic citric acid cycle, the major source of gluconeogenic carbons and a key regulatory site for gluconeogenesis [3][4][5][6][7]. We recently demonstrated that NMR analysis of plasma glucose enriched with a combination of 2 H and 13 C tracers provides a comprehensive picture of glycogenolysis, gluconeogenesis, and citric acid cycle flux in humans [6]. ...
Mammalian liver disposes of acetaminophen and other ingested xenobiotics by forming soluble glucuronides that are subsequently removed via renal filtration. When given in combination with the stable isotopes 2H and 13C, the glucuronide of acetaminophen isolated from urine provides a convenient "chemical biopsy" for evaluating intermediary metabolism in the liver. Here, we describe isolation and purification of urinary acetaminophen glucuronide and its conversion to monoacetone glucose (MAG). Subsequent 2H and 13C NMR analysis of MAG from normal volunteers after ingestion of 2H2O and [U-13C3]propionate allowed a noninvasive profiling of hepatic gluconeogenic pathways. The method should find use in metabolic studies of infants and other populations where blood sampling is either limited or problematic.
... One important advance was the ability to monitor glycogen stores in vivo by 13 C NMR, which provides a direct measure of glycogenolysis (10,18,25). Other methods to characterize glucose production require only analysis of blood samples (15). Jin et al. (14) recently introduced the use of 13 C NMR coupled with [3,4-13 C 2 ]glucose to measure glucose turnover. ...
The metabolic mechanism of hepatic glucose overproduction was investigated in 3,3'-5-triiodo-l-thyronine (T3)-treated rats and Zucker diabetic fatty (ZDF) rats (fa/fa) after a 24-h fast. 2H2O and [U-13C3]propionate were administered intraperitoneally, and [3,4-13C2]glucose was administered as a primed infusion for 90 min under ketamine-xylazine anesthesia. 13C NMR analysis of monoacetone glucose derived from plasma glucose indicated that hepatic glucose production was twofold higher in both T3-treated rats and ZDF rats compared with controls, yet the sources of glucose overproduction differed significantly in the two models by 2H NMR analysis. In T3-treated rats, the hepatic glycogen content and hence the contribution of glycogenolysis to glucose production was essentially zero; in this case, excess glucose production was due to a dramatic increase in gluconeogenesis from TCA cycle intermediates. 13C NMR analysis also revealed increased phosphoenolpyruvate carboxykinase flux (4x), increased pyruvate cycling flux (4x), and increased TCA flux (5x) in T3-treated animals. ZDF rats had substantial glycogen stores after a 24-h fast, and consequently nearly 50% of plasma glucose originated from glycogenolysis; other fluxes related to the TCA cycle were not different from controls. The differing mechanisms of excess glucose production in these models were easily distinguished by integrated 2H and 13C NMR analysis of plasma glucose.
A wide variety of methods, such as enzymatic methods, LC-MS, and LC-MS/MS, are currently available for the concentration determination of plasma glucose in studies of diabetes, obesity, exercise, etc. However, these methods rarely discriminate endogenous and exogenous glucose in plasma. A novel NMR strategy for discriminative quantification of the endogenous and exogenous glucose in plasma has been developed using an adapted isotope dilution 1H–13C heteronuclear single-quantum correlation (ID-HSQC) with uniformly 13C-labeled glucose as a tracer of exogenous glucose. This method takes advantage of the distinct 1H–13C chemical shifts of the hemiacetal group of the α-D-glucopyranose and makes use of the 13C–13C one-bond J-coupling (1JCC) in uniformly 13C-labeled glucose to differentiate the 1H–13C HSQC signal of labeled glucose from that of its natural counterpart when data are acquired in high-resolution mode. The molar ratio between the endogenous and exogenous plasma glucose can then be calculated from the peak intensities of the natural and labeled glucose. The accuracy and precision of the method were evaluated using a series of standard mixtures of natural and uniformly 13C-labeled glucose with varied but known concentrations. Application of this method is demonstrated for the quantification of endogenous and exogenous glucose in plasma derived from healthy and diabetic cynomolgus monkeys. The results nicely agree with our previous LC-MS/MS results. Considering the natural abundance of 13C isotope at the level of 1.1% in endogenous glucose, comparable peak intensities of quantitatively measurable signals derived from natural and labeled glucose imply that the ID-HSQC can tolerate a significantly high ratio of isotope dilution, with labeled/natural glucose at ~ 1%. We expect that the ID-HSQC method can serve as an alternative approach to the biomedical or clinical studies of glucose metabolism.
C metabolic flux analysis (MFA) provides a rigorous approach to quantify intracellular metabolism. This technology has found broad use in metabolic engineering and physiologic investigations where there is a need to assess metabolic fluxes inside living cells and tissues. Because fluxes cannot be unequivocally inferred from other complementary methods, 13C MFA provides a unique perspective of how metabolic pathways are natively regulated and how they can be manipulated to achieve a desired outcome. This chapter highlights the methodological advances, biotechnology and biomedical applications, and emerging challenges that have appeared in the flux analysis literature over the past 15 years.
The rapid adaptation of hepatic carbohydrate metabolism to feeding and fasting states is a key component of glucose homeostasis. Hepatic metabolic fluxes that are involved in glucose production and disposal can be studied in great detail by combining stable isotope tracers with noninvasive sampling of hepatic UDP-glucose via glucuronidation probes. These methods are readily applicable to the clinical setting and pose negligible risk to human subjects. For patients with diabetes, these measurements inform abnormalities in hepatic glucose and glycogen metabolism and to what extent they are normalized by novel therapeutic approaches such as automated insulin delivery.
Introduction13C Tracers for Analysis of Metabolic NetworksMethods: 13C and 2H NMRRelating isotopomers to metabolism: Entry of 13C-enriched acetyl-CoA into the TCA cycleIsotopomer analysis in gluconeogenic tissuesAddition of 2H NMR data: Identifying the sources of liver glucoseThe Gluconeogenic Network: Conversion of 2H and 13C NMR data to absolute fluxesOne Interesting Example: The Case of Liver-Specific PEPCK Knockout MouseFuture Directions: Comprehensive, High-Throughput Metabolic Analyses
13C NMR isotopomer analysis is a powerful method for measuring metabolic fluxes through pathways intersecting in the tricarboxylic acid cycle. However, the inherent insensitivity of 13C NMR spectroscopy makes application of isotopomer analysis to small tissue samples (mouse tissue, human biopsies, or cells grown in tissue culture) problematic. 1H NMR is intrinsically more sensitive than 13C NMR and can potentially supply the same information via indirect detection of 13C providing that isotopomer information can be preserved. We report here the use of J-resolved HSQC (J-HSQC) for 13C isotopomer analysis of tissue samples. We show that J-HSQC reports isotopomer multiplet patterns identical to those reported by direct 13C detection but with improved sensitivity.
Research reports suggest that vitamin D affects glucose and insulin metabolism; however, the exact mechanisms are unclear. ²H NMR analysis of monoacetone glucose (MAG) after tracer administration provides a non-invasive method of profiling hepatic glucose metabolism. This study examined the effects of supplementation with vitamin D₃ on contribution of glycogenolysis to glucose production.
Tracer administration and biofluid collections were performed with eight healthy females before and following a 4-wk vitamin D₃ administration period. Following an overnight fast subjects ingested deuterated water and acetaminophen. Full void urine samples were collected after 4 h. ²H NMR spectra of urinary monoacetone glucose were acquired to determine the contribution of glycogenolysis to glucose production. The mean contribution of glycogenolysis to glucose production was 60±13%. Supplementation with vitamin D₃ had no effect on hepatic glucose production. Regression analysis revealed a significant relationship between carbohydrate intake and the contribution of glycogenolysis (β=0.914, p=0.004).
In conclusion, we saw no changes in the percentage contribution of glycogenolysis following supplementation with vitamin D₃. The reproducibility of our results and the non-invasive nature of the method highlight the potential for this method in assessing mechanistic modes of action in future nutritional interventions.
Proteolytic and cataplerotic sources of hepatic glutamine were determined by (2)H NMR analysis of urinary phenylacetylglutamine (PAGN) (2)H-enrichments in eight healthy subjects after (2)H(2)O and phenylbutyric acid ingestion. Body water enrichment was 0.49+/-0.03%. PAGN was enriched to lower levels with significant differences between the various glutamine positions. PAGN position 2 enrichment=0.33+/-0.02%; 3R=0.27+/-0.02%; 3S=0.27+/-0.02% and position 4=0.17+/-0.01%. Position 3R,S enrichments are conditional with the net conversion of citrate to glutamate and are therefore markers of cataplerosis. From the ratio of positions 3R,S to body water enrichment, 55+/-3% of hepatic glutamine was derived from cataplerosis and 45+/-3% from proteolysis. In conclusion, enrichment of PAGN 3R,S hydrogens relative to that of body water reflects the contribution of cataplerotic and proteolytic sources to hepatic glutamine.
Endogenous glucose production (EGP), gluconeogenic and glycogenolytic fluxes by analysis of a single (2)H-NMR spectrum is demonstrated with 6-hr and 24-hr fasted rats. Animals were administered [1-(2)H, 1-(13)C]glucose, a novel tracer of glucose turnover, and (2)H(2)O. Plasma glucose enrichment from both tracers was quantified by (2)H-NMR analysis of monoacetone glucose. The 6-hr fasted group (n = 7) had EGP rates of 95.6 +/- 13.3 micromol/kg/min, where 56.2 +/- 7.9 micromol/kg/min were derived from PEP; 12.1 +/- 2.1 micromol/kg/min from glycerol, and 32.1 +/- 4.9 micromol/kg/min from glycogen. The 24-hr fasted group (n = 7) had significantly lower EGP rates (52.8 +/- 7.2 micromol/kg/min, P = 0.004 vs. 6 hr) mediated by a significantly reduced contribution from glycogen (4.7 +/- 5.9 micromol/kg/min, P = 0.02 vs. 6 hr) while PEP and glycerol contributions were not significantly different (39.5 +/- 3.9 and 8.5 +/- 1.2 micromol/kg/min, respectively). These estimates agree with previous assays of EGP fluxes in fasted rats obtained by multinuclear NMR analyses of plasma glucose enrichment from (2)H(2)O and (13)C-glucose tracers.
Glucose is stored in mammalian tissues in the form of glycogen. Glycogen levels are markedly reduced in liver or muscle cells of patients with insulin-resistant or insulin-deficient forms of diabetes, suggesting that impaired glycogen synthesis may contribute to development of hyperglycemia. Recently, interest in this area has been further stimulated by new insights into the spatial organization of metabolic enzymes within cells and the importance of such organization in regulation of glycogen metabolism. It is now clear that a four-member family of glycogen targeting subunits of protein phosphatase-1 (PP1) plays a major role in coordinating these events. These proteins target PP1 to the glycogen particle and also bind differentially to glycogen synthase, glycogen phosphorylase, and phosphorylase kinase, thereby serving as molecular scaffolds. Moreover, the various glycogen-targeting subunits have distinct tissue expression patterns and can influence regulation of glycogen metabolism in response to glycogenic and glycogenolytic signals. The purpose of this article is to summarize new insights into the structure, function, regulation, and metabolic effects of the glycogen-targeting subunits of PP1 and to evaluate the possibility that these molecules could serve as therapeutic targets for lowering of blood glucose in diabetes.
Acute liver failure was induced in rats by CCl4 administration and its effects on the hepatic Krebs cycle and gluconeogenic fluxes were evaluated in situ by 13C NMR isotopomer analysis of hepatic glucose following infusion of [U-13C]propionate. In fed animals, CCl4 injury caused a significant increase in relative gluconeogenic flux from 0.80+/-0.10 to 1.34 +/-0.24 times the flux through citrate synthase (p<0.01). In 24-h fasted animals, CCl4-injury also significantly increased relative gluconeogenic flux from 1.36+/-0.16 to 1.80+/-0.22 times the flux through citrate synthase (p<0.01). Recycling of PEP via pyruvate and oxaloacetate was extensive under all conditions and was not significantly altered by CCl4 injury. CCl4 injury significantly reduced hepatic glucose output by 26% (42.8+/-7.3 vs 58.1+/-2.4 micromol/kg/min, p=0.005), which was attributed to a 26% decrease in absolute gluconeogenic flux from PEP (85.6+/-14.6 vs 116+/-4.8 micromol/kg/min, p<0.01). These changes were accompanied by a 47% reduction in absolute citrate synthase flux (90.6+/-8.0 to 47.6+/-8.0 micromol/kg/min, p<0.005), indicating that oxidative Krebs cycle flux was more susceptible to CCl4 injury. The reduction in absolute fluxes indicate a significant loss of hepatic metabolic capacity, while the significant increases in relative gluconeogenic fluxes suggest a reorganization of metabolic activity towards preserving hepatic glucose output.
The rate of endogenous glucose production (EGP) is important in understanding the pathophysiology of Type II (non-insulin-dependent) diabetes mellitus, the aetiology of its complications, and the identification of potential therapeutic targets. A great deal of effort has therefore been expended in its evaluation. Most measurements in humans have been made using tracers, or labelled analogues of glucose. Experimental strategies have included the injection and the infusion of such tracers which were often primed to achieve constant concentrations of the label more quickly. Primers have either been fixed or adjusted to the ambient glycaemia in each diabetic subject. Analyses were carried out using steady-state or non-steady-state calculations, the latter based on a one-compartment model or higher order systems. The principal finding of this review is that all approaches yield the same EGP when an appropriate model of the system is used. Under basal conditions, a single compartment model is sufficient to evaluate EGP, but the estimation of the volume of distribution, V, from individual data is critical in obtaining consistent results. Other sources of variation arose from the length of the fasting period and the patient population being studied. Overall, in Type II diabetes, EGP is frequently high in the morning and decreases gradually to rates comparable to healthy control subjects. This can be a very delayed response to a preceding meal, but more likely corresponds to an accentuated circadian rhythm in glucose production. Metabolic clearance of glucose, on the other hand, is decreased in diabetes, and remains so during the course of the day.
Understanding the metabolic and regulatory pathways of hepatocytes is important for biotechnological applications involving liver cells, including the development of bioartificial liver (BAL) devices. To characterize intermediary metabolism in the hepatocytes, metabolic flux analysis (MFA) was applied to elucidate the changes in intracellular pathway fluxes of primary rat hepatocytes exposed to human plasma and to provide a comprehensive snapshot of the hepatic metabolic profile. In the current study, the combination of preconditioning and plasma supplementation produced distinct metabolic states. Combining the metabolic flux distribution obtained by MFA with methodologies such as Fisher discriminant analysis (FDA) and partial least squares or projection to latent structures (PLS) provided insights into the underlying structure and causal relationship within the data. With the aid of these analyses, patterns in the cellular response of the hepatocytes that contributed to the separation of the different hepatic states were identified. Of particular interest was the recognition of distal pathways that strongly correlated with a particular hepatic function. The hepatic functions investigated were intracellular triglyceride accumulation and urea production. This study illustrates a framework for optimizing hepatic function and a possibility of identifying potential targets for improving hepatic functions.
An ion-exchange chromatography method was established for simultaneously analyzing the tricarboxylic acid (TCA) cycle acids and other related substances in cultured mammalian cells, including citrate, cis-aconitate, isocitrate, alpha-ketoglutarate, succinate, malate, fumarate, oxaloacetate, trans-aconitate, phosphate, lactate and pyruvate. A Dionex 600 ion chromatograph with an ion suppressor and a conductivity detector, and an IonPac AS11-HC analytical column were employed. An NaOH gradient elution containing 13.5% methanol contributed to sufficient separation of target substances. The stability of carboxylic acids was investigated and oxaloacetate was found to be extremely unstable especially at pH 3. TCA cycle acids and other related substances in Chinese hamster ovary (CHO) cells were separated completely, and lactate, malate, phosphate, citrate and cis-aconitate were quantified due to their higher concentrations. In the quantification of the five substances, detection limits (S/N=3) ranged from 0.12 to 0.48 microM, the correlation coefficients from 0.9982 to 1.0000 in their linear ranges of concentration, and the recoveries from 87 to 95%. The metabolic status of CHO cells was analyzed on the basis of the intracellular concentrations of TCA cycle acids.
The field of metabolic engineering encompasses a powerful set of tools that can be divided into (a) methods to model complex metabolic pathways and (b) techniques to manipulate these pathways for a desired metabolic outcome. These tools have recently seen increased utility in the medical arena, and this paper aims to review significant accomplishments made using these approaches. The modeling of metabolic pathways has been applied to better understand disease-state physiology in a variety of cellar, subcellular, and organ systems, including the liver, heart, mitochondria, and cancerous cells. Metabolic pathway engineering has been used to generate cells with novel biochemical functions for therapeutic use, and specific examples are provided in the areas of glycosylation engineering and dopamine-replacement therapy. In order to document the potential of applying both metabolic modeling and pathway manipulation, we describe pertinent advances in the field of diabetes research. Undoubtedly, as the field of metabolic engineering matures and is applied to a wider array of problems, new advances and therapeutic strategies will follow.
MR imaging and MR spectroscopy are fundamental techniques for molecular and cellular imaging. The implementation of molecular imaging techniques for MR imaging has been facilitated by the development of targeted contrast agents for detection of gene expression, surface receptors, or enzyme activity. This article reviews recent advances in targeting strategies for MR molecular imaging focusing on the two main classes of MR contrast agents, paramagnetic gadolinium chelates and superparamagnetic iron oxide compounds, including methods for improving MR contrast through modulation of relaxivity in response to specific biologic interactions.
A recently introduced tracer, [3,4-(13)C(2)]glucose, was compared to the widely used tracer, [6,6-(2)H(2)]glucose, for measurement of whole-body glucose turnover. The rate of glucose production (GP) was measured in rats after primed infusions of [3,4-(13)C(2)]glucose, [6,6-(2)H(2)]glucose, or both tracers simultaneously followed by a constant infusion of tracer(s) over 90 min. Blood glucose was purified and converted into monoacetone glucose for analysis by (13)C NMR (for [3,4-(13)C(2)]glucose) or (1)H and (2)H NMR (for [6,6-(2)H(2)]glucose). The values of GP measured during infusion of each single tracer were not significantly different. In rats infused with both tracers simultaneously, GP was identical as reported by each tracer, 42 +/- 4 micromol/kg/min. Since (2)H and (13)C enrichment in glucose is typically much less than 2% for in vivo studies, [3,4-(13)C(2)]glucose does not interfere with measurements of (13)C or (2)H enrichment patterns and therefore is valuable when multiple metabolic pathways are being evaluated simultaneously.
Tissue engineering is a rapidly expanding, multi-disciplinary field in biomedicine. It provides
the ability to manipulate living cells and biomaterials for the purpose of restoring, maintaining, and enhancing
tissue and organ function. Scientists have engineered various tissues in the body, from skin substitutes
to artificial nerves to heart tissues, with varying degrees of success. Although the field of tissue engineering
has come a long way since its first successful demonstration by Bisceglie in the 1930s, methods of
coaxing them into functional tissues have been predominantly empirical to date. To successfully develop
tissue-engineered organs, it is important to understand how to maintain the cells under conditions that
maximize their ability to perform their physiological roles, regardless of their environment. In that context,
a methodology that combines empirical data with mathematical and statistical techniques, such as metabolic
engineering and cellular informatics, to systematically determine the optimal (1) type of cell to use, (2)
scaffold properties and the corresponding processing conditions to achieve those properties, and (3) the
required types and levels of environmental factors and the operating conditions needed in the bioreactor,
will enable the design of viable and functional tissues tailored to the specific requirements of individual
situations.
The design of strategies that improve the absorption of insulin through the gastrointestinal tract is a considerable challenge in the pharmaceutical sciences and would significantly enhance the treatment of diabetes mellitus. Several strategies have been devised to overcome physiologic and morphologic barriers to insulin absorption, including the inhibition of acidic and enzymatic degradation, enhancement of membrane permeability or widening of tight junctions, chemical modification of insulin, and the formulation of carrier systems. In particular, the concept of nanoparticulate carriers for oral insulin delivery has evolved through remarkable advances in nanotechnology. Investigations focused on uptake and translocation via Peyer's patches have demonstrated high levels of nanoparticle absorption based on significant alterations in the glycemic response to various glucogenic sources. This paper reviews the mechanisms for insulin and particle uptake and translocation through the gastrointestinal tract, and the potential barriers to this, outlines the design of nanoparticulate carriers for the oral delivery of insulin, and presents prospects for its clinical application.
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.
To determine whether the source of carbon for the indirect pathway of hepatic glycogen synthesis differs between the periportal and pericentral zones, we studied seven 24-h-fasted conscious rats given a constant 2-h intraduodenal infusion of glucose, 40% labeled with [U-13C]glucose (99% 13C enriched), to raise and maintain plasma glucose concentration at approximately 10 mM. Glycogen, glutamate, aspartate, and alanine were selectively sampled from the periportal and pericentral zones of the liver by the dual-digitonin pulse technique and analyzed by 13C-NMR for positional isotopomer distribution and by gas chromatography-mass spectrometry for mass isotopomer distribution. Plasma glucose mass isotopomer distribution was determined from gas chromatography-mass spectrometry. The isotopomer distribution indicates that there was no significant difference between the zones with respect to 1) percent direct flux of glucose into the glycogen (periportal, 34 +/- 4; pericentral, 38 +/- 4), 2) extent of oxaloacetate/fumarate equilibration (periportal, 0.54 +/- .01;, pericentral, 0.53 +/- 0.01), 3) dilution of tracer in oxaloacetate (periportal, 0.64 +/- 0.07;, pericentral, 0.64 +/- 0.07), or 4) inflow of pyruvate versus tricarboxylic acid cycle flux (periportal, 0.70 +/- 0.20; pericentral, 0.68 +/- 0.16). Positional isotopomer populations, determined from the 13C-13C splitting in C3 and C4 of periportal and pericentral glycogen, were indistinguishable, indicating no significant differences in the source of the 3-carbon precursors for hepatic glycogen synthesis by the indirect pathway. In conclusion, glucose metabolism is the same in the periportal and pericentral zones with regard to 1) the relative flux of carbon via the direct/indirect pathways, 2) the source of the 3-carbon precursor used in the indirect pathway of glycogen synthesis, and 3) the flux of the 3-carbon precursors through the tricarboxylic acid cycle.
We conducted an extensive mass isotopomer analysis of citric acid cycle and gluconeogenic metabolites isolated from livers of overnight fasted rats perfused with 4 mM glucose, 0.2 mM octanoate, 1 mM [U-13C3]lactate, and 0.2 mM [U-13C3]pyruvate, in the anterograde or retrograde mode. In both perfusion modes, two distinct isotopomer patterns were observed: (i) those of phosphoenolpyruvate, glucose, malate, and aspartate and (ii) those of citrate, alpha-ketoglutarate, glutamate, and glutamine. Key citric acid cycle parameters and, hence, rates of gluconeogenesis, calculated (Lee, W.-N.P. (1989) J. Biol. Chem. 264, 13002-13004 and Lee, W.-N.P. (1993) J. Biol. Chem. 268, 25522-25526) from our mass isotopomer data did not only vary, but lead to conclusions inconsistent with Lee's citric acid cycle model. Compared to lactate and pyruvate uptake, which sets an upper limit to glucose production, rates of gluconeogenesis calculated (i) with the phosphoenolpyruvate and citrate data were similar, but those calculated (ii) with the glutamate data amounted to only 60%, which is unlikely. All these conclusions are independent of the perfusion modes. We provide evidence that the following processes contribute to the observed labeling discrepancy: (i) the reversibility of the isocitrate dehydrogenase reaction and (ii) an active citrate cleavage pathway for the transfer of the oxaloacetate carbon skeleton from mitochondria to the cytosol. Also, a good fit of our labeling data was obtained with a model of citric acid cycle and gluconeogenesis which we developed to incorporate the above reactions (Fernandez, C.A., and Des Rosiers, C. (1995) J. Biol. Chem. 270, 10037-10042). The following conclusions can be drawn from the calculated reaction rates: (i) about half of the lactate conversion to glucose occurs via the citrate cleavage pathway, (ii) the flux through the reversal of the isocitrate dehydrogenase reaction is almost as fast as that through the citrate synthase reaction, and (iii) the flux through citrate synthase and alpha-ketoglutarate dehydrogenase is 1.6- and 3.2-fold that through pyruvate carboxylase, respectively.
Fasted rats were intragastrically infused with either [2,3-13C]lactate or [1,2,3-13C]lactate. The infusate also contained 14C-labeled lactate and [3-3H]glucose. Glucose, alanine, glutamate, and glutamine were isolated from liver and blood. There was near complete equilibration of lactate and alanine, and the relative specific activities and relative enrichments were the same in blood and liver. Glucose was cleaved enzymatically to lactate. The compounds were examined by gas chromatography-mass spectroscopy. From the mass isotopomer spectra of the lactate, glutamate, and glutamine and their cleavage fragments the positional isotopomer composition of these compounds was obtained. The enrichment and isotopomer pattern in the lactate from cleaved glucose represents that in phosphoenolpyruvate (PEP). When [1,2,3-13C]lactate was infused the mass isotopomer spectrum of glutamates consisted only of compounds containing either one, two, or three 13C carbons per molecule (m1, m2, and m3). There was little 13C in C-4 and C-5 of glutamate. The rate of pyruvate decarboxylation is low, and 3-4% of the acetyl-CoA flux in the Krebs cycle is contributed by lactate carbon. The major isotopomers in lactate, alanine, and PEP were m3 and m2 with 13C in C-2 and C-3. The predominant isotopomer in PEP from [2,3-13C]lactate was m2 with 13C in C-2 and C-3. There was much more of m1 isotopomer with 13C in C-3 and C-2 than the m1 isotopomer with 13C in C-1. There was very little m3, the isotopomer with 13C in all three carbons. Most of the 13C in C-3 and C-4 of glucose and C-1 of glutamate was introduced via 13CO2 fixation. From the isotopomer distribution and the rate of glucose turnover we deduced, applying the analysis described in the "Appendix," the absolute rates of gluconeogenic pathways, recycling of PEP and the Cori cycle, and flux in the Krebs cycle. The flux from oxaloacetate (OAA)-->PEP was seven times that of OAA-->citrate, and about half of PEP was recycled to pyruvate via pyruvate kinase. The mass isotopomer patterns in glutamate and glutamine were similar but differed from those of lactate and glucose. It appears that the glutamates are derived from alpha-ketoglutarate from a different Krebs cycle pool than PEP. The flux from OAA to PEP in this pool was two to three times that of OAA to citrate.(ABSTRACT TRUNCATED AT 400 WORDS)
Absolute rates of gluconeogenesis and of the citric acid cycle were assessed in livers isolated from 24-h starved rats, perfused with physiological concentrations of [3-13C]lactate and [3-13C]pyruvate +/- 0.2 mM octanoate. Calculations are based on (i) the 13C-labeling pattern of glutamate determined by gas chromatography-mass spectrometry combined with isotopomer analysis, (ii) substrate balance, and (iii) equations developed by Magnusson et al. (Magnusson, I., Schumann, W. C., Bartsch, G. E., Chandramouli, V., Kumaran, K., Wahren, J., and Landau, B. R. (1991) J. Biol. Chem. 266, 6975-6984) based on a citric acid cycle model proposed by Katz (Katz, J. (1985) Am. J. Physiol. 248, R391-R399). Glutamate, isolated from liver extracts, is enzymatically or chemically converted to gamma-aminobutyrate, alpha-hydroxyglutarate, isocitrate, and glutamine before mass spectrometric analysis. General equations have been developed ("Appendix I") to determine the isotopic enrichment of each carbon of glutamate from the isotopic enrichment of fragments obtained from the mass spectra of trimethylsilyl or t-butyldimethylsilyl derivatives of glutamate and of derived compounds ("Appendix II"). In the presence of octanoate, (i) the rate of the citric acid cycle decreases from 0.25 to 0.13 mumol/min x g wet weight which are one-third and one-sixth of the rate of pyruvate carboxylation, and (ii) the rate of gluconeogenesis increases from 0.65 to 0.83 mumol/min x g wet weight. The rate of pyruvate carboxylation is 13 and 34-fold faster than that of pyruvate dehydrogenation in the absence or presence of octanoate, respectively. The rate of oxaloacetate to fumarate interconversion is at least six times greater than that of the citric acid cycle. Our data closely agree with those obtained by Magnusson et al. who used a non-invasive "chemical biopsy" of the human liver and support the use of labeled lactate and/or pyruvate for tracing hepatic metabolism in vivo.
Six normal subjects (NL group) and 13 cancer patients (CAI and CAII groups) were fasted overnight and infused with [U-(13)C]glucose (0.016-0.058 mg x min(-1) x kg(-1)). Plasma glucose and lactate were isolated, and mass isotopomer distributions were determined by gas chromatography-mass spectroscopy. Applying equations modified from those previously described [J. A. Tayek and J. Katz. Am. J. Physiol. 270 (Endocrinol. Metab. 33): E709-E717, 1996], we determined glucose production (GP), recycling of glucose carbons, fraction of recycled molecules in blood glucose (Cori cycle), formation of pyruvate from unlabeled carbons, dilution of pyruvate via the tricarboxylic acid cycle and other reactions, and rate of gluconeogenesis. Glucose production was similar in all groups: 2.4 +/- 0.2 mg x min(-1) x kg (-1). The fraction of recycled carbon and of the Cori cycle were elevated in the CAI group vs. the CAII and NL groups: 15 and 33% vs. 7.8 and 19%, respectively (P = 0.01). Gluconeogenesis was 1.9 +/- 0.1, 1.0 +/- 0.1, and 0.83 +/- 0.11 mg x min(-1) x kg(-1) in the CAI, CAII, and NL groups, respectively. In the NL and CAII groups, 20% of GP is via recycling, 20% from unlabeled carbon sources (muscle glycogen, amino acids), and 60% from hepatic glycogenolysis; in the CAI group, 30% is from recycling, 50% from unlabeled carbon, and 20% from glycogen and other sources. Serum cortisol was elevated in the CAI group vs. the CAII group: 11.2 +/- 1.2 vs. 7.7 +/- 1.2 microg/dl (P < 0.05). There was a strong correlation between plasma cortisol and Cori cycle in the NL (r = 0.963) and CAI and CAII groups (r = 0.771). Serum cortisol was directly, and insulin was inversely, correlated with gluconeogenesis in the NL (r2 = 0.967) and CAI and CAII groups (r2 = 0.727). We conclude that whereas the cancer population is heterogeneous with respect to gluconeogenesis, many cancer patients derive their GP predominantly from gluconeogenesis compared with healthy controls, who derive less than one-half of their GP from gluconeogenesis.
Fluxes through intrahepatic glucose-producing metabolic pathways were measured in normal humans during overnight or prolonged (60 h) fasting. The glucuronate probe was used to measure the turnover and sources of hepatic UDP-glucose; mass isotopomer distribution analysis from [2-13C1]glycerol for gluconeogenesis and UDP-gluconeogenesis; [U-13C6]glucose for glucose production (GP) and the direct UDP-glucose pathway; and [1-2H1]galactose for UDP-glucose flux and retention in hepatic glycogen. After overnight fasting, GP (fluxes in milligram per kilogram per minute) was 2.19+/-0.09, of which 0.79 (36%) was from gluconeogenesis, 1.40 was from glycogenolysis, 0.30 was retained in glycogen via UDP-gluconeogenesis, and 0.17 entered hepatic UDP-glucose by the direct pathway. Thus, total flux through the gluconeogenic pathway (1.09) represented 54% of extrahepatic glucose disposal (2.02) and the net hepatic glycogen depletion rate was 0.93 (46%). Prolonging [2-13C1]glycerol infusion slowly increased measured fractional gluconeogenesis. In response to prolonged fasting, GP was lower (1. 43+/-0.06) and fractional and absolute gluconeogenesis were higher (78+/-2% and 1.11+/-0.07, respectively). The small but nonzero glycogen input to plasma glucose (0.32+/-0.03) was completely balanced by retained UDP-gluconeogenesis (0.31+/-0.02). Total gluconeogenic pathway flux therefore accounted for 99+/-2% of GP, but with a glycogen cycle interposed. Prolonging isotope infusion to 10 h increased measured fractional gluconeogenesis and UDP-gluconeogenesis to 84-96%, implying replacement of glycogen by gluconeogenic-labeled glucose. Moreover, after glucagon administration, GP (1.65), recovery of [1-2H1]galactose label in plasma glucose (25%) and fractional gluconeogenesis (91%) increased, such that 78% (0.45/0.59) of glycogen released was labeled (i.e., of recent gluconeogenic origin). In conclusion, hepatic gluconeogenic flux into glycogen and glycogen turnover persist during fasting in humans, reconciling inconsistencies in the literature and interposing another locus of control in the normal pathway of GP.
Rats were infused with glucose at 30 mg/min, containing 18% enriched [U-C-13]glucose and [1-C-14]- and [3-H-3]glucose. The mass isotopomer patterns of C-13-labeled blood glucose and liver glycogen were determined by gas chromatography/mass spectroscopy. The contribution of the direct pathway to glycogen was calculated from the three tracers, and the values by all three were nearly identical, about 50%. The C-14 specific activity in carbon 6 of glycogen glucose was about 6% that of carbon 1. The [H-3]glucose/[1-C-14]glucose ratio in glycogen was 80-90% that in blood glucose. The enrichment of C-13 and the specific activity of C-14 in glycogen formed by the indirect path were 20-25% of glycogen formed directly from glucose. The dilution is of two kinds: (i) an exchange of labeled carbon with unlabeled carbon in the tricarboxylic acid cycle and (ii) dilution by unlabeled nonglucose carbon. Methods to calculate the two types of dilution are presented. In control rats the dilution factor by exchange in the tricarboxylic acid cycle is 1.4, and the dilution by unlabeled carbon is 2.5- to 3.0-fold, with the overall dilution about 4-fold. In rats preinjected with glucagon, the dilution through the tricarboxylic acid cycle was unaffected but that by nonglucose carbon was decreased.
Simple equations that relate glucose and glutamate 13C-NMR multiplet areas to gluconeogenesis and pyruvate recycling during metabolism of [1,2,3-13C3]propionate are presented. In isolated rat livers, gluconeogenic flux was 1.2 times TCA cycle flux and about 40% of the oxaloacetate pool underwent recycling to pyruvate prior to formation of glucose. The 13C spectra of glucose collected from rats after gastric versus intravenous administration of [1,2,3-13C3]propionate indicated that pyruvate recycling was slightly higher in vivo (49%) while glucose production was unchanged. This indicates that a direct measure of gluconeogenesis and pyruvate recycling may be obtained from a single 13C-NMR spectrum of blood collected after oral administration of enriched propionate.
Early assignments of the ¹³C resonances in the natural abundance ¹³C NMR spectra of monosaccharides have been reevaluated in light of recent coupling data from the spectra of ¹³C-1 labeled sugars. The technique of specific ¹³C enrichment not only identifies the labeled carbon unambiguously but can be used to assign more remote carbon resonances due to scalar carbon-carbon coupling. The pattern of carbon-carbon coupling observed in all of the sugars thus far studied is remarkably constant. In addition to the large (approximately 46 Hz) one-bond coupling between C-1 and C-2, C-3 exhibits a coupling to C-1 only in the ..beta.. anomer (approximately 4 Hz) while C-5 is coupled to C-1 only in the ..cap alpha.. anomer (approximately 2 Hz). In addition, C-6 is coupled to C-1 in both anomers and C-4 shows no evidence of coupling to C-1 in any of the sugars examined. These couplings have been used to reassign several resonances and the original assignments are discussed in terms of the predictive rules used for resonance assignments in carbohydrates. The vicinal couplings of C-6 and C-4 to C-1 appear to obey a Karplus-type relationship. The geminal ²J/sub CCC/ and ²J/sub COC/ couplings are discussed in terms of a dihedral angle dependence where the angle is defined by the relative orientations of C-3 or C-5 and the electronegative oxygen substituents on C-1. Additional data on ²J/sub CCH/ couplings involving C-1 and H-2 are also readily obtained with the C-1 labeled sugars.
Starved and starved-refed rats were injected intravenously with labelled glucose (a mixture of [2-3H]-, [3-3H]- and [U-14C]glucose with either [5-3H]- or [6-3H]glucose), and the decay of the specific activity of [14C]glucose followed. Glucose was degraded to obtain the 3H/14C ratios for 3 isotope combinations in the same sample. The apparent rates of replacements, apparent carbon recycling, and the body glucose mass were calculated for the different tracers. The 3H/14C ratio from [2-3H, -U-14C]glucose declined much faster than that of the other tracers. Apparent recycling as calculated in fasted rats was 28% for [2-3H, U-14C]- 18% for [5-3H,-U-14C]- 17% for [3-3H, U-14C]- and 14% for [6-3H,U-14C]glucoses. The values in fed rats showed a similar pattern. We estimate that in fasted rats 85 to 90% of the 3HOH liberated from injected [2-3H]glucose is formed by catabolism in the periphery and the rest by recycling in the liver between glucose and glucose 6-P. Detritiation of other labels by hepatic recycling accounts for a very small fraction of the total 3HOH yield.
The glucose replacement rate, percent carbon recycling, mean glucose transit time, and the glucose mass were determined in fasted unanesthetized rabbits after administration of [2-3H,U-14C]-, [3-3H,U-14C]-, [5-3H,U-14C]- or [6-3H,U-14C]glucose using the procedures of Katz et al. (10). The glucose replacement rates and carbon recycling determined with [2-3H,U-14C] and [5-3H,U-14C]glucose are equivalent and greater than those obtained with [3-3H,U-14C]- and [6-3H,U-14C]glucose. Although the means of the glucose replacement rates and percent carbon recycling obtained using [3-3H,U-14C]- and [6-3H,U-14C]glucose are similar, greater variation resulted using the former tracer. Comparisons of detritiation rates and percent carbon recycling using [2-3H,U-14C]- and [6-3H,U-14C]glucose suggest that about 10% of tritium is lost from carbon 2 via futile cycling at the glucose 6-phosphate level. Similarly, comparisons of [5-3H,U-14C]- and [6-3H,U-14C]glucose metabolism suggest that about 10% of tritium lost from carbon 5 occurs via futile cycling at the fructose diphosphate level and/or via the transaldolase reaction. Our results indicate that [6-3H,U-14C]glucose is the more suitable tracer for determining the glucose replacement rate and carbon recycling in vivo.
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)
Rats were infused with glucose at 30 mg/min, containing 18% enriched [U-13C]glucose and [1-14C]- and [3-3H]glucose. The mass isotopomer patterns of 13C-labeled blood glucose and liver glycogen were determined by gas chromatography/mass spectroscopy. The contribution of the direct pathway to glycogen was calculated from the three tracers, and the values by all three were nearly identical, about 50%. The 14C specific activity in carbon 6 of glycogen glucose was about 6% that of carbon 1. The [3H]glucose/[1-14C]glucose ratio in glycogen was 80-90% that in blood glucose. The enrichment of 13C and the specific activity of 14C in glycogen formed by the indirect path were 20-25% of glycogen formed directly from glucose. The dilution is of two kinds: (i) an exchange of labeled carbon with unlabeled carbon in the tricarboxylic acid cycle and (ii) dilution by unlabeled nonglucose carbon. Methods to calculate the two types of dilution are presented. In control rats the dilution factor by exchange in the tricarboxylic acid cycle is 1.4, and the dilution by unlabeled carbon is 2.5- to 3.0-fold, with the overall dilution about 4-fold. In rats preinjected with glucagon, the dilution through the tricarboxylic acid cycle was unaffected but that by nonglucose carbon was decreased.
Experimental determinations of glucose carbon recycling using 14C or 13C glucose tracer often underestimate true Cori cycle activity because of dilution and exchange of isotope tracer through the tricarboxylic acid (TCA) cycle. The term glucose isotope recycling therefore is used to distinguish recycling of isotope from recycling of glucose carbon, the actual quantity of circulating glucose recycled. Recently, per-labeled glucose ([U-13C6]glucose) has been used to estimate glucose appearance rate and glucose isotope recycling. Chemical structural information determined by mass isotopomer analysis has been used to correct for dilution of isotope through the TCA cycle. In this report, we present experiments in the study of glucose turnover and recycling using [U-13C6]glucose. Methods of single injection and continuous infusion of [U-13C6]glucose are compared. A formula for the calculation of a dilution factor using TCA cycle parameters is derived. In this study of six rabbits, glucose turnover rate ranged from 3.4 to 8.8 mg/kg/min, and glucose m + 3 mass isotopomer recycling from 7 to 12%. The rate of pyruvate carboxylation (Y) was comparable to that of citrate synthetase, having an average relative flux of 0.89. Applying the correction factor for tracer dilution to the observed mass isotopomer recycling, we determined glucose carbon recycling (or Cori cycle activity) to be 22-35% of hepatic glucose output.
A significant problem which may be encountered in 13C NMR studies of metabolism is the contribution that background levels of 13C may make to the observed spectra when low or tracer levels of the 13C label are used. We propose that the introduction of two or more labeled sites in the same tracer molecule is an effective strategy for eliminating or reducing this difficulty and demonstrate its feasibility in an isotope dilution study of glucose turnover in a human volunteer. This approach has two significant advantages over the more common use of a singly enriched labeling strategy: (i) as a consequence of the scalar coupling interactions, multiple-labeled metabolites will yield spectra distinct from those containing natural abundance 13C, and (ii) at a 99% level of enrichment for the precursor, concentration levels which are approximately 1% of the endogenous pools can be detected with approximately equal sensitivity. As a demonstration of this strategy, glucose production in a human subject was determined by continuous infusion of tracer levels of [U-13C6]glucose over a 4-h period and subsequent analysis of plasma levels of the tracer in vitro by NMR. Mass spectroscopy was used on the same samples to provide a basis for comparison of the precision and accuracy of the NMR technique. The results demonstrate the feasibility of the multiply labeled approach for detection by NMR of tracer amounts of label in the presence of a much larger endogenous pool of glucose. The NMR and mass spectrometric data gave quantitatively identical results for the glucose production rate demonstrating that equivalent data may be obtained by both methods.(ABSTRACT TRUNCATED AT 250 WORDS)
13C NMR has been used to study the competition of pyruvate dehydrogenase with pyruvate carboxylase for entry of pyruvate into the tricarboxylic acid (TCA) cycle in perfused liver from streptozotocin-diabetic and normal donor rats. The relative proportion of pyruvate entering the TCA cycle by these two routes was estimated from the 13C enrichments at the individual carbons of glutamate when [3-13C]alanine was the only exogenous substrate present. In this way, the proportion of pyruvate entering by the pyruvate dehydrogenase route relative to the pyruvate carboxylase route was determined to be 1:1.2 +/- 0.1 in liver from fed controls, 1:7.7 +/- 2 in liver from 24-fasted controls, and 1:2.6 +/- 0.3 in diabetic liver. Pursuant to this observation that conversion of pyruvate to acetyl coenzyme A (acetyl-CoA) was greatest in perfused liver from fed controls, the incorporation of 13C label into fatty acids was monitored in this liver preparation. Livers were perfused under steady-state conditions with labeled substrates that are converted to either [2-13C]acetyl-CoA or [1-13C]acetyl-CoA, which in the de novo synthesis pathway label alternate carbons in fatty acids. With the exception of the repeating methylene carbons, fatty acyl carbons labeled by [1-13C]acetyl-CoA (from [2-13C]pyruvate) gave rise to resonances distinguishable on the basis of chemical shift from those observed when label was introduced by [3-13C]alanine plus [2-13C]ethanol, which are converted to [2-13C]acetyl-CoA. Thus, measurement of 13C enrichment at several specific sites in the fatty acyl chains in time-resolved spectra of perfused liver offers a novel way of monitoring the kinetics of the biosynthesis of fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)
We have developed and validated a new method to measure simultaneously glucose turnover, alanine turnover, and gluconeogenesis in human, in steady and non-steady states, using a double stable-isotope-labeled tracer infusion and GC-MS analysis. The method is based on the concomitant infusion and dilution of D-[2,3,4,6,6-2H5]glucose and L-[1,2,3-13C3]alanine. The choice of the tracers was done on the basis of a minimal overlap between the ions of interest and those arising from natural isotopic abundances. Alanine was chosen as the gluconeogenic substrate because it is the major gluconeogenic amino acid extracted by the liver and, with lactate, constitutes the bulk of the gluconeogenic precursors. The method was validated by comparing the results obtained during simultaneous infusion of trace amounts of both stable isotope labeled compounds with the radioactive tracers (D-[3-3H]glucose and L-[1,2,3-14C3]alanine) in a normal and a diabetic subject; the radiolabeled tracers were used as the accepted reference procedure. A slight overestimation of glucose turnover (7.3 versus 6.8 in normal and 10.8 versus 9.2 mumol/kg min in diabetic subject) was noticed when the stable isotope-labeled tracers were used. For the basal turnover rate of alanine, similar values were obtained with both methods (6.2 mumol/kg min). For gluconeogenesis, higher values were observed in the basal state with the stable isotopes (0.42 versus 0.21 mumol/kg min); however, these differences disappeared in the postprandial period after the ingestion of a mixed meal. Despite those minor differences, the overall correlation with the reference method was excellent for glucose turnover (r = 0.87) and gluconeogenesis (r = 0.86).(ABSTRACT TRUNCATED AT 250 WORDS)
1. Glucose labelled with (3)H in position 2 and uniformly with (14)C was administered simultaneously to rabbits and rats either as a single injection or by continuous infusion. Plasma glucose specific radioactivity and the yield of (3)H in the plasma water were monitored. 2. The rates of synthesis, recycling of carbon and total body mass of glucose were calculated, without assuming a multicompartmental model and without fitting data by exponential expressions. 3. The rate of synthesis of glucose in starved-overnight rabbits was 4mg/min per kg (range 3-4.5mg/min per kg) and 25-35% of the glucose carbon was recycled. The mass of total body glucose in starved rabbits was 290mg/kg (range 220-390mg/kg). About one-third of the total body glucose equilibrates nearly instantaneously with plasma glucose. 4. In rats starved overnight, glucose synthesis was about 10mg/min per kg and recycling of carbon ranged from 30-40%. Total body mass (per kg body weight) is similar to that in rabbits. 5. The activity in plasma water after injection of [2-(3)H]glucose was determined. The initial rate of (3)H(2)O formation is rapid, indicating that the major site of glucose catabolism is in the rapidly mixing pool. The curve of total body glucose radioactivity was obtained from the (3)H(2)O yield, and total mass of glucose was calculated. This agrees with that obtained from the (3)H specific-radioactivity curve.
The characteristics of the digestive and hepatic metabolism of glucogenic and ketogenic substrates were studied in vivo in fed or starved rats. For this purpose, a procedure for blood flow measurements in the splanchnic area was developed, based on an indicator-dilution technique. Hepatic blood flow (HBF) was markedly reduced after 24 h starvation, mainly corresponding to the decrease of portal blood supply; however, HBF expressed per gram liver was almost unchanged. In overnight-fed rats, glucose absorption was limited and glucose was released by the liver, essentially after gluconeogenesis from C3 units: alanine was the main glucogenic substrate removed by the liver, then propionate and lactate, whereas only a slight release of pyruvate occurred. As large amounts of lactate were released by the digestive tract, there was a net production of lactate in the splanchnic area. In contrast, in starved rats, lactate became the main glucogenic substrate removed by the liver as its fractional extraction was raised from 7% (fed) to 58% (starved) whereas the contribution of alanine and propionate to gluconeogenesis was limited by their availability, their hepatic extraction being highly efficient in fed and in starved rats. The present results are consistent with the view that glucose turnover was practically halved during starvation and suggest that net glucose cycling via lactate was very low in fed rats but could correspond to about 40% of produced glucose in starved rats. Besides propionate, acetate and butyrate made a significant contribution to fuel supply for hepatic metabolism in fed rats, acetate availability for extrasplanchnic tissues remained relatively constant. FFA were extensively removed by the liver (50%) in starved rats and ketogenesis could account for 68% of removed FFA. In spite of low concentrations in the artery, acetoacetate was released by the liver at a higher rate than 3-hydroxybutyrate. This process could correspond to a higher turnover rate for acetoacetate, nevertheless not evident in portal-drained viscera where net 3-hydroxybutyrate uptake was observed only.
The functional integrity of the gluconeogenic pathway was measured in nine term infants, four appropriate-for-gestational age (AGA), and five normoglycemic small-for-gestational age (SGA), by determination of 13C2 enrichment in blood glucose during the constant infusion of tracer [2,3]13C2]alanine between 4 and 8 h of postnatal age. Alanine flux, calculated from the steady-state blood [2,3-13C2]alanine enrichment was 16.6 +/- 1.3 (SE) (mumol.kg-1.min-1 in the AGA infants and not statistically different from the value of 15.3 +/- 0.7 mumol.kg-1.min-1 in the SGA infants. Alanine flux did not correlate with blood alanine level in either group. By 6 h of age, the earliest sampling time, there was 13C2 enrichment of blood glucose in every infant studied, indicating that the gluconeogenic pathway was functionally intact by that time and implying that it was operative sooner. At 8 h of age, 9.3 +/- 2.3% of blood glucose was derived from alanine in the AGA group and 12.9 +/- 2.4% in the SGA group, values not statistically different. These data indicate that the term human newborn has a functional gluconeogenic pathway very early in postnatal life and that intrauterine growth retardation per se does not impair maturation of the system. Furthermore, the plasma alanine level alone is a poor index of gluconeogenic carbon flow in these infants.
To compare two methods of estimating systemic glucose production rates and to quantify carbon tracer recycling, six newborn infants, aged 2 h to 3 days, were infused simultaneously with [1-13C]glucose and [6,6-2H2]glucose tracers. The older infants were studied 6 h after a meal. [1-13C]Glucose was infused at 6 microgram/kg.min. Systemic glucose production rates were calculated from tracer dilution, assuming steady state kinetics. Although 13C was expected to randomize away from the C-1 of glucose, recycling occurred and was estimated from the difference in the rate of systemic glucose production quantified by the dilution of the two tracers. Systemic glucose production rates ranged from 4.2--5.4 mg/kg.min. Recycling on the glucose C-1 was 3--20% of the systemic glucose production rate and did not change with the age of the infant. Because recycling of glucose carbon signifies gluconeogenesis from lactate or pyruvate, it is concluded that the human newborn is able to initiate gluconeogenesis soon after birth.
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%.
We developed gas chromatography-mass spectrometric methods for assaying the enrichment of 99 at.% [6,6-2H2]glucose and 30 at.% [U-13C6]glucose, although both tracers are mostly M + 2. 13C enrichment is determined either by the C-1 to C-5 fragment of glucose aldonitrile pentaacetate or by oxidation of glucose to glucarate. 2H enrichment is assayed as the difference between the 13C enrichment of glucarate and the 2H + 13C enrichment of glucose. The techniques, which were validated in in vivo experiments, are applicable to the determination of simultaneous or sequential measurements of the rate of glucose appearance before and after an intervention. They could also be applied to the simultaneous determination of (i) gluconeogenesis by incorporation of a 13C-labeled precursor into glucose and (ii) the rate of glucose appearance by [6,6-2H2]glucose infusion.
13C NMR analysis of 13C-enriched glucose containing multiple isotopomers is hampered by chemical shift similarities of several carbon resonances and by the presence of two anomeric forms. A convenient and quantitative method of enzymatically oxidizing glucose to gluconate in tissue and perfusate extracts is presented. The six carbon resonances of the resulting 13C-enriched gluconate are fully resolved at high pH, thereby allowing a determination of the fractional population of each 13C isotopomer by 13C NMR. The utility of this method is demonstrated using the effluent from an isolated perfused liver containing 13C-enriched glucose produced by hepatic metabolism of sodium [1,2,3-13C3]propionate via the citric acid cycle and gluconeogenesis. An analysis of the gluconate C2 and C5 resonances in this sample showed that pentose phosphate activity was insignificant during this perfusion protocol. As demonstrated, this method provides a means of fully describing 13C isotopomer populations in enriched glucose samples where isotope may be derived from multiple metabolic pathways, thus expanding the scope of experimental design and enrichment strategies.
We developed a gas chromatography-mass spectrometric method which allows to determine the complete 13C-labeling pattern of glucose. The method uses four derivatives of glucose (methyloxime trimethylsilyl, bisbutylboronate acetate, aldonitrile pentaacetate, and permethyl) and selective analysis of fragment ions retaining specific carbon atoms. The technique was tested by analyzing glucose from rat livers perfused with various 13C tracers. The labeling patterns agree with theoretical calculations and with literature reports where [14C]glucose was analyzed by degradation and [13C]glucose was analyzed by NMR.
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.
Mass isotopomer distribution analysis (MIDA) of glucose during infusion of [2-13C]glycerol is a new method for measuring total gluconeogenesis (GNG). Since this method relies on calculation of the isotopic enrichment (IE) of hepatic triose phosphates (TP), the results should be independent of the sites of tracer infusion and blood sampling. Postabsorptive and starved rats were infused with [2-13C]glycerol and sampled either in the arterial-venous (A-V) or venous-arterial (V-A) modes. Blood was also sampled from the portal vein. In both postabsorptive and starved rats, glycerol turnover rate (Rt) and the percent contribution of glycerol to total glucose production were higher in the A-V mode than in the V-A mode (P < .05). Glycerol IE in portal venous blood was intermediate between IE values observed in peripheral arterial and venous blood. Its use for calculating the contribution of glycerol to glucose production reconciled the results obtained with the two infusion-sampling modes in both postabsorptive and starved rats; this contribution was increased by starvation (P < .01). In postabsorptive rats, total GNG calculated from MIDA of glucose accounted for approximately 50% of glucose production whatever the infusion-sampling mode (A-V, 48.8% +/- 4.7%; V-A, 52.2% +/- 3.9%). This contribution increased to 90% in starved rats, again, with no difference between A-V (95.2% +/- 1.8%) and V-A (89.2% +/- 1.3%) modes. In conclusion, during infusion of [2-13C]glycerol, total GNG measured from MIDA of glucose is independent of the infusion-sampling mode, contrary to calculations of Rt and GNG from glycerol. Measurement of glycerol IE in portal venous blood reconciles the results obtained with the two modes with respect to the contribution of glycerol to GNG.
Eight normal controls and nine non-insulin-dependent diabetes mellitus diabetics were, after an overnight fast, infused for 3 h with [6-3H]- and with [U-13C]glucose with six 13C carbons at rates from 0.03 to 0.15 mg.kg-1.min-1. Plasma glucose and lactate were assayed by gas chromatography-mass spectroscopy. Several parameters of glucose metabolism were calculated from the mass isotopomer distribution. Glucose production (GP) determined with [6-3H]- and [U-13C]glucose agreed closely. GP was 1.9 +/- 0.16 (range 1.3-2.5) mg.kg-1.min-1 in controls and 2.8 +/- 0.29 (1.7-4.5) mg.kg-1.min-1 in diabetics (P < 0.05). The correlation in diabetes between plasma glucose and GP (r = 0.911, P < 0.01) was close. Recycling of carbon (8 vs 7%) dilution by unlabeled carbon (2- vs 2.3-fold), and dilution via the tricarboxylic acid cycle (1.5-fold) were similar in controls and diabetics. Gluconeogenesis was 0.90 +/- 0.08 (0.5-1.3) mg.kg-1.min-1 in controls and 1.30 +/- 0.13 (0.8-1.9) mg.kg-1.min-1 in diabetics (P < 0.05). Gluconeogenesis contributions to GP were 46.6 +/- 4.0% (26-61%) in the controls and 48.8 +/- 5.7% (32-83%) in diabetics. We show that, using [U-13C]glucose infusion of 2-5% of glucose turnover (0.03-0.10 mg.kg-1.min-1), a large number of parameters of glucose metabolism may be determined in humans.
In this study, we investigated the contribution of different subtypes of voltage-dependent Ca2+ channels to changes in cytosolic free Ca2+ ([Ca2+]i) and secretion in noradrenergic and adrenergic bovine chromaffin cells. In single immunocytochemically identified chromaffin cells, [Ca2+]i increased transiently during high K+ depolarization. Furnidipine and BAY K 8644, L-type Ca2+ channel blocker and activator, respectively, affected the [Ca2+]i rise more in noradrenergic than in adrenergic cells. In contrast, the Q-type Ca2+ channel blocker omega-conotoxin MVIIC inhibited the [Ca2+]i rise more in adrenergic cells. omega-Agatoxin IVA (30 nM), which blocks P-type Ca2+ channels, had little effect on the [Ca2+]i signal. The N-type Ca2+ channel blocker omega-conotoxin GVIA similarly inhibited the [Ca2+]i rise in both cell types. The effects of furnidipine, BAY K 8644, and omega-conotoxin MVIIC on K+-evoked norepinephrine and epinephrine release paralleled those effects on [Ca2+]i signals. However, omega-conotoxin GVIA and 30 nM omega-agatoxin IVA did not affect the secretion of either amine. The data suggest that, in the bovine adrenal medulla, the release of epinephrine and norepinephrine are preferentially controlled by Q- and L-type Ca2+ channels, respectively. P- and N-type Ca2+ channels do not seem to control the secretion of either catecholamine.
- S C Kalhan
- D M Bier
- S M Savin
Kalhan, S. C., Bier, D. M., Savin, S. M., and Adam, P. A. (1980)
J. Clin. Endocrinol. Metab. 50, 456 – 460.
- J Katz
- A Dunn
- M Chenoweth
- S Golden
Katz, J., Dunn, A., Chenoweth, M., and Golden, S. (1974) Biochem. J. 142, 171–183.
- Des Rosiers
- Di Donato
- L Comte
- B Laplante
- A Marcoux
- C David
- F Fernandez
- C A Brunengraber
Des Rosiers, C., Di Donato, L., Comte, B., Laplante, A., Marcoux,
C., David, F., Fernandez, C. A., and Brunengraber, H. (1995)
J. Biol. Chem. 270, 10027–10036.
- Di Donato
- L Des Rosiers
- C Montgomery
- J A David
- F Garneau
- M Brunengraber
Di Donato, L., Des Rosiers, C., Montgomery, J. A., David, F.,
Garneau, M., and Brunengraber, H. (1993) J. Biol. Chem. 268,
4170 – 4180.
- A Martineau
- L Lecavalier
- P Falardeau
- J L And Chiasson
Martineau, A., Lecavalier, L., Falardeau, P., and Chiasson, J. L.
(1985) Anal. Biochem. 151, 495–503.
- J Katz
- P Wals
Katz, J., Wals, P., and Lee, W. N. (1993) J. Biol. Chem. 268,
25509 –25521.