Limitations of the Mass Isotopomer Distribution Analysis of Glucose to Study Gluconeogenesis (1998)

We previously reported (Previs, S. F., Fernandez, C. A., Yang, D., Soloviev, M. V., David, F., and Brunengraber, H. (1995) J. Biol. Chem. 270, 19806–19815) that glucose made in isolated livers from starved rats perfused with physiological concentrations of lactate, pyruvate, and either [2-13C]- or [U-13C3]glycerol had a mass isotopomer distribution incompatible with glucose being made from a homogeneously labeled pool of triose phosphates. Similar data were obtained in live rats infused with [U-13C3]glycerol. We ascribed the labeling heterogeneity to major decreases in glycerol concentration and enrichment across the liver. We concluded that [13C]glycerol is unsuitable for tracing the contribution of gluconeogenesis to total glucose production. We now report isotopic heterogeneity of gluconeogenesis in hepatocytes, even when all cells are in contact with identical concentrations and enrichments of gluconeogenic substrates. Total rat hepatocytes were incubated with concentrations of glycerol, lactate, and pyruvate that were kept constant by substrate infusions. To modulate competition between substrates, the (glycerol)/(lactate + pyruvate) infusion ratio ranged from 0.23 to 3.60. Metabolic and isotopic steady states were achieved in all cases. The apparent contribution of gluconeogenesis to glucose production (f) was calculated from the mass isotopomer distribution of glucose. When all substrates were13C-labeled, f was 97%, as expected in glycogen-deprived hepatocytes. As the infusion ratio ([13C]glycerol)/(lactate + pyruvate) increased,f increased from 73% to 94%. In contrast, as the infusion ratio (glycerol)/([13C]lactate + [13C]pyruvate) increased, f decreased from 93% to 76%. In all cases, f increased with the rate of supply of the substrate that was labeled. Variations in fshow that the 13C labeling of triose phosphates was not equal in all hepatocytes, even when exposed to the same substrate concentrations and enrichments. We also showed that zonation of glycerol kinase activity is minor in rat liver. We conclude that zonation of other processes than glycerol phosphorylation contributes to the heterogeneity of triose phosphate labeling from glycerol in rat liver. We previously reported (Previs, S. F., Fernandez, C. A., Yang, D., Soloviev, M. V., David, F., and Brunengraber, H. (1995) J. Biol. Chem. 270, 19806–19815) that glucose made in isolated livers from starved rats perfused with physiological concentrations of lactate, pyruvate, and either [2-13C]- or [U-13C3]glycerol had a mass isotopomer distribution incompatible with glucose being made from a homogeneously labeled pool of triose phosphates. Similar data were obtained in live rats infused with [U-13C3]glycerol. We ascribed the labeling heterogeneity to major decreases in glycerol concentration and enrichment across the liver. We concluded that [13C]glycerol is unsuitable for tracing the contribution of gluconeogenesis to total glucose production. We now report isotopic heterogeneity of gluconeogenesis in hepatocytes, even when all cells are in contact with identical concentrations and enrichments of gluconeogenic substrates. Total rat hepatocytes were incubated with concentrations of glycerol, lactate, and pyruvate that were kept constant by substrate infusions. To modulate competition between substrates, the (glycerol)/(lactate + pyruvate) infusion ratio ranged from 0.23 to 3.60. Metabolic and isotopic steady states were achieved in all cases. The apparent contribution of gluconeogenesis to glucose production (f) was calculated from the mass isotopomer distribution of glucose. When all substrates were13C-labeled, f was 97%, as expected in glycogen-deprived hepatocytes. As the infusion ratio ([13C]glycerol)/(lactate + pyruvate) increased,f increased from 73% to 94%. In contrast, as the infusion ratio (glycerol)/([13C]lactate + [13C]pyruvate) increased, f decreased from 93% to 76%. In all cases, f increased with the rate of supply of the substrate that was labeled. Variations in fshow that the 13C labeling of triose phosphates was not equal in all hepatocytes, even when exposed to the same substrate concentrations and enrichments. We also showed that zonation of glycerol kinase activity is minor in rat liver. We conclude that zonation of other processes than glycerol phosphorylation contributes to the heterogeneity of triose phosphate labeling from glycerol in rat liver. When a polymer is synthesized from a monomeric compound labeled with a stable isotope (13C, 2H,15N, or 18O), the mass isotopomer distribution (MID) 1The abbreviations are: MID, mass isotopomer distribution; GNG, gluconeogenesis; MIDA, mass isotopomer distribution analysis; PEP, phosphoenolpyruvate carboxykinase. 1The abbreviations are: MID, mass isotopomer distribution; GNG, gluconeogenesis; MIDA, mass isotopomer distribution analysis; PEP, phosphoenolpyruvate carboxykinase. of the polymer, measured by gas chromatography-mass spectrometry, is a unique function of the isotopic enrichment (p) of the precursor subunit (1Lee W.-N.P. Bergner F.A. Guo Z. Biol. Mass. Spectrom. 1992; 21: 114-122Crossref PubMed Scopus (77) Google Scholar, 2Hellerstein M.K. Neese R. Am. J. Physiol. 1992; 263: E988-E1001PubMed Google Scholar, 3Brunengraber H. Kelleher J.K. Des Rosiers C. Annu. Rev. Nutrition. 1997; 17: 559-596Crossref PubMed Scopus (94) Google Scholar, 4Kelleher J.K. Kharroubi A.T. Aldaghlas T.A. Shambat I.B. Kennedy K.A. Holleran A.L. Masterson T.M. Am. J. Physiol. 1994; 266: E384-E395Crossref PubMed Google Scholar). This was validated in chemical models, such as (i) glucose penta-[13C]acetate, made by reacting glucose with samples of [13C]acetic anhydride of varying enrichments (1Lee W.-N.P. Bergner F.A. Guo Z. Biol. Mass. Spectrom. 1992; 21: 114-122Crossref PubMed Scopus (77) Google Scholar), and (ii) hexamethylenetetramine made from either [13C]formaldehyde and NH4Cl, or formaldehyde and 15NH4Cl (3Brunengraber H. Kelleher J.K. Des Rosiers C. Annu. Rev. Nutrition. 1997; 17: 559-596Crossref PubMed Scopus (94) Google Scholar). The MID of the polymer can be predicted from probability analysis (1Lee W.-N.P. Bergner F.A. Guo Z. Biol. Mass. Spectrom. 1992; 21: 114-122Crossref PubMed Scopus (77) Google Scholar, 2Hellerstein M.K. Neese R. Am. J. Physiol. 1992; 263: E988-E1001PubMed Google Scholar, 3Brunengraber H. Kelleher J.K. Des Rosiers C. Annu. Rev. Nutrition. 1997; 17: 559-596Crossref PubMed Scopus (94) Google Scholar, 4Kelleher J.K. Kharroubi A.T. Aldaghlas T.A. Shambat I.B. Kennedy K.A. Holleran A.L. Masterson T.M. Am. J. Physiol. 1994; 266: E384-E395Crossref PubMed Google Scholar). Conversely, computation of the MID of the polymer yields the enrichment of the monomer.Mass isotopomer distribution analysis (MIDA) was proposed (2Hellerstein M.K. Neese R. Am. J. Physiol. 1992; 263: E988-E1001PubMed Google Scholar) as a method for estimating the fractional synthesis rate of various biopolymers (fatty acids, cholesterol, glucose) synthesized in vivo and in vitro in the presence of 13C tracers that label the appropriate monomer (acetyl-CoA or triose phosphates). Computation of the MID of the biopolymer yields both the fractional synthetic rate and the enrichment of the monomeric precursor. A variant of MIDA, isotopomer spectral analysis, was presented by Kelleher et al. (4Kelleher J.K. Kharroubi A.T. Aldaghlas T.A. Shambat I.B. Kennedy K.A. Holleran A.L. Masterson T.M. Am. J. Physiol. 1994; 266: E384-E395Crossref PubMed Google Scholar).The validity of MIDA and isotopomer spectral analysis requires that two parameters be constant in all cells making the polymer, namely the fractional synthetic rate and the enrichment of the monomeric precursor. When the enrichment of the precursor is not constant, computation of the MID of the polymer yields erroneous values for the enrichment of the monomeric precursor and for the fractional synthesis. In fact, the calculated fractional synthesis is not the integrated average of this parameter. This is because the precursor enrichment appears in the equations for the amount of each isotopomer 2In the context of this report, the word “isotopomer” refers to “mass isotopomer.” Positional isotopomers are not considered here. 2In the context of this report, the word “isotopomer” refers to “mass isotopomer.” Positional isotopomers are not considered here. as a nonlinear parameter. For a detailed discussion, see Ref. 3Brunengraber H. Kelleher J.K. Des Rosiers C. Annu. Rev. Nutrition. 1997; 17: 559-596Crossref PubMed Scopus (94) Google Scholar.Glucose can be considered as a dimer formed from the condensation of two triose phosphate molecules. Thus, MIDA of glucose labeled from13C-labeled precursors was proposed to estimate the contribution of gluconeogenesis (GNG) to total glucose production (f) (2Hellerstein M.K. Neese R. Am. J. Physiol. 1992; 263: E988-E1001PubMed Google Scholar, 5Neese R.A. Schwartz J.M. Faix D. Turner S.M. Letscher A. Vu D. Hellerstein M.K. J. Biol. Chem. 1995; 270: 14452-14463Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 6Péroni O. Large V. Odéon M. Beylot M. Am. J. Physiol. 1995; 269: E516-E523PubMed Google Scholar, 7Péroni O. Large V. Odéon M. Beylot M. Metabolism. 1996; 45: 897-901Abstract Full Text PDF PubMed Scopus (29) Google Scholar, 8Lee W.-N.P. Bassilian S. Bergner E.A. Wals P. Katz J. Clin. Res. 1994; 42: 28AGoogle Scholar). Calculations of f would not be subject to artifacts of isotopic exchange which affects calculations of GNG from the amount of 13C tracers incorporated into glucose (9Kelleher J.K. Am. J. Physiol. 1986; 250: E296-E305PubMed Google Scholar, 10Landau B.R. Adv. Exp. Med. Biol. 1993; 334: 209-220Crossref PubMed Scopus (22) Google Scholar). However, the main underlying assumption of MIDA is that the triose phosphate pool(s) of all gluconeogenic cells must be at about the same enrichment. To the extent that the triose phosphates are not equally labeled, fis underestimated.Investigations of GNG have been conducted in which MIDA was applied for determining f. Some investigators have concluded that MIDA does provide a reliable estimation of f during the infusion of [13C]glycerol (5Neese R.A. Schwartz J.M. Faix D. Turner S.M. Letscher A. Vu D. Hellerstein M.K. J. Biol. Chem. 1995; 270: 14452-14463Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 6Péroni O. Large V. Odéon M. Beylot M. Am. J. Physiol. 1995; 269: E516-E523PubMed Google Scholar, 7Péroni O. Large V. Odéon M. Beylot M. Metabolism. 1996; 45: 897-901Abstract Full Text PDF PubMed Scopus (29) Google Scholar) or [13C]lactate (5Neese R.A. Schwartz J.M. Faix D. Turner S.M. Letscher A. Vu D. Hellerstein M.K. J. Biol. Chem. 1995; 270: 14452-14463Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar,8Lee W.-N.P. Bassilian S. Bergner E.A. Wals P. Katz J. Clin. Res. 1994; 42: 28AGoogle Scholar, 11Previs S.F. Fernandez C.A. Yang D. Soloviev M.V. David F. Brunengraber H. J. Biol. Chem. 1995; 270: 19806-19815Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). However, we demonstrated previously that the use of [13C] glycerol and MIDA is limited for estimatingf (11Previs S.F. Fernandez C.A. Yang D. Soloviev M.V. David F. Brunengraber H. J. Biol. Chem. 1995; 270: 19806-19815Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 12Landau B.R. Fernandez C.A. Previs S.F. Ekberg K. Chandramouli V. Wahren J. Kalhan S.C. Brunengraber H. Am. J. Physiol. 1995; 269: E18-E26PubMed Google Scholar). We concluded that the major mechanisms responsible for our observations were (i) a gradient of glycerol concentration and (ii) a gradient of the 13C enrichment of glycerol across the liver (11Previs S.F. Fernandez C.A. Yang D. Soloviev M.V. David F. Brunengraber H. J. Biol. Chem. 1995; 270: 19806-19815Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 12Landau B.R. Fernandez C.A. Previs S.F. Ekberg K. Chandramouli V. Wahren J. Kalhan S.C. Brunengraber H. Am. J. Physiol. 1995; 269: E18-E26PubMed Google Scholar, 13Previs S.F. Martin S.K. Hazey J.W. Soloviev M.V. Keating A.P. Lucas D. David F. Kirschenbaum D. Tserng K.-Y. Brunengraber H. Am. J. Physiol. 1996; 271: E1118-E1124PubMed Google Scholar, 14Landau B.R. Wahren J. Previs S.F. Ekberg K. Chandramouli V. Brunengraber H. Am. J. Physiol. 1996; 271: E1110-E1117PubMed Google Scholar). Thus, most likely, triose phosphates were not equally labeled from [13C]glycerol in all hepatocytes. Therefore, the assumption of a triose phosphate pool of homogeneous enrichment is probably not valid for all conditions. Indeed, Péroni et al. (15Péroni O. Large V. Diraison F. Beylot M. Metabolism. 1997; 46: 1358-1363Abstract Full Text PDF PubMed Scopus (23) Google Scholar) infused [2-13C]glycerol to 2-day-starved diabetic rats with undetectable liver glycogen and calculated an impossibly lowf of 43%.In an attempt to further test our hypothesis, i.e. thatf was underestimated due to heterogeneous triose phosphate labeling induced by substrate gradients across the liver, hepatocytes from 48-h fasted rats were incubated in a medium that contained glycerol, lactate and pyruvate. Under these conditions, GNG should account for 100% of glucose production. The substrates were maintained at steady state concentrations by constant infusions following loading boluses. The substrate infusion ratio glycerol/(lactate + pyruvate) was varied to simulate gluconeogenic conditions favoring either glycerol or (lactate + pyruvate). Glycerol, lactate, and pyruvate were present in all experiments; however, the source of 13C was alternated in different experiments (either glycerol or (lactate + pyruvate)). Both singly 13C- and U-13C3-labeled substrates were tested. This design guaranteed that all cells had equal exposure to all substrates and 13C labeling. Our data show that calculations of f via MIDA are limited by variations in the relative contribution of glycerol versus lactate + pyruvate to triose phosphates in gluconeogenic hepatocytes. This finding is true whether one uses singly 13C- or uniformly13C3-labeled tracers. In addition, we found minimal zonation of hepatic glycerokinase activity in 48-h fasted rats.RESULTSSteady state concentrations of lactate, pyruvate, and glycerol were achieved under all conditions tested. As an example, Fig. 1 shows these concentrations under the conditions of row 3 of Table I. Glucose production was linear (r = 0.99; Fig. 1 and last column of Table I). In control flasks incubated without any substrates, concentrations of glycerol, lactate, pyruvate, and glucose were very low (bottom row of Table I). Thus, our incubation conditions allow to keep hepatocytes under physiological concentrations of substrates without having to resort to perifusion techniques (25McMenamy R.H. Kleineke J. Roil W. Soling H.D. Anal. Biochem. 1981; 112: 117-127Crossref PubMed Scopus (12) Google Scholar).Table I (right column) shows that the production of glucose, expressed as a percent of the maximum possible production from the infused substrates, increased as the substrate infusion ratio glycerol/(lactate + pyruvate) increased. At a low infusion ratio, when lactate/pyruvate is the dominant gluconeogenic precursor, a fraction of the substrate must be oxidized in the citric acid cycle to provide energy for glucose production (6 ATP per glucose formed from lactate). Similar partial oxidation of lactate has been reported in kidney slices synthesizing glucose (26Weidemann M.J. Krebs H.A. Biochem. J. 1969; 112: 149-166Crossref PubMed Scopus (169) Google Scholar). In contrast, when glycerol is the dominant gluconeogenic substrate, the yield of glucose production is much higher because the conversion of glycerol to glucose generates ATP (4 or 2 ATP per glucose formed from glycerol, depending on whether the conversion of α-glycerophosphate to dihydroxyacetone phosphate involves reduction of NAD+ or FAD).Isotopic steady state of glucose labeling was reached within the first 5 min of the incubation. As an example, Fig. 2 shows parameter f, which is the apparent contribution of GNG to glucose production under the conditions of row 3 of Table I. Under these conditions, when all the substrates were U-13C3-labeled, fwas 97%, as expected in glycogen-depleted hepatocytes. However, when glycerol was U-13C3-labeled and lactate and pyruvate were unlabeled, f was 93%. Additionally, when lactate and pyruvate were U-13C3-labeled and glycerol was unlabeled, f was 78%. In the case when lactate and pyruvate were U-13C3-labeled, the first point, obtained at 5 min during the incubation, is slightly lower than the average of the other points (i.e. 71% versus78%). This does not significantly impact on the calculation off from glucose obtained at 45 min during the incubation. This is because (i) the f calculated at 5 min (i.e. 71%) is within 91% of the final f(i.e. 78%) and (ii) the amount of glucose produced after only 5 min represents less than 10% of the total glucose produced during the incubation. Similar isotopic steady states were maintained using the other substrate infusion ratios (data not shown).Figure 2Isotopic steady state achieved during the hepatocyte incubations.View Large Image Figure ViewerDownload (PPT)The enrichment of glycerol, lactate and pyruvate was measured in samples obtained at the end of the incubation. When glycerol and [3-13C]lactate + [3-13C]pyruvate were infused, the glycerol pool actually became 13C-labeled during the incubation. As the substrate infusion ratio (glycerol)/([3-13C]lactate + [3-13C]pyruvate) increased from 0.23 to 0.61, 1.4, and 3.6, the enrichment of glycerol at 40 min was 12 ± 3%, 5 ± 2%, 3 ± 1%, and 1 ± 1%, respectively. When [2-13C]glycerol and (lactate + pyruvate) were infused, labeling of the (lactate + pyruvate) pool (4 ± 2%) was observed only at the highest substrate infusion ratio ([2-13C]glycerol)/(lactate + pyruvate) of 3.60.Fig. 3 shows f calculated in all protocols that used U-13C3-labeled substrates. When all substrates were uniformly 13C-labeled,f was 97%, as expected in glycogen-deprived hepatocytes. As the infusion ratio ([U-13C3]glycerol)/(lactate + pyruvate) increased, f increased from 73% to 94%. In contrast, as the infusion ratio (glycerol)/([U-13C3]lactate + [U-13C3]pyruvate) increased, fdecreased from 93% to 76%. In all cases, f increased with the rate of supply of the substrate that was labeled.Figure 3Apparent contribution of GNG (f) in experiments with U-13C3-labeled substrates. f was calculated from the mass isotopomer distribution of glucose isolated at the end of experiments conducted with the substrate combinations and infusion ratios indicated. Data are shown as mean ± S.E. (n = 4–7).View Large Image Figure ViewerDownload (PPT)We compared the f values calculated from U-13C3-labeled substrates with those calculated from singly 13C-labeled substrates. Fig. 4 shows that practically identicalf were calculated from either U-13C3- or singly 13C-labeled substrates.Figure 4Comparison of fcalculated from U-13C3-labeled substrates or singly13C-labeled substrates. The solid circlesrepresent experiments in which [13C]glycerol plus unlabeled lactate and pyruvate were infused (Y = 0.95X + 5.5; r2 = 0.99). Theopen circles represent experiments in which unlabeled glycerol plus [13C]lactate and [13C]pyruvate were infused (Y = 0.95X + 3.2; r2 = 0.99).View Large Image Figure ViewerDownload (PPT)Fig. 5 shows the degree of triose phosphate isotopic equilibration in hepatocytes, as reflected by the (C1-C3)/(C4-C6) labeling ratio of glucose formed in experiments with singly 13C-labeled substrates (see “Experimental Procedures”). As the infusion ratio ([2-13C]glycerol)/(lactate + pyruvate) increased, triose phosphate equilibration increased from 89 to 95%. In contrast, as the infusion ratio (glycerol)/([13C]lactate + [13C]pyruvate) increased, triose phosphate equilibration decreased from 89 to 77%. In all cases, triose phosphate equilibration increased with the rate of supply of the substrate that was labeled.Figure 5Triose phosphate equilibration calculated from either [2-13C]glycerol and lactate + pyruvate or glycerol and [3-13C]lactate + [3-13C]pyruvate at the different substrate infusion ratios. The percent of triose phosphate equilibration is 100·(13CC4-C6/13CC1-C3) when glucose is labeled from [2-13C] glycerol (solid circles), and 100·(13CC1-C3/13CC4-C6) when glucose is labeled from [3-13C]lactate + [3-13C]pyruvate (open circles).View Large Image Figure ViewerDownload (PPT)To test the linearity of our measurements of isotopomer distributions, for each labeling pattern, we pooled final samples of incubation medium from experiments in which the (glycerol)/(lactate + pyruvate) infusion ratio was 1.40. After measurement of the MID of each pool, seven increasing dilutions of the [13C]glucose (up to 20-fold) were made by adding increasing amounts of a solution of unlabeled glucose. Then, after purification on resins, the MID of the diluted glucose samples were measured, converted to f values that were compared with f values calculated using the unlabeled glucose dilution factor. The regression slopes of the seven points of each comparison ranged from 0.965 to 0.99, withr2 > 0.99. Thus, we are confident of the precision and linearity of our measurements of the MID of glucose, whether labeled from singly or uniformly 13C-labeled substrates.To test whether low values of f obtained in previousin vivo and perfused rat liver experiments with [13C]glycerol (11Previs S.F. Fernandez C.A. Yang D. Soloviev M.V. David F. Brunengraber H. J. Biol. Chem. 1995; 270: 19806-19815Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 12Landau B.R. Fernandez C.A. Previs S.F. Ekberg K. Chandramouli V. Wahren J. Kalhan S.C. Brunengraber H. Am. J. Physiol. 1995; 269: E18-E26PubMed Google Scholar) resulted from zonation of glycerol kinase, we isolated hepatocytes from the periportal and pericentral areas of the liver. Table II shows, for livers of 2-day-starved rats, the total activity and zonal distribution of glutamine synthetase, lactate dehydrogenase, and glycerol kinase. Data for zonation of glutamine synthetase and lactate dehydrogenase, as well as total activity of glycerol kinase, are in agreement with published reports (27Jungermann K. Kietzmann T. Annu. Rev. Nutr. 1996; 16: 179-203Crossref PubMed Scopus (410) Google Scholar, 28Sestoft L. Fleron P. Biochim. Biophys. Acta. 1975; 375: 462-471Crossref PubMed Scopus (27) Google Scholar). We found no sizeable zonation of glycerol kinase activity in hepatocytes (Table II).Table IIDistribution of enzymatic activities in periportal vs perivenous hepatocytesEnzymeActivityPeriportal/perivenous activity ratioLiver homogenateTotal hepatocytesμmol · min−1g−1Glutamine synthetase74 ± 872 ± 74.08 ± 0.46Lactate dehydrogenase55 ± 1146 ± 70.71 ± 0.09Glycerol kinase2.8 ± 0.22.3 ± 0.30.89 ± 0.02Activities were measured in liver homogenates and in hepatocytes (total, periportal, and perivenous). Data are presented as mean ± S.E. (n = 4). Open table in a new tab DISCUSSIONMIDA was proposed by Neese et al. (5Neese R.A. Schwartz J.M. Faix D. Turner S.M. Letscher A. Vu D. Hellerstein M.K. J. Biol. Chem. 1995; 270: 14452-14463Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar) as an effective and reliable method for estimating f, the contribution of GNG to total glucose production. Glucose can be considered as a dimer of triose phosphates, the 13C enrichment of which should be very similar under most conditions. Neese et al. showed, with mathematical simulations, that a lack of perfect equilibration between the enrichments of the triose phosphates has minor impact on calculations of f, unless the ratio of enrichments of glyceraldehyde 3-phosphate and dihydroxyacetone phosphate is greater than 1.6 or smaller than 0.65. Thus, MIDA should be applicable to studies of GNG provided that (i) f has the same value in all gluconeogenic cells, and (ii) that the enrichment of triose phosphates is the same in all these cells.These two conditions may not always be fulfilled because of the metabolic zonation of the liver lobule. When we infused [U-13C3]glycerol or [U-13C3]lactate to 2-day-starved rats (11Previs S.F. Fernandez C.A. Yang D. Soloviev M.V. David F. Brunengraber H. J. Biol. Chem. 1995; 270: 19806-19815Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), the MID of plasma glucose yielded f values of and 97%, respectively. The value of f obtained with [U-13C3]lactate was expected as the liver of these rats was and GNG was the only source of glucose. However, the low values of f measured with [U-13C3]glycerol were Similar low values of f had been measured in infused with [U-13C3]glycerol B.R. Fernandez C.A. Previs S.F. Ekberg K. Chandramouli V. Wahren J. Kalhan S.C. Brunengraber H. Am. J. Physiol. 1995; 269: E18-E26PubMed Google Scholar). Additionally, in in one contribution of to glucose [U-13C3]glycerol and [U-13C3]lactate yielded f values of and (11Previs S.F. Fernandez C.A. Yang D. Soloviev M.V. David F. Brunengraber H. J. Biol. Chem. 1995; 270: 19806-19815Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The low f calculated with [U-13C3]glycerol with this the enrichment of triose phosphates across the liver was not This from zonation of glycerol kinase or from a in glycerol concentration across the liver lobule. We found no data on the zonation of glycerol kinase in the We showed that the concentration of glycerol decreases to across the liver of S.F. Martin S.K. Hazey J.W. Soloviev M.V. Keating A.P. Lucas D. David F. Kirschenbaum D. Tserng K.-Y. Brunengraber H. Am. J. Physiol. 1996; 271: E1118-E1124PubMed Google Scholar), and to across the of B.R. Wahren J. Previs S.F. Ekberg K. Chandramouli V. Brunengraber H. Am. J. Physiol. 1996; 271: E1110-E1117PubMed Google Scholar). In addition, we showed (i) an to in glycerol enrichment across the liver of S.F. Martin S.K. Hazey J.W. Soloviev M.V. Keating A.P. Lucas D. David F. Kirschenbaum D. Tserng K.-Y. Brunengraber H. Am. J. Physiol. 1996; 271: E1118-E1124PubMed Google Scholar), and (ii) an to in glycerol enrichment across the of B.R. Wahren J. Previs S.F. Ekberg K. Chandramouli V. Brunengraber H. Am. J. Physiol. 1996; 271: E1110-E1117PubMed Google Scholar), the production of glycerol by the liver. The of physiological concentrations of glycerol across liver and the production of glycerol by the liver were in perfused rat liver experiments (11Previs S.F. Fernandez C.A. Yang D. Soloviev M.V. David F. Brunengraber H. J. Biol. Chem. 1995; 270: 19806-19815Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). on the we concluded that [U-13C3]glycerol is not for tracing GNG in that [U-13C3]lactate be at under conditions. found (11Previs S.F. Fernandez C.A. Yang D. Soloviev M.V. David F. Brunengraber H. J. Biol. Chem. 1995; 270: 19806-19815Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) similar values of f in infused with [U-13C3]lactate or [3-13C]lactate In to our Neese et al. (5Neese R.A. Schwartz J.M. Faix D. Turner S.M. Letscher A. Vu D. Hellerstein M.K. J. Biol. Chem. 1995; 270: 14452-14463Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar) and Péroni et al. O. Large V. Odéon M. Beylot M. Am. J. Physiol. 1995; 269: E516-E523PubMed Google Scholar, 7Péroni O. Large V. Odéon M. Beylot M. Metabolism. 1996; 45: 897-901Abstract Full Text PDF PubMed Scopus (29) Google Scholar) reported values of and in 2-day-starved rats infused with concluded that this [2-13C] glycerol is for tracing GNG in The between our (11Previs S.F. Fernandez C.A. Yang D. Soloviev M.V. David F. Brunengraber H. J. Biol. Chem. 1995; 270: 19806-19815Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) and those of al. (5Neese R.A. Schwartz J.M. Faix D. Turner S.M. Letscher A. Vu D. Hellerstein M.K. J. Biol. Chem. 1995; 270: 14452-14463Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar) and Péroni et al. O. Large V. Odéon M. Beylot M. Am. J. Physiol. 1995; 269: E516-E523PubMed Google Scholar, 7Péroni O. Large V. Odéon M. Beylot M. Metabolism. 1996; 45: 897-901Abstract Full Text PDF PubMed Scopus (29) Google Scholar) may from the that infused amounts of [2-13C]glycerol in plasma glycerol enrichment of about which decreased the concentration and enrichment gradients of glycerol across the liver. Indeed, we found (11Previs S.F. Fernandez C.A. Yang D. Soloviev M.V. David F. Brunengraber H. J. Biol. Chem. 1995; 270: 19806-19815Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) that f increased from to when the concentration of glycerol in liver was increased from to Péroni et al. (15Péroni O. Large V.