©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Gluconeogenesis and Intrahepatic Triose Phosphate Flux in Response to Fasting or Substrate Loads
APPLICATION OF THE MASS ISOTOPOMER DISTRIBUTION ANALYSIS TECHNIQUE WITH TESTING OF ASSUMPTIONS AND POTENTIAL PROBLEMS (*)

Richard A. Neese (1) (2), Jean-Marc Schwarz (1)(§), Dennis Faix (1), Scott Turner (1), Amy Letscher (1), Danae Vu (1), Marc K. Hellerstein (1) (2)(¶)

From the (1)Department of Nutritional Sciences, University of California, Berkeley, California 94720 and the (2)Division of Endocrinology and Metabolism, Department of Medicine, San Francisco General Hospital, University of California, San Francisco, California 94101

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We measured gluconeogenesis (GNG) in rats by mass isotopomer distribution analysis, which allows enrichment of the true biosynthetic precursor pool (hepatic cytosolic triose phosphates) to be determined. Fractional GNG from infused [3-C]lactate, [1-C]lactate, and [2-C]glycerol was 88 ± 2, 89 ± 3, and 87 ± 2%, respectively, after 48 h of fasting. [2-C]Glycerol was the most efficient label and allowed measurement of rate of appearance of intrahepatic triose phosphate (Ra triose-P), by dilution. IV fructose (10-15 mg/kg/min) increased absolute GNG by 81-147%. Ra triose-P increased proportionately, but endogenous Ra triose-P was almost completely suppressed, suggesting feedback control. Interestingly, 15-17% of fructose was directly converted to glucose without entering hepatic triose-P. IV glucose reduced GNG and Ra triose-P. 24-h fasting reduced hepatic glucose production by half, but absolute GNG was unchanged due to increased fractional GNG (51-87%). Reduced hepatic glucose production was entirely due to decreased glycogen input, from 7.3 ± 1.8 to 1.1 ± 0.2 mg/kg/min. Ra triose-P fell during fasting, but efficiency of triose-P disposal into GNG increased, maintaining GNG constant. Secreted glucuronyl conjugates and plasma glucose results correlated closely. In summary, GNG and intrahepatic triose-P flux can be measured by mass isotopomer distribution analysis with [2-C]glycerol.


INTRODUCTION

As pointed out by Krebs et al.(1) , measurement of gluconeogenesis (GNG)()with isotopes is complicated by the fact that the metabolic pathway from pyruvate proceeds through mitochondrial oxalacetate (Fig. 1), which is exposed to numerous metabolic sources of carbon dilution during an isotope labeling experiment(2, 3, 4, 5, 6) . This is a problem because in order to interpret incorporation from labeled GNG substrates such as lactate or alanine into glucose or glycogen, it is necessary to establish the isotope content (specific activity or enrichment) of the true biosynthetic precursor. For GNG, the true all-source precursor pool is most accurately considered to be the triose phosphate (triose-P) pool, consisting of glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP) (Fig. 1). Several solutions to the problem of dilution in GNG intermediates have been proposed, including the [C]acetate method developed by Weinman et al.(2) , which has since been modified by Hetenyi(3) , Katz(4) , and Kelleher(5) . This approach has recently been applied in humans (6). The technique remains open to severe criticisms(7, 8, 9) , however. A stable isotope method for estimating the dilution of recycled [C]glucose in GNG intermediates after passage through the indirect pathway of glycogen synthesis has been proposed by Katz and Lee(10) . This approach is attractive but is currently limited to recycled glucose and cannot address incorporation from ``new'' (nonrecycled) gluconeogenic precursors. Thus, despite its importance in intermediary metabolism, there currently exists no satisfactory technique for measuring GNG in vivo.


Figure 1: Pathways of GNG and hepatic triose-P metabolism in liver. Abbreviations: AcCoA, acetyl-CoA; OAA, oxalacetate; TCA, tricarboxylic acid cycle; Pyr, pyruvate; PEP, phosphoenolpyruvate; -Glyc-P, -glycerol phosphate; G-3P, glyceraldehyde 3-phosphate; Fru-1,6-P, fructose 1,6-bisphosphate; Glc-6P, glucose 6-phosphate; Glc-1-P, glucose 1-phosphate; GlcUA, glucuronate. At metabolic steady state, the sum of metabolic inputs into triose-P (solidlines) equals the sum of outflows (dashedlines) equals Ra triose-P.



We (11, 12, 13, 14, 15) recently described a general technique for measuring the synthesis of biological polymers in vivo, which we have called mass isotopomer distribution analysis (MIDA). This technique uses probability logic with mass spectrometric analysis to calculate the isotopic enrichment of the true precursor subunits from which a polymer was synthesized. In principle, this approach can be used for any polymer composed of repeating monomeric subunits(11, 12) . Once the true precursor enrichment is determined, calculation of the fractional synthesis of a polymer is straightforward(11, 12, 13, 14) . In principle, the MIDA technique can be used for measurement of GNG and the isotopic enrichment of the hepatic triose-P pool, if glucose is considered to be a polymer composed of two triose subunits. Any carbon-labeled substrate that enters the triose-P precursor pool can then be used to measure GNG.

If flux through GNG could be measured and if access to hepatic triose-P labeling were achieved, a number of questions might be answered concerning the integration of biochemical fluxes within the mammalian liver in response to changes in substrate availability. An area of great interest in this regard is the phenomenon of autoregulation, or intrahepatic metabolic adjustments to substrate loads, proposed by Soskin and Levine (15) many years ago. Several studies in humans and experimental animals have affirmed the importance of hepatic autoregulation to gluconeogenic substrate loads. Infusion of glycerol at 1.5 mg/kg/min to fasted humans does not increase hepatic glucose production (HGP) from basal rates of approximately 2 mg/kg/min(16) . Infusions of lactate or glycerol also do not alter HGP, even when glucose, insulin, growth hormone, glucagon and cortisol concentrations are held constant(17) . Similar results have been reported in other species(18, 19, 20) . The mechanisms allowing the normal liver to absorb large substrate loads for GNG without altering HGP are not understood, however, nor is the response of the liver to progressive glycogen depletion (the fed-to-fasted transition) well understood. In particular, the mechanism by which GNG from amino acids is reduced, thereby minimizing nitrogen losses, is not known. Is this due to reduced entry of precursors for GNG into the triose-P precursor pool, more efficient channeling of triose-P into GNG, or both?

In this study, we use the MIDA technique to measure GNG. We also present an isotope dilution method for measuring the flux through the hepatic triose-P pool or the rate of appearance of triose-P (Ra triose-p) and from this the fate of triose-P flux under varied conditions. The assumptions of the GNG and Ra triose-P techniques are tested, and the hepatic metabolic response to substrate loads (fructose or glucose) and progressive fasting are examined.


MATERIALS AND METHODS

Male Sprague-Dawley rats weighing 250-350 g were used. Animals were housed in individual cages and maintained on a 12-h light/12-h. dark cycle. Rats were fed adlibitum (Purina® rat chow). Some animals underwent progressive fasts (removal of food at 6:00 a.m.), while other animals were infused with intravenous substrates (fructose at 10-15 mg/kg/min or glucose at 15-30 mg/kg/min) after a variable period of fasting (Fig. 2). [3-C]Lactic acid and [1-C]lactic acid were purchased from Isotec, Inc. (Miamisburg, OH). [2-C]Glycerol was purchased from Tracer Technologies (Somerville, MA), Cambridge Isotope Laboratories (Woburn, MA) and Isotec, Inc. All labeled substrates were >98% enriched. The [C]lactates and [2-C]glycerol were infused at 0.011 mmol/kg/min (1 mg/kg/min). During infusions of fructose, the rate of [2-C]glycerol infusion was increased to 0.022 mmol/kg/min (2 mg/kg/min). Some animals received concurrent intravenous infusions of [6-H]glucose (DuPont NEN) at 10 µCi/h and/or acetaminophen (40 mg/kg/h) as described previously(23) . Authentic - and -anomers of D-glucose pentaacetate for use as standards were purchased commercially (from Aldrich). Acetaminophen for parenteral administration was prepared by Dr. L. Tomimatsu of the University of California San Francisco School of Pharmacy.


Figure 2: Metabolic infusion protocol in catheterized rats.



Catheterization of rats for constant infusions of labeled precursors was as described previously (23, 24) with the addition of an indwelling carotid artery catheter for blood sampling during infusions. All procedures were carried out with the approval of the Office of Laboratory Animal Care of the University of California at Berkeley. Water was available adlibitum during periods of fasting. The carotid artery catheter for blood sampling was placed contralateral to the jugular venous infusion catheter. Sequential samples of 0.3-0.5 ml were drawn prior to sacrifice, acid precipitated, and centrifuged for measurement of blood metabolite enrichments.

Isolation and Preparation of Metabolites for Mass Spectrometric Analyses

Glucose and glycerol were isolated from deproteinized plasma by ion-exchange chromatography (23) and then derivatized with acetic anhydride in pyridine(25) . Liver glycogen was precipitated from liver by the method of Good et al.(26) and then was hydrolyzed in 6 N HCl (110 °C, 2 h) prior to neutralization and derivatization. Plasma very low density lipoprotein was isolated by ultracentrifugation, and the glycerol remaining after transmethylation of fatty acids in methanolic HCl (13, 27) was derivatized as described above. Acetaminophen-glucuronate (GlcA) was isolated from urine by high performance liquid chromatography(23) . The GlcA moiety was transmethylated in methanolic HCl (28) prior to acetylation with acetic anhydride in pyridine.

Gas Chromatography/Mass Spectrometry (GC/MS)

GC/MS analyses were with an HP model 5971 instrument (Hewlett Packard, Palo Alto, CA). For lactate, selected ion monitoring (SIM) and electron impact ionization were used. Lactate was analyzed as the heptafluorobutyrl n-propylamide derivative (m/z 327 and 328) as described elsewhere(29) . For glucose, chemical ionization with methane gas and GC/MS was used. A 60-m DB17 column (0.25 mm inner diameter; 0.25 µm film thickness; J and W Scientific, Folsom, CA) was used in a temperature-programmed mode starting at 150 °C, rising at 40 °C/min to 270 °C. Glucose was derivatized with acetic anhydride in pyridine, and the tetraacetate fragment (m/z 331-333) was analyzed using SIM. For analysis of undifferentiated three-carbon fragments of glucose, the isolated glucose was reduced to sorbitol using sodium borohydride in anhydrous dimethyl sulfoxide (30) and then peracetylated with acetic anhydride/pyridine. Carbons 1-3 are indistinguishable from carbons 4-6 in the triose fragment in the mass spectrum(31) . The fragment at m/z 217-219, representing glucose carbons 1-3 and 4-6, was analyzed by EI with SIM. For comparison of the carbon 1-3 to the carbon 4-6 moieties of glucose, in order to assess triose-P isotopic equilibrium, the aldonitrile, pentaacetate derivative was synthesized as described by Guo et al.(32) . Fragments at m/z 187-189 (representing carbons 3-6) and m/z 243-245 (carbons 1-4) were analyzed by SIM. The difference between these fragments represents carbons 5-6 versus carbons 1-2, where most or all C from [2-C]glycerol is likely to be. Glycerol from plasma was isolated by ion-exchange chromatography, as described above for glucose, peracetylated with acetic anhydride/pyridine, and analyzed by SIM as m/z 159 and 160, using chemical ionization with methane gas. Blood glycerol and lactate enrichments were calculated by comparison to standard curves, prepared by mixing labeled and natural abundance materials. Measurement of blood glucose-specific activities during [H]glucose infusions was by liquid scintillation counting and enzymatic glucose measurements on the eluate collected from the ion-exchange column(23) . Acetaminophen-GlcA was isolated from urine as described previously (23) and then transmethylated in methanolic/HCl and peracetylated in acetic anhydride/pyridine. GC/MS of the acetylated methyl ester of GlcA was under the same conditions as for glucose pentaacetate with SIM of m/z 317, which contains all six carbons of GlcA. All analyses for MIDA calculations were performed in quadruplicate, with frequent interjection of base-line measurements (e.g. every fourth sample) throughout the GC/MS run. When base-line mass isotopomer fractional abundances were not within ± 0.0050 molar excess of theoretical values expected based on the elemental composition of the ion analyzed (see below and ``Appendix''), a run was rejected. In the case of the m/z 331-333 ions for glucose tetraacetate, for example (elemental composition CHO), calculated isotopomeric fractional abundances are M (m/z 331) = 0.8396, M (m/z 332) = 0.1348, and M ( m/z 333) = 0.0256. For the m/z 317-319 ions for methyl, triacetyl-GlcA, (elemental composition CHO), calculated fractional isotopomeric abundances are M (m/z 317) = 0.8489, M (m/z 318) = 0.1267, and M (m/z 319) = 0.0244. If persistent divergence of base-line fractional abundances from expected values was observed, the instrument was retuned, or the source was cleaned. Also, samples were preinjected, and volumes were then adjusted to achieve similar absolute areas for the most abundant ion in all samples, in order to minimize the possibility of detector nonlinearity or compromised mass resolution due to spill-over between adjacent masses.

Application of MIDA for Measurement of GNG

The principle of MIDA and its application for measuring biosynthesis of a variety of polymers have been discussed in detail elsewhere(11, 12, 13, 14, 33) . In brief, the technique consists of quantifying mass isotopomer fractional abundances in an intact polymer or fragment that contains two or more repeats of a precursor monomeric subunit. A stable isotope-labeled substrate that is capable of perturbing the isotope abundances of the monomeric precursor pool is administered, and the mass isotopomeric fractional abundances in the polymer after labeling are compared with the natural fractional abundance (base line) values. The pattern or distribution of mass isotopomeric excesses ( fractional abundances) in the polymer reveals the proportion (p) of the biosynthetic precursor units entering the polymer that were isotopically labeled,()using formulae derived from the binomial or multinomial expansions (Refs. 11 and 12 and see ``Appendix,'' ). Once p is established, the precursor-product relationship can be applied for kinetic analysis or for calculating the fraction (f) of polymers that were derived from the endogenous biosynthetic pathway during the labeling period. In the case of GNG (for glucose or GlcA), the ratio of excess M (EM) to excess M (EM) fractional abundances()reveals the isotopic abundance of the true precursor pool (hepatic triose-P, Fig. 1), from which f can be calculated. For GNG, however, there are several potential problems that might influence the use of MIDA: 1) the possibility that CO reincorporation into oxalacetate (or C entry through other metabolic pathways) could result in double-labeled triose-P, which could confound interpretation of isotopomer ratios in glucose; 2) the possibility of isotopic disequilibrium between the two triose-P, glyceraldehyde 3-phosphate and DHAP (Fig. 1), in which case the assumption of a single precursor enrichment would not be correct; 3) the short chain length of glucose as a polymer, containing only n = 2 subunits, which reduces the likelihood of double-labeled glucose molecules being formed during an isotope-incorporation experiment and thereby could represent a practical limitation to the method, if analytic precision is insufficient. These potential problems are discussed below.

Calculations

Metabolite Fluxes, GNG, and HGP

Calculations of precursor enrichment (p) and fractional GNG are described in the ``Appendix.''

Ra glucose was calculated from the dilution of blood [H]glucose during constant infusions of [3-H]glucose and Ra glycerol from the dilution of blood glycerol during constant infusions of [2-C]glycerol.

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

where I is the isotopic tracer infusion rate. Contribution of carbon from precursors (glycerol, lactate) to triose-P was calculated by comparison of their respective isotopic enrichments, each expressed in comparable terms (i.e. enrichment of plasma precursors was corrected by a standard curve to represent the proportion of labeled to total molecules present). Fractional GNG (the fraction of blood glucose that came from the GNG pathway) was calculated by MIDA. The ratio of double-labeled to single-labeled glucose molecules, after subtraction of the frequency of each species due to natural abundance molecules, reveals p and the asymptotic isotope enrichment of new glucose molecules (A) synthesized at this value of p(11, 12) . The actual enrichment of labeled glucose molecules (EM) is then compared with the asymptotic value for new glucose molecules (A) to calculate fractional GNG (f).

On-line formulae not verified for accuracy

Absolute GNG (the chemical flux through the pathway from triose-P to plasma glucose) was calculated from f multiplied times the turnover rate of plasma glucose.

On-line formulae not verified for accuracy

The portion of HGP not due to GNG, which reflects the contribution from hepatic glycogenolysis under postabsorptive conditions, was calculated from the difference between Ra glucose and absolute GNG.

On-line formulae not verified for accuracy

During infusions of intravenous glucose, the measured rate of exogenous glucose infusion was subtracted from Ra glucose to calculate HGP and non-GNG HGP. Fractional UDPGNG was calculated by isolation of secreted urinary acetaminophen-GlcA (19, 25, 26) with application of the MIDA algorithm to fragments of the GlcA derivative analyzed by GC/MS.

Ra Triose-P

The turnover of this intracellular metabolite pool was determined by use of the isotope dilution principle. Isotope dilution techniques are based on three central requirements or assumptions(34) : first, that a labeled metabolite can be injected into a metabolic pool at a known rate; second, that the isotopic content (enrichment or specific activity) of the metabolite in the pool can be measured; and third, that an isotopic steady state is attained and can be documented in the metabolite pool. The first requirement can be fulfilled for the hepatic triose-P pool by administration of [C]glycerol at a constant rate intravenously, if all infused [C]glycerol is taken up by the liver (35, 36) and metabolized via hepatic triose-P (Fig. 1). Ra triose-P will be over-estimated in proportion to extrahepatic utilization of labeled glycerol. This assumption can be tested experimentally (see below). The second requirement can be fulfilled by application of MIDA on a polymeric end product derived from the hepatic triose-P pool, such as secreted glucose or GlcA-conjugates(11, 34, 37) to infer p of the precursor pool. The third requirement is readily tested by performing serial measurements on the end product. If these requirements are met, the dilution principle can be applied to the hepatic triose-P pool.

On-line formulae not verified for accuracy

Endogenous Ra triose-P was calculated as the difference between Ra triose-P and the rate of direct entry of exogenous fructose and/or labeled glycerol into the hepatic triose-P pool (Fig. 1).

On-line formulae not verified for accuracy

Correction was necessary if any direct conversion of fructose to glucose without passage through the triose-P pool was documented (see below). It should be noted that recycling of label after leaving the triose-P pool (e.g. by entry into plasma lactate and return of the [C]lactate to the hepatic triose-P pool), will result in proportional underestimation of Ra triose-P (see below).

Isotopic Contribution of Plasma Glycerol to Hepatic Triose-P

The proportion of the hepatic triose-P pool derived from plasma glycerol was estimated based on the precursor-product relationship

On-line formulae not verified for accuracy

where plasma glycerol enrichment was expressed in the same manner as p, as the proportion of labeled molecules, by use of standard curves (see above). [C]Glycerol Recovery in Plasma Glucose or Other End Products-The rate of labeled glycerol entry into plasma glucose was calculated by using a stoichiometric or isotope balance approach. Absolute GNG was multiplied times the proportion of the triose-P pool that was labeled (enriched), to determine the flux of C atoms into glucose.

On-line formulae not verified for accuracy

A similar calculation was used for C recovery in liver glycogen, secreted GlcA-conjugates, and plasma triglyceride-glycerol. For glycogen, the final concentration in liver was multiplied by f and then p; for GlcA, the urinary excretion rate was multiplied by f and then p; for triglyceride-glycerol, the estimated(38, 39) triglyceride production rate was multiplied by very low density lipoprotein-glycerol enrichment.

Non-GNG Triose-P Disposal

The disposal of triose-P flux into non-glucose pathways was also calculated.

On-line formulae not verified for accuracy


RESULTS

Gas Chromatographic Separation of Glucose Anomers

Two peaks with identical mass spectra were observed when derivatized glucose standards were analyzed by GC/MS (not shown). Injection of authentic - and -anomers of glucose-pentaacetate confirmed the identity of these two peaks. D-Galactose, D-mannose, D-fructose, and other monosaccharides are separated from the glucose peaks under these GC conditions. The existence of dual peaks is useful because they can be used as internal replicates for quantification of mass isotopomeric abundances (see below).

Comparison of GNG from [3-C]Lactate, [1-C]Lactate, and [2-C]Glycerol during Fasting

Rats were given intravenous infusions of sodium [3-C]lactate or sodium [1-C]lactate (60-70 mg/kg/h of the lactate moiety, or 0.67-0.78 mmol/kg/h) or [2-C]glycerol (60-70 mg/kg/h) during fasts of 36-48-h duration. The value for p generally reached a fairly stable value within 2-3 h of starting a constant infusion of [3-C]lactate or [2-C]glycerol (Fig. 3). Blood lactate enrichments of 0.155 ± 0.019 were associated with p = 0.085 ± 0.014, for a dilution from blood lactate to the true precursor of 46 ± 6% (). The range of dilutions was from 31 to 72%. Measurement of f was performed from [3-C]lactate (). After 36-48 h of food deprivation, f was 87 ± 2% (n = 22 animals). With [1-C]lactate, f was 89 ± 3% (n = 4) (not shown).


Figure 3: Values for EM, EM, p and f over time during infusions of [3-C]lactate (60 mg/kg/h) or [2-C]glycerol (60 mg/kg/h) in 24-h fasted rats. EM, EM and p are higher with labeled glycerol than with labeled lactate, but calculated values for f are not different from the two labeled substrates.



With [2-C]glycerol infused at the same rate, values for p were higher (0.12-0.18 ME), but the values for f were identical at 88 ± 2% ( and Fig. 3). Mass spectrometric data and their conversion into p and f values in representative individual animals administered [2-C]glycerol are shown (). The constancy of fractional abundances for - and -anomers of glucose and for consecutive time points and the reproducibility of derived values for p and f are clearly apparent when [2-C]glycerol is used under these conditions. Because of the superiority of [2-C]glycerol for labeling the triose-P precursor pool, further studies used this labeled substrate for measuring GNG.

The fractional contribution to GNG from plasma glycerol can also be calculated from the [2-C]glycerol data. Isotope enrichments of plasma glycerol were about 3 times the calculated triose-P enrichments (0.440 ± 0.029 versus 0.148 ± 0.008, respectively). The ratio of triose-P/glycerol enrichments was 34 ± 1%, (range of 30-37%) in 48-h fasted rats (). The roughly 3-fold dilution between plasma glycerol and the hepatic triose phosphate pool indicates that about one-third of the subunits in the triose-P pool for GNG came directly or indirectly from circulating glycerol. Ra glucose was 7 mg/kg/min, and GNG provided approximately 88% of glucose flux (), so the glycerol contribution to GNG was approximately 2.1 mg/kg/min (7 mg/kg/min 0.88 0.34). Comparison of these values to plasma glycerol flux is of interest. The average Ra glycerol in these animals was 2.3 mg/kg/min, so about 90% of glycerol entering the circulation could be accounted for by entry into blood glucose, under conditions of 36-48 h fasting.

Fed to Fasted Transition

HGP was 14.3 ± 1.4 mg/kg/min 6 h after food was removed and fell to 10.3 ± 0.5 mg/kg/min at 10-11 h and 7.6 ± 0.5 mg/kg/min at 24 h (Fig. 4A). Food is absent from the stomachs of these adlibitum fed rats within 4-5 h of the start of fasting, so Ra glucose represents HGP under these conditions. Concurrent with the fall in HGP was an increase in f, from 51.3 ± 7.5 to 78.9 ± 8.1 and to 87.0 ± 1.5% at 6, 11, and 24 h of fasting, respectively (p < 0.05 for 11 and 24 h versus 6 h values, Fig. 4B). Absolute GNG was unchanged at 7.0 ± 0.7, 8.0 ± 0.6, and 6.9 ± 0.3 mg/kg/min (Fig. 4C), whereas non-GNG HGP fell significantly from 7.3 ± 1.8 to 2.6 ± 0.9 and 1.1 ± 0.2 mg/kg/min at the three time points, respectively (p < 0.05 for 11 and 24 h versus 6 h values, Fig. 4D). Thus, the fall in HGP during progressive 24-h fasting was entirely due to decreased non-GNG HGP, representing glycogenolysis, whereas GNG remained constant.


Figure 4: Effects of progressive fasting on GNG and related parameters. A, HGP (mg/kg/min); B, f (percent); C, absolute (Abs) GNG (mg/kg/min); D, non-GNG HGP (mg/kg/min); *, p < 0.01 versus 5-6 value; **, p < 0.05 versus 10-11 h value. The metabolic infusion protocol and measurements are described in the text.



Ra triose-P fell from 9.9 ± 0.9 mg/kg/min at 5-6 h of fasting and 9.3 ± 0.7 mg/kg/min at 10-11 h to 7.6 ± 0.2 mg/kg/min at 20-24 h (p < 0.05 for 20-24 h value versus other two time points, Fig. 5A). The proportion of the triose-P pool derived from plasma glycerol increased significantly from 11 to 24 h of fasting, (24.2 ± 3.2 to 37.3 ± 1.3%) (p < 0.05). Accordingly, the non-glycerol Ra triose-P fell by 37% during progressive fasting, from 7.5 ± 0.9 to 4.8 ± 0.2 mg/kg/min (p < 0.05). Moreover, the efficiency by which triose-P flux was converted to plasma glucose (Ra triose-P minus absolute GNG) also increased between 6 and 24 h of fasting (Fig. 5B), from 70.8 ± 7.3 to 88.0 ± 2.6% (p < 0.05). Thus, at 6 h of fasting, 3.0 ± 0.8 mg/kg/min of triose-P flux was not accounted for as flux into GNG; at 10 h, the value was 1.3 ± 0.6 mg/kg/min (nonsignificantly different versus 6 h), and at 24 h, all but 0.7 ± 0.3 mg/kg/min of flux into the triose-P pool was directed into plasma glucose (p < 0.01 versus 6 h) (Fig. 5C).


Figure 5: Effect of progressive fasting on metabolic parameters relating to triose-P flux. A, Ra triose-P (mg/kg/min); B, efficiency of triose-P disposal into glucose (percent triose-P flux into GNG); C, nongluconeogenic triose-P disposal (mg/kg/min); *, p < 0.05 versus 5-6 h value;**, p < 0.05 versus 10-11 h value.



Effect of Substrate Loads

IV Fructose Administration

Next, the effect of substrate loads on fractional and absolute GNG was studied. Rats were infused intravenously with fructose at 10-15 mg/kg/min after a preceding 24-h fast. HGP increased by 80-149% (I), from 8.6 ± 0.9 to 15.5 ± 1.3 mg/kg/min at fructose 10 mg/kg/min (p < 0.01 versus base line) and from 6.9 ± 1.5 to 17.2 ± 3.0 mg/kg/min at fructose 15 mg/kg/min (p < 0.01). The infusion rate of [C]glycerol was doubled during fructose infusions to maintain p at high values, and in fact p remained fairly constant (not shown). The basal value of f did not change during fructose infusion (84.7 ± 3.0 to 84.4 ± 3.2% and 80.7 ± 1.5 to 79.9 ± 3.6% at fructose 10 and 15 mg/kg/min, respectively), while absolute GNG increased by 81-147% (from 7.2 ± 0.5 to 13.0 ± 0.8 mg/kg/min and from 5.5 ± 1.1 to 13.6 ± 2.1 mg/kg/min, respectively, p < 0.01 versus basal for each). Interestingly, non-GNG HGP also increased, from 1.4 ± 0.4 to 2.5 ± 0.6 mg/kg/min (nonsignificantly different versus base line) and from 1.4 ± 0.4 to 3.6 ± 1.0 mg/kg/min (p < 0.05 versus base line). Since these animals are depleted of glycogen, the most likely explanation for increased non-GNG HGP is direct conversion of 15-17% of the fructose load to glucose, without having passed through triose-P and GNG(27) .

Ra triose-P increased markedly and in a graded fashion during intravenous infusions of fructose in 20-h fasted rats (I). Base-line values of 7.0 ± 0.4 mg/kg/min increased to 13.6 ± 0.4 mg/kg/min at fructose 10 mg/kg/min (p < 0.01 versus base line) and from 6.6 ± 0.6 to 16.4 ± 0.7 mg/kg/min at fructose 15 mg/kg/min (p < 0.01 versus base line, p < 0.05 versus fructose 10 mg/kg/min). The increases in Ra triose-P were 94-148% relative to base-line values, for fructose 10 and 15 mg/kg/min, respectively.

The input from noninfused sources of hepatic triose-P (endogenous Ra triose-P) was also calculated by subtracting from Ra triose-P the exogenous infusion rate of labeled glycerol and the rate of unlabeled fructose entry into triose-P. Direct conversion of fructose to glucose had to be subtracted from the fructose infusion rate to determine the rate at which exogenous fructose entered the hepatic triose-P pool. Endogenous Ra triose-P calculated in this manner fell markedly during fructose infusions (I), from 6.0 ± 0.4 to 1.9 ± 0.4 mg/kg/min (p < 0.01) at fructose 10 mg/kg/min and from 5.6 ± 0.6 to -0.6 ± 0.7 mg/kg/min (p < 0.01) at fructose 15 mg/kg/min (the negative flux rate is not significantly different from zero).

The efficiency of conversion of hepatic triose-P flux to glucose remained high during fructose infusions (I). Absolute GNG accounted for 80-100% of triose-P disposal under conditions of 24-h fasting, and subsequent intravenous fructose infusions did not alter the fractional disposal of triose-P flux into GNG.

Comparison of plasma glycerol enrichments to p demonstrated that circulating glycerol provided 38-44% of triose-P subunits at base line, compared with 20-23% during fructose administration (I). Plasma glycerol flux (Ra glycerol) did not decrease during fructose infusion. The proportion of plasma glycerol flux entering GNG remained >85% during fructose infusions (not shown). The reduced fractional contribution from plasma glycerol to p therefore reflects an increased input from other sources (fructose) rather than reduced input from glycerol.

IV Glucose Administration

Administration of intravenous glucose at 15-30 mg/kg/min had a very different effect than intravenous fructose on GNG, reducing f to 23 and 13%, respectively (I). Ra glucose increased during infusions of exogenous glucose, so that absolute GNG was 4.3 ± 0.6 and 5.0 ± 0.6 mg/kg/min, while HGP was calculated to be 3.6 ± 1.2 and 6.3 ± 2.0 mg/kg/min. These values were significantly reduced from base-line fasted values (p < 0.01). Plasma glycerol flux was reduced by 29-35%, from 2.86 ± 0.12 to 1.85 ± 0.14 mg/kg/min and from 2.58 ± 0.11 to 1.83 ± 0.25 mg/kg/min, respectively, (p < 0.05 for each) during intravenous glucose infusions at 15 and 30 mg/kg/min, respectively. The proportion of triose-P subunits derived from glycerol was also nonsignificantly reduced during cold glucose infusions (I). The proportion of plasma glycerol flux entering plasma glucose was also significantly reduced (to 66 and 71%) compared with fasted or intravenous fructose-infused animals (p < 0.05).

At glucose 15 mg/kg/min, Ra triose-P decreased from 8.6 ± 0.4 to 6.8 ± 0.4 mg/kg/min (p < 0.05 versus base line, p < 0.01 versus intravenous fructose 15 mg/kg/min). At glucose 30 mg/kg/min, Ra triose-P fell from 8.5 ± 0.7 to 7.0 ± 0.3 mg/kg/min (p < 0.05 mg base line, p < 0.01 versus fructose 15 mg/kg/min). Thus, intravenous glucose infusions consistently increased hepatic triose-P enrichments, resulting in a lower Ra triose-P (I). Plasma glycerol flux was significantly reduced by intravenous glucose (see above). The reduction in plasma glycerol flux could account for 56 and 50% of the reduced Ra triose-P at glucose 15 and 30 mg/kg/min, respectively.

Intravenous glucose also reduced the efficiency of triose-P disposal into plasma glucose. The fraction of triose-P flux not entering glucose increased from 19.1 ± 1.3 to 36.3 ± 8.0% (p < 0.01) at intravenous glucose 15 mg/kg/min and to 30.2 ± 1.9% (p < 0.01) at intravenous glucose 30 mg/kg/min (I).

Quantitative Recovery of C in End Products of Hepatic Triose-P Metabolism

The quantitative accuracy of the Ra triose-P method is dependent upon the assumption that all of the infused [C]glycerol in fact enters the hepatic triose-P pool sampled (see above). Although it is generally accepted that the liver accounts for the great majority of glycerol disposal in mammals(33, 34) , the quantitative validity of this assumption needed to be tested under the experimental conditions present in these studies. The effect of administering different unlabeled substrates in particular had to be evaluated. We addressed this difficult question by performing an isotope accounting. The rate of entry of C atoms into glucose and other selected end products of hepatic triose-P metabolism (glycogen, secreted GlcA, plasma triglyceride-glycerol) was tabulated and compared with the rate of C infusion (). The rate of C entry into each end product was calculated as the flux from triose-P into the product (the production rate) multiplied times the fraction of triose-P labeled with C (see above). In the case of plasma glucose, this calculation reduces to absolute GNG times p. For liver glycogen, it is the glycogen accumulation rate times the fraction from GNG times p of glycogen, and for GlcA it is the urinary excretion rate times the fraction from GNG times p of GlcA. For triglyceride-glycerol, it is the triglyceride production rate (estimated from literature values, Refs. 38 and 39) times the enrichment of glycerol isolated from plasma triglycerides. It should be noted that release from the liver as lactate or other glycolytic end products was not quantitatively included in this estimate. Also not included is de novo hepatic lipogenesis, which is quantitatively minor under most conditions(13, 40) .

After 11-24 h of fasting, the recovery of C in these end products was 91.4 ± 6.8 to 95.5 ± 2.7% (). With fructose infusions at 10-15 mg/kg/min, C recovery was not significantly different (99.7-102.3%). During intravenous glucose infusions, however, C recovery was slightly lower (79.7-83.7%), perhaps reflecting release of glycolytic end products from the liver. The observation that triose-P enrichments were higher during intravenous glucose infusions (I) argues against the lower C recovery being due to a reduction in the rate of [C]glycerol entry into the hepatic triose-P pool during intravenous glucose administration, which would have tended to decrease triose-P enrichments.

The high recovery of C in these end products of triose-P metabolism under conditions of GNG is direct evidence in support of the central assumption of this dilution method, namely that the administered label enters the pool of interest. Based on these results, the Ra triose-P estimates are unlikely to be overestimated by more than about 5-10%, if at all, due to incomplete isotope entry into the hepatic triose-P pool.

The question of recycling of label was also considered. The enrichment of plasma lactate during infusions of [2-C]glycerol was 20-25% of the hepatic triose-P enrichments (not shown). Since lactate contributes about 50% of the carbon in triose-P (see ) under fasting conditions, recycled [C]lactate contributes about 10-12% of triose-P enrichment. Put differently, Ra triose-P might be underestimated by about 10% due to the reentry of substrate that is labeled rather than being a source of dilution.

Reproducibility of Estimates

We evaluated experimental reproducibility of estimates by two strategies.

Comparison of - and -Anomers of Glucose

The calculated values for p and f were compared in - and -anomers of glucose from all analyses made. The ratio of their values for p was 1.009 ± 0.056 and for f was 0.995 ± 0.047 (mean ± S.D.). The correlation coefficient was also very high (r = 0.99 for both p and f) for - versus -anomers. This degree of reproducibility was achieved by use of quadruplicate analyses, by preinjection to achieve similar peak areas in sequential samples, and by paying careful attention to the closeness of base-line fractional abundances to expected values (as discussed above).

Comparison of Plasma Glucose to Secreted GlcA Conjugates

We also applied the MIDA approach to secreted acetaminophen-GlcA, which has been used as a noninvasive index of hepatic UDP-glucose labeling by us (23, 28, 34) and others(41, 42) . GlcA should have a similar labeling pattern as plasma glucose if glucuronidation occurs in the liver from a shared pool of hexose phosphates. The latter assumption has been controversial(23, 41, 42) . MIDA provides an interesting way to approach this question, since the enrichment of two end products might differ for physiologic reasons (contributions from unlabeled glycogen, galactose, etc.), but the molecules of each product, newly synthesized from the GNG pathway, should derive from the same triose-P pool. The enrichment of the triose-P pools feeding the two end products can be measured by MIDA. We compared p in GlcA to blood glucose under conditions of 24 h fasting (Fig. 6). The ratio of GlcA/glucose for p was 0.996 ± 0.026, with r = 0.973. These results support the analytic reproducibility of the MIDA technique as well as the identity of (or isotopic equilibrium within) both the triose-P and glucose 6-phosphate precursor pools for glucose and secreted GlcA-conjugates.


Figure 6: Comparison of values for p between plasma glucose and secreted urinary GlcA (Glc-UA) (from acetaminophen-GlcA). Line of identity is shown. Ratio of GlcA/glucose = 0.996 ± 0.026 (r = 0.973).



Evaluation of Potential Deviations from Model

Impact of Isotopic Disequilibrium between Triose Phosphates on MIDA Calculations

Theoretical Considerations

Use of the binomial expansion assumes that there exists a single p (proportion of subunits undergoing combination that are isotopically labeled, Refs. 11-13). The value for p may change over time, but at any moment it is assumed that there exists a single value for the isotope enrichment of the precursor subunit. The same applies for the multinomial expansion; more than two choices are available, but their probabilities are the same for each trial. In the case of GNG, this assumption may be called into question because there is not in fact a single biochemical triose phosphate precursor for GNG but a pair of precursors (DHAP and glyceraldehyde 3-phosphate) that combine to form the glucose ``polymer'' (Fig. 1). The assumption of complete isotopic equilibrium between these two triose phosphates has not been fully evaluated and may not be justified under all conditions. Many, but not all, studies have indicated that isotopic equilibrium is present between the triose phosphates(7, 32) , based on comparisons of labeling in glucose carbons 1-3 (representing DHAP) and carbons 4-6 (representing glyceraldehyde-3-phosphate). It can be shown, however, that divergence from isotopic equilibrium between DHAP and glyceraldehyde 3-phosphate generally has only a very minor impact on MIDA calculations for glucose (see ``Appendix''). Unless the isotopic disequilibrium is substantial (e.g. one triose-P enrichment is less than 40-50% of the other), effect on calculation of p and f is minor ( Fig. 1of the ``Appendix'').

Experimental Testing

To evaluate the presence or absence of isotopic equilibrium experimentally, we used the GC/MS technique described by Guo et al.(32) for fragmenting glucose into carbon 1-4 and carbon 3-6 moities. The aldonitrile, pentaacetate derivative is analyzed for this purpose. Under electron-impact ionization, the fragment at m/z 187 represents carbons 3-6 and m/z 242 represents carbons 1-4(24) . Differences in isotopic enrichments between these two fragments reveal differences in carbons 5-6 versus carbons 1-2 (with [2-C]glycerol more highly labeling carbons 1-2 and [3-C]lactate labeling carbons 5-6, if disequilibrium exists). Since [2-C]glycerol primarily labels positions C-2 and C-5 of glucose and secondarily randomizes into C-1 and C-6 in the fumarase equilibrium, only entering C-3 and C-4 of glucose after sequential turns of the tricarboxylic acid cycle, comparison of these two fragments provides a quantitative index of isotopic equilibrium between DHAP and glyceraldehyde 3-phosphate (represented by carbons 1-2 and carbons 5-6, respectively). First, in order to test the accuracy of the aldonitrile fragmentation technique, we mixed known ratios of [1-C]glucose and [6-C]glucose and compared measured with expected ratios of carbons 3-6/carbons 1-4. The observed results were very close to expected (ratio of measured/prepared = 0.975 ± 0.060, r = 0.997). Next, the aldonitrile, pentaacetate derivative of plasma glucose was analyzed by electron-impact ionization/mass spectometry in fasted and fructose-infused rats (). The ratio between the carbon 3-6/carbon 1-4 moieties of the glucose molecule was 1.16-1.17. The impact of this degree of disequilibrium on calculated parameters (p and f) is <1% (0.7% at a ratio of 1.17). A quantitatively important degree of isotopic disequilibrium therefore is not present under these conditions. Consistent with theoretical expectations (Fig. 1), the ratio of carbons 3-6/carbons 1-4 was slightly less than 1.0 from [C]lactate (0.93, ), but again this is not significant in terms of quantitative impact on calculated parameters.

COReincorporation and Double-labeling of the Hepatic Triose Phosphate Pool

Theoretical Considerations

The fixation of a CO by [3-C]pyruvate molecule results in double-labeling in the hepatic triose phosphate precursor pool after randomization of oxalacetate. Combination of [2-C]acetyl-CoA (e.g. after decarboxylation of [3-C]lactate) with [3-C]oxalacetate (e.g. from carboxylation of [3-C]pyruvate) could also result in double-labeled triose phosphates (e.g. derived from [1,3-C]oxalacetate), although this route has a much lower probability of occurring. Other low probability pathways exist that might result in double-labeled triose subunits (e.g. GlcA to xylulose followed by nonoxidative portion of pentose phosphate pathway). Doubly-labeled triose subunits would complicate MIDA, because the assumption of the MIDA technique using [3-C]lactate or [2-C]glycerol is that double-labeled glucose molecules represent the combination of two single-labeled precursor triose-P subunits. Double labeling of an individual triose-P subunit, if present, would increase the EM/EM ratio in glucose, thereby artifactually increasing the estimated p and reducing estimated fractional GNG. The CO enrichment in hepatic mitochondria under these conditions is difficult to predict, so the probability of CO combining with [C]pyruvate cannot be excluded on theoretical grounds. The analytic strategy and calculations used to address this question are described in the ``Appendix.''

Experimental Testing

In order to test whether double C-labeling of triose units occurred, we asked whether the measured M isotopomers of the triose fragment of sorbitol (m/z 219) could be completely accounted for on the basis of natural abundance isotope distributions combined with incorporation of a single C-labeled atom or whether double-labeled [C]triose units needed to be invoked (see ``Appendix''). Measurements were carried out on 22 plasma glucose samples after reduction to sorbitol and GC/MS analysis of the m/z 217-219 fragment ion envelope, from rats infused with [2-C]glycerol or [3-C]lactate (). Enrichments of EM in the triose fragment were high (0.0400-0.1500 ME) and the divergence of observed from expected EM values was very small (-0.0005 ± 0.0001).

These results exclude double C-labeling of triose units as a significant process during infusions of [2-C]glycerol or [3-C]lactate. A second experimental strategy to exclude CO reincorporation or other sources of triose double-labeling was also employed. [1-C]Lactate cannot become double-labeled from the simple CO reincorporation/carboxyl group randomization/decarboxylation sequence or from the pyruvate decarboxylation/acetyl-CoA recombination sequence, because loss of either carboxyl group will result in loss of C. As noted above, when [1-C]lactate was infused to 48-h fasted rats, the value for f was 89 ± 3%, identical to results from [3-C]lactate, which is further evidence against an important role for any artifact due to double labeling in the triose phosphate pool.

Error-Sensitivity Analysis

Theoretical Considerations

See ``Appendix'' for theoretical considerations.

Experimental Testing

Evidence for experimental reproducibility of estimates is presented above (comparisons between and -anomers of glucose and between glucose and GlcA). The need for analytic accuracy as well as precision, if MIDA is to be applied reliably to a short chain (n = 2) polymer like glucose, must be emphasized.


DISCUSSION

We have applied the MIDA technique to the long standing problem of measuring GNG. The results can be discussed in terms of their methodologic and their metabolic implications. With regard to methodology, MIDA has previously been used by us for measurement of polymers composed of larger numbers of repeating monomeric subunits (11-14, 25, 27, 33, 40). Others (43, 44, 45) have also applied probability analysis to the precursor-product relationship for long chain polymers. Measurement of GNG presented some unique methodologic challenges. Glucose contains the smallest number of monomeric subunits possible for application of combinatorial analysis (n = 2). In addition, the possibility that the precursor pool might not be well mixed (i.e. that there could be isotopic disequilibrium between DHAP and glyceraldehyde 3-phosphate (Fig. 1)) could in theory render the binomial or multinomial model inapplicable in this system. Other potentially confounding factors such as the generation of double-labeled triose subunits by reincorporation of CO also had to be considered. Nevertheless, the widely recognized absence of an alternative method for measuring GNG(2, 3, 4, 5, 6, 7, 8, 9, 10) and various advantages of MIDA (that it measures all-source GNG rather than only PEP-GNG and that the intracellular triose-P enrichment can be calculated) motivated the attempt to apply MIDA to GNG, as did the large number of related metabolic questions that might be addressed if GNG and hepatic triose-P enrichments could be measured in vivo.

The potential methodologic problems in application of MIDA to GNG were addressed. Based on both theoretical and experimental results, we conclude that isotopic disequilibrium within the triose-P pool is not a serious problem under most conditions because, first, even an isotopic gradient of 2:1 has only a relatively minor effect on calculated GNG (overestimation of f by about 12-13%, see ``Appendix'') and, second, much less disequilibrium (1.17:1) is in fact present in the triose-P pool when tested experimentally (). Moreover, double-labeled triose-P do not occur sufficiently frequently to have a significant quantitative impact and error sensitivity analysis revealed acceptably low levels of error amplification in derived parameters ifp is maintained at 0.08 or greater (``Appendix''). We have also calculated the effect of inconstancy of p in space, e.g. extrahepatic GNG.()As long as the second source provides less than about 20% of GNG and the ratio of values for p is 2:1 or closer, effects on calculated p and f are minor. Finally, various technical aspects of the mass spectrometric analyses can help maintain both precision and accuracy of quantification. This last point is critical for successful application of MIDA to an n = 2 polymer like glucose, since the abundance of the M-isotopomer will tend to be lower than with longer chain length polymers and therefore must be quantified with a high degree of precision and accuracy.

With regard to the Ra triose-P technique for measuring flux through this intrahepatic metabolic pool, the first requirement was to establish the validity of the technique. An important metabolic assumption of the Ra triose-P measurement is that the infused [C]glycerol enters the hepatic triose-P pool in a quantitative fashion (Fig. 1). Isotope recovery studies demonstrated that, in the prolonged fasted state, about 85-90% of infused [C]glycerol could be accounted for as entry into plasma glucose via the hepatic triose phosphate pool. Another approximately 1-2% of [C]glycerol could be accounted for by secretion of [C]glycerol into very low density lipoprotein-glycerol and about 2-3% as entry into the GlcA moiety of infused acetaminophen (). Thus, even without considering possible entry of C from glycerol into lactate, acetyl-CoA, or fat (all likely to be minor in 24-h fasted rats), 90-95% of labeled glycerol was documented to pass through the hepatic triose-P pool used for GNG. Brief fasting was associated with a lower recovery of [C]glycerol in plasma glucose compared with more prolonged fasting, as might have been anticipated. Administration of fructose at 10-15 mg/kg/min to previously fasted rats resulted in 82-90% recovery as plasma glucose, and >95% total recovery in end products of hepatic triose-P metabolism. These results all support the expectation that label from glycerol enters the hepatic triose-P pool in rats in a nearly quantitative way(35, 36) , here documented to be 90% or greater.

Several metabolic results of interest emerged, based on this methodologic foundation. The mechanism by which HGP falls during the first 24 h of a fast was determined. There was no effect on absolute GNG, but a 90% reduction in non-GNG HGP (Fig. 4). The latter presumably reflects reduced glycogen to glucose flux as liver glycogen becomes depleted. A similar sequence has been reported in humans, based on in vivo NMR estimates of hepatic glycogenolysis(46) . The reduction of non-GNG HGP became apparent as early as the interval from the 6th to the 11th h of fasting. GNG was maintained constant during a fast despite a reduction in Ra triose-P, by two mechanisms. First, the efficiency at which triose-P flux was disposed into plasma glucose increased. After 6 h, 3.0 mg/kg/min of triose-P flux did not enter GNG, while this value fell to 0.7 mg/kg/min at 24 h of fasting. In addition, the contribution from plasma glycerol to hepatic triose-P increased significantly, thereby preventing a further fall in Ra triose-P while sparing amino acids and other limited sources of precursors for GNG. The liver thereby becomes more efficient in terms of both the fate and the sources of hepatic triose as liver glycogen becomes depleted.

A quantitative result with interesting implications was that intravenous fructose slightly but consistently increased non-GNG HGP in glycogen-depleted rats (I). This finding is consistent with the report of Gopher et al.(47) . They analyzed plasma glucose by NMR after administration of [U-C]fructose in normal children and found that at least 50% of fructose was directly converted to glucose without being broken down to the 3-carbon level (i.e. not proceeding via fructose-1-phosphate aldolase). Our results in rats given higher rates of intravenous fructose also indicate that some direct conversion of fructose to glucose occurs, representing roughly 15-17% of the fructose load. Direct conversion may occur by phosphorylation of fructose 1-phosphate by 1-phosphofructokinase, but this has not been established. Chandramouli et al.(48) did not find evidence for substantial direct conversion of fructose to glucose (0-15%, mean 5.1% of the load) when fructose was administered at much lower doses (0.3 mg/kg/min) to adult humans. Fructose load, species, age, route of administration, and other factors may explain different activities of this pathway.

Our results also suggest (I) that flux into the hepatic triose-P pool is regulated in a feedback manner in response to variations in exogenous inputs, much as Ra of plasma glucose is regulated in response to exogenous glucose infusions. Most impressive was the effect of loads of exogenous gluconeogenic precursors to almost completely suppress endogenous Ra triose-P. A dose-response relationship between exogenous load and suppression of Ra triose-P was also apparent (I). Since >80% of triose-P flux enters plasma glucose under these conditions, the reduction in endogenous Ra triose-P by 4.1-6.2 mg/kg/min during fructose infusions prevented Ra glucose from rising by an additional 4-5 mg/kg/min. The mechanism by which one source of triose-P reduces input from other sources cannot be stated at present. Feedback inhibition by a triose-P or related metabolite on phosphoenolpyruvate carboxykinase or phosphofructosekinase, or activation of pyruvate kinase or pyruvate dehydrogenase(49, 50) could be responsible. Since phosphoenolpyruvate carboxykinase is often considered to be a regulatory step in GNG(51, 52) , the role of altered phosphoenolpyruvate carboxykinase activity in the reduced Ra triose-P is of great interest.

Our observation that HGP increased during infusions of fructose differs from the lack of increase in HGP observed by others (16, 17, 18) during infusions of gluconeogenic substrates. There are several possible explanations for the difference, in addition to species differences. The rats in our studies were previously fasted so there was little or no glycogenolysis to suppress, which is one potential mechanism of intrahepatic adaptation to substrate loads(17, 53) . Moreover, the substrate loads that we administered (10-15 mg/kg/min) were greater than basal HGP (7 mg/kg/min), so that even complete suppression of endogenous Ra triose-P and other endogenous sources of HGP could not fully compensate for the exogenous load. Finally, the low insulin state in these 24-h fasted animals may have limited channeling of GNG into liver glycogen(54, 55, 56) . It will be necessary to perform studies in liver glycogen replete rats at substrate loads lower than HGP to address the efficiency and mechanism of other hepatic adaptations besides changes in triose-P input under these conditions.

The lack of an increase in Ra triose-P during intravenous glucose infusions (I) was somewhat surprising, in view of the role of the indirect pathway for hepatic glycogen synthesis from glucose(23, 57, 58) . About 50% of the reduced triose-P flux was attributed to reduced contribution from plasma glycerol due to a decrease in plasma glycerol flux, but the explanation for the remaining 50% was not determined. Several possibilities might be considered. Increases in plasma glucose and insulin concentrations could reduce the contribution from glycogen to hepatic triose-P, although the amount of liver glycogen in these 24-h fasted rats is quite low. Intravenous glucose infusion could direct some 3-carbon metabolites away from the triose-P pool, by activating pyruvate dehydrogenase (49) or pyruvate kinase (19, 59). Alternatively, liver glycogen deposition by the indirect pathway during glucose infusions may not involve an increase in carbon flux into the triose-P pool, but a redirection of GNG flux away from plasma glucose and into glycogen (54, 55, 56) along with a replacement of endogenous sources of triose-P by glucose.

Administration of glucose had relatively minor effects on Ra triose-P and on the contribution of plasma glycerol to the triose-P pool, but it suppressed GNG, HGP, and non-GNG HGP while increasing flux to glycogen (Tables III and IV). The major consequence of glucose infusion was therefore on the disposal of triose-P rather than on its production. Intravenous fructose infusion at a high rate (15 mg/kg/min) increased non-GNG triose-P disposal to 2.8 mg/kg/min. If this all represented glycogen synthesis, it could provide about 4 mg of glycogen/g of liver/h via the ``indirect'' pathway. The difference between Ra triose-P and absolute GNG may provide an index of hepatic indirect glycogen synthesis under certain refeeding conditions. Our measurements clearly demonstrate that the disposal as well as production of hepatic triose-P adapts to metabolic conditions.

We observed fractional GNG to be consistently about 90% after a 24-h fast, leaving 10% unaccounted for. Although we did not determine other sources of glucose experimentally, some possibilities include turnover of glycoprotein carbohydrates (galactose, mannose, glucosamine, etc.), a persistent small contribution from liver glycogen, release of free glucose from muscle or other glycogen-containing tissues, or input from gluconeogenic pools to which labeled glycerol or lactate do not have access.

Finally, the hexose phosphate pool from which glucuronidation of xenobiotics occurs had been an area of uncertainty(23, 41, 42) . MIDA provides an interesting additional tool to address this question. Under conditions of fasting or fructose infusion, the isotopic enrichment of the triose-P pool for GlcA was identical to that for blood glucose (Fig. 6). One can infer that glucuronidation and glucose secretion derive from the same tissue and the same pool (or at least isotopically well mixed pools) under these conditions. Other end products can be compared in an analogous fashion, to evaluate compartmentalization of ostensibly shared precursor pools (e.g. GlcA and glycogen under various refeeding conditions).

In conclusion, the ability to quantify fluxes into hepatic GNG in vivo opens up a wide variety of metabolic regulatory questions to experimental analysis. Our results indicate that the liver exerts control over GNG, the metabolic sources of triose-P, flux into the triose-P pool, the efficiency of triose-P disposal into GNG, and the contribution from glycogen to plasma glucose, in response to alterations in substrate availability. Since MIDA does not involve radiation exposure, it is also suitable for measurement of GNG and related parameters in humans.

  
Table: Incorporation from [3-C]lactate and [2-C]glycerol into hepatic triose-P precursor pool

Plasma metabolite refers to plasma lactate in studies infusing [3-C]lactate and plasma glycerol in studies infusing [2-C]glycerol. Rats were fasted for 36-48 h prior to infusion of labeled substrate. Number of rats studied in each group given by n. Experimental details are described in the text.


  
Table: Primary data and derived GNG parameters in three rats infused with [2-C]glycerol


  
Table: ±1.9

  
Table: Recovery of [C]glycerol in various end products of hepatic triose-P metabolism

Values shown are means ± S.E.M. Calculations of C-recovery in various end-products are described in text. VLDL, very low density lipoprotein.


  
Table: Quantification of isotopic disequilibrium in triose-P pool under various experimental conditions

Experimental details are described in the text. The ratio of DHAP/G3P was determined by analysis of the m/z 242 to the m/z 187 ion abundances from the aldonitrile, pentaacetate derivative of glucose by GC/MS (24).


  
Table: 0p4in Extra M residual (double labeled) = labeled (residual) - Expected M residual.(119)


FOOTNOTES

*
This work was supported in part by Grant DK-40995 from the National Institutes of Health, Grant 87-51 (BRSG) from the University of California at Berkeley, a clinical research grant from the American Diabetes Association, and Grant IRT475 from the Tobacco Related Disease Research Program of the State of California. Portions of this work have appeared previously in abstract form (21, 22). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by the Fonds National Suisse de la Recherche Scientifique, by the Fondation Suisse de Bourses de Medicine et Biologie, and by the Fonds Reymond-Berger.

To whom correspondence should be addressed: 325 Morgan Hall, Dept. of Nutritional Sciences, University of California, Berkeley, CA 94720-3104. Tel.: 510-642-0646; Fax: 510-642-0535.

The abbreviations used are: GNG, gluconeogenesis; triose-P, triose phosphate; DHAP, dihydroxyacetone phosphate; MIDA, mass isotopomer distribution analysis; HGP, hepatic glucose production; Ra triose-P, rate of appearance of triose-P; GC/MS, gas chromatography/mass spectrometry; SIM, selected ion monitoring; ME, molar excess; PEP, phosphoenolpyruvate.

The word labeled as used here refers to isotopic enrichment above that due to natural isotope abundances, by analogy to radioisotope labeling. Thus, the presence of C or H does not by itself make a subunit labeled, by the following definition:

On-line formulae not verified for accuracy

In accordance with recent usage (10-12), we define M as the monoisotopic mass + 0; M is the monoisotopic mass + 1; etc.

R. Neese, D. Faix, K. Caldwell, and M. K. Hellerstein, unpublished observations.


ACKNOWLEDGEMENTS

-We thank Christina Papageorgopoulos for helpful discussions and Cici Hyde for preparation of the manuscript.


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