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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Mitochondrial fatty acid oxidation (mFAO) is considered to be essential for driving gluconeogenesis (GNG) during fasting. However, quantitative in vivo data on de novo synthesis of glucose-6-phosphate upon acute inhibition of mFAO are lacking. We assessed hepatic glucose metabolism in vivo after acute inhibition of mFAO by 30 mg kg−1 2-tetradecylglycidic acid (TDGA) in hypoketotic hypoglycemic male C57BL/6J mice by the infusion of [U-13C]glucose, [2-13C]glycerol, [1-2H]galactose, and paracetamol for 6 hours, which was followed by mass isotopomer distribution analysis in blood glucose and urinary paracetamol-glucuronide. During TDGA treatment, endogenous glucose production was unaffected (127 ± 10 versus 118 ± 7 μmol kg−1 minute−1, control versus TDGA, not significant), but the metabolic clearance rate of glucose was significantly enhanced (15.9 ± 0.9 versus 26.3 ± 1.1 mL kg−1 minute−1, control versus TDGA,P < 0.05). In comparison with control mice, de novo synthesis of glucose-6-phosphate (G6P) was slightly decreased in TDGA-treated mice (108 ± 19 versus 85 ± 6 μmol kg−1 minute−1, control versus TDGA, P < 0.05). Recycling of glucose was decreased upon TDGA treatment (26 ± 14 versus 12 ± 4 μmol kg−1 minute−1, control versus TDGA, P < 0.05). Hepatic messenger RNA (mRNA) levels of genes encoding enzymes involved in de novo G6P synthesis were unaltered, whereas glucose-6-phosphate hydrolase mRNA expressions were increased in TDGA-treated mice. Glucokinase and pyruvate kinase mRNA levels were significantly decreased, whereas pyruvate dehydrogenase kinase isozyme 4 expression was increased 30-fold; this suggested decreased glycolytic activity. Conclusion: Acute pharmacological inhibition of mFAO using TDGA had no effect on endogenous glucose production and only a marginal effect on de novo G6P synthesis. Hence, fully active mFAO is not essential for maintenance of hepatic GNG in vivo in fasted mice.(HEPATOLOGY 2008.)

The pioneering experiments by Williamson et al.1 in isolated perfused livers showed that gluconeogenesis (GNG) is stimulated upon addition of fatty acids. Moreover, changes in the rate of GNG were observed after modulation of mitochondrial fatty acid oxidation (mFAO) in vitro and in vivo.2–5 Energy, acetyl coenzyme A, and reducing equivalents are all necessary for the conversion of pyruvate into glucose, and these compounds are thought to be generated by mFAO.6 As a result of inherited disorders of mFAO, patients suffer the inability to oxidize fatty acids, and consequently, hepatic ketogenesis is absent or limited.7 Biochemically, such an acute presentation is usually characterized by hypoketotic hypoglycemia and clinical features such as hepatic steatosis with or without cardiac and skeletal muscle involvement. These patients usually present during infancy with acute life-threatening events following a period of metabolic stress.

Kinetic data on the consequences of impairments of mFAO on glucose metabolism are scarce. Therefore, the etiology of hypoglycemia in human disorders of mFAO is still not completely understood. With multiple stable isotopes, metabolic flux rates through the separate pathways can now be quantified in vivo. In an earlier study, we investigated glucose metabolism in peroxisome proliferator-activated receptor α (PPARα)–deficient mice.8 Regulation of mFAO is perturbed in these mice, and prolonged fasting results in severe hypoglycemia.9 By the use of mass isotopomer distribution analysis (MIDA) calculations in short-term–fasted PPARα-deficient mice, it was observed that the de novo synthesis of glucose-6-phosphate (G6P) was unaffected but was preferentially directed toward glycogen rather than toward blood glucose; this accounted for a 15% decrease of the endogenous glucose production.8 Yet, it should be realized that in PPARα-deficient mice, mFAO is reduced but not absent and that PPARα deficiency may have metabolic consequences not related to impairment of mFAO.

Transport of fatty acyl residues into the mitochondria is brought about by the concerted action of three enzymes, that is, carnitine palmitoyltransferase I (CPT-I), carnitine acylcarnitine translocase, and CPT-II. The first enzyme is CPT-I (EC 2.3.1.21) and is thought to be rate-controlling for the entire process.10, 11 2-Tetradecylglycidic acid (TDGA) has been demonstrated to act as an irreversible inhibitor of mFAO at the level of CPT-I both in vitro and in vivo, with plasma glucose concentration–lowering consequences in the latter situation.12–14 We have recently evaluated hepatic insulin sensitivity in steatotic livers induced by acute inhibition of mFAO using TDGA in short-term–fasted mice.15 During these experiments, mice became significantly hypoglycemic already at 3 hours after treatment with a single dose of 30 mg kg−1 TDGA.

In the present study, we quantified important pathways of glucose metabolism in vivo after acute inhibition of mFAO by TDGA in fasting male C75BL/6J mice. We addressed the following questions: (1) does acute inhibition of mFAO affect de novo synthesis of G6P or glycogenolysis and thereby endogenous glucose production and (2) does acute inhibition of mFAO affect the metabolic clearance rate (MCR) of glucose. We addressed these questions by infusing [U-13C]glucose, [2-13C]glycerol, [1-2H]galactose, and paracetamol for 6 hours, collecting serial blood and urine spots on filter paper, and measuring the mass isotopomer distribution in blood glucose and urinary paracetamol-glucuronide (Par-GlcUA). Calculated metabolic fluxes were compared with hepatic messenger RNA (mRNA) expression levels of genes encoding crucial enzymes involved in hepatic glucose metabolism and mFAO. Our data surprisingly indicate that de novo synthesis of G6P was suppressed by only 22%, whereas endogenous glucose production remained unaffected, when mFAO was suppressed. Therefore, hypoglycemia was entirely due to a strongly enhanced MCR, and this indicates that the paradigm regarding the relationship between hepatic mFAO and GNG requires revision.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Animals and Materials

Male C75BL/6J mice (∼22 g) were obtained from Harlan Laboratories (Zeist, The Netherlands). The mice were housed in Plexiglas cages in a temperature-controlled room (20°C) on a controlled light-dark regime (10 hours of darkness and 14 hours of light). For the stable isotope infusion experiments, mice were equipped with a permanent jugular catheter in the right jugular vein, which was attached to the skull with acrylic glue, as described in rats by Kuipers et al.16 Mice were allowed to recover from surgery for at least 4 days. Experiments were approved by the Ethical Committee for Animal Experiments of the University of Groningen.

The sodium salt of TDGA was a kind gift from Dr. P. J. Voshol (LUMC, Leiden, The Netherlands). Before each experiment, a TDGA solution was freshly prepared, as described previously.15 All solvents and reagents used were of analytical grade.

Animal Experiments

The response to short-term fasting in TDGA-treated mice was determined first. Food was removed, and the mice were injected intraperitoneally with either vehicle or 30 mg kg−1 TDGA. After 12 hours, a large blood sample was taken by cardiac puncture from deeply isoflurane-anaesthetized mice, and the plasma was stored at −20°C until analysis. Livers were rapidly removed, and small aliquots were frozen in liquid N2 for mRNA expression studies.

Stable isotope infusion experiments were performed as described previously, with slight modifications.17 In short, food was removed 9 hours before the start of the infusion, and the mice were injected with either vehicle or 30 mg kg−1 TDGA. The mice were placed in individual metabolic cages. Bloodspots obtained by tail bleeding for gas chromatography–mass spectrometry (GC-MS) measurements were collected on filter paper (no. 2992, Schleicher en Schuell, ‘s Hertogenbosch, The Netherlands) before the start of the infusion and hourly for 6 hours thereafter. Mice were intravenously infused with a sterile solution containing [U-13C]glucose (7.0 μmol mL−1), [2-13C]glycerol (81 μmol mL−1), [1-2H]galactose (17 μmol mL−1), and paracetamol (1 mg mL−1) at a rate of 0.6 mL hour−1 for 6 hours. At the start of the infusion experiment, filter paper was placed under the wired floor of the cage to collect urine samples and was replaced at hourly intervals. At the end of the infusion period, the animals were deeply anesthetized with isoflurane and sacrificed by heart puncture.

Metabolite Analysis

Glucose, lactate, and 3-hydroxybutyric acid were determined with standard laboratory methods. Plasma free fatty acids were determined with a commercial kit (Wako Chemicals, Neuss, Germany). Carnitine and acylcarnitines were extracted from dried filter paper blood spots with methanol and analyzed with an electrospray ionization tandem mass spectrometer, as described previously.18

mRNA Expression Studies

Total RNA was isolated from approximately 30 mg of liver tissue with the Trizol method (Invitrogen, Paisley, United Kingdom), and RNA was converted into complementary DNA (cDNA) with M-Mulv-RT (Roche Diagnostics, Mannheim, Germany) with random primers according to the manufacturer’s protocol. cDNA levels of the genes of interest were measured by real-time polymerase chain reaction, and calibration curves were run on serial dilutions of pooled cDNA solutions as used in the assay. Data processing was performed with the ABI-Prism 7700 sequence detection system (Applied Biosystems, Foster City, CA), and the expression levels were quantified within the linear part of the calibration curves. Polymerase chain reaction results were normalized for β-actin + 18S mRNA levels. The sequences of most primers and probes used are available online (http://www.labpediatricsrug.nl), except those for fructose-1,6-biphosphatase-1 (Fbp1) and pyruvate carboxylase (Pc). For these genes, the following forward primers, reverse primers, and probe sequences were used: for Fbp-1, GCG ATC AAA GCC ATC TCG TC, GTC ATT GGA AAG TAT GTC CAG CTT C, and CCC CAG TCA CAT TGG TTG AGC CAG C, and for Pc, GCC AGA GGC AGG TGT TCT TT, CAT GGC CTG GGT GTC TTT AAC, and ACT CAA TGG GCA GCT TCG ATC CAT TC, respectively.

MIDA

Isolation and Derivatization.

Analytical procedures for extraction of blood glucose and urinary Par-GlcUA, their derivatization, and the GC-MS measurements of derivatives were according to Van Dijk et al.,17, 19 with modifications, and have been described previously. In short, after a disk was punched out of a bloodspot on filter paper, glucose was extracted by incubation of the disk in ethanol/water (10/1 vol/vol). After drying at 60°C under steam of N2, glucose was derivatized into both pentaacetate and aldonitrile pentaacetate esters. The derivates were dissolved in 100 μL ethyl acetate before injection onto the GC-MS. In contrast to previous publications, isotopic analysis of Par-GlcUA was done on glucose obtained from urinary Par-GlcUA. For this purpose, urine spots on filter paper were cut out, and Par-GlcUA was extracted by incubation in methanol-water (3/1 vol/vol). After drying at 60°C under a stream of N2, glucose was obtained after methylation, reduction by sodium borohydride, subsequent HPLC isolation, and enzymic hydrolysis with β-glucuronidase. Glucose was derivatized into its pentaacetate and aldonitrile pentaacetate esters and analyzed by GC-MS.

GC-MS.

All samples were analyzed by GC-MS (SSQ7000, ThermoFinnigan, San Jose, CA) with an AT-5MS 30 m × 0.25 mm column (Altech, Breda, The Netherlands). For calculating the mass isotopomer distributions, Excaliber software (ThermoFinnigan) was used. Mass spectrometric analysis of glucose pentaacetate and glucose aldonitrile pentaacetate was performed by positive ion chemical ionization with methane. The ions monitored were m/z 331–337 for glucose pentaacetate and m/z 328–334 for glucose aldonitrile pentaacetate, corresponding to the m0-m6 mass isotopomers. A series of measurements was accepted for further calculations only when all required conditions were met, as described by Van Dijk et al.17 (see the supplemental material for a detailed description of the analytical procedures)

Calculations of Metabolic Fluxes.

The calculations of the parameters and fluxes describing glucose metabolism were done essentially according to Hellerstein et al.20, 21 as described by Van Dijk et al.19 with slight modifications. Figure 1 depicts the isotopic model used, and the corresponding equations are given in Table 1. In short, the measured mass isotopomer clusters (m0-m6) were normalized and corrected for the naturally occurring abundance of 13C to obtain the excess fractional mass isotopomer distribution (M0-M6) due to incorporations of infused labeled compounds ([U-13C]glucose, [2-13C]glycerol, and [1-2H]galactose). The distribution obtained for glucose aldonitrile pentaacetate was used in the 13C calculations. The difference in single-labeled fractional isotopomer contributions of glucose pentaacetate and glucose aldonitrile pentaacetate was used in 2H calculations. All rates were calculated by isotope dilution with primary isotopic parameters (Table 1). For the estimation of the fractional contribution of newly synthesized glucose to plasma glucose [f(glc)] and fractional contribution of newly synthesized glucose to the uridine diphosphoglucose pool [f(UDPglc); Table 1], MIDA algorithms of 13C-isotope incorporation were used according to Hellerstein et al.20, 21 as described by Van Dijk et al.19 The various rates describing whole body glucose metabolism and the individual fluxes through the relevant enzymes were calculated with the appropriate equations given in Table 1 (see the supplemental material for a more detailed presentation).

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Figure 1. Schematic model of hepatic glucose metabolism. Major metabolic pathways and enzymatic reactions are depicted, despite glycolysis, sharing glucose-6-phosphate as a central metabolite. The pathways included are (1) GNG(G6P), (2) GP, (3) G6Pase, (4) GK, and (5) GS. The mice received an infusion containing [U-13C]glucose, [2-13C]glycerol, [1-2H]galactose, and paracetamol (depicted in italics) for 6 hours, and after serial collection of blood and urine samples, mass isotopomer distribution analysis was applied on blood glucose and urinary paracetamol-glucuronide. G6Pase indicates glucose-6-phosphatase flux; GK, glucokinase flux; GNG(G6P), total flux of glucose-6-phosphate de novo synthesis corrected for the exchange between plasma glucose and uridine diphosphoglucose pools; GP, glycogen phosphorylase flux; GS, glycogen synthase flux; and UDP, uridine diphosphate.

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Table 1. Parameters and Equations Used in the Calculations of Glucose Metabolism
ParameterEquation
  1. c(glc) indicates fractional contribution of plasma glucose to uridine diphosphoglucose formation; c(UDPglc), fractional contribution of uridine diphosphoglucose to blood glucose formation; d(glc), fractional contribution of infused glucose to blood glucose; d(UDPglc), fractional contribution of infused galactose to uridine diphosphoglucose; f(glc), fractional contribution of newly synthesized glucose to plasma glucose; f(UDPglc), fractional contribution of newly synthesized glucose to uridine diphosphoglucose pool; G6Pase, glucose-6-phosphatase flux; GK, glucokinase flux; [glc], blood glucose concentration (μmol mL−1); GLY(glc), rate of glycogenolysis contributing to plasma glucose formation; GLY(UDPglc), rate of glycogenolysis contributing to uridine diphosphoglucose formation; GNG(G6P), total flux of glucose-6-phosphate de novo synthesis corrected for the exchange between plasma glucose and uridine diphosphoglucose pools; GNG(glc), rate of gluconeogenesis into plasma glucose; GNG(UDPglc), rate of gluconeogenesis into uridine diphosphoglucose; GP, glycogen phosphorylase flux; GS, glycogen synthase flux; Inf(gal), rate of infusion of [1-2H]galactose (μmol kg−1 minute−1); Inf(glc), rate of infusion of [U-13C]glucose (μmol kg−1 minute−1); M1(gal)infusate, mole percent enrichment of [1-2H]galactose in the infusate; M1(glc)blood, mole percent enrichment of [1-2H]-glucose in blood; M1(pGlcUA)urine, mole percent enrichment of [1-2H]-UDPglc measured in urinary paracetamol-glucuronide; M2(FBP:MIDA)glc, theoretical mole percent enrichment of [13C2]-FBP calculated by mass isotopomer distribution analysis with 13C-enrichment data of glucose; M2(FBP:MIDA)pGlcUA, theoretical mole percent enrichment of [13C2]-FBP calculated by mass isotopomer distribution analysis with 13C-enrichment data of urinary paracetamol-glucuronide; M2(glc)blood, mole percent enrichment of [13C2]-glucose in blood; M2(pGlcUA)urine, mole percent enrichment of [13C2]-UDPglc sampled as urinary paracetamol-glucuronide; M6(glc)blood, mole percent enrichment of [U-13C]glucose in blood; M6(glc)infusate, mole percent enrichment of [U-13C]glucose in the infusate; M6(pGlcUA)urine, mole percent enrichment of [U-13C]-UDPglc measured in urinary paracetamol-glucuronide; MCR(glc), metabolic clearance rate of blood glucose; Ra(glc;endo), rate of endogenous plasma glucose appearance, not corrected for recycling of tracer; Ra(glc;whole body), whole body rate of appearance of glucose in the blood glucose pool; Ra(UDPglc;endo), rate of endogenous uridine diphosphoglucose appearance, not corrected for recycling of tracer; Ra(UDPglc;whole body), whole body rate of appearance of uridine diphosphoglucose; Rr(glc), rate of recycling of glucose tracer; Rr(UDPglc), rate of recycling of uridine diphosphoglucose tracer ([1-2H]-gal); totalRa(glc;endo), total endogenous glucose production, including recycling of tracer; and totalRa(UDPglc), total endogenous uridine diphosphoglucose production, including recycling of tracer.

Primary Parameters
 d(glc)M6(glc)blood/M6(glc)infuse
 d(UDPglc)M1(pGlcUA)urine/M1(gal)infuse
 c(glc)M6(pGlcUA)urine/M6(glc)blood
 c(UDPglc)M1(glc)blood/M1(pGlcUA)urine
 f(glc)M2(glc)blood/M2(FBP:MIDA)glc
 f(UDPglc)M2(pGlcUA)urine/M2(FBP:MIDA)pGlcUA
Whole Body Glucose Metabolism
 Ra(glc;whole body)Inf(glc)/d(glc)
 Ra(UDPglc;whole body)Inf(gal)/d(UDPglc)
 MCR(glc)Ra(glc;whole body)/[glc]
 Ra(glc;endo)Ra(glc;whole body) − Inf(gal)
 Ra(UDPglc;endo)Ra(UDPglc;whole body) − Inf(gal)
 Rr(glc){c(glc)/[1 − c(glc)]} × Ra(glc;endo)
 Rr(UDPglc){c(UDPglc)/[1 − c(UDPglc)]} × Ra(UDPglc;endo)
 TotalRa(glc;endo)Ra(glc;endo) + Rr(glc)
 TotalRa(UDPglc;endo)Ra(UDPglc;endo) + Rr(UDPglc)
 GNG(glc)f(glc) × [Ra(glc;whole body) + Rr(glc)]
 GNG(UDPglc)f(UDPglc) × [Ra(UDPglc;whole body) + Rr(UDPglc)]
 GLY(glc)Total Ra(glc;endo) − GNG(glc)
 GLY(UDPglc)[1 − c(glc)]totalRa(UDPglc;endo) − GNG(UDPglc)
Associated Individual Fluxes Through Enzymes Involved in Glucose Metabolism
 GNG(G6P){[1 − c(glc)] × GNG(glc)} + {[1 − c(UDPglc)] × GNG(UDPglc)}
 GKc(glc) × total Ra(UDPglc) + R(glc)
 G6PaseTotal Ra(glc;endo) = GNG(glc) + GLY(glc)
 GSTotalRa(UDPglc) = GNG(UDPglc) + GLY(UDPglc)
 GPGLY(glc) + GLY(UDPglc)

Statistics

For metabolite and mRNA levels, values are presented as mean ± standard error of the mean, and the level of significance was determined with the Student t test, with P < 0.05 considered significant. For fluxes, values are presented as mean ± standard error of the mean, and statistical significance was determined with multiple measurements of analysis of variance, with P < 0.05 considered significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Response on TDGA During Fasting

Biometric and plasma parameters in control mice and TDGA-treated mice are presented in Table 2. As published previously,15 blood glucose concentrations were statistically significantly lower in TDGA-treated mice compared to control mice from 3 hours onward. Moreover, the hepatic content of G6P decreased upon treatment [50.6 ± 15.6 versus 25.7 ± 2.6 nmol (g liver)−1, control versus TDGA, P < 0.05].15 The biochemical characteristics of the model show a resemblance to those in patients with acute presentation of a disorder of mFAO, which is also reflected by hypoketosis and an impaired free fatty acid/3-hydroxybutyrate ratio, which is known to be indicative for a pathological response to fasting.22

Table 2. Biometrical and Biochemical Parameters After 12 Hours of Fasting in Control and TDGA-Treated Mice
 ControlTDGA
  • Mice were injected with vehicle (controls) or 30 mg/kg TDGA intraperitoneally and fasted for 12 hours. Subsequently, they were anesthetized, and blood was obtained by cardiac puncture. Analyses of the various parameters were performed as described in the Materials and Methods section. Data are presented as mean (standard error of the mean) from at least 6 animals in each group. BOHB indicates 3-hydroxybutyrate; FFA, free fatty acid; FFA/BOHB, ratio of free fatty acids over 3-hydroxybutyrate; and TDGA, 2-tetradecylglycidic acid.

  • *

    P < 0.05.

Biometric parameters  
 Initial body weight (g)23.4 (0.4)23.8 (0.4)
 End body weight (g)21.1 (0.3)21.4 (0.3)
 Decreased body weight (%)10 (0)10 (1)
 Liver weight (g)0.98 (0.03)1.13 (0.02)*
 Relative liver weight (%)4.6 (0.1)5.3 (0.1)*
Plasma parameters  
 Glucose (mmol L−1)6.3 (0.6)3.4 (0.2)*
 Lactate (mmol L−1)2.1 (0.3)1.2 (0.1)
 FFA (umol L−1)827 (131)1577 (76)*
 BOHB (mmol L−1)1.88 (0.27)0.37 (0.06)*
 FFA/BOHB0.55 (0.04)4.65 (0.63)*
 Total carnitine (μmol L−1)25.7 (2.2)21.8 (0.6)
 Free carnitine (μmol L−1)4.4 (0.2)5.1 (0.3)
 Acylcarnitine (μmol L−1)21.3 (2.1)16.6 (0.4)

Optimization of the Experimental Approach for Measuring Hepatic Glucose Metabolism Under Hypoglycemic Conditions

Using the infusion protocol as described previously,17 we found that the blood glucose concentrations only slightly increased in fasting control mice, that is, from 6.6 ± 0.2 to 7.3 ± 0.2 mmol L−1 after 6 hours. However, in TDGA-treated hypoglycemic mice, blood glucose concentrations increased from 5.0 ± 0.5 to 6.7 ± 0.4 mmol L−1 during the infusion (P < 0.05), suggesting interference by the infusion. Therefore, to study hepatic glucose metabolism under hypoglycemic conditions, we adapted our infusion protocol. Optimal results were obtained with a solution containing 7.0 μmol mL−1 [U-13C]glucose, 81 μmol mL−1 [2-13C]glycerol, 17 μmol mL−1 [1-2H]galactose, and 1 mg mL−1 paracetamol at an infusion rate of 0.6 mL hour−1. With the new infusion protocol, the TDGA-treated mice remained hypoglycemic throughout the infusion experiment (Fig. 2). The start of the infusion experiment (t = 0) corresponds with 9 hours of fasting. Importantly, analytical criteria were still fulfilled as described previously17 (data not shown). In the steady state, the enrichment of blood glucose due to 13C incorporation derived from [2-13C]-glycerol was 21.4% ± 0.6% and 2.3% ± 0.1% for singly and doubly labeled glucose molecules in control mice and 19.5% ± 0.7% and 2.1% ± 0.1% in TDGA-treated mice. Similarly, the values for Par-GlcUA were 18.0% ± 0.6% and 1.8% ± 0.1% in control mice and 15.0% ± 0.7% and 1.5% ± 0.1% in TDGA-treated mice. All experiments were performed with this optimized protocol.

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Figure 2. Blood glucose concentrations measured during the infusion experiment in (○) control and (●) 2-tetradecylglycidic acid–treated mice. Mice were injected with vehicle (controls) or 30 mg/kg 2-tetradecylglycidic acid intraperitoneally and fasted for 9 hours. Subsequently, the 6-hour triple-label infusion experiment was performed, and blood glucose was measured as described in the Materials and Methods section. Data are presented as mean (standard error of the mean) for at least 6 animals in each group. *P < 0.05.

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Alterations in Hepatic Glucose Metabolism

In Table 3, the primary parameters are given. In the calculations of f(glc) and f(UDPglc), the theoretical enrichment of [13C2]-FBP during infusion of [2-13C]-glycerol is an estimation using MIDA algorithms. In control animals, the calculated enrichment of [13C2]-FBP was 3.2% ± 0.2% {theoretical mole percent enrichment of [13C2]-FBP calculated by mass isotopomer distribution analysis with 13C-enrichment data of glucose [M2(FBP:MIDA)glc]} and 2.8% ± 0.2% {theoretical mole percent enrichment of [13C2]-FBP calculated by mass isotopomer distribution analysis with 13C-enrichment data of urinary paracetamol-glucuronide [M2(FBP:MIDA)pGlcUA]} with blood glucose and Par-GlcUa, respectively, and in TDGA-treated animals, it was 3.1% ± 0.2% and 2.8% ± 0.2%, respectively. Figure 3 illustrates the total endogenous glucose production [totalRa(glc;endo); Fig. 3A] and MCR of glucose (Fig. 3B) measured during the infusion experiment. TotalRa(glc;endo) remained unaffected upon TDGA treatment (127 ± 10 versus 118 ± 7 μmol kg−1 minute−1, control versus TDGA, not significant). In contrast, MCR of glucose was significantly enhanced in TDGA-treated mice (15.9 ± 0.9 versus 26.3 ± 1.1 mL kg−1 minute−1, control versus TDGA, P < 0.05). The total rate of the de novo synthesis of G6P in TDGA-treated mice was suppressed by only ∼21%, but the difference was statistically significant (108 ± 19 versus 85 ± 6 μmol kg−1 minute−1, control versus TDGA, P < 0.05). Figure 4 illustrates that the partitioning of newly synthesized G6P toward blood glucose was significantly enhanced in TDGA-treated mice compared to control mice (69% ± 5% versus 78% ± 3%, control versus TDGA, P < 0.05). Glucose recycling (rate of recycling of the glucose tracer) decreased upon TDGA (26 ± 14 versus 12 ± 4 μmol kg−1 minute−1, control versus TDGA, P < 0.05), and Fig. 5 illustrates that in TDGA-treated mice, glucose recycling contributed less to endogenous glucose production than in control mice (20% ± 3% versus 10% ± 1%, control versus TDGA, P < 0.05). Figure 6 presents the calculated mean values for the individual fluxes through the relevant enzyme systems in G6P metabolism during the last 3 hours of the infusion experiment. As is clear from this figure, glycogen synthase flux (total endogenous uridine diphosphoglucose production, including recycling of tracer) was significantly decreased upon TDGA, whereas the flux through glycogen phosphorylase remained unaltered. Finally, the flux through glucokinase, catalyzing the first step of glycolysis and glucose recycling, was strongly decreased in TDGA-treated mice.

Table 3. Primary Isotopic Data in the Steady State During the Triple-Label Infusion Experiment in 9-Hour–Fasted Control and TDGA-Treated Mice
 ControlTDGA
  • Mice were injected with vehicle (controls) or 30 mg/kg TDGA intraperitoneally and fasted for 9 hours. Subsequently, the infusion experiment was performed. Analyses of the isotopic enrichment and subsequent calculations were performed as described in the Materials and Methods section. Data are presented as mean (standard error of the mean) from 7 animals in each group. c(glc) indicates fractional contribution of plasma glucose to uridine diphosphoglucose formation; c(UDPglc), fractional contribution of uridine diphosphoglucose to blood glucose formation; d(glc), fractional contribution of infused glucose to blood glucose; d(UDPglc), fractional contribution of infused galactose to uridine diphosphoglucose; f(glc), fractional contribution of newly synthesized glucose to plasma glucose; f(UDPglc), fractional contribution of newly synthesized glucose to the uridine diphosphoglucose pool; MIDA, mass isotopomer distribution analysis; and TDGA, 2-tetradecylglycidic acid.

  • *

    P < 0.05.

Isotope dilution  
 d(glc)0.029 (0.002)0.027 (0.001)
 d(UDPglc)0.152 (0.008)0.221 (0.010)*
Isotope exchange  
 c(glc)0.19 (0.03)0.10 (0.01)*
 c(UDPglc)0.23 (0.02)0.20 (0.02)
MIDA  
 f(glc)0.73 (0.02)0.67 (0.03)
 f(UDPglc)0.65 (0.02)0.54 (0.01)*
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Figure 3. (A) Total endogenous glucose production and (B) metabolic clearance rate of glucose measured during the infusion experiment in (○) control and (●) 2-tetradecylglycidic acid–treated mice. Mice were injected with vehicle (controls) or 30 mg/kg 2-tetradecylglycidic acid intraperitoneally and fasted for 9 hours. Subsequently, the 6-hour infusion experiment was performed, and blood glucose was measured as described in the Materials and Methods section. Data are presented as mean (standard error of the mean) for at least 6 animals in each group. *P < 0.05.

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Figure 4. Partitioning of the rates of the de novo synthesis of glucose-6-phosphate (G6P) to (□) glucose and (▪) uridine diphosphoglucose during the last 3 hours of the infusion experiment in control and 2-tetradecylglycidic acid (TDGA)–treated mice. Mice were injected with vehicle (controls) or 30 mg/kg TDGA intraperitoneally and fasted for 9 hours. Subsequently, the 6-hour infusion experiment was performed as described in the Materials and Methods section. Data are presented as mean (standard error of the mean) for at least 6 animals in each group. *P < 0.05.

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Figure 5. Partitioning of the rates of the cycling between (□) glucose and(▪) glucose-6-phosphate during the last 3 hours of the infusion experiment in control and 2-tetradecylglycidic acid (TDGA)–treated mice. Mice were injected with vehicle (controls) or 30 mg/kg TDGA intraperitoneally and fasted for 9 hours. Subsequently, the 6-hour infusion experiment was performed, and blood glucose was measured as described in the Materials and Methods section. Data are presented as mean (standard error of the mean) for at least 6 animals in each group. *P < 0.05.

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Figure 6. Metabolic fluxes through the relevant enzymes involved in hepatic glucose metabolism during the last 3 hours of the infusion experiment in (□) control and(▪) 2-tetradecylglycidic acid (TDGA)–treated mice. Mice were injected with vehicle (controls) or 30 mg/kg TDGA intraperitoneally and fasted for 9 hours. Subsequently, the 6-hour infusion experiment was performed, and blood glucose was measured as described in the Materials and Methods section. Data are presented as mean (standard error of the mean) for at least 6 animals in each group. *P < 0.05.

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Alterations in Hepatic mRNA Expressions

Expression levels of hepatic genes encoding enzymes involved in glucose metabolism are presented in Fig. 7. Expression of glucokinase flux and pyruvate kinase (Pk) were significantly decreased. Pyruvate dehydrogenase kinase isozyme 4 expression was increased 30-fold (data not shown). Together, these observations reflect a decreased glycolytic capacity. The expression levels of glucose transporter 2 and glycogen phosphorylase flux were significantly reduced in livers of TDGA-treated mice. Interestingly, expressions of genes encoding the key enzymes involved in de novo synthesis of G6P [for example, Pc, phosphopyruvate carboxykinase (Pepck), and Fbp-1] remained unaltered upon TDGA treatment. TDGA treatment enhanced the expression of glucose-6-phosphatase hydrolase in contrast to that of glucose-6-phosphatase translocase. The expressions of genes encoding enzymes involved in mFAO, such as Pparα, Octn2, Cpt1a, Cact, Cpt2, Lcad, Mcad, and Hmgcoas, were all significantly increased by 2- to 4-fold.23

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Figure 7. Changes in hepatic relative gene expression of enzymes involved in glucose metabolism in (□) control and (▪) 2-tetradecylglycidic acid–treated mice. Mice were injected with vehicle (controls) or 30 mg/kg 2-tetradecylglycidic acid intraperitoneally and fasted for 12 hours. Livers were removed, and hepatic mRNA levels were measured by real-time reverse-transcription polymerase chain reaction as described in the Materials and Methods section. Results were normalized to β-actin and 18S mRNA levels; data from control mice were defined as 1. Each value represents the mean ± standard error of the mean for 7 animals in each group; the P value represents the statistical level of significance. Fbp1 indicates fructose-1,6-bisphosphatase-1; G6ph, glucose-6-phosphatase hydrolase; G6pt, glucose-6-phosphatase translocase; Gk, glucokinase flux; Glut2, glucose transporter 2; Gp, glycogen phosphorylase flux; Gs, glycogen synthase flux; mRNA, messenger RNA; Pc, pyruvate carboxylase; Pepck, phosphopyruvate carboxykinase; and Pk, pyruvate kinase.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

TDGA has been used successfully to study the effects of acute inhibition of mFAO both in vitro and in vivo12–14 and has allowed us to decipher changes in glucose metabolism underlying hypoglycemia during inhibition of mFAO. For the first time, we present an integrated in vivo model of hepatic carbohydrate flux distributions upon the blocking of mFAO by TDGA. The results surprisingly show that, after acute inhibition of mFAO in short-term–fasted mice, endogenous glucose production remained largely unaffected. De novo synthesis of G6P was only slightly suppressed, but glucose cycling was drastically decreased. Importantly, MCR of glucose was significantly enhanced under the conditions induced by TDGA treatment.

Different approaches are available to measure GNG. Like any approach, the MIDA approach combined with the isotopic model is based on certain assumptions. The validity of the MIDA approach has been substantiated in various studies.8, 17, 19, 24 Still, some controversy remains, particularly concerning the use of [2-13C]-glycerol and the precursor pool from which both blood glucose and Par-GlcUA are produced.25, 26 In the MIDA approach, [2-13C]-glycerol is used to label the triose phosphate pool, out of which [13C1]-FBP and [13C2]-FBP are synthesized. Consequently, only glucose-producing cells containing glycerol kinase will contribute to the estimation of GNG. Therefore, the calculated de novo synthesis of blood glucose and uridine diphosphoglucose (UDPglc) will tend to be an underestimate. In this respect, it is encouraging that both the 2H2O method and the MIDA approach give similar values for the rate of GNG, for instance, in healthy volunteers after a prolonged fast.20, 27 In the isotopic model, FBP is the common precursor for both blood glucose and UDPglc. In other words, cells that produce blood glucose are assumed to produce Par-GlcUA as well. Consequently, the MIDA calculations should give very similar values of the enrichment of [13C2]-FBP whether blood glucose or Par-GlcUA is used. As is clear, M2(FBP:MIDA)glc is not significantly different from M2(FBP:MIDA)pGlcUA, although the latter tended to be higher both in control and TDGA-treated animals. Apparently, most of the newly synthesized blood glucose and Par-GlcUA do derive from a common precursor pool of FBP. Some debate exists concerning how to calculate the endogenous glucose production from isotope dilution data.28 With [U-13C]-glucose, a recycling label, the initial calculated endogenous production of glucose and UDPglc {rate of endogenous plasma glucose appearance, not corrected for recycling of tracer [Ra(glc;endo)], and rate of endogenous uridine diphosphoglucose appearance, not corrected for recycling of tracer [Ra(UDPglc;endo)], respectively} underestimates the real endogenous production rates by a factor equal to the rate of recycling of the label. Therefore, Ra(glc;endo) and Ra(UDPglc;endo) should be corrected for this recycling to obtain an appropriate estimation of the endogenous production [in this study, totalRa(glc;endo) and totalRa(UDPglc;endo)].

Contrary to what was expected, acute inhibition of mFAO in short-term–fasted mice by TDGA did not affect endogenous glucose production, nor was a major decrease of the de novo synthesis of G6P observed (Fig. 6). The minor decrease in de novo synthesis of G6P was compensated by a significant increase in the partitioning of newly synthesized G6P toward blood glucose (Fig. 4) combined with decreased glucose cycling upon TDGA treatment (Fig. 5). It is noteworthy that Spiekerkoetter et al.29 speculated that endogenous glucose production might be impaired in mice deficient in very long chain acyl coenzyme A dehydrogenase, the first enzyme in mFAO following CPT-II.29 They observed a decrease of the hepatic content of intermediates of GNG, such as G6P (similar to our experiments), glycerol-3-phosphate, and dihydroxyacetone-phosphate during hypoglycemia, whereas the in vitro measured activity of glucose-6-phosphatase (the combined activity of G6P-hydrolase and G6P-transporter) remained unaffected. Our measurements of unaffected endogenous glucose production during hypoglycemia with mFAO inhibited by TDGA treatment do not support this speculation. In contrast, hypoglycemia was accompanied by a significantly increased MCR of glucose; this value doubled upon mFAO blockade (Fig. 3B). Although insulin was not measured in this series of experiments, previous measurements of insulin concentrations in TDGA-treated ApoE*3Leiden mice showed a decrease of insulin concentration immediately after treatment with TDGA.30 Taking this observation into consideration, we anticipated that in hypoglycemic TDGA-treated mice, peripheral glucose consumption would be decreased. Apparently, mechanisms other than hormonal regulation are operative in TDGA-treated mice. We observed an enhanced peripheral glucose utilization, most likely compensating for the lack of peripheral mFAO and/or hepatic ketogenesis. This seems to be the major cause of hypoglycemia associated with impaired mFAO.

It becomes clear that, in vivo, constant endogenous glucose production may be ensured by several adaptive mechanisms in the liver. This study shows major changes in G6P partitioning and decreased glucose cycling in mice after acute inhibition of mFAO. Xu et al.31 observed a switch in preferred gluconeogenic substrates, that is, from lactate to glycerol, in PPARα-deficient mice. Recently, Burgess et al.32 showed that altering the cataplerotic flux of oxaloacetate out of the Krebs cycle into GNG provides another mechanism to ensure endogenous glucose production. This observation is in line with earlier data, indicating a role for both Pk33 and Pc2 in the partitioning of pyruvate into oxaloacetate and thereby into GNG. Quantification of hepatic mRNA levels revealed no significant changes in expression of key genes implicated in de novo synthesis of G6P, that is, Pepck, Pc, and Fbp1 (Fig. 7). However, the mRNA levels of glucose-6-phosphatase hydrolase, encoding part of the glucose-6-phosphatase complex, were significantly enhanced. This observation is in line with the alterations in G6P partitioning during the infusion experiment.

In speculation about the etiology underlying hypoglycemia in human disorders of mFAO, several issues should be addressed. First, an acute, pharmacologically induced inhibition of mFAO in mice does not equal an acute presentation of a permanent enzyme defect in humans. Second, acute presentation with hypoketotic hypoglycemia in patients is often associated with additional metabolic stress, such as an infection or extensive exercise. Taking these considerations into account, we describe now an in vivo mouse model characterized by significant hypoketotic hypoglycemia and a decreased liver G6P content. In our opinion, the model resembles the biochemical characteristics in patients with acute presentation of disorders of mFAO.7 We speculate that peripheral effects (that is, the inhibition of mFAO and/or ketogenesis) leading to increased peripheral glucose utilization rather than hepatic effects underly hypoglycemia associated with mFAO defects. Bougneres et al.6 reported an infant with impaired mFAO caused by CPT-I deficiency. The authors observed hypoketotic hypoglycemia upon prolonged fasting, which was reversed by infusion of medium-chain-length triglycerides. The authors hypothesized that the impairment of mFAO led to a decrease in energy, acetyl coenzyme A, and reducing equivalents and consequently to the GNG suppression. However, it is known that mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A synthase deficiency, a human disorder of ketogenesis, is sufficient to induce hypoketotic hypoglycemia without defective mFAO.34, 35 Hence, peripheral effects may also explain the observations by Bougneres et al.

In conclusion, this study provides evidence for only a weak metabolic link between mFAO and glucose metabolism in vivo: upon acute inhibition of mFAO in mice, endogenous glucose production was unaffected, and de novo synthesis of G6P was only slightly suppressed. This was associated with a decreased cycling of glucose and enhanced partitioning of newly synthesized G6P toward blood glucose. However, MCR of glucose strongly increased during mFAO blockade, reflecting alternative substrate utilization by peripheral tissues resulting in hypoglycemia in the hypoketotic state. These results indicate that the textbook paradigm that links mFAO to GNG and fasting-induced hypoglycemia requires revision.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We thank R. Havinga, A. Gerding, K. Bijsterveld, and J. F. Baller for excellent technical assistance.

References

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  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

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