1 Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee
2 Mary Nell and Ralph B. Rogers Magnetic Resonance Center, Department of Radiology, Veteran Affairs Medical Center, University of Texas Southwestern Medical Center, Dallas, Texas
3 Department of Surgery, Vanderbilt University School of Medicine, Nashville, Tennessee
4 Vanderbilt Diabetes Center, Vanderbilt University School of Medicine, Nashville, Tennessee
5 Department of Internal Medicine, Veteran Affairs Medical Center, University of Texas Southwestern Medical Center, Dallas, Texas
6 Department of Chemistry, University of Texas at Dallas, Dallas, Texas
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ABSTRACT |
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Glucose homeostasis requires a precise balance between glucose production and utilization. The liver plays a key role in maintaining this balance during fasting by being the major site for both glycogenolysis and gluconeogenesis. During a prolonged fast, when glycogen stores are exhausted, hepatic gluconeogenesis is considered essential for maintaining glucose homeostasis. The conversion of oxaloacetate to phosphoenolpyruvate (PEP) by PEPCK has long been thought to play a key role in this process (1,2), as the enzyme provides the only known path whereby tricarboxylic acid (TCA) cycle intermediates can be converted to glucose. PEPCK is highly expressed in the liver, where it is adaptively regulated by a variety of different hormones and other agents in a manner that parallels gluconeogenic flux (2,3). Moreover, techniques of cross-organ substrate balance plus use of radioactive tracers in humans and dogs have clearly demonstrated that the liver is the major site of glucose production during a fast (4,5).
Previously, we examined the effects of either globally reducing PEPCK gene expression or eliminating PEPCK in a liver-specific manner on glucose homeostasis. We found that 1) mice that totally lack PEPCK die within 3 days of birth, apparently from hypoglycemia; 2) mice with a 50, 90, and 95% global reduction in PEPCK gene expression maintain a normal blood glucose concentration after a 24-h fast; 3) resting mice with a liver-specific knockout of the enzyme also maintain fasting euglycemia; and 4) mice with markedly diminished PEPCK gene expression develop profound abnormalities in lipid metabolism, including hepatic steatosis, after fasting (6).
Because our previous findings challenged current concepts of PEPCKs role in gluconeogenesis, as well as the exclusive role of the liver in maintaining glucose homeostasis during a fast, we sought to determine the mechanisms whereby mice with a liver-specific knockout of PEPCK are able to maintain fasting euglycemia. Here we describe studies that combined the use of isotopic tracers, biochemical analysis, and nuclear magnetic resonance (NMR) spectroscopy to measure the contributions from glycogenolysis, glycerol, PEP, and TCA cycle activity and fluxes of some pathways associated with the TCA cycle (7,8) to gluconeogenic flux, both in the presence and absence of hepatic PEPCK. Together, the results of these studies indicate the existence of multiple compensatory mechanisms, both intra- and extrahepatic, that act to preserve normal fasting blood glucose in the absence of hepatic PEPCK.
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RESEARCH DESIGN AND METHODS |
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Liver perfusion.
Isolated livers were perfused with Krebs-Henseleit bicarbonate buffer alone, buffer containing 5 mmol/l lactate and 0.5 mmol/l pyruvate, or buffer containing 5 mmol/l glycerol, as previously described (9). The flow rate into the cannulated portal vein was 8 ml · min-1 · liver-1. Postperfusion samples were collected from inferior vena cava.
Basal in vivo isotopic kinetic experiments.
All experiments were performed on awake, unrestrained, unstressed mice. Catheterizations and isotopic infusions were performed as previously described (6,10). Each experiment consisted of a 70-min tracer equilibration period (-100 to -30 min), a 30-min control period (-30 to 0 min), and a 120-min test period (0 to 120 min). Blood was collected from a carotid catheter at 30, 0, 30, 60, 90, and 120 min. Basal glucose turnover rates were determined by a 5-µCi bolus injection followed by a continuous infusion at 0.05 µCi/min of [3-3H]glucose, purified by high-performance liquid chromatography (HPLC), for 220 min. At the end of each experiment, the livers were excised and immediately freeze-clamped in liquid nitrogen for analysis of [3H]-specific activities of UDP-glucose and concentrations of hepatic metabolites.
To determine glucose cycling rates, HPLC-purified [2-3H]glucose, [6-3H]glucose, and [U-14C]glucose were bolus-injected at 20, 5, and 3 µCi, respectively, then continuously infused at 0.2, 0.05, and 0.03 µCi/min, respectively, into a jugular catheter throughout the experiment. Deproteinized plasma was passed through two columns, a cation-exchange column (Dowex 50Wx8200; Sigma, St. Louis, MO) and an anion-exchange column (Amberlite IRA-67; Sigma), and glucose was eluted with water. The elutes were treated with hexokinase, ATP, and phosphoglucose-isomerase to selectively remove 3H from carbon 2 of [2-3H]glucose. Glucose turnover rates measured using three types of tracer glucose were then calculated.
Glycerol turnover was determined by continuous infusion of [d5]glycerol (Cambridge Isotope Laboratories, Woburn, MA) into the jugular vein at 0.48 µmol · kg-1 · min-1 for 220 min. After a 70-min tracer equilibration period, six blood samples were collected from the carotid catheter at 30-min intervals. Two consecutive 30-µl samples were pooled, [d5]-glycerol enrichment was determined using gas chromatography-mass spectrometry (GCMS; Hewlett-Packard, Palo Alto, CA), and the glycerol turnover rate was calculated, as previously described (11). HPLC-purified [6-3H]glucose was also simultaneously infused in the experiment with [d5]-glycerol infusion to obtain the glucose turnover rate in the presence of glycerol.
Determination of flux alterations in whole-body and hepatic glucose metabolism during hyperglycemic clamp.
Hyperglycemic clamp studies were performed together with infusions of [3-3H]glucose, as previously described (10). At the end of each experiment, whole liver and hindlimb muscle samples were collected for glycogen and UDP-glucose analysis. The total hepatic glycogen synthesis rate (micromoles per kilogram per minute) was calculated as (total tritium counts in hepatic glycogen)/(hepatic UDP-glucose tritium specific activity [SA] per body weight per 120 min). The rate of hepatic glycogen synthesis from the direct pathway was calculated as (total tritium counts in hepatic glycogen)/(plasma glucose tritium SA per body weight per 120 min). Muscle glycogen synthesis was expressed as glucose incorporation into glycogen (tritium counts per gram tissue per plasma glucose tritium SA).
Infusion of stable isotopes for metabolic flux profiling by NMR.
Total body water was enriched to 3% by direct intraperitoneal injection of 0.8 ml of D2O 30 min before infusion of 13C-labeled tracers (Cambridge Isotope Laboratories). 2H enrichment in plasma water reaches a steady state in <30 min using this protocol (S.B., C.R.M., A.D.S., unpublished observations). A continuous infusion of [3-3H]glucose dissolved in D2O was started 10 min after the intraperitoneal injection of D2O and continued for 80 min to determine glucose turnover rate. At 30 min, a mixture of unenriched glycerol and 99% [U-13C3]propionate (12 mg each substrate/30 g) dissolved in saline was infused (
1 µl/min adjusted for variations in body weight) into a jugular vein catheter for 60 min. At the end of the experiment,
1 ml of blood was collected via the carotid artery catheter; 60 µl of plasma were used for determining 2H enrichment and 10 µl were used to measure the SA of [3H]glucose. Glucose was extracted from the remaining plasma by ion-exchange chromatography then converted to 1,2-isopropylidene glucofuranose (monoacetone glucose [MAG]), as described elsewhere (12)
NMR spectroscopy.
2H and 13C NMR spectra were collected using a 14.1 T Inova NMR spectrometer (Varian Instruments, Palo Alto, CA), 3-mm sample probes, and parameters previously described (7,13). Multiplet areas in the 13C spectra were measured using the line fitting routine in the PC-based NMR program, NUTS (Acorn NMR, Fremont, CA).
Analytical procedures.
Plasma glucose was measured using hexokinase and glucose-6-phosphate (G6P) dehydrogenase (14). Glucose in samples from the liver perfusion studies was measured using a glucose kit (Trinder; Sigma). Blood glucose was measured using a glucose meter (HemoCue, Mission Viejo, CA). Hepatic glucokinase and glucose-6-phosphatase (G6Pase) activities were measured as previously described (15). Hepatic UDP-glucose- and UDP-galactose-specific activities were measured with a chromatographic and HPLC method using a reverse-phase Supelcosil C18 column (Waters, Milford, MA). All results are presented as means ± SE. Statistical significance was determined by one-way ANOVA. P < 0.05 was considered statistically significant.
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RESULTS |
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High resolution 2H and 13C NMR of MAG derived from plasma glucose from control and knockout mice are compared in Fig. 2. Although the 2H spectra were nearly identical, differences were readily apparent in the two 13C spectra. As shown previously, the distribution of 2H in H2, H5, and H6S provides a measure of glycogenolysis and gluconeogenesis from glycerol versus the TCA cycle (8). The H5/H2 ratio in spectra of MAG from control (n = 4) and knockout (n = 4) animals averaged 1.02 ± 0.09 and 0.94 ± 0.08, respectively. This confirmed that the contribution of glycogen to plasma glucose was insignificant in both 24-h-fasted control and knockout mice. Similarly, the gluconeogenic contribution coming from the level of the TCA cycle was given by the H6S/H2 intensity ratios, 0.78 ± 0.15 in controls and 0.60 ± 0.15 in knockouts (P ≤ 0.128), with the contribution of glycerol to glucose production being determined by the difference (0.24 ± 0.08 in controls and 0.34 ± 0.15 in knockouts; P ≤ 0.286). Although the tissue origin of this gluconeogenic flux could not be determined from these data, the 2H NMR results clearly indicated that the TCA cycle contributes somewhat more to overall glucose production in control versus knockout animals. Interestingly, the glycerol contribution to gluconeogenesis was only slightly higher in knockout animals in vivo, even though the perfusion experiments showed that livers from knockout mice have a much greater capacity to make glucose (Fig. 1). It is also interesting to note that glucose production from glycerol in control animals infused with the additional 1 µmol/min glycerol did not differ from that in control animals without glycerol co-infusion (glycerol typically contributes
25% in mice without infusion of any substrates; S.B., C.R.M., A.D.S., unpublished observations).
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DISCUSSION |
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During prolonged starvation, when hepatic glycogenolysis is negligible, hepatic G6P is produced only from gluconeogenesis and by glucose phosphorylation, mostly via glucokinase. The six-fold higher [3H] SA ratio of hepatic UDP-glucose (taken as reflection of the hepatic G6P pool) (16) to plasma glucose in the knockout mice indicates there was either a marked increase in the rate of hepatic glucose phosphorylation, a marked decrease in the amount of gluconeogenesis, or both. The consistent differences in [2-3H]glucose- and [6-3H]glucose-derived glucose turnover rates between the knockout and control mice (Table 2) and the 50% decrease in glucokinase activity in the liver of the knockout animals (Table 1) strongly suggest that both glucose phosphorylation and G6P synthesis by gluconeogenesis was lower in the mice lacking hepatic PEPCK. Furthermore, the 33% decrease in G6Pase Vmax and the 90% decrease in hepatic G6P concentration also point to a marked decrease in G6Pase-mediated flux to glucose. Taken together, these results indicate markedly diminished hepatic glucose production in these knockout mice, even though endogenous glucose production rates were near normal.
One might anticipate that hepatic gluconeogenesis from glycerol would be elevated in the knockout mice, especially as they have an elevated hepatic gluconeogenic capacity from glycerol, as shown by liver perfusion. Based on the glycerol turnover rate of 31.4 µmol · kg-1 · min-1 in the knockout mice (no different from control values) (Table 2), glycerol could contribute as much as 15.7 µmol · kg-1 · min-1 or 35% of total body glucose production, assuming that the glycerol turnover rate equals the gluconeogenic rate from glycerol. This value is consistent with the NMR-derived result (Fig. 3) that gluconeogenesis from glycerol in animals lacking hepatic PEPCK is 19 ± 7 µmol glucose µmol · kg-1 · min-1 or 34% of total glucose turnover. It is interesting that this consistency was maintained despite the fact that glycerol was included in the infusate in the NMR experiment, but not in the [d5]-glycerol turnover experiment. Furthermore, the contribution of glycerol to gluconeogenesis as measured by 2H NMR was the same in control and knockout animals. Thus, one can conclude that glucose production from glycerol in mice must occur largely in tissues other than liver and that, in the absence of hepatic PEPCK, the liver does not produce more glucose from glycerol in vivo, despite the increased metabolic capacity in vitro (Fig. 1).
Compensatory extrahepatic gluconeogenesis in mice lacking hepatic PEPCK.
Given that the liver makes only a small amount of glucose without hepatic PEPCK, it follows that gluconeogenesis by extrahepatic tissues is responsible for a majority of glucose synthesized in mice lacking PEPCK. Although these data do not identify which organs are responsible for this compensatory gluconeogenesis, the kidney (17) and/or intestine (18) are likely sources as both express PEPCK and other gluconeogenic enzymes. Based on our finding of a twofold increase in plasma glutamine (892.2 ± 9.1 vs. 444.6 ± 6.9 µmol/l in knockout versus control mice, respectively; P < 0.001; n = 7), it is possible that glutamine serves as a major gluconeogenic substrate in the intestine and kidney of PEPCK knockout mice (1820). Although the majority of glucose produced in these animals was derived from an unlabeled gluconeogenic substrate ([1,2,3-13C]propionate was present only at tracer levels), the NMR experiment did not provide information about the identity of the gluconeogenic substrate.
The 13C NMR analysis of plasma glucose revealed important differences in these animals: total anaplerosis from the TCA cycle was reduced by 37% (P ≤ 0.02) and pyruvate cycling was reduced by 52% (P ≤ 0.059), whereas TCA cycle flux was actually higher by 49% (P ≤ 0.047) in knockout animals compared to controls. Thus, although mice lacking hepatic PEPCK were able to circumvent their inability to convert oxaloacetate to PEP in the liver, the compensatory mechanisms involved may have required additional energy expenditure. This observation is consistent with our previous finding that the expression of genes encoding hepatic pyruvate carboxylase, alanine aminotransferase, and enzymes in both the malate-aspartate shuttle and TCA cycle are increased in these animals (6). Moreover, the abundance of mRNA encoding the mitochondrial dicarboxylate carrier protein, a component of the inner mitochondria membrane responsible for transporting malate and succinate across the inner membrane in exchange for phosphate, sulfate, or thiosulfate (21), is increased 3.5-fold during fasting (data not shown). Because the operation of anaplerotic reactions requires the coexistence of cataplerotic reactions (22), it is likely that the elevated hepatic gene expression for the above proteins leads to a net release of glutamine from the liver to the blood as a response to the blockage of hepatic gluconeogenesis from TCA cycle intermediates in these knockout animals. Thus, although the liver is incapable of producing much glucose in the absence of hepatic PEPCK, this does not mean that it ceases to be metabolically involved in maintaining glucose homeostasis.
Diminished glucose utilization in mice lacking hepatic PEPCK.
The slight decrease in the basal glucose turnover and glucose clearance rates in the presence of similar levels of plasma insulin hint at a defect in glucose disposal. The hyperglycemic clamp study, which showed a marked decrease in glycogen synthesis from glucose in both liver and skeletal muscle, revealed that there are coexisting defects in glucose utilization in the knockout mice. In the case of the liver, our finding of a 46% decrease in hepatic glucokinase activity in the knockout mice is consistent with decreased hepatic glucose utilization, as glucose phosphorylation by this enzyme is essential (23).
Muscle glycogen synthesis was also found to be impaired in knockout mice, and thus is another indicator of impaired peripheral glucose utilization. This may reflect the markedly abnormal lipid metabolism that occurs in these mice during fasting. Even though the overall lipolytic rate was not altered, as suggested by similar glycerol turnover rates between the control and knockout mice, the fasting plasma free fatty acid and triglyceride concentrations were elevated. Furthermore, severe hepatic steatosis occurred in the knockout mice, which likely reflected an inhibition of hepatic fatty acid oxidation despite upregulated gene expression of fatty acid oxidation enzymes (6). Moreover, hepatic mRNAs for apolipoprotein CII, a specific activator of lipoprotein lipase, and CD36, a membrane fatty acid translocase, were increased 3.5- and 6-fold, respectively, in the fasted knockout mice (data not shown). The net effect of the changes would be elevated lipid delivery from secreted hepatic VLDL into the muscle by lipoprotein lipase, which would increase muscle concentrations of triglycerides and fatty acyl-CoA associated with impaired glucose utilization in muscle.
In summary, mice lacking hepatic PEPCK have a markedly impaired hepatic gluconeogenesis, but maintain fasting euglycemia through greatly increased extrahepatic gluconeogenesis coupled with mildly diminished peripheral glucose utilization. 2H and 13C NMR analysis of plasma glucose, reported here for the first time in mice, revealed that euglycemia in these fasted animals is achieved at the expense of marked perturbations in whole-body energy metabolism.
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ACKNOWLEDGMENTS |
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We thank C. Malabanan, K.D. Shelton, J. Lindner, D. Barrick, M. Zheng, Y. Fujimoto, W. Snead, and Y. Yang for excellent technical assistance, and A.D. Cherrington for constructive discussions and proofing of the manuscript.
Address correspondence and reprint requests to Dr. Mark A. Magnuson, 702 Light Hall, Vanderbilt University School of Medicine, Nashville, TN 37232-0615. E-mail: mark.magnuson{at}vanderbilt.edu
Received for publication July 12, 2002 and accepted in revised form March 13, 2003
G6P, glucose-6-phosphate; G6Pase, glucose-6-phosphatase; GCMS, gas chromatography-mass spectrometry; HPLC, high-performance liquid chromatography; MAG, monoacetone glucose; NMR, nuclear magnetic resonance; PEP, phosphoenolpyruvate; SA, specific activity; TCA, tricarboxylic acid
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REFERENCES |
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