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Deficiency Diminishes Insulin-Responsiveness of Gluconeogenic/Glycolytic/Pentose Gene Expression and Substrate Cycle Flux
Department of Medicine (J.X., V.C., C.T., M.F.S., I.J.K.) and Laboratory of Metabolomics (J.X., V.C., C.T., I.J.K.), David Geffen School of Medicine, and Departments of Biological Chemistry (J.X.) and Pathology (S.B.J.) and Molecular Biology Institute (I.J.K.), University of California, Los Angeles, California 90095; and Department of Pediatrics, Harbor-University of California-Los Angeles Research and Education Institute (S.B., W.N.P.L.), Torrance, California 90502
Address all correspondence and requests for reprints to: Dr. Irwin J. Kurland, Molecular Biology Institute, University of California, Los Angeles, California 90095. E-mail: irwinjk{at}earthlink.net.
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Abstract |
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(PPAR) modulates insulin action in a compensatory fashion for hepatic glucose balance vs. peripheral glucose disposal. Therefore, we have examined the expression of insulin-dependent gluconeogenic/glycolytic/pentose cycle enzymes and compared these to insulin responsiveness for peripheral vs. hepatic substrate flux and futile cycling in the PPAR knockout mouse. Hepatic gluconeogenic flux, glucose absorption, clearance and recycling, as well as in vivo glucose disposal were evaluated using new mass isotopomer methods. Insulin-dependent gluconeogenic/glycolytic/pentose cycle enzyme expression and glucose futile cycling were diminished; however, glucose disappearance was increased. This supports the hypothesis of hepatic insulin resistance and increased peripheral glucose uptake as compensatory events secondary to the decrease in fatty acid oxidation characteristic of the PPAR knockout. We conclude that 1) the loss of PPAR results in lower expression levels and diminished response to meal regulation for gluconeogenic/glycolytic enzyme expression; and 2) consequently, substrate/futile cycling of glucose is decreased when PPAR is absent despite increased gluconeogenesis. The compensatory changes in liver and peripheral tissue substrate flux and the resultant adaptation for enzyme expression in the liver to have a diminished insulin dependence reflect the loosely linked correlation between phenotype and genotype in hepatic glucose metabolism. |
Introduction |
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(PPAR) knockout (KO) mouse is a model of fasting hypoglycemia due to disordered fatty acid metabolism (1, 2). We previously characterized interorgan flux regulation in the absence of PPAR using stable isotopes and Alza osmotic pumps to examine muscle/liver Cori cycling and adipose/liver glucose/glycerol cycling in clearly defined fasted and fed states (3). The hypoglycemia evident in the fasted state, despite elevated hepatic glucose production, suggested increased peripheral glucose utilization as the etiology of hypoglycemia in the PPAR KO mouse (3). However, the elevated glucose production and gluconeogenesis were resistant to the suppression by insulin, suggesting hepatic insulin resistance (3). In addition, the high glucose production and nonsuppressed gluconeogenesis by meal was sustained by a high glycerol turnover, probably from lipolysis, suggesting adipocyte insulin resistance (3). Hepatic insulin resistance in PPAR KO mice was also suggested by studies of insulin action on fatty acid metabolism by Sugden et al. (4). Hepatic pyruvate dehydrogenase kinase isoenzyme 4 (PDK4), which regulates the activity of pyruvate dehydrogenase, is normally induced with fasting and is suppressed after feeding; however, this regulation was found to be attenuated in the PPAR KO mice (4).
Previously, insulin tolerance test or ip glucose tolerance test (IPGTT) was used to investigate the role of PPAR in glucose homeostasis in terms of insulin sensitivity or resistance (5, 6). A high fat diet was necessary to elicit an observable metabolic phenotype in PPAR KO, giving rise to the idea of a silent phenotype under normal dietary conditions. To understand the role of PPAR in modulating tissue-specific insulin action, we have examined hepatic expression of insulin-dependent gluconeogenic/glycolytic enzymes and those of the pentose cycle. To understand how intraorgan flux regulation is affected by changes in insulin-responsive gene expression in the PPAR KO mouse, we measured hepatic gluconeogenic flux, glucose absorption, clearance, and recycling using newly developed stable isotope IPGTTs to examine intraorgan flux regulation in the fasted to fed transition. These stable isotope studies together with enzyme expression analyses allow us to begin to understand the molecular mechanism for the compensation underlying the hepatic and peripheral metabolic response to the absence of PPAR and may shed light on the effector mechanism(s) by which fatty acids influence tissue-specific insulin sensitivity.
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Materials and Methods |
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-/- (PPAR KO) mice on a C57BL/6 background were a gift from Frank J. Gonzalez (5). The wild-type (WT; C57BL/6) mice were obtained from colonies maintained by the NIH. Stable isotopes, [1,2-13C2]-, or [2-2H]-, and [6, 6-2H2]glucose (all isotopes were 99% enriched), were purchased from Cambridge Isotope (Andover, MA). Animal studies were conducted in accordance with the institutional laboratory animal care and use committee guidelines and the protocol approved by the institutional review board. Mice were maintained on standard chow (NIH-31 sterilizable diet 7013, purchased from Harlan Teklad, Madison, WI). All experiments were performed with male mice ranging from 16–18 wk in age, except for the deuterated glucose IPGTT (Fig. 5
), where the mice were all males, but between 11 and 13 months of age. The average mouse weight of the 16- to 18-wk-old mice was 31 ± 1.9 g for C57BL/6 mice and 30 ± 3.5 g for PPAR KO mice. The average weight for 11- to 13-month-old PPAR KO mice was 35.94 ± 1.53 g, and that for C57BL/6 mice was 34.17 ± 1.84 g. For IPGTT studies, fasting of both groups of mice was initiated at 2000 h and continued until noon the following day. To minimize stress on the animals, plasma glucose was sampled at only two time points for each mouse during the experiment, and the time zero (basal-fasting) point was drawn on the entire group of WT and PPAR KO mice 3 wk before the stable isotope tracer experiments. Four groups of mice (n = 4 in each group) were used to complete the IPGTT study. [1,2-13C2]Glucose or a 1:1 mixture of [2-2H]- and [6, 6-2H2]glucose, was administered at 1 mg glucose/g body weight by ip injection as in our previous study (7). Blood was sampled at 0, 0.5, 1, 2, and 3 h for [1, 2-13C2]glucose isotopomer analysis and at 0, 0.5, 1, and 2 h for deuterated glucose isotopomer analysis.
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RT-PCR methods
Total RNA was prepared from approximately 100 mg liver after extraction with a guanidinium HCl/phenol mixture (8) following the Roche Tripure Isolation Reagent protocol. Typically, 100 µg total RNA are obtained from one extraction.
Assessment of glucokinase (GK), glucose-6-phosphatase (G6Pase), pyruvate kinase (PK), pyruvate carboxylase (PC), phosphoenolpyruvate (PEP) carboxykinase (PEPCK), glucose-6-phosphate dehydrogenase, and transaldolase (TA) enzyme enzyme mRNA levels was accomplished by TaqMan (PE Applied Biosystems, Foster City, CA) RT-PCR. The TaqMan system is designed to have a fluorescent and quencher-tagged probe bind to the region amplified between the forward and reverse primers for the cDNA amplification reaction (after the RT step). The Taq polymerase hydrolyzes the probe during the extension reaction, releasing the fluorescent tag from the proximity of the quencher, and thereby each amplification cycle can be detected, allowing for the accurate, quantitative determination of the linear region of RT-PCR amplification. Specifically, the number of cycles necessary to reach the threshold for linear amplification of the cDNA (the CT value) is obtained. The 2-CT reflects the total mRNA abundance for a given target RNA; the higher the CT, the lower the abundance of target mRNA. Normalization of the target mRNA to a housekeeping reference is given by 2-CT/2-CR or 2(CR- CT) = 2-
CT. When this result is normalized to a baseline, as for example, a dimethylsulfoxide (DMSO) control for an experiment with insulin signaling inhibitors dissolved in DMSO, the relative abundance of the target mRNA to the housekeeping reference mRNA, normalized to the DMSO control, is 2-CT condition/2-CT DMSO = 2CT. This RT-PCR system affords easy screening with only approximately 50 ng needed for each measurement, which are performed in triplicate for accuracy. In all RT-PCR assessments, measures were taken to avoid amplification of a region of genomic DNA or other contaminants. Target and reference RT-PCR reactions were run singly and then together as a multiplex reaction. The primer concentrations were adjusted in the multiplex reaction tube such that accurate CTs are obtained, but soon after, the exhaustion of primers defines the end of the reaction. In this way, amplification of the majority species is stopped before it can limit reactants available for amplification of the minority species. Table 1 shows the target primer pairs and labeled probes used in these TaqMan RT-PCR studies. The TaqMan ribosomal RNA control reagents were used for the reference and are designed to detect the 18S ribosomal RNA gene (ABI catalog no. 4308329).
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GC/MS conditions
All isotopomeric determinations were performed on a Hewlett-Packard Mass Selective Detector (model 5973A, Hewlett-Packard, Palo Alto, CA) connected to a Hewlett-Packard gas chromatograph (model 6890) using chemical ionization with 20% methane (10). The glucose derivative was isolated on an HP5 capillary column, 30 m x 250 µm internal diameter. GC conditions were helium as carrier gas at a flow rate of 1.0 ml/min, and sample injector temperature of 250 C.
For glucose derivatives, the oven temperature was programmed from 220–250 C at a ramp of 10 C/min, and the retention time was 2.9 min. The glucose aldonitrile pentaacetate derivative gives the molecular ion (C1–C6) of the glucose molecule at mass to charge ratio (m/z) of 328. Electron impact ionization of the aldonitrile derivative was used to characterize glucose positional isotopomers at m/z 187 for C3–C6 and m/z 242 for C1–C4 fragments. Isotope enrichment in these fragments was used to determine symmetry of the C1–C3 vs. C4–C6 glucose carbons. Selected ion monitoring was used to follow specific ions. For glucose isotopomer determination, the ion clusters monitored were from m/z 327 to m/z 336, with a fragment of m/z 328 corresponding to unlabeled glucose.
Data calculation and interpretation
Mass isotopomer distribution was determined using the method of Katz et al. (11, 12) that corrects contribution of derivatizing agent and natural 13C abundance to mass isotopomer distribution of the compound of interest. Results of the mass isotopomers in glucose (enrichment of glucose isotopomers) are reported as molar fractions of M0, M1, M2, etc. according to the number of labeled carbons or hydrogen in the molecule (11, 12). The sum of all isotopomers of the glucose molecules, 
Mi for i = 0 to n (n = 6 for glucose), is equal to 1 or 100%.
IPGTT with [1,2-13C2]glucose
When [1,2-13C2]glucose (an M2 glucose isotopomer) is administered ip, its dynamic change during an IPGTT time course can be interpreted as follows.
Plasma M2 glucose.
The appearance of M2 glucose in blood is a direct evidence of absorption of administered [1, 2-13C2]glucose. The levels of plasma M2 glucose during the IPGTT time course depend on the balance between glucose absorption and glucose disposal.
Plasma M1 glucose.
Plasma M2 glucose can be recycled via liver back to plasma as M1 glucose due to the loss of a 13C at the first position of the glucose in the reaction catalyzed by glucose-6-phosphate dehydrogenase (G6PDH) of the pentose cycle. Alternatively, the plasma M1 glucose can be produced via the Cori and tricarboxylic acid (TCA) cycles. In this case, the [1,2-13C2]glucose is first converted to [2,3-13C2]lactate (an M2 lactate isotopomer) through the glycolytic pathway. The M2 lactate generated, via the Cori cycle, is converted to m1 PEP via the TCA cycle and then M1 glucose by the gluoneogenic pathway. Here, the loss of a 13C in the M2 lactate is catalyzed by the exchange reactions of the TCA cycle and PEPCK (13). Thus, the appearance of plasma M1 glucose is the result of the recycling of plasma M2 glucose through the pentose cycle and/or the Cori cycle described above.
Plasma M0 glucose.
M0 glucose is an unlabeled glucose. It can be generated by glycogenolysis from unlabeled glycogen or gluconeogenesis from unlabeled substrates. In this study, M0 could also be produced by the recycling of M1 glucose due to the metabolism/recycling of the [1,2-13C2]glucose used (M2
M1M0) or recycling of unlabeled glucose itself (M0trioseM0 recycling).
Glucose-dependent futile recycling rate constant, kHR
The recycling of hepatic glucose, leading to the appearance of M1 glucose in blood, occurs when glucose traverses the glucose <-> glucose-6-phosphate (G-6-P), pentose phosphate pathway, and possibly the TCA cycle. The relationship of M2 to M1 glucose conversion has been shown to be dependent on glucose concentration (7). Thus, when glucose M1/M2 ratios are plotted as a function of time, a linear dependence with time is revealed. The slope of the increase in this ratio gives the in vivo glucose-dependent futile recycling rate constant of glucose through G-6-P, kHR, defined as the fraction of total glucose uptake that recirculates through the hepatic pentose cycle, and/or through the gluconeogenic and TCA cycles (7).
IPGTT with deuterated glucose
Hepatic uptake of [2-2H]glucose generally leads to the loss of deuterium label at the C2 position due to isomerization between G-6-P and fructose-6-phosphate (F-6-P). Hepatic glucose uptake of [6, 6-2H2]glucose generally leads to loss of the deuterium label in part between the interconversion of pyruvate to lactate and in part between pyruvate and oxaloacetate. When [2-2H]- and [6,6-2H2]glucose are administered as a 1:1 mixture, the disappearance of the two isotopes, [2-2H]- and [6,6-2H2]glucose can be determined by mass fragmentography by assessing the M1 label in the C1–C4 fragment (for [2-2H]glucose) and the M2 label in the C3–C6 fragment (for [6,6-2H2]glucose) by the electron impact ionization mass spectrometry. The difference between the two disappearance rates has long been recognized as the standard measure of futile cycling (i.e. glucose to glucose-6-phosphate and back) (14): relative rate of glucose/G-6-P futile cycling = % difference in plasma [2-2H]glucose vs. [6,6-2H2]glucose enrichments = (% [6,6-2H2]glucose - % [2-2H]glucose)/(% [6,6-2H2]glucose).
Statistical analyses
All data were expressed as the mean ± SD. Analyses of the significance of differences were performed using t test or z test. The slope of the M1/M2 ([13C]glucose) against time and the percent difference between the plasma [2-2H]- and [6,6-2H2]glucose enrichments against time during the IPGTT were determined using linear regression analysis.
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Results |
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control of insulin-dependent hepatic gluconeogenic/glycolytic/pentose enzyme expression KO mouse (3). These findings of hypoglycemia and the lack of suppression of HGP and gluconeogenesis in the transition from the fasted to the fed state suggest a complex metabolic phenotype, characterized by an increase in peripheral glucose utilization in the presence of hepatic insulin resistance when PPAR is absent.
To understand how the expression of insulin-responsive enzymes may be controlled by PPAR, we performed TaqMan expression analysis on key enzymes in glycolytic/gluconeogenic/pentose pathways in the liver (Fig. 1), especially those involved in gluconeogenesis and substrate cycling, in both the 17-h fasted and 5-h refed states. Figure 2 shows the expression of the p46 G6Pase translocase subunit, part of the G6Pase multienzyme complex (15). There was at most a mild decrease in the expression of the p46 G6Pase translocase subunit in the PPAR KO mice. In contrast to the meal suppression of PEPCK or p36 G6Pase catalytic subunit expression in the WT mice, there was no significant change in PEPCK or p36 G6Pase catalytic subunit expression in the fasted or fed state when PPAR was absent (data not shown), consistent with previous reports (16, 17). However, a 32-fold decrease in the expression of PC was seen in both the fasted and fed states in PPAR KO mice. PC is a key enzyme for the conversion of lactate to glucose via the TCA cycle, and our finding of diminished expression of PC supports our previous finding of decreased HGP from lactate in the PPAR KO mouse (3). Figure 2 also illustrates the profound changes in the expression of key glycolytic and pentose cycle enzymes when PPAR is absent that can serve to diminish futile cycling. PK is 4-fold diminished in the fasted and fed states, which can intensify the decrease in PEP/pyruvate futile cycling caused by the decreased expression of PC. GK expression normally increases 32-fold in the transition between the 17- and 5-h refed state for the C57BL/6 WT control. However, although GK expression is unchanged in the fasted state, there is no induction of GK in the transition from the fasted to the fed state, indicating a profound unresponsiveness to hepatic insulin signaling in the PPAR KO mouse. This lack of hepatic insulin action is further evident by the 32-fold decrease in expression of the rate-limiting enzyme of the oxidative limb of the pentose cycle, G6PDH, seen in the both the fasted and fed states (Fig. 2). TA, a key enzyme for regulating flux from the triose phosphate pool through the nonoxidative limb of the pentose cycle, was also down- regulated approximately 50- to 100-fold in the fasted and fed states when PPAR was absent (Fig. 2). As shown in Fig. 2, the pattern of expression changes in GK, PK, G6Pase, PC, TA, and G6PDH is consistent with optimizing gluconeogenesis from triose phosphates to hexose phosphates with as little energy wastage as possible from futile/substrate cycling through the glycolytic/gluconeogenic/pentose or TCA cycles.
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KO mouse KO and the WT are shown in Figs. 3 and 4, respectively. The integrated changes in plasma glucose and insulin are expressed as the area under the curve (AUC) and are presented in Table 2. The whole body insulin resistance can be defined as the glucose AUC (change from basal) for a given change in the insulin AUC. The AUC of glucose over the 3-h time course of IPGTT for C57BL/6 was higher than that for PPAR KO mice (P < 0.001), whereas the AUC of insulin was not statistically different between the two groups (Table 2). These results are consistent with our previous steady state measurements of glucose and insulin in these mice, which showed lower blood glucose values for PPAR KO mice at the same insulin values as for C57BL/6 controls in both the fasted and fed states (3).
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unlabeled trioseglucose). Thus, plasma M0 glucose levels during the IPGTT reflect the balance between glucose utilization, glucose recycling, and HGP. Figure 3B
indicates that a significant difference exists in the time course of the level of M0 glucose during the IPGTT. After the injection, the increase in plasma M0 glucose for the PPAR KO mice was less than that for the C57BL/6 control (Fig. 3B). The AUC for M0 for the PPAR KO mouse is approximately half that for the C57BL/6 control (Table 2). The M0 time course reflects the integrated responses of liver and the periphery to the action of insulin during the IPGTT. In a previous study we showed that HGP and gluconeogenesis were increased secondary to increased glucose-glycerol cycling between liver and adipose tissue and decreased Cori cycling between liver and muscles (3). Thus, the lower levels of plasma M0 glucose in PPAR KO mice were not caused by the increase in HGP and gluconeogenesis (3), but, rather, by a decrease in glucose cycling and/or increased glucose utilization. The results in Fig. 3B also suggest that whole body insulin sensitivity, on the average, is greater in the PPAR KO mouse.
Peripheral disposal of plasma glucose in PPAR KO mice
The smaller AUC of plasma glucose for the PPAR KO mouse can be due to either decreased absorption of administered M2 glucose and/or increased disposal of plasma glucose. As glucose uptake does not distinguish between tracer (M2 glucose) and tracee (endogenous M0 glucose), the rate of appearance of ip injected [1,2-13C2]glucose (an M2 glucose isotopomer) in blood reflects the rate of glucose absorption (13). As M2 is not produced endogenously, the fall in tracer concentration after a bolus dose is entirely due to irreversible loss of the glucose/tracer from the plasma pool. Therefore, the rate of disappearance of M2 glucose from plasma reflects the rate of overall glucose clearance. For both C57BL/6 and PPAR KO mice, the initial rise of plasma M2 glucose to the same level at 0.5 h indicated a similar absorption rate of administered M2 glucose. The fall in plasma M2 levels between 0.5 and 3.0 h was 32% faster (P < 0.001) in PPAR KO mice than in C57BL/6 mice (Fig. 3D), indicating a greater rate of overall glucose clearance when PPAR is absent. The overall change in AUC of plasma M2 glucose was also lower in PPAR KO mice, as shown in Table 2.
Pathways of glucose carbon recycling of [1,2-13C2]glucose in the PPAR KO mouse
The appearance of M1 glucose during the [1,2-13C2]glucose IPGTT study is the consequence of modification of plasma M2 glucose via the oxidative limb of the pentose cycle (G6PDH) or Cori/TCA cycles. The modified labeled glucose can be recycled back to plasma as M1 glucose via hepatic futile cycling (glucose
G6P). As shown in Table 2, the AUC of plasma M1 glucose over the 3-h time course of IPGTT in C57BL/6 mice was 37% higher than that in PPAR KO mice (P < 0.01). At 0.5 h, the rise of plasma M1 glucose in PPAR KO mice reached its plateau, whereas plasma M1 glucose in C57BL/6 mice kept rising until 2 h (Fig. 3C). The generation of plasma M1 glucose in both groups of mice indicates active glucose recycling during the IPGTT, with a lower degree of recycling when PPAR is absent.
Figure 5 shows a plot of the ratio of M1 to M2 plasma glucose against time during the [1,2-13C2]glucose IPGTT in PPAR KO and C57BL/6 mice. The M1/M2 glucose ratio for PPAR KO mice exhibited a time-dependent linearity (Fig. 5), as previously shown for C57BL/6 mice (7). The slope of the linear plot gives a glucose recycling rate constant, kHR. The rate of glucose recycling can be expressed as the product of plasma glucose concentration and the recycling rate constant, kHR (see Materials and Methods). The slope for the line, kHR, was determined by regression analysis to be 0.1086 ± 0.0049/h for C57BL/6, which was significantly higher than the slope of 0.0790 ± 0.0064/h for the PPAR KO mice (P < 0.025). The time-dependent linearity of the M1/M2 glucose ratio is the consequence of two factors: 1) a change in plasma M2 enrichment with time, and 2) the return of a constant fraction of glucose uptake by the liver in futile recycling. The change in M2 glucose enrichment with time is directly dependent on both peripheral and hepatic glucose uptake. The time course of M1 glucose enrichment is dependent on its generation via hepatic recycling of plasma M2 glucose taken up by the liver via the pentose cycle, with some contribution from lactate generated from peripheral M2 glucose uptake via the Cori cycle. Thus, the kHR takes on the meaning of the fraction of glucose uptake that is returned through hepatic glucose recycling (including the pentose phosphate pathway, and theoretically via the TCA and gluconegenic cycles) per hour. This constant is apparently a physiological property of the liver in response to a glucose challenge. In the case of C57BL/6, 10.86% of the total glucose uptake was returned in hepatic glucose recycling during the IPGTT. In contrast, PPAR KO mice returned only 7.9%.
Determination of hepatic futile cycling
The hepatic glucose carbon recycling is the sum process of the flux recycling through the TCA cycle, the pentose phosphate pathway, and the glucose/G-6-P, F-6-P/fructose-1,6-P2 and PEP/pyruvate futile cycles (Fig. 1). Hepatic glucose cycling at the level of glucose/G-6-P has been traditionally determined using separate infusions of [2-3H]glucose and [6-3H]glucose tracers (14). The infusion of [2-3H]glucose is known to provide a different estimate of glucose turnover rate than that from the infusion of [6-3H]glucose. The difference is attributed to the fact that tritium in the carbon-2 position of glucose is lost in the equilibrium reaction of isomerization of G-6-P to F-6-P, whereas tritium in the carbon-6 position is retained. Glucose/G-6-P futile cycling equals the difference between glucose turnover measured by the two tracers. Figure 6 shows a modified IPGTT based on these tests, using a 1 mg/g glucose bolus injection composed of equal amounts of the deuterium-labeled stable isotopes [2-2H]glucose and [6,6-2H2]glucose. Figure 6 shows that at all times during the [2-2H]-/[6,6-2H2]glucose IPGTT, the percent difference between the plasma enrichments of the two tracers is greater for the WT than for the PPAR KO mouse, indicating a much smaller amount of glucose/G-6-P futile cycling when PPAR is deficient.
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Discussion |
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KO mice. To further characterize the role of PPAR in modulating insulin action on glucose metabolism, we have examined the expression of insulin-dependent gluconeogenic/glycolytic enzymes and those of the pentose cycle. RT-PCR expression analysis indicated a slight decrease (<4-fold) in gluconeogenic gene expression for p46 G6Pase translocase and PEPCK in the PPAR KO mouse, but a greater than 32-fold decrease in pyruvate carboxylase expression. Glycolytic and pentose cycle enzymes, glucokinase, G-6-P dehydrogenase, and transaldolase were significantly reduced (>32-fold) in PPAR KO mice compared with C57BL/6 mice (WT). More importantly, differences in insulin-responsive gene expression between the fasted and fed states were lost when PPAR was absent. These observations are consistent with the observation of PPAR dependence of PDK4 and support the concept of PPAR modulating the compensatory mechanism(s) for the responsiveness of insulin-dependent gluconeogenic/glycolytic/lipogenic enzymes as well as the mechanism(s) regulating peripheral glucose uptake. We have further studied the metabolic consequences of these compensatory/adaptive changes using stable isotope flux measurements. The [1,2-13C2]glucose and [2-2H]/[6,6-2H2] IPGTTs are unique in that recycling of the original tracer after hepatic modification (to M1 or M0 glucose) or differences in the rate of exchange of 2-2H vs. 6,6-2H2 (from glucose to water) can be quantitatively detected (7). As hepatic glucose/G-6-P recycling is dependent on insulin’s regulation of phosphoinositol 3-kinase-dependent enzyme expression, such as, for example, glucokinase, this study showcases an easily performed, mass spectrometry-based glucose tolerance test that can indicate a specific locus for hepatic insulin resistance by making only noninvasive, peripheral measurements of glucose isotopomers. This article also demonstrates the first use of the [1,2-13C2]glucose tolerance test (7) in a targeted gene KO mouse model. The predictions of changes in glucose recycling due to changes in hepatic pentose cycle flux were validated by measurements of hepatic gene expression. This again is novel, in that peripheral plasma glucose isotopomer measurements are shown to reflect specific changes in the expression of hepatic insulin-sensitive gene expression.
We were able to compare glucose absorption and clearance by following the M2 glucose isotopomer using [1,2-13C2]glucose. The peak plasma M2 glucose concentrations were not different between PPAR KO and C57BL/6 mice, whereas the clearance of plasma M2 glucose was slower in C57BL/6 WT compared with PPAR KO mice. Therefore, injected glucose was absorbed at similar rate, but was cleared differently in these mice. For the major hepatic glycolytic/gluconeogenic futile cycles, the net flux through the glucose/G-6-P cycle determines the net production or uptake of glucose and is thus key in determining blood glucose levels and tolerance to a glucose load (18). Increased glucose futile recycling has been shown to be associated with overall insulin resistance, mild hyperglycemia, and type II diabetes (19, 20). The background strain, C57BL/6, has a higher rate of hepatic futile cycling, as evidenced in the higher levels of plasma M0 and M1 glucose and a higher kHR, and a greater relative exchange rate of [2-2H] vs. [6,6-2H2] from glucose to water (19). In the PPAR KO mouse, however, decreased hepatic futile cycling of glucose was observed, resulting in increased hepatic glucose production that partially compensated for the increased peripheral glucose clearance in the PPAR KO mouse. The glucose AUC was lower in the PPAR KO mouse for the same insulin AUC as the WT during the IPGTT, indicating a higher whole body average insulin sensitivity when PPAR was absent.
The whole body average insulin sensitivity is the end result of peripheral and hepatic insulin action. Studies of animal models with tissue-specific disruption of insulin receptor, or of disruptions in the gene coding for the insulin receptor substrate, have adequately demonstrated that tissue-specific resistance to insulin action may result in varying presentations of glucose tolerance and insulin sensitivity/resistance (21, 22). The ultimate phenotype depends on the specific role of insulin in the various tissue types (liver, muscle, or fat). Although the peripheral action of insulin affects tissue uptake of glucose, and therefore the glucose clearance, hepatic insulin action affects the net balance of gluconeogenic vs. glycolytic flux and is dependent on substrate futile cycling. Hepatic insulin resistance in the PPAR KO mouse was supported by the observed diminished response in the expression of insulin-dependent gluconeogenic/glycolytic enzymes as well as those of the pentose cycle. From these results and those of our previous study of glucose production in the PPAR KO mouse, we conclude that 1) the loss of PPAR results in lower expression levels and diminished response to meal regulation of gluconeogenic/glycolytic enzyme expression; 2) consequently, substrate cycling, including futile cycling of glucose, is decreased when PPAR is absent despite increased gluconeogenesis; and 3) the changes in substrate cycle fluxes reflect changes in the direction, but not the magnitude, of enzyme expression levels. Substrate flux between liver and peripheral tissues and futile cycling of glucose metabolites in the liver are two important factors that feed back to influence insulin action in the regulation of glucose metabolism. Measurements of substrate flux between liver and peripheral tissues and futile cycling of glucose metabolites in the liver are necessary to understand how hepatic and peripheral insulin resistance interact to regulate glucose production and utilization when a single gene is overexpressed in transgenic animals, or when a single gene is knocked out, as in the PPAR KO mouse (3).
This work implicates PPAR in setting the tone for the responsiveness of insulin-responsive hepatic gene expression. The diminished hepatic insulin responsiveness shown in this study might result from impaired signaling within the insulin signaling pathway, secondary to a compensatory mechanism within the whole body glucose metabolic network for substrate utilization, or both. The relationship between insulin signaling and the impaired hepatic insulin responsiveness for gluconeogenic/glycolytic/pentose gene expression in the PPAR KO mouse will be the subject of future work. Evidence exists for some impairment in hepatic insulin signaling related to the absence of PPAR, as it has been observed by Sugden et al. (4) that at all concentrations of plasma insulin seen during their fasting and refeeding studies, mRNA expression of sterol regulatory element binding protein (SREBP)-1c was lower in the livers of PPAR KO mice than in those of WT mice. SREBP-1c has been proposed to be a major mediator of insulin action on hepatic glycolytic and lipogenic gene expression (23). As hepatic SREBP-1c induction by insulin is dependent on stimulation of the phosphoinositol 3-kinase pathway (23, 24, 25, 26), as is glucokinase gene expression (25, 26, 27), shown here to be drastically decreased in the fed state, impairment of phosphoinositol 3-kinase pathway effector(s) activation by insulin may be part of the mechanism by which the absence of PPAR impairs hepatic responsiveness to insulin.
Future studies examining the molecular and feedback mechanisms underlying diminished hepatic responsiveness to insulin will provide new insight into the mechanisms of the in vivo glucose metabolic network (coordinated, compensatory, or both) and how the downstream targets of insulin signaling may fail to respond when orphan nuclear receptor action is dysregulated.
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Acknowledgments |
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KO mice. |
Footnotes |
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Equal work was performed by J.X. and V.C.
Abbreviations: AUC, Area under the curve; DMSO, dimethylsulfoxide; F-6-P, fructose-6-phosphate; GC/MS, gas chromatography/mass spectrometry; GK, glucokinase; G-6-P, glucose-6-phosphate; G6Pase, glucose-6-phosphatase; G6PDH, glucose-6-phosphate dehydrogenase; HGP, hepatic glucose production; IPGTT, ip glucose tolerance test; KO, knockout; m/z, mass to charge ratio; PC, pyruvate carboxylase; PDK4, pyruvate dehydrogenase kinase isoenzyme 4; PEP, phosphoenolpyruvate; PEPCK, phosphoenolpyruvate carboxykinase; PK, pyruvate kinase; PPAR, peroxisomal proliferator-activated receptor; SREBP, sterol regulatory element binding protein; TA, transaldolase; TCA, tricarboxylic acid; WT, wild-type.
Received September 5, 2003.
Accepted for publication November 17, 2003.
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References |
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