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Originally published In Press as doi:10.1074/jbc.M413948200 on May 11, 2005

J. Biol. Chem., Vol. 280, Issue 27, 25396-25402, July 8, 2005
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Physiologic and Pharmacologic Factors Influencing Glyceroneogenic Contribution to Triacylglyceride Glycerol Measured by Mass Isotopomer Distribution Analysis*

Jerry L. Chen{ddagger}, Erin Peacock{ddagger}, Waheeda Samady{ddagger}, Scott M. Turner{ddagger}, Richard A. Neese{ddagger}§, Marc K. Hellerstein{ddagger}§, and Elizabeth J. Murphy{ddagger}§

From the {ddagger}Department of Nutritional Sciences & Toxicology, University of California-Berkeley, Berkeley, California 94720 and the §Department of Medicine, San Francisco General Hospital, University of California-San Francisco, San Francisco, California 94110

Received for publication, December 13, 2004 , and in revised form, May 11, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
An imbalance between triacylglycerol synthesis and breakdown is necessary for the development of obesity. The direct precursor for triacylglycerol biosynthesis is {alpha}-glycerol phosphate, which can have glycolytic and glyceroneogenic origins. We present a technique for determining the relative glyceroneogenic contribution to triacylglyceride glycerol by labeling the glycerol moiety with 2H2O. The number of hydrogen atoms (n) incorporated from H2O into C–H bonds reflects the metabolic source of {alpha}-glycerol phosphate and can be calculated by combinatorial analysis of the distribution of mass isotopomers in triacylglyceride glycerol. Three physiological settings with potential effects on glyceroneogenesis and glycolysis were studied in rodents. Adipose tissue acylglyceride glycerol in mice fed a low carbohydrate diet had significantly higher values of n than in mice fed a high carbohydrate diet, suggesting an increased contribution from glyceroneogenesis of from 17 to 50% on the low carbohydrate diet. Similarly, mice administered rosiglitazone had a significant relative increase in glyceroneogenesis (from 17 to 53%), indicated by an increase in adipose acylglyceride glycerol n. Fructose infusion in overnight fasted rats rapidly lowered plasma triacylglyceride glycerol n, reflecting a decreased contribution from glyceroneogenesis (from 66 to 34%) presumably because of increased glycolytic input. In conclusion, we demonstrate that the number of C–H atoms derived from cellular H2O in triacylglyceride glycerol is an informative indicator of {alpha}-glycerol phosphate origin and, ultimately, triacylglycerol metabolism. Under certain physiological conditions, glyceroneogenesis can be up-regulated in adipose (e.g. low carbohydrate diet) or down-regulated in liver (e.g. fructose infusion). Additionally, stimulation of glyceroneogenesis by rosiglitazone in adipose tissue may be an important factor in the antilipolytic actions of thiazolidinediones.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The prevalence of obesity in both developed and developing countries is steadily on the rise (1). With this worldwide health problem comes a new urgency in understanding the fundamentals behind the regulation of triacylglycerol (TG)1 synthesis. One such question is the relative contributions from glyceroneogenesis and glycolysis to the synthesis of {alpha}-glycerol phosphate ({alpha}-GP), the direct intracellular precursor for TG synthesis. There are three primary sources of {alpha}-GP for TG synthesis (Fig. 1): plasma glycerol, via direct phosphorylation by glycerol kinase (traditionally thought not to be present to a significant extent in adipose tissue); glucose, via glycolysis to the level of triose phosphate; and glyceroneogenesis, which is defined as the synthesis of {alpha}-GP from gluconeogenic precursors (i.e. precursors other than glucose or glycerol) (2). A better understanding of the relative contributions of these pathways to TG synthesis under different conditions could aid our understanding of TG metabolism.

Recently, methods have been presented for the in vivo measurement of TG synthesis using heavy water (2H2O) (3, 4). We present here the application of 2H2O labeling combined with mass isotopomer distribution analysis (MIDA), to the discernment of sources of {alpha}-GP. MIDA involves quantifying, by mass spectrometry, the relative abundances of molecular species of a polymer differing only in mass (mass isotopomers), after introduction of a stable isotope-labeled precursor. The degree and pattern of incorporation of 2H into C–H bonds of {alpha}-GP is dependent on two factors: the 2H enrichment of body water and the number of C–H bonds in {alpha}-GP that were derived from body water. The latter is dependent on the biochemistry of hydrogen incorporation from solvent H2O into glyceroneogenic and glycolytic intermediates (Figs. 1 and 2) (5). The maximal theoretical number of hydrogen atoms (n) that can be incorporated from H2O into C–H bonds is 5 (italicized in Fig. 1). Any {alpha}-GP generated via glyceroneogenesis (i.e. through the tricarboxylic acid cycle and "bottom" portion of the gluconeogenesis pathway, from pyruvate or oxaloacetate) will have all five hydrogens incorporated from body water (Figs. 1 and 2A, italicized). The {alpha}-GP generated via glycolysis from unlabeled glucose, in contrast, has an n of at most 3.5 (i.e. at most three and a half hydrogen atoms are incorporated from body water (Figs. 1 and 2B, bold)). This non-integer value derives from the phosphoglucose isomerase step of glycolysis, which adds label to C1 (Fig. 2B) (6). This results in a labeled hydrogen on the phosphate carbon of dihydroxyacetate phosphate but not glyceraldehyde 3-phosphate. Of the two {alpha}-GPs generated from one glucose, one will therefore have an n of 3 and one will have an n of 4 (average = 3.5). {alpha}-GP generated in the liver from unlabeled glycerol via glycerol kinase could theoretically have an n of 0 (Fig. 1). However, it has been shown previously that there is rapid exchange of {alpha}-GP with glycolytic intermediates (e.g. in the triose phosphate pool) in liver (7, 8) resulting in an n closer to 3.



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  		FIG. 1.
Labeling pathways of 2H incorporation into C–H bonds of the glycerol moiety of TG. The pathway of labeled hydrogen incorporation from 2H2O from either glucose (bold H, TG hydrogens 1, 2, 3, and half of 4), glyceroneogenesis (italic H, TG hydrogens 1, 2, 3, 4, and 5) or glycerol (TG hydrogens 1, 2, 3). Hydrogen exchange between water and C–H bonds in {alpha}-GP does not occur during the subsequent synthesis of TG.

 
Previously, we have shown experimentally that n calculated from adipose TG is ~4.0 and from liver TG ~4.7 (4). These estimates were based upon both plateau label incorporation after long term 2H2O administration and combinatorial labeling patterns using MIDA. We hypothesized that experimentally observed values of n less than 5 could be explained by contributions from non-glyceroneogenic sources of {alpha}-GP (Fig. 1). By this interpretation, the resulting experimentally determined n reflects the mixture of TG-glycerol from glycolysis (glyc3.5), glyceroneogenesis (glyc5), and glycerol kinase (glyc3). The different values of n observed in different tissues, with a higher n observed in liver compared with adipose tissue, were consistent with the known higher activity of glyceroneogenesis in liver (8, 9). If this is, in fact, the physiological explanation for a measured value of n less than 5, we reasoned that n in TG-glycerol might be informative in its own right as a parameter of cellular metabolism and glyceroneogenesis.

The goal of the present study was to test this hypothesis. Three experimental conditions were imposed. First, the effect of a high fat, low carbohydrate (LC) diet on adipose TG n was studied in mice. Reduced dietary glucose input in animals fed a LC diet should result in a lesser contribution to TG-glycerol from uptake and glycolytic metabolism of dietary glucose (glyc3.5) and a greater contribution from the glyceroneogenic pathway (glyc5), resulting in a higher measured n. Second, we investigated the effect of fructose infusion on hepatic TG n, sampled from plasma TG-glycerol in rats. Fructose floods the hepatic triose phosphate pool from the glycolytic direction (glyc3) and might result in a lowering of plasma TG-glycerol n. Finally, having established the informative value of TG-glycerol n, we evaluated the effect on glyceroneogenesis of rosiglitazone, a PPAR{gamma} agonist known to increase adipose TG deposition and alter adipose tissue glycerol metabolism (10). The relative importance of rosiglitazone induced increases in PEPCK versus glycerol kinase expression and activity in adipose tissue has been controversial (11). If an increase in PEPCK was the predominant mechanism involved in rosiglitazone-induced {alpha}-GP synthesis (and hence TG synthesis), the measured n should increase with increased contribution from glyc5 (i.e. glyceroneogenesis). In contrast, a significant increase in glycerol kinase activity would result in a decrease in measured n (glyc3 or less). We show here that measurement of n in acylglyceride glycerol can be informative as an index of glyceroneogenesis, and, ultimately, TG metabolism under different conditions.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Animal Studies
All procedures were approved by the University of California, Berkeley, Animal Use Committee.

Mice—4-week-old male C57Bl/6J mice (16–18 g; Jackson Laboratories, Bar Harbor, ME) were used. Mice were fed ad libitum a high carbohydrate, low fat (HC, 70% carbohydrate, 10% fat) diet or a low carbohydrate, high fat diet (LC, 35% carbohydrate, 45% fat) (Research Diets Inc., New Brunswick, NJ). An additional group of mice were fed an HC diet containing rosiglitazone (6.34 mg/kcal diet), which resulted in a dose of ~3 mcg/kg mouse/day (Research Diets Inc., New Brunswick, NJ). After 11 days on the diet, all animals were given a priming dose of 99.8% 2H2O in saline via intraperitoneal injection to achieve ~4.8% 2H2O enrichment in body water (30 µl/g mouse) followed by administration of 8% 2H2O in drinking water. Mice (n = 6 per group) were sacrificed after either 15 or 64 days on heavy water. Mesenteric, epididymal, retroperitoneal, and inguinal adipose tissue depots were removed, and blood and urine samples were obtained.

In an additional experiment to investigate potential changes in deuterium enrichment in glucose, mice, as above, were put on the LC diet, HC diet, or HC diet with rosiglitazone for 2 weeks with 2H2O administered as above during the final week (n = 10 per group). Half the animals in each group were sacrificed in the morning (7:30–9:30 AM) and half at the end of the day (4:30–6:15 PM), roughly corresponding to the start of the light and dark cycles (7 AM–7 PM). Animals were ad libitum fed throughout. Blood samples were obtained at the time of sacrifice.

Rats—Sprague-Dawley rats (400–500 g, Charles River Laboratories, Wilmington, MA) were purchased with indwelling jugular vein and carotid artery catheters in place. Rats (n = 4) were fed ad libitum a Purina chow diet. After an overnight fast, animals were anesthetized with isoflurane and a priming dose of 99.8% [2H2O]saline was given via intraperitoneal injection, as above. After a minimum of 60 min, to allow for 2H2O equilibration with body water, two baseline blood samples were collected (times –30 and –15 min). At time 0, fructose was infused intravenously (16–19 mg/kg/min for 4 h). Blood samples were taken after 3 and 4 h of fructose administration.

Isolation of Acylglyceride Glycerol from Adipose Tissue—Lipids from adipose tissue were extracted by a modified Folch extraction (12). The lipid fraction was transesterified by incubation with 3 N methanolic HCl (Sigma-Aldrich) at 55 °C for 60 min. Fatty acid methyl esters were separated from glycerol by the Folch technique. The aqueous phase containing glycerol was lyophilized, and glycerol was converted to glycerol triacetate by incubation with acetic anhydride-pyridine (2:1) as described elsewhere (13).

Isolation of TG-glycerol from Plasma—Plasma, obtained from fresh whole blood, was extracted by the Folch technique. TG was isolated by TLC as described previously (14). Glycerol isolation and derivatization were then performed as described above.

Isolation of Glucose from Plasma—Plasma glucose was isolated using the method of Van Dijk et al. (15). Briefly glucose was extracted from plasma spotted on filter paper using an ethanol/water extraction. Glucose was then derivatized to the aldonitrile tetra-acetate derivative as described previously (13).

Measurements of 2H2O Enrichment in Body Water—2H2O enrichment in body water (from plasma or urine) was measured by one of two methods. Briefly, 15–20 µl of plasma or urine were reacted in an evacuated GC vial with calcium carbide to produce acetylene. The acetylene gas was then removed with a syringe and injected into a GC vial containing 10% bromine in carbon tetrachloride and incubated at room temperature for 2 h to produce tetrabromoethane. Excess bromine was neutralized with 25 µl of 10% cyclohexene, and the sample was suspended in ethyl acetate (16). Alternatively, the acetylene gas was directly measured by a new mass spectrometric method (17). Briefly, 25 µl of sample were injected into a closed Exitainer vial containing calcium carbide in a dry helium atmosphere. A small amount (0.5 ml) of the acetylene gas generated from the reaction was removed and injected into another closed vial with a helium atmosphere for direct analysis. The two methods, used with standard curves, give identical results.2 However, the direct acetylene method is less time consuming and, thus, became the preferred method during the course of this study.



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  		FIG. 2.
Incorporation of 2H into {alpha}-GP from glyceroneogenesis and glycolysis. In the presence of 2H2O, 2His incorporated into C–H bonds in {alpha}-GP. Label incorporation is shown via glyceroneogenesis (A, italicized H) and glycolysis (B, bold H). Two molecules of {alpha}-GP with unequal labeling are generated from each glucose molecule resulting in an n of 3.5. Carbon numbering shown is based on glucose carbons.

 

GC-MS Analyses—Glycerol triacetate and glucose aldonitrile were analyzed for isotope enrichment by GC-MS as described previously (13). Mass isotopomer abundances were analyzed by selected ion monitoring of mass-to-charge ratios (m/z) 159–161 (M0-M2) for glycerol and 328–331 (M0-M3) for glucose. Tetrabromoethane was analyzed for isotope enrichment by GC-MS method as described previously (18). The isotopic enrichment of acetylene (m/z 26 and 27) was measured by cycloidal mass spectrometry (Monitor Instruments, Pittsburgh, PA).

Calculations
Body 2H2O Enrichment—Isotopic enrichment of body 2H2O was calculated by comparison to a standard curve prepared gravimetrically from natural abundance water and 2H2O.

[2H]Glycerol Enrichment—Isotope enrichments of [2H]glycerol derived from acylglycerides were calculated by subtraction of mass isotopomer abundances in unlabeled glycerol standards (19). EM1 and EM2 were calculated as a fraction of the sum of mass isotopomers M0-M2, as previously described for MIDA calculations (4).

[2H]Glucose Enrichment—Glucose labeled with 2H via gluconeogenesis could result in a glycerol n of greater than the theoretical glycolytic value of 3.5, thereby resulting in an overestimation of the contribution of glyceroneogenesis to adipose TG. Therefore we measured 2H glucose enrichments at two different times of the day for each dietary group. 2H label was calculated using a modification of Lee et al. (20) as shown in Equation 1,

(Eq. 1)
where EMi represents the excess fraction of single-, double-, or triple-labeled glucose molecules present and ni represents the number of labeled hydrogens on each of the M1–M3 molecular species (i.e. 1, 2, and 3). Similar net [2H]glucose enrichment would indicate a similar contribution of gluconeogenic glucose to circulating glucose. Any changes observed would therefore be unrelated to changes in glucose enrichment.

MIDA Calculations of n and A{infty}1—MIDA is a technique based on combinatorial analysis of the labeling patterns present in polymers (4, 19, 20). The EM2 to EM1 ratio (R) is one embodiment of this labeling pattern. R is dependent on two factors: the proportion (p) of labeled hydrogen atoms present in tissue water (i.e. the enrichment of 2H2O in tissue water) and the possible number of C–H bonds in glycerol that are derived from this tissue water (n). If one assumes that the 2H isotopic enrichment (p) of hydrogen in each actively incorporated C–H of {alpha}-GP is equal to the 2H enrichment of body water and that 2H2O enrichment is constant during the labeling period, n can be calculated from the measured p in body water and the measured R (4, 19, 20).

Using calculation algorithms based on combinatorial probabilities as previously described (4, 19), a "lookup" table can be generated, describing R over a given range of values of p for discrete values of n (n = 3, n = 4, n = 5) (e.g. Table I). By using the value of p from measured body water enrichment, the expected R across the range of values for n is compared with the measured value of R (based on the lookup table, e.g. p = 4.00% yields R = 0.121 for n = 3, R = 0.143 for n = 4, R = 0.166 for n = 5). Because physiologic samples contain a mixture of glyc3.5 and glyc5 (see below), we treat n as a non-integral value for this modeling. A linear regression equation is then generated, reflecting the relationship between R and n at the experimentally determined p (the latter based on measured 2H2O enrichment). The value for n is then calculated from the measured R (e.g. if p = 4.00%, the equation is n = 43.86R–2.29, so for R = 0.152, n = 4.38) (Fig. 3).


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  		TABLE I
Sample lookup table illustrating relationship between R, p, and n

R (ratio of EM2/EM1) generated by MIDA for a given precursor enrichment (p) and different n representing different possible labeling patterns for TG-glycerol (e.g. glyc3, glyc4, glyc5).

 



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  		FIG. 3.
Sample linear regression plots for determination of n. Plots illustrating the relationship between n and R (EM2/EM1) for three different values of p (body water 2H enrichment) are shown along with the linear regression generated from the data.

 
Once n is established, one can calculate A{infty}1or the theoretical asymptotic value for fully labeled TG-glycerol. Accurate determination of this asymptotic or plateau value requires knowledge of n and is necessary for the determination of fractional synthesis (f) using the precursor product or rise-to-plateau approach (19) in Equation 2,

(Eq. 2)
where EM1 represents the isotopic enrichment of the mass +1 labeled species of glycerol (i.e. the measured abundance in excess of natural abundance) and A{infty}1 represents the asymptotic or plateau value possible for the isotopic enrichment of the mass +1 species of glycerol, calculated from p and a non-integer value of n.

Relative Contribution of Glyceroneogenesis to Acylglyceride Glycerol— The term "n" is a probability parameter; therefore, we also calculated a measure with more physiological meaning, by translating n into a relative contribution from glyceroneogenesis to acylglyceride glycerol (a percentage). Assuming no contribution from glycerol kinase, n can be assumed to come from only two sources (glyc5 and glyc3.5). Thus n = 5x + 3.5(1–x), and in Equation 3, solving for x.

(Eq. 3)
Because glucose 2H-labeled via gluconeogenesis can contribute to glyc5, this percentage represents a maximum possible contribution.

Statistical Analyses—A one-way analysis of variance with planned pair-wise comparisons was used with p < 0.05 as the criterion for significance for comparison of adipose n. Glucose enrichment was analyzed using a Kruskal-Wallis non-parametric analysis of variance (p < 0.05 criteria) followed by Mann-Whitney pair-wise comparisons. An independent group Student's t test and a nonparametric Mann-Whitney test with p < 0.05 as the criterion for significance was used for the fructose infusion comparison.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of LC Diet—After 26 days on the diet (15 days on 2H2O), n was significantly higher in the LC group compared with the HC group in retroperitoneal and inguinal adipose depots (Fig. 4A), and there was a nonsignificant trend in the same direction in mesenteric and epididymal adipose depots. By 75 days on the diet (64 days on 2H2O), n was significantly higher in all adipose depots in the LC group (Fig. 4B). At that point, the body water enrichments were 4.46% ± 0.11% in the LC group and 4.63% ± 0.05% in the HC group (not significantly different). R in the LC group ranged from 0.149 to 0.162 and in the HC group from 0.146 to 0.152. From these measured values, the n in the LC group ranged from 3.92 to 4.40 and in the HC group from 3.68 to 3.86. The relative maximal contributions from glyceroneogenesis in each fat pad after 75 days on the diet are presented in Table II showing an increase with an LC diet from 17 to 50%. There were no significant differences in n between any adipose depots within either group at either time point.



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  		FIG. 4.
Low carbohydrate diet increases relative contribution from glyceroneogenesis to adipose tissue TG. Experimentally determined n in different adipose depots in mice (n = 6) on low fat, high carbohydrate diet or high fat, low carbohydrate diet for 26 days (A) or 75 days (B). Data are mean ± S.E. M, mesenteric; E, epididymal; R, retroperitoneal; I, inguinal. *, p < 0.05; **, p < 0.001; comparing diets.

 


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  		TABLE II
Relative maximal contribution from glyceroneogenesis to adipose TG-glycerol

Percent contribution of glyceroneogenesis after 75 days of diet or drug treatment. Fat pad abbreviations are defined as in the legend to Fig. 4. Data are mean ± S.E.

 
Effect of Rosiglitazone—The addition of rosiglitazone to an HC diet significantly increased n in the epididymal and inguinal adipose depots after 26 days of the diet and 15 days of 2H2O (Fig. 5A). A similar, nonsignificant trend was seen in the other adipose depots. By 75 days of diet, n was significantly higher in all adipose depots in the rosiglitazone group (Fig. 5B). Body water enrichments were 4.67% ± 0.15% in the rosiglitazone group and 4.63% ± 0.05% in the untreated HC group (not significantly different). R in the rosiglitazone group ranged from 0.153 to 0.168. From these measured values, the n in the rosiglitazone group ranged from 3.90 to 4.44 compared with an n ranging from 3.68 to 3.86 in the control HC group. This corresponds to an increase in relative, maximal contribution from glyceroneogenesis after 75 days of rosiglitazone treatment, from 17 to 53% (Table II). There were no significant differences in n between any adipose depots within either group at either time point.

Effect of Fructose Infusion—Body water enrichment for rats 5 h after an intraperitoneal bolus of 2H2O was 4.67 ± 0.21%. In the fasting state, prior to fructose infusion, values of n from plasma TG-glycerol were 4.51 ± 0.20 at –30 min and 4.41 ± .15 at –15 min, reflecting a stable n (Fig. 6) and an average relative contribution of glyceroneogenesis of 66%. After 3 h of fructose infusion, n in plasma TG-glycerol dropped significantly to 3.84 ± 0.08 (p < 0.01). After 4 h of infusion, n remained significantly suppressed from baseline at 4.01 ± 0.08, corresponding to a relative contribution from glyceroneogenesis of 34%.

Plasma Glucose Enrichment—2H enrichments for glucose were determined in both the morning and evening. Results from the morning and evening groups were pooled resulting in 10 measures for each diet/treatment group. The LC group resulted in the highest net [2H]glucose enrichment (3.2 ± 0.5), which was significantly (p = 0.004) higher than the rosiglitazone treatment group (2.3 ± 0.6) and minimally significantly higher (p = 0.03) than the HC group (2.6 ± 0.6). Therefore increased glucose enrichment from increased gluconeogenesis on a low carbohydrate diet could contribute to the increased n seen with the LC diet. The decrease in glucose enrichment with rosiglitazone treatment suggests all the increase in n is caused by increased glyceroneogenesis, and in fact its contribution if anything would be underestimated in this group.



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  		FIG. 5.
Rosiglitazone increases relative contribution from glyceroneogenesis to adipose tissue TG. Experimentally determined n in different adipose depots in mice (n = 6) with or without treatment with rosiglitazone for 26 days (A) or 75 days (B). M, mesenteric; E, epididymal; R, retroperitoneal; I, inguinal. Data are mean ± S.E. *, p < 0.01; **, p < 0.002 rosiglitazone versus controls.

 



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  		FIG. 6.
Fructose infusion reduces relative contribution from glyceroneogenesis to liver TG. Experimentally determined n in plasma TG-glycerol in overnight fasted rats before and during a fructose infusion (16–19 mg/kg/min) (n = 4). Data are mean ± S.E. *, p < 0.05 versus baseline.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, the physiological basis and the informational value of n in TG-glycerol was explored. The experimentally determined values of n under normal conditions in adipose TG-glycerol (3.7–3.9) and during a fast in liver TG-glycerol (4.4–4.5) were comparable to previously measured values (3.8–4.0 and 4.6–4.8, respectively) (4). These values indicate that under normal conditions, with a standard HC chow diet, the majority (83%) of TG-glycerol present in adipose tissue is derived from glycolytic metabolism of unlabeled glucose (i.e. glyc3.5). In contrast, in a fasted liver, the majority of acylglyceride glycerol (66%) is derived from glyceroneogenesis (i.e. glyc5). These results are consistent with the traditional view that glucose is the major precursor for the adipose tissue TG-glycerol used for storage of TG and that glyceroneogenesis is the predominant source in the fasted liver. The finding that n in adipose tissue TG-glycerol (3.7–3.9) is relatively close to the theoretical value from glycolysis of unlabeled, dietary glucose (up to 3.5) is also consistent with the notion that most TG deposition in adipose tissue occurs in the fed state, when unlabeled glucose is present, as opposed to the fasted state, when blood glucose contains a higher proportion of gluconeogenically derived, labeled glucose.

What had been less well recognized is the role that glyceroneogenesis may play in adipose tissue TG synthesis under altered physiologic states or after pharmacologic treatment. Glyceroneogenesis, a process first described almost 40 years ago (21–23), has been shown to be up-regulated during periods of decreased glucose availability such as during starvation (2) or feeding of a carbohydrate-free diet (24). We show here, in vivo, that an LC diet increases adipose TG-glycerol n, suggestive of increased glyceroneogenesis. It has previously been shown that a carbohydrate-free diet results in an increased PEPCK activity in adipose tissue (25), which could directly account for the increase in glyc5 contribution seen. The LC diet we used has been shown to increase gluconeogenesis in liver (26), and we did see an increase in plasma glucose enrichment. If plasma glucose derived from gluconeogenesis were subsequently taken up by adipose tissue and metabolized through glycolysis, this could also contribute to increased values of n. However, LC diets also reduce glucose uptake in adipose tissue in C57Bl/6J mice (27) and as shown by our data and others, glucose is preferentially taken up in the postprandial period when unlabeled dietary glucose predominates. Nonetheless, increased gluconeogenesis on the LC diet is clearly a confounder, and therefore, the percent contribution from glyceroneogenesis should be seen as a maximum possible contribution.

We also asked whether it was possible to experimentally decrease n in liver TG, by infusion of fructose. A fructose load directly contributes glycolytically derived {alpha}-GP (glyc3 as there is no contribution of 0.5 H-atom from the phosphoglucose isomerase reaction) and suppresses the gluconeogenic contribution to triose phosphate formation in liver (28). The decreased liver TG-glycerol n that we observed experimentally is consistent with a simultaneous effect of increasing glyc3 contribution (both from increased glycolysis and potentially increased contribution from recycled unlabeled glycerol via glycerol kinase) and decreasing glyc5 contribution by suppression of glyceroneogenesis.

Having shown under two conditions that the triglyceride n reflects expected physiology and can be altered in predictable ways, we explored the role of glyceroneogenesis in adipose TG synthesis following PPAR{gamma} agonist treatment. PPAR{gamma} agonists, such as rosiglitazone, are insulin-sensitizing agents that induce a number of actions, including stimulation of adipogenesis and increased fat storage. PPAR{gamma} is required for the transcription of the PEPCK gene in adipocytes (29), and rosiglitazone has been shown to increase PEPCK expression and activity in adipose tissue (30–32). Conversion of oxaloacetate to phosphoenolpyruvate via PEPCK is generally accepted as the rate-limiting step in glyceroneogenesis (11) (Fig. 2A). Controversy has recently emerged about the effect of the PPAR{gamma} agonist thiazolidinediones on glyceroneogenesis and their role in promoting insulin sensitivity and reducing net lipolysis. Rosiglitazone has been reported to increase glycerol kinase activity in isolated adipocytes (33). However, despite this increase in glycerol kinase activity, it was subsequently shown in vitro by Tordjman et al. (32) that flux through glycerol kinase in rosiglitazone-treated adipocytes remains a quantitatively minor contributor to {alpha}-GP synthesis and therefore to suppression of fatty acid release. A physiologically significant contribution to adipogenesis from increased glycerol kinase activity would result in a decrease in n, whereas an increased contribution from glyceroneogenesis should increase n (Fig. 1). The latter was clearly observed during rosiglitazone treatment (Fig. 5). There was a nonsignificant decrease in net [2H]glucose enrichment with rosiglitazone treatment, and therefore the increase in n cannot be accounted for and is not confounded by an increased contribution from labeled glucose. If, rosiglitazone treatment does significantly increase glycerol kinase activity there could be recycling of previously labeled glycerol. However that recycled glycerol would simply reflect its prior origin (e.g. glyceroneogenesis or glycolysis). Whereas we would not be able to detect that it had gone through glycerol kinase, we would know that it had already gone through glyceroneogenesis and, as a matter of definition, we consider that as glyceroneogenesis. If unlabeled glycerol were recycled, that would appear with primarily an n of 3 and would look more like glycolysis glycerol, which was not what we observed. The observed increase in n with rosiglitazone treatment provides in vivo evidence for greater significance of up-regulation of glyceroneogenesis, presumably via increased PEPCK expression, compared with up-regulation of glycerol kinase.

In vitro studies have suggested a greater increase in rosiglitazone-induced glyceroneogenesis in visceral adipose (retroperitoneal and mesenteric) compared with subcutaneous depots (34). We observed no significant differences in n between different adipose depots in vivo. It is possible that this difference was because of the duration of our study, which was weeks as opposed to hours in the in vitro study.

The observed stimulation of glyceroneogenic activity in adipose tissue may be important to the mechanism of action of PPAR{gamma} agonists. Glyceroneogenic-induced increases in {alpha}-GP availability favor fatty acid re-esterification in adipose tissue and inhibition of fatty acid release into plasma. This is believed to play a key role in the insulin-sensitizing action of PPAR{gamma} agonists (10). Measurement of the glyceroneogenic contribution to adipose tissue {alpha}-GP, based on a change in the value of n, may therefore provide a novel measure of in vivo net antilipolytic activity for PPAR{gamma} agonists or other agents that reduce free fatty acid release into the plasma.

Our interpretation of these results could be altered by several metabolic processes. First, we assume that {alpha}-GP derived from glycerol kinase is in rapid exchange with glycolytic intermediates in the triose phosphate pool, resulting in an n of 3 rather than 0. This is not entirely accurate. It has been shown in humans (7), that of the plasma glycerol that is incorporated via glycerol kinase into very low density lipoprotein TG (i.e. TG synthesized in liver), ~5% does not have any exchange into the triose phosphate pool (i.e. retained all 5 2H labels from [2H5]glycerol infused). In the presence of 2H2O, this would result in a small percentage of n = 0 (glyc0). However, hepatic {alpha}-GP generated via glycerol kinase represents <20% of the total contribution to TG-glycerol under normal conditions (28, 35), so that 5% of this relatively small number should not alter our quantitative interpretation. In any case, since TG-glycerol from this source would be unlabeled, the measured EM2/EM1 ratio is not affected and would not affect calculated n (19). There is also potentially labeled glycerol being recycled via glycerol kinase and {alpha}-GP. We will consider the original source of glycerol (e.g. glyceroneogenesis or glycolysis) for our calculation.

Second, glycolytic cycling of glucose to the level of pyruvate or oxaloacetate then back to {alpha}-GP can contribute to TG (7, 36). Cycling of this type converts glyc3.5 to glyc5, and thus can complicate estimates of the proportion of TG-glycerol derived from glycolysis. As a matter of definition, therefore, glyceroneogenesis must be recognized to include glycolytic cycling or any other prior metabolic source of pyruvate or oxaloacetate. Last, contributions to the triose phosphate pool from other pathways such as the non-oxidative arm of the pentose phosphate cycle can introduce non-glyc5 species to TG-glycerol. Those pathways make a relatively small contribution relative to glycolytic flux (37); however these complexities of intracellular metabolism make precise calculations of the quantitative sources of {alpha}-GP difficult.

The results presented here have implications for the measurement of TG synthesis from 2H. We report here that n can range from 3.73 to 4.44 in adipose tissue. The calculation of the theoretical plateau or asymptotic value (A{infty}1) for fully labeled TG-glycerol depends on the value of n, and accurate determination of this plateau is required to measure fractional synthesis (f) of TG using the precursor product or rise-to-plateau approach (4, 19). Calculations of A{infty}1 based on the range of n determined here could cause A{infty}1 to vary from 12.95% to 14.90% for p = 4.8%, as an example. This range of calculated A{infty}1 can lead to a difference in calculated f of up to 13%. Thus, considering the mutability of n demonstrated in this study, experimental measurement of n by the technique described here is optimal when calculating TG-glycerol fractional synthesis, particularly under new experimental conditions.

In summary, we have demonstrated that values of n in TG-glycerol can be calculated by MIDA and are influenced by physiological and pharmacologic conditions. The variations observed were consistent with expected differences in the glycolytic versus glyceroneogenic origins of {alpha}-GP. Our findings also underscore the important role that glyceroneogenesis serves in maintaining TG homeostasis in adipose tissue and liver. In particular, stimulation of glyceroneogenesis may play a central role in the antilipolytic actions of thiazolidinediones on adipose tissue. Thus, measurements of n can serve as an informative indicator of {alpha}-GP and, ultimately, TG metabolism under different physiological conditions.


   FOOTNOTES
 
* These studies were supported in part by National Institutes of Health Grant NHLBI HL65919 (to M. H. K.), College of Natural Resources at UC Berkeley Hatch award (to M. K. H.), and a Pfizer post-doctoral fellowship (to E. J. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: San Francisco General Hospital, 1001 Potrero Ave., Bldg. 30, Rm. 3501-K, San Francisco, CA 94110. E-mail: smurph{at}itsa.ucsf.edu.

1 The abbreviations used are: TG, triacylglycerol; GP, glycerol phosphate; MIDA, mass isotopomer distribution analysis; LC, low carbohydrate; HC, high carbohydrate; GC-MS, gas chromatography-mass spectrometry; PEPCK, phosphoenolpyruvate carboxykinase; PPAR{gamma}, peroxisome proliferator-activated {gamma}. Back

2 R. Neese, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank Alan Bostrom for statistical assistance. Calcium carbide was the kind gift of George Houghton at Carbide Industries, Louisville, KY.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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