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Am J Physiol Endocrinol Metab 290: E448-E455, 2006. First published October 18, 2005; doi:10.1152/ajpendo.00139.2005
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Accelerated triacylglycerol turnover kinetics in hearts of diabetic rats include evidence for compartmented lipid storage

J. Michael O’Donnell,1,2 Manuela Zampino,1,2 Nathaniel M. Alpert,3 Matthew J. Fasano,1,2 David L. Geenen,2 and E.Douglas Lewandowski1,2

1Program in Integrative Cardiac Metabolism and 2Center for Cardiovascular Research, University of Illinois at Chicago, College of Medicine, Chicago, Illinois; and 3Department of Radiology, Massachusetts General Hospital, Boston, Massachusetts

Submitted 29 March 2005 ; accepted in final form 12 October 2005


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Triacylglycerol (TAG) storage and turnover rates in the intact, beating rat heart were determined for the first time using dynamic mode 13C- NMR spectroscopy to elucidate profound differences between hearts from diabetic rats (DR, streptozotocin treatment) and normal rats (NR). The incorporation of [2,4,6,8,10,12,14,16-13C8]palmitate into the TAG pool was monitored in isolated hearts perfused with physiological (0.5 mM palmitate, 5 mM glucose) and elevated substrate levels (1.2 mM palmitate, 11 mM glucose) characteristic of the diabetic condition. Surprisingly, although the normal hearts were enriched at a near-linear profile for ≥2 h before exponential characterization, exponential enrichment of TAG in diabetic hearts reached steady state after only 45 min. Consequently, TAG turnover rate was determined by fitting an exponential model to enrichment data rather than conventional two-point linear analysis. In the high-substrate group, both turnover rate (DR 820 ± 330, NR 190 ± 150 nmol·min–1·g–1 dry wt; P < 0.001) and [TAG] content (DR 78 ± 10, NR 32 ± 4 µmol/g dry wt; P < 0.001) were greater in the diabetic group. At lower substrate concentrations, turnover was greater in diabetics (DR 530 ± 300, NR 160 ± 30; P < 0.05). However, this could not be explained by simple mass action, because [TAG] content was similar between groups [DR 34 ± 7, NR 39 ± 9 µmol/g dry wt; not significant (NS)]. Consistent with exponential enrichment data, 13C fractional enrichment of TAG was lower in diabetics (low- substrate groups: DR 4 ± 1%, NR 10 ± 4%, P < 0.05; high-substrate groups: DR 8 ± 3%, NR 14 ± 9%, NS), thereby supporting earlier speculation that TAG is compartmentalized in the diabetic heart.

metabolism; nuclear magnetic resonance; palmitate; triglyceride; fatty acids

UNCONTROLLED DIABETES IS CHARACTERIZED by high levels of circulating fatty acids and glucose (32, 37). Although it has been shown that high levels of exogenous fats can drive fatty acid oxidation and simultaneously block glucose oxidation (35), high levels of plasma glucose are not enough to augment glucose utilization in diabetes. Glucose oxidation is limited, in part, by a reduction in GLUT1–4 transporter proteins and mRNA levels (7, 37). With diabetes, glucose utilization is significantly reduced, such that the heart relies almost exclusively on fatty acid oxidation to meet energy demands (2, 17, 32, 37). An increased reliance on fatty acids has since been linked to functional abnormalities of the heart (2, 24, 32).

Metabolic derangements are not explained entirely by alterations in circulating substrates and membrane transport activity. The heart of a diabetic is also characterized metabolically by 1) a reduction in the dephosphorylated active form of pyruvate dehydrogenase (12, 39), 2) an alteration in creatine kinase enzyme kinetics (36), and 3) an activation of the PPAR{alpha} and PPAR{alpha} coactivator-1a (PGC-1{alpha}) gene-regulatory system (8). Although these are key processes linked to the balance between carbohydrate and fatty acid oxidation, there remain numerous metabolic processes yet to be characterized in the diabetic.

We postulate that changes in glucose and fatty acid oxidation may also be linked to changes in turnover and the kinetics of fatty acid storage. Whereas circulating fatty acids provide the majority of substrates for mitochondrial metabolism, endogenous triacylglycerol (TAG) pools are also an important source of energy. With diabetes, TAG pools accumulate (28), and the intermediates of lipid storage can have potentially deleterious effects (30, 40, 42). The importance of this TAG pool as a substrate source and its regulatory control over exogenous substrate oxidation have not been well characterized for diabetes.

In this study, we present the first kinetic model of TAG turnover from 13C-NMR isotopic enrichment data. This approach holds a distinct advantage over the traditional radiolabeled experiments used to estimate TAG turnover. Whereas 14C-3H studies rely on two-point linear analysis from heart extracts or effluent, 13C enrichment of the TAG pool can be monitored continuously in the intact beating heart (25). This enabled us to assess palmitate storage, TAG turnover, and palmitate oxidation simultaneously in individual hearts.

Our objectives were to establish 13C-NMR analysis of TAG turnover in the intact beating heart and determine the effects of 18 days of streptozotocin (STZ)-induced diabetes on myocardial fatty acid storage and turnover in the intact functioning heart. To provide a complete assessment of lipid utilization, turnover and storage were assessed in parallel to measures of glucose, glycogen, palmitate, and endogenous TAG oxidation. This study denotes the first direct assessment of TAG kinetics in the intact beating heart. Most importantly, these experiments have yielded surprising new findings of lipid turnover and oxidation in hearts from diabetic rats (DR).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Induction of diabetes mellitus. Type 1 diabetes (insulin deficient) was induced in female rats of the Sprague-Dawley strain (250 g body wt) by a single intravenous injection of STZ (60 mg/kg) (20). STZ was dissolved in a 0.01 M citrate buffer, pH 4.5, and injected within 5 min. Normal rats (NR) were injected with citrate buffer. After 48 h, blood samples were obtained for determination of glucose levels, and rats with glucose levels greater than 350 mg/dl were held for the diabetic group. The protocol was approved by the Animal Care Policies and Procedures Committee at the University of Illinois at Chicago (Institutional Animal Care and Use Committee accredited), and animals used were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, revised 1996).

This model of diabetes displays many of the features seen in human subjects with uncontrolled diabetes mellitus (type 1), including hyperglycemia, hypertriglyceridemia, hypoinsulinemia, increased urinary glucose levels, and, consequently, polyuria, as well as weight loss (26, 31). At a moderate dose of STZ (60 mg/kg), reductions in heart rate (HR) and diastolic performance are observed within 3 days, whereas systolic dysfunction develops after 35 days (11, 22).

Isolated-heart experiments. After 18 days, the animals were heparinized (500 U/100 g ip) and anesthetized with pentobarbital sodium (100 mg/kg ip). Rats were weighed and blood samples were taken to measure glucose levels. The rats were nonfasted (standard rat chow), and hearts were excised for perfusion experiments between 10 AM and 5 PM. The heart was immediately excised and immersed in a solution containing 20 mM KCl and 120 mM NaCl for cardioplegia at 0°C. The aorta was cannulated for retrograde perfusion at 100 cm hydrostatic pressure, with modified Krebs-Henseleit buffer equilibrated with 95% O2-5% CO2 at 37°C. The buffer contained (in mM): 116 NaCl, 4 KCl, 1.5 CaCl2, 1.2 MgSO4, 1.2 NaH2PO4, and 25 NaHCO3. During preparation, spontaneously beating hearts were perfused with buffer containing 5 mM glucose and 2.5 mM butyrate. A latex balloon was placed in the left ventricle and connected to a pressure transducer line for physiological measurements (PowerLab; AD Instruments, Colorado Springs, CO). The balloon was inflated with water to create a diastolic pressure of 5–10 mmHg. Left ventricular developed pressure (LVDP) and HR were continuously measured and recorded with the intraventricular balloon. Rate pressure product (RPP = HR x LVDP) was used as an index of mechanical work. Myocardial oxygen consumption was calculated from the difference in O2 content of perfusion medium in the supply line and coronary effluent collected from the pulmonary artery (23).

Protocols. The hearts were first perfused for 30 min with medium containing unlabeled substrates. Then, one protocol provided normal hearts (n = 6) and hearts from diabetics (n = 6) with recirculated medium containing 0.5 mM [2,4,6,8,10,12,14,16-13C8]palmitate plus 5 mM unlabeled glucose. The concentrations of substrates were selected to represent near-normal physiological levels. Sequential 13C-NMR spectra were collected every 2 s and averaged every 2 or 4 min to follow the incorporation of labeled substrate into the TAG and glutamate pools. After 2 h, the hearts were freeze-clamped for additional analysis. The protocol was repeated for a second group of normal (n = 8) and diabetic hearts (n = 8) perfused with medium containing concentrations of substrates more characteristic of the in vivo diabetic state (1.2 mM [2,4,6,8,10,12,14,16-13C8]palmitate plus 11 mM unlabeled glucose).

In all experiments, the sodium salt of palmitic acid was complexed to albumin in a 3:1 molar ratio and dialyzed before use (25). Palmitate, a 16-carbon-length chain, was selected as the representative long-chain fatty acid on the basis of the equimolar presence in plasma, along with the 18-carbon-chain oleate. Additionally, a large body of literature (19, 28, 33, 34) that describes the metabolism of palmitate in the heart is already available. An additional practical consideration for the use of [2,4,6,8,10,12,14,16-13C8]palmitate in this study is the relative cost of synthesis and commercial availability compared with oleate.

To access the oxidation of exogenous glucose, the protocol was repeated with perfusate containing 13C-labeled glucose (5 mM [1,6-13C2]glucose) and unlabeled 0.5 mM palmitate. Together, the two labeling protocols enabled profiles of substrate selection in normal and diabetic groups.

NMR measurements. NMR parameters required for the acquisition of 13C-NMR spectra from isolated hearts are as previously reported (25). Briefly, perfused hearts were positioned in a 20-mm broadband probe in a 9.4-T/89-mm vertical bore, superconducting NMR magnet. Magnetic field homogeneity was optimized by shimming to a proton line width of 10–20 Hz. Carbon spectra were then acquired at 100 MHz, with bilevel broadband decoupling, and subtracted from naturally abundant endogenous 13C signal.

High-resolution 13C- and 1H-NMR spectra of heart samples (perchloric acid extracts) reconstituted in 0.5 ml of 2H2O were obtained with a 5-mm probe placed in a Bruker 14-T magnet, as previously reported (6, 14, 15). In vitro 13C spectra were collected over 3 to 12-K scans (45° pulse, 30,000 Hz sweep width, 32 K data set, 2 s recycle time) with broadband proton decoupling. The multiplet structures of the glutamate carbon resonance signals allowed the fraction of [2-13C] acetyl-CoA entering the TCA cycle to be calculated (21).

In vitro 1H spectra were collected over 500 scans (45° pulse, 12,000 Hz sweep width, 32 K data set, 2 s repetition). The incorporation of 13C produced scalar (J 13C-1H) coupling that resulted in multiplet signals (signal splitting) of the alanine and lactate methyl group proton resonances, assigned at 1.5 and 1.35 ppm, respectively. The splitting of signals from protons covalently bonded to 13C from the central resonance of protons bonded to 12C signal were used to determine the fractional 13C enrichment of both metabolites (6).

Kinetic analysis. As depicted in Fig. 1, [13C]palmitate crosses the cell membrane and is either stored in the endogenous TAG pool or oxidized via the mitochondria. The incorporation of labeled palmitate into the TAG pool was assessed based on the 13C-NMR detection of the TAG methylene resonances at 31 ppm (25).


Figure 1
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  		Fig. 1. Schematic representation of palmitate and glucose uptake and incorporation into intermediary metabolite pools. Storage of palmitate into triacylglycerol (TAG) pool can be assessed based on 13C-NMR enrichment of TAG. Oxidation of palmitate through mitochondrial processes is assessed based on the final enrichment of the NMR detectable glutamate pool. Likewise, labeled glucose utilization is assessed based on NMR analysis of alanine (ALA) and glutamate enrichment. LAC, lactate; PYR, pyruvate.

 
A monoexponential equation of TAG enrichment was fitted to the dynamic 13C-NMR data collected for TAG from each heart, and solutions to the equation gave the rate constant of enrichment and turnover rate. The exponential equation listed below is used as the model of TAG 13C enrichment:

Formula
where TAG*(t) is the fractional enrichment of the TAG pool at time (t), Fc is the fractional enrichment of palmitate (100%), RT is the flux of palmitate entering the TAG pool, and k2 is the rate constant for palmitate efflux from the TAG pool. The turnover rate of the cytosolic TAG pool can be determined by nonlinear least-square fitting of the equation to 13C-NMR data of TAG enrichment. The rate of TAG efflux, or turnover, is RTAG = k2* [TAG]/3 (nmol·min–1·g dry wt–1). [TAG] is the concentration in µmol/g dry wt, and the turnover is divided by three to account for the three fatty acids that exchange with one TAG molecule.

Lipid extract data. Lipid extracts were prepared from heart samples for mass spec analysis and [TAG] content as previously described (1, 3). One hundred milligrams of frozen heart tissue were homogenized and extracted in 20 ml of chloroform-methanol (2:1), followed by an addition of 3 ml of methanol, and the extract was vortexed. After 30 min, the sample was centrifuged at 3,000 rpm for 12 min. The pellet was discarded, and 0.04% CaCl2 was added to the supernatant, which was then centrifuged at 1,000 rpm for 20 min. The upper phase was removed, and the lower phases were washed three times with solvent (1.5 ml chloroform, 24 ml methanol, and 23.5 ml H2O). The final wash was removed, and 0.5 ml of methanol was added to obtain one phase. The samples were dried under N2 gas at 55°C and redissolved in 3:2 methyl-propanol and Triton X-100. Cardiac TAG was then quantified colorimetrically by enzymatic assay (Sigma).

TAG was isolated and saponified, and fractional 13C enrichment of the fatty acids was assessed by mass spectrometry analysis. First, the lipid samples were passed through a silicic adsorption chromatography column and flushed with 20 ml of chloroform to separate TAG from phospholipids (Bio-Sil HA gel, 325 mesh) (18). The TAG fraction was evaporated under N2 gas at 55°C and resuspended in 95% ethanol-5% KCl, for saponification at 70°C for 1 h. The fraction of fatty acid chains labeled with 13C was determined by mass spectrometry (Waters X-terra C18MS column; MS:scan m/z 100–600 Fragmentor 75V Negative ESI).

Substrate oxidation. The high-resolution NMR analysis of tissue extracts provided the relative contribution of glucose, palmitate, glycogen, and endogenous fats to acetyl-CoA formation and mitochondrial oxidative metabolism. With both glucose and palmitate contributing to the formation of mitochondrial acetyl-CoA (see Fig. 1), the oxidation of either substrate was assessed by following the incorporation of 13C label from either glucose or palmitate into the acetyl-CoA pool. The Fc was determined by standard isotopomer analysis from glutamate resonances (21).

The contribution of endogenous glycogen to mitochondrial metabolism was assessed on the basis of 1H-NMR analysis of alanine enrichment in hearts oxidizing [1,6-13C2]glucose and unlabeled palmitate. As shown in Fig. 1, labeled glucose and endogenous glycogen contribute to the formation of both pyruvate and alanine via glycolysis. Although intracellular pyruvate content is too low for NMR detection, isotopic equilibrium with the readily NMR-detected alanine pool indicates the enrichment of glycolytic end products (6, 14, 15, 29). Thus the labeled fraction of alanine (FA) corresponds to exogenous [13C]glucose utilization, and the unlabeled fraction of alanine (1-FA) corresponds to the endogenous glycogen utilization. Having measured the contribution of [1,6-13C2]glucose to mitochondrial metabolism based on acetyl-CoA enrichment (Fc, described above), the subsequent contribution of endogenous glycogen to mitochondrial acetyl-CoA and metabolism can be calculated as Fc(1-FA)/FA.

Statistical analysis. Data are presented as means ± SD unless otherwise stated. Data set comparisons were performed with Student’s unpaired, two-tailed t-test. Differences in mean values were considered statistically significant at a probability level of <5% (P < 0.05).


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Characteristics of acutely DR. Survival rates were 90% for the STZ-treated rats and 100% for the citrate buffer control group. Plasma glucose and body weights were measured in hearts from the NR and DR groups. In rats injected 18 days earlier with a single intravenous dose of STZ, blood glucose levels were 390 ± 19 mg/dl in DR (n = 12) compared with 126 ± 9 mg/dl in NR (n = 12, P < 0.005), whereas body weights were 205 ± 20 and 264 ± 63 g (NS), respectively.

Heart function in the isolated hearts was monitored for both diabetic and normal groups throughout the perfusion protocol. Table 1 shows HR, LVDP, and RPP. As expected, a decrease in HR was seen in the DR hearts compared with NR (4, 34), whereas developed pressure was similar between groups for the duration of the 2-h perfusion protocol. Oxygen consumption was not significantly different between groups (low-substrate groups: DR 22.9 ± 6.1, NR 20.5 ± 6.5 µmol·min–1·g dry wt–1; high-substrate groups: DR 21.0 ± 3.0, NR 19.0 ± 3.5 µmol·min–1·g dry wt–1; NS). Thus TAG turnover and energy substrate utilization measurements made in this study were not complicated by major changes in heart function.


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  		Table 1. Effects of streptozotocin-induced diabetes on body weight, blood glucose, and physiological function of the isolated retrograde perfused rat heart

 
TAG content in heart tissue was measured both in a subset of animals at the time of death and from all hearts at the end of the perfusion protocol. As expected, TAG content was significantly greater in the DR group compared with NRs at the time of death: 53 ± 7 (DR, n = 5) vs. 36 ± 10 µmol/g dry wt (NR, n = 5, P < 0.05). In normal and diabetic hearts perfused with medium containing normal substrate levels (0.5 mM palmitate and 5 mM glucose), TAG content was similar between groups at the end of the perfusion protocol: 34 ± 7 µmol/g dry wt in DRs (n = 5) vs. 39 ± 9 µmol/g dry wt in NRs (n = 6, P > 0.05).

The lower TAG content seen in the diabetic group at the end of the protocol, compared with at the time of excision, is consistent with the literature (5, 24). That is, TAG content drops in heart from diabetic animals during the first 10–20 min after excision when perfused with medium containing normal levels of substrates: 0.5 mM palmitate and 5 mM glucose. For this reason, we collected 13C-NMR data after allowing for this initial 30-min equilibration period on unlabeled substrates. Importantly, the 13C-NMR data revealed no change in total TAG content during the 2-h NMR acquisition period. That is, a further drop in the endogenous TAG pool would have appeared as negative resonances in the NMR spectra after subtracting the initial background signal. Furthermore, additional hearts supplied unlabeled palmitate, and unlabeled glucose confirmed no change in the natural abundance signal from endogenous TAG after the initial 30-min equilibration period (data not shown).

In normal and diabetic hearts perfused with medium containing substrate levels more characteristic of the diabetic condition (1.2 mM palmitate and 11 mM glucose), TAG content was significantly greater in the diabetic group (78 ± 10 µmol/g dry wt) compared with normal hearts (32 ± 4 µmol/g dry wt, P < 0.005). This expected result matches earlier reports by Lopaschuk and colleagues (19, 34).

Labeling heart lipids and glutamate with [13C]palmitate. Proton-decoupled 13C spectra of an isolated heart perfused with [2,4,6,8,10,12,14,16-13C8]palmitate and unlabeled glucose are shown in Fig. 2. Peak assignments include glutamate carbons C2, C4, and C3 at 56, 34, and 28 ppm, respectively. The incorporation of 13C-labeled palmitate into the endogenous TAG pool is also apparent in the 13C NMR spectra. Five of the eight labeled carbons of palmitate are assigned to 31 ppm, whereas the other three labeled carbons are assigned at 14, 33, and 38 ppm. The assignment of labeled TAG is based on an earlier study of high-resolution 13C NMR spectra from labeled palmitate and TLC-mass spec analysis of lipid extracts prepared from heart samples (25).


Figure 2
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  		Fig. 2. Selected 13C spectra from dynamic data set obtained from isolated rat heart perfused with 0.5 mM [2,4,6,8,10,12,14,16-13C8]palmitate and 5 mM glucose demonstrate labeling of both glutamate and TAG pools. Peak assignments are glutamate carbon-2 (GLU-C2), -3 (GLU-C3), and -4 (GLU-C4). Resonances detected at 15, 30, and 32 ppm correspond to enrichment of TAGs.

 
The enrichment of the TAG pool with 13C-labeled palmitate is plotted in Fig. 3 as a function of time. The NMR data is normalized to the final fractional enrichment of the TAG pool. The final enrichment was measured by mass spectrometry. Surprisingly, 13C NMR enrichment of the TAG pool in the diabetic groups reaches steady state within the first hour, whereas the TAG enrichment did not reach steady state in the normal group, even after 2 h. Even though the diabetic group showed a leveling off of TAG enrichment, results from mass spec analysis showed that only 4 ± 1% (n = 5) of the total pool was enriched by 2 h in the low substrate group, whereas in the hearts from healthy rats, enrichment was 10 ± 4% (n = 6, P < 0.05) after 2 h. With elevated substrates (1.2 mM palmitate and 11 mM glucose), enrichment was 8 ± 3% (n = 8) for diabetics and 14 ± 9% (n = 8, NS) for normals. Note that, because each 13C-labeled carbon of palmitate is paired with an unlabeled carbon, we simply double the 13C fractional enrichment values reported here to define the fraction of TAG synthesized from exogenous palmitate.


Figure 3
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  		Fig. 3. Representative time course of TAG 13C enrichment from exogenous [2,4,6,8,10,12,14,16-13C8]palmitate in isolated perfused hearts from nondiabetic and diabetic rats. Left: perfusion medium included 0.5 mM labeled palmitate and 5 mM unlabeled glucose; right: perfusate included 1.2 mM labeled palmitate and 11 mM unlabeled glucose. Enrichment of the TAG pool reached steady state by 1 h in the diabetic group with only 4–8% of the total pool labeled, whereas enrichment continued to climb in the healthy group, even after 2 h (values are means ± SE).

 
We perfused an additional four normal hearts with labeled palmitate and unlabeled glucose for 4 h. NMR enrichment data from two of the hearts showed the beginning signs of leveling off at 4 h (i.e., steady-state 13C enrichment), whereas the signal from the other two hearts was still linear at 4 h (data not shown).

TAG turnover in NR and DR hearts. Myocardial TAG was labeled by perfusing hearts for 2 h with buffer containing [2,4,6,8,10,12,14,16-13C8]palmitate and unlabeled glucose. The kinetic model of TAG turnover was fitted to the resultant 13C enrichment curves of Fig. 3 to give the rate constant of enrichment and TAG turnover rate. Solutions to the fit are listed in Table 2. In the diabetic groups, the rate constant of enrichment was 0.044 for diabetics, and the turnover rate was 530 nmol·min–1·g dry wt–1 (n = 5). As described, the 13C enrichment of the nondiabetic normal group did not reach steady state after perfusion of the heart for 2–4 h with [13C]palmitate. However, the model was fitted to these data, and solutions to the fit yielded a rate constant of 0.013 and a TAG turnover of 160 nmol·min–1·g dry wt–1 (n = 6, P < 0.05). Importantly, additional simulations from the model indicated that the rate constant could be ≤0.013. Higher constants would have resulted in the enrichment reaching steady state earlier than observed.


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  		Table 2. Analysis of [TAG] content, enrichment, and turnover in heart

 
At high levels of substrates (1.2 mM palmitate and 11 mM glucose), we observed a greater turnover rate in the diabetics (n = 8) compared with normals (n = 8; Table 2). This is consistent with earlier reports (17, 19, 34). In addition, the rate constant of enrichment was similar between diabetics at low and high substrate levels. Therefore, the greater turnover rate of the high-substrate group can be attributed to the higher endogenous [TAG] pool.

Substrate selection. To provide a complete assessment of lipid utilization, turnover and storage were determined in parallel to measures of glucose, glycogen, palmitate, and endogenous TAG oxidation (low-substrate group only). Here, substrate oxidation is the contribution of the substrate to mitochondria ATP production. As expected (17, 19, 34), the percent contribution of glucose to mitochondrial metabolism was lower in the diabetic group (2.9 ± 1.2% contribution) relative to the normal group (6.4 ± 2.5%, P < 0.05). Palmitate oxidation was also significantly lower in the diabetic group (72.2 ± 7.5%) compared with normals (85.8 ± 4.3%, P < 0.005). The percent contribution of endogenous glycogen to mitochondrial metabolism was not significantly different between groups: 7.6 ± 3.1% in normal hearts and 8.1 ± 4.0% in diabetic. The balance of oxidized substrate shown in Fig. 4 is attributed to the oxidation of endogenous fats. Endogenous fats contributed significantly to overall oxidative metabolism in the diabetic group (16.8 ± 12.0%, n = 5), consistent with the higher turnover rate. In the normal group, endogenous TAG oxidation was not detected (n = 6).


Figure 4
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  		Fig. 4. Relative contribution (%) to mitochondrial ATP production by [13C]palmitate, [13C]glucose, glycogen, and endogenous TAGs. Glucose contributions were lower in the diabetic group relative to the nondiabetic group, as expected. Interestingly, endogenous fatty acid TAG contributed significantly more in the diabetic group, consistent with a higher TAG turnover relative to the normal group. (buffer contained 0.5 mM palmitate and 5 mM glucose).

 
Alanine and lactate enrichment. Whereas the labeling of the small intracellular pool of the glycolytic end product pyruvate is below the sensitivity of NMR detection, the unlabeled fraction of the alanine pool denotes glycogen contributions to glycolysis. 13C enrichment of the glycolytic end products lactate and alanine were determined from proton NMR spectroscopy of extracts of hearts perfused with buffer containing labeled glucose and unlabeled palmitate (6, 15). Alanine enrichment was 45.0 ± 10.4% in the normal group vs. 23.2 ± 8.5% (P < 0.05) in the diabetics. The fractional enrichment of lactate in the normal group was 40.7 ± 16.9% (n = 5) compared with 21.4 ± 13.1% (n = 5, P < 0.05) in the diabetic group. Despite the fact that in these experiments the isotopic enrichment of lactate is relatively similar to that of alanine, lactate is not in chemical or isotopic equilibrium with pyruvate and cannot be reliably used as a measure of pyruvate labeling. Rather, alanine more closely represents the isotopic labeling of pyruvate (6, 29).


   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We show here the first approach toward a comprehensive, 13C-NMR analysis of long-chain fatty acid handling in the heart of diabetic rat. The advantage of NMR over traditional methods is that the sequential enrichment of the TAG pool is directly observed with time for each individual heart with concurrent measurements of fatty acid entry into, and oxidation by, mitochondria. The normal and diabetic data shown here represent the first continuous detection of long-chain fatty acid incorporation into the TAG pool that is characterized by an exponential enrichment pattern. Although many of our data for substrate oxidation correlated well with other reports, we have discerned profound differences in lipid turnover between the diabetic and nondiabetic normal heart. The greater TAG turnover would account for the greater oxidation of endogenous fats found in the hearts of diabetics. In addition, even though TAG content was similar between groups after an initial equilibration period in the low-substrate group (0.5 mM palmitate and 5 mM glucose), TAG enrichment reached steady state much earlier in the diabetic group relative to the normals. We postulate whether this original observation is a consequence of changes in TAG compartmentation between diabetics and normal myocardium.

In earlier studies, the contribution of myocardial TAG to oxidative metabolism was inferred from changes in TAG content or by measuring glycerol release [see reviews (28, 34)]. However, measurement of myocardial TAG content does not provide a measurement of the relationship between TAG lipolysis and synthesis (i.e., turnover) (34), and glycerol release is not a good measure of TAG lipolysis, because it can originate from other sources (13). More recent studies measured myocardial TAG turnover and fatty acid oxidation in isolated perfused hearts by following the oxidation of radiolabeled long-chain fatty acids (10, 19, 28, 34). As an example, the isolated heart was retrogradely perfused with buffer containing [14C]palmitate for 1 h to label the endogenous lipid pools. The heart was then either freeze-clamped to assess incorporation of the labeled substrate into the TAG pool or perfused for a second hour with [3H]palmitate. 14CO2 and 3H2O release during the second hour provided a measure of endogenous and exogenous fatty acid oxidation, respectively. Linear analysis of the enrichment data at the end of the first hour gave TAG synthesis rates (i.e., rate = change in enrichment/time). If the study is completed under steady-state conditions, TAG synthesis equals lipolysis.

Although the radiolabeled studies are clearly a substantial improvement over earlier approaches, the turnover of the TAG pool is at best an estimate by this technique. The synthesis rate reported assumed linear enrichment with time and is calculated solely on the final enrichment of the TAG pool at the time the heart is frozen. Several groups (33) have shown, however, that shorter enrichment periods yielded greater turnover rates when based on end-point linear enrichment. This change in turnover is not possible if it is assessed for a single pool under steady-state conditions. Conversely, we collected an NMR-free induction decay signal every 2 s and averaged the data over 2- to 4-min time intervals to assess TAG enrichment. Over a 2-h period, we found that the enrichment is more appropriately defined by an exponential. As expected, this finding is consistent with previous isotopic labeling profiles of nonlipid metabolic pools under steady-state conditions (41). Our results also explain why others saw greater turnover rates with shorter enrichment periods (5, 33). A linear analysis of an exponential enrichment profile yields greater slopes at earlier time points.

Fitting our exponential model of TAG turnover to the NMR data gave a turnover rate of 530 nmol·min–1·g dry wt–1 in the diabetics (low-substrate group). A linear fit to the same final 13C data would have yielded a significantly lower rate. In the normal group, TAG turnover rate was 160 nmol·min–1·g dry wt–1 despite a similar [TAG] pool to diabetics. At high levels of substrates (1.2 mM palmitate and 11 mM glucose), we observed a greater turnover rate in the diabetics compared with normals. This is consistent with earlier reports (17, 19, 28, 34). Unlike the low-substrate group, the higher turnover rates seen in this group of diabetics includes a component of mass action, because TAG content was significantly greater in the diabetic group (see Table 2).

The incorporation of labeled palmitate into the endogenous TAG pool was significantly different between groups. In the diabetic group, the 13C fractional enrichment of TAG reached steady state within 1 h with only 4–8% of the total pool labeled, whereas enrichment continued to climb to 10–14% in the healthy group, even after 2 h. As previously stated, we need only double the 13C fractional enrichment values to describe the fraction of TAG synthesized from the exogenous palmitate (i.e., each 13C carbon of palmitate is paired with an unlabeled carbon). Thus 8–16% of the TAG pool was synthesized from the exogenous palmitate in the diabetic group and 20–28% in the normal groups. These values are low compared with the 17–58% reported by others (19, 34) using 14C and 3H methods. However, the radiolabeling methods do not provide a direct measurement of the actual fraction of molecules that are labeled. Rather, the radioisotope method relies on specific activity, a measure of disintegrations per minute per unit of mass or moles and not a direct measurement of a labeled and unlabeled mass. A separate TAG measurement is required to determine total fat based on glycerol content after saponification. By contrast, the use of stable isotopes with mass spectrometry provides direct measurement of both labeled and unlabeled fractions of fats simultaneously from a given sample after phospholipids have been removed. Thus the radiolabeled approach is indirect, whereas the mass spectrometry method is a direct analysis of the fractional isotopic enrichment.

Our observation that TAG reached steady-state enrichment in the diabetic group with only 4–8% of the total TAG pool enriched suggests that multiple pools of TAG exist in the myocardium. Other laboratories have also suggested multiple pools of cardiac TAG, with each pool having different turnover rates (5, 9, 16, 27, 33, 38). For example, Stein and Stein (38) pulsed labeled perfused hearts with [9,10-3H]oleic acid and showed changes in the subcellular localization of the esterified fatty acid with time, using radioautographic techniques. Separate studies by Crass (5) and Saddik and Lopaschuk (33), using the prelabeling approaches, also obtained data that suggested the presence of multiple pools of TAG. Further evidence has been obtained in heart perfused in the absence of exogenous substrates. After eventual arrest of the heart, nearly one-half of the TAG remained in the tissue, indicating that only about one-half of the lipid was available for energy metabolism (9, 27).

With respect to fatty acid vs. glucose oxidation, we were able to assess the contribution from glucose, glycogen, palmitate, and endogenous fatty acids to mitochondrial ATP production on the basis of high resolution 13C-NMR analysis of metabolite intermediate labeling from heart extracts. In agreement with other reports (32, 37), glucose contributed less to mitochondrial ATP production in the diabetic group relative to nondiabetics, whereas glycogen contributions were unchanged. Interestingly, the contribution from exogenous palmitate was also lower in the diabetic group (17, 19, 34). The balance of oxidized substrate, contributing to the formation of acetyl-CoA, originated from unlabeled endogenous sources and would reflect increased oxidation of TAG. This would be a requisite for the mechanism of a higher turnover of the TAG pool observed here and would support data presented by Paulson and Crass (28). They found increased lipolysis and TAG fatty acid oxidation in hearts from diabetics perfused under substrate conditions similar to ours (0.5 mM palmitate). On the other hand, Saddik and Lopaschuk (34) reported similar oxidation rates of endogenous TAG fatty acids in diabetics relative to normals. They showed that the majority of fatty acids derived from TAG lipolysis in the diabetic group was released into the perfusate. The difference between our finding and was that of Saddik and Lopaschuk may reflect the higher concentrations of exogenous palmitate that they used in their study to assess oxidation. Without this elevated source of exogenous fatty acids, the heart would rely on endogenous sources to meet a similar energy demand (33).

Importantly, we included a group of normal and diabetic rat hearts perfused with physiological levels of fats (0.5 mM palmitate) and carbohydrates (5 mM glucose). Kinetic analysis of TAG turnover was assessed after TAG content had equilibrated and reached similar levels between these two groups. This eliminated the confounding variable of pool size between experimental groups so that a mechanistic conclusion could be established. Turnover was greater in the diabetic group despite similar [TAG] between groups. Therefore, the difference in turnover is not explained by simple mass action of a higher [TAG] pool reported for the diabetic group in vivo, but rather demonstrates a mechanism of known changes in fatty acid oxidation enzymes that impact on TAG turnover rates.

In summary, three important conclusions can be drawn from the new data. 1) NMR can be used to directly monitor TAG turnover in intact functioning heart; thereby, this is the first study to correctly demonstrate an exponential enrichment profile of TAG. 2) The turnover rate of the endogenous TAG pool was significantly greater in the diabetics; consistent with this finding, the oxidation of endogenous unlabeled fatty acids was significantly greater in the diabetic group relative to the nondiabetics under the conditions of our protocol. 3) 13C-NMR enrichment of the TAG pool reached steady state far earlier in the diabetic group and at a much lower final fractional enrichment compared with normals. This finding supports earlier suggestions that lipid storage may be compartmentalized in the myocardium (5, 9, 16, 27, 33, 38), and not all of the lipid pools are coupled to oxidative processes of the mitochondria.


   GRANTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by National Heart, Lung, and Blood Institute Grant nos. R37-HL-49244 and RO1-HL-62702.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. D. Lewandowski, Program in Integrative Cardiac Metabolism, Dept. of Physiology and Biophysics, Univ. of Illinois at Chicago, 835 South Wolcott Ave., Chicago, IL 60612 (e-mail: dougl{at}uic.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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