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Am J Physiol Heart Circ Physiol 282: H717-H725, 2002; doi:10.1152/ajpheart.00257.2001
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Vol. 282, Issue 2, H717-H725, February 2002

Palmitate-induced cardiac apoptosis is mediated through CPT-1 but not influenced by glucose and insulin

Jennifer Y. Kong and Simon W. Rabkin

Division of Cardiology, Department of Medicine, University of British Columbia, Vancouver, British Columbia, Canada V5Z 3J5


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To test the hypothesis that regulation of palmitate metabolism, through carnitine palmitoyl transferase-1 (CPT-1) or through alterations of glycolysis, was involved in the pathway of palmitate-mediated cell death, cardiomyocytes were cultured from 7-day-old chick embryos. Palmitate-induced cell death, assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, was enhanced by carnitine, a cofactor needed for palmitate transport into mitochondria via CPT-1. Carnitine co-incubation with palmitate significantly (P < 0.01) increased the amount of apoptotic cells, assessed by propidium iodine staining and fluorescent-activated cell sorting analysis compared with treatment with either palmitate or carnitine alone. The CPT-1 inhibitor oxfenicine significantly (P < 0.05) blocked the cell death induced by the combination of palmitate and carnitine. The short-chain saturated fatty acid capric acid (100 µM), which is not likely transported by CPT-1, did not significantly affect cell viability, whereas the C18 saturated fatty acid stearic (100 µM) significantly (P < 0.01) reduced cell viability and to a similar extent as palmitate. In contrast, there was no significant alteration of palmitate-induced cell death by cotreatment with 100 nM insulin + 2 g/l glucose or 1 mM lactate, which promote ATP generation by glycolysis rather than fatty acid oxidation. Fumonisin did not alter palmitate-induced cell death or apoptosis, suggesting that the effect of palmitate was not operative through increased ceramide synthesis. These results suggest that oxidation of palmitate through CPT-1 is involved in the production of apoptosis in cardiomyocytes.

carnitine; fatty acid metabolism; oxfenicine; fumonisin; palmitate; capric acid


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

FATTY ACIDS ARE A PRINCIPAL source of energy for the heart, and the metabolism of fatty acids is of fundamental importance for cardiac function (for reviews, see Refs. 23, 24, 48). Briefly, fatty acid metabolism involves fatty acids binding to carrier binding proteins that are transported across the sarcolemmal membrane, and once inside the cell are metabolized to long chain acyl-CoA by acyl-CoA synthetase (23, 24, 48). The acyl moieties are transferred to the mitochondria by a series of enzymes involving carnitine palmitoyl transferase-1 and -2 (CPT-1 and -2) as well as carnitine-acylcarnitine translocase. In the mitochondria, long-chain acyl-CoA is subjected to beta -oxidation-producing acetyl CoA, a fatty acyl moiety less two carbons, and the by-products, reduced flavin adenine dinucleotide and reduced nicotinamide adenine dinucleotide. Fatty acid oxidation also occurs in peroxisomes (23, 24, 48) but the amount is small (9).

High concentrations of certain fatty acids can be damaging to the heart. Palmitate, a 16-carbon saturated fatty acid, is one of the most common fatty acids and induces apoptotic cell death in various cell types, including the myocyte element of the heart (12, 20, 37, 44, 55). The mechanism of palmitate-induced cell death is not completely understood, yet it is of considerable importance because high levels of fatty acids are present in patients with acute myocardial infarction and accentuate the extent of the myocardial injury (36, 52).

Several hypotheses have been advanced to explain palmitate-induced cell death. Palmitate is incorporated into de novo synthesis of ceramide (31), a lipid-signaling molecule implicated in the induction of apoptosis (35), so that excess palmitate may induce cell death through increased intracellular ceramide concentration (37). An equally attractive hypothesis, which may be especially relevant for the heart, is that the adverse effect of excess fatty acids is due to metabolic factors, specifically, palmitate may induce cell death by its inability to be completely metabolized, hence, producing partially metabolized fatty acids that are toxic (17, 22). This postulate leads to consideration of the regulatory control of fatty acid metabolism and the possibility that changes in its regulation may modulate palmitate-induced cell death. Fatty acid oxidation is subject to glycolysis-initiated control (23, 24). Excess acetyl CoA is transported out of the mitochondria and is converted to malonyl CoA in the cytosol by acetyl CoA carboxylase (ACC) (27). Malonyl CoA can directly inhibit CPT-1 (4). Hence, there are two points of glycolysis-initiated control of fatty acid oxidation: generation of malonyl CoA and ACC activity. Upregulation of glycolysis can occur by the exogenous addition of glucose. ACC activity is sensitive to hormones such as insulin and glucagon (29). Thus we hypothesized that insulin and glucose, through inhibition of palmitate oxidation, might modulate palmitate-induced cell death. Another approach to examine the same mechanism is through the ability of lactate, a product of glycolysis, to inhibit palmitate oxidation in cardiomyocytes (3). Lactate is readily converted to pyruvate. The advantage of administration of exogenous lactate is that lactate produces pyruvate without the energy-requiring step of glucose phosphorylation and dephosphorylation. The pyruvate that is derived from lactate is then converted by pyruvate dehydrogenase complex to acetyl-CoA within the mitochondria. An increase in acetyl-CoA production from pyruvate dehydrogenase complex will result in a shuttling of acetyl groups into the cytoplasm, producing an increase in malonyl-CoA production, which, in turn, will inhibit fatty acid oxidation (for a review, see Ref. 23). The embryonic heart is more dependent on glycolysis than the adult heart (15, 41). This maybe attributed to fewer and smaller mitochondria, with a lower density of cristae, lighter matrices, and more variable inner membrane configurations in the fetal heart than in the adult heart (45). Thus the embryonic heart is ideal to test the hypothesis that regulatory control of fatty acid metabolism by glycolysis modulates palmitate-induced cell toxicity.

Another step in the regulatory control of palmitate metabolism that may modulate palmitate-induced cell death is CPT-1. Because CPT-1 is integral to fatty acid metabolism and governs a rate-limiting step of fatty acid transport into the mitochondria for its oxidation (30), we sought to investigate the role of CPT-1 in palmitate-induced cell death. This was examined by two approaches. Carnitine is a substrate for and an important regulator of CPT-1. It is a necessary co-factor for fatty acyl CoA translocation, and its intramitochondrial concentration governs the enzyme kinetics of CPT-1 (10). In contrast, fatty acyl-CoA does not alter CPT-1 kinetics (10). Various agents, including oxfenicine (hydroxyphenylglycine), also inhibit CPT-1 (28, 49). In the present study, carnitine was combined with palmitate to test the hypothesis that increasing exogenous carnitine, which translocates more palmitoyl CoA into the mitochondria so as to enhance fatty acid oxidation, will augment palmitate-induced cell death. Because CPT-1 may interact with Bcl-2, which is involved in the inhibition of apoptosis (37), we further sought to determine whether the CPT-1 inhibitor oxfenicine would modulate palmitate-induced cell death.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell cultures. Chick embryonic ventricular cells were cultured from 7-day-old chick embryos from white Leghorn eggs using previously described methods (20, 40). The protocol was approved by the University Committee on Use of Animals for Research. Myocytes were maintained in culture in medium 818A composed of 73% DBSK [(in mM) 116 NaCl, 0.8 MgSO4, 0.9 NaH2PO4, 5.5 dextrose, 1.8 CaCl2, 26 NaHCO3,] 20% medium 199, 2 or 6% fetal calf serum, and 1% antibiotic-antimycotic (10,000 mg/ml streptomycin sulfate, 10,000 U/ml penicillin G sodium, and 25 µg/ml amphotericin B) for 72 h before the experiment. The proportion of myocytes at this time was >90%, as verified by the proportion of cells showing spontaneous contraction or displaying muscle-specific markers (myosin) on immunohistological examination.

MTT assay. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, an index of cell viability and cell growth, is based on the ability of viable cells to reduce MTT from a yellow water-soluble dye to a dark blue insoluble formazan product (34). Cardiomyocytes were grown in multiwell microtiter plates (Falcon 3072, Becton Dickinson; Lincoln Park, NJ) for 72 h. They were treated with various concentrations of palmitate and/or other agents. After the appropriate time, MTT dye was added to cardiomyocytes, and the plates were incubated at 37°C for 4 h. Solubilization reagent was added and the absorbance was determined at 570 nm (model 3550, Bio-Rad; Mississauga, Canada). The background absorbance of medium in the absence of cells was subtracted. There is a highly significant linear relationship between cell number and absorbance (40).

Flow cytometry. Cardiomyocytes, at 72 h of culture, were exposed to palmitate and/or other agents for 24 h. The reaction was stopped by the removal of media, followed by brief exposure to trypsin (0.01% in 0.68% NaCl, 0.006% NaH2PO4, 0.027% Na2HPO4, 0.04% KCl) to suspend the adherent cells. Trypsinization was stopped by dilution with 818A media containing 6% fetal calf serum. The suspended cardiomyocytes were gently spun down and washed with phosphate-buffered saline. The cells were fixed in 75% ethanol for 30 min at room temperature and then stained with propidium iodide (PI) staining buffer (Triton X-100, EDTA, RNAse A, and PI). Cells were washed twice and then resuspended in phosphate-buffered saline for flow cytometric analysis. Cardiomyocytes (exactly 10,000) were aspirated into a fluorescent activated cell sorting (FACS) machine (model Epics XL MCL, Coulter Electronics; Burlington, Canada) and examined for fluorescence at a wavelength >600 nm on fluorescence channel 3, as previously described (20, 40). The cells are examined on a flow cytometer on the fluorescence channel 3, which measures the wavelength of PI. The resulting histogram is a measure of PI staining, which is indicative of the amount of DNA in the nucleus that the PI is bound to. Hence, the histogram of PI staining is a reflection of DNA content.

Materials, drugs, and chemicals. Culture media, fetal calf serum, antibiotics, and antimycotics were obtained from GIBCO (Burlington, Canada). Palmitate, capric acid, carnitine, glucose, lactate, oxfenicine, and PI were from Sigma (St. Louis, MO). Fumonisin B1 was from Calbiochem (La Jolla, CA). All other chemicals for flow cytometry were from VWR (Mississauga, ON, Canada).

Data analysis. Data are presented as means ± SE. Hypothesis testing used one-way analysis of variance. When the overall difference in means was significant, Tukey's test was used for comparison of between group data. The null hypothesis was rejected if the probability of a type I error was <5% (P < 0.05).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Palmitate induces a loss of cell viability. Cell viability was assessed by the MTT assay that is based on the ability of viable cells to have sufficient mitochondrial dehydrogenase activity to reduce MTT (19, 34). Considering the linear relationship between cell number and absorbance (40), palmitate (100 µM for 24 h) induced a significant (P < 0.01) reduction in absorbance from control and produced a significant (P < 0.01) loss of cell viability (Fig. 1). We (39) have demonstrated that the effect of palmitate to induce cell death using the MTT assay was similar to the extent of cell death determined by the trypan blue assay. This effect, which can be attributed directly to palmitate as a nonspecific effect of fatty acids to induce cell death in cardiomyocytes, has been previously excluded because not all fatty acids induce cell death (12, 39). We sought to determine whether other fatty acids of different chain length with similar or different dependency on CPT-1 would induce cell death. The short-chain fatty acid capric (n-decanoic acid), a C10 saturated fatty acid that is likely not transported by CPT-1, did not significantly affect cell viability. In contrast, the C18 saturated fatty acid stearic (n-octadecanoic acid; 100 µM) significantly (P < 0.01) reduced cell viability to a similar extent as palmitate.


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  		Fig. 1.   Palmitic and stearic acid, but not capric acid, induces a loss of cell viability. Cardiomyocytes, grown for 72 h, were treated with palmitic (100 µM, n = 9), stearic (100 µM, n = 10), capric acid (100 µM, n = 5), or the diluent (control) for 24 h. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) dye was added for the final 4 h. Reactions were stopped and the plate read at 570 nm. The loss of absorbance (A570) is consistent with the loss of viable cells, which is shown as percent change from control. Values are means ± SE. **P < 0.01, significant difference between some treated and control cells.

Fumonisin does not block palmitate-induced cell death. To determine whether palmitate-induced apoptotic cell death might be mediated through an increased synthesis of ceramide, cardiomyocytes were treated with palmitate plus the ceramide synthetase inhibitor fumonisin B1 (32), beginning 30 min before and continuing with palmitate for 24 h. Fumonisin (10 µM) was selected because this concentration inhibits ceramide synthesis (37). Fumonisin did not alter palmitate-induced loss of absorbance (A570) and hence loss of cell viability (Fig. 2).


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  		Fig. 2.   Fumonisin did not block palmitate-induced loss of cell viability. Cardiomyocytes, grown for 72 h, were treated with 100 µM palmitate (n = 13), its diluent (control, n = 13), 10 µM fumonisin B1 (Fumonisin; n = 13), or the combination of palmitate and fumonisin B1 (Fumon + Palm; n = 13) for 24 h. Cell viability was assessed by the MTT assay. Values are means ± SE. **P < 0.01, significant decrease from control cells.

Carnitine enhances palmitate-induced cell death. Because palmitate is transported into mitochondria with carnitine, we sought to determine whether simultaneous exposure of cardiomyocytes to palmitate and carnitine would alter cell viability. Low concentrations of L-carnitine that were without effect on cell viability were chosen and co-incubated with 100 µM palmitate for 24 h (Fig. 3). These carnitine concentrations did not significantly alter A570. However, in combination with palmitate, there was a further reduction in A570 (Fig. 3A). L-carnitine (1 mM) reduced A570 for palmitate (100 mM) from 0.18 ± 0.02 to 0.16 ± 0.03 (P < 0.05) for co-incubation of palmitate and L-carnitine. L-Carnitine (5 mM) significantly (P < 0.05) reduced A570 for palmitate (100 mM) from 0.18 ± 0.02 to 0.11 ± 0.03 for co-incubation of palmitate and L-carnitine. Compared with palmitate-induced loss of cell viability of 43.4 ± 7.7% (of control), L-carnitine (1 and 5 mM) co-incubation with palmitate induced a significant (P < 0.05) loss of cell viability to 52.5 ± 9.7% and 65.3 ± 10.2%, respectively (Fig. 3B).


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  		Fig. 3.   Carnitine co-incubation with palmitate (P) enhances loss of cell viability. Cardiomyocytes, grown for 72 h, were treated with 1 mM L-carnitine (L-C; n = 9) or 5 mM (n = 8) L-C or the combination of palmitate with carnitine (L-C+P) (1 or 5 mM, n = 8 each) for 24 h and compared with 100 µM palmitate (n = 9). Cell viability was assessed by the MTT assay. A: for 1 mM L-carnitine, there was a significant overall effect (F = 15.5, P < 0.01) and for 5 mM (F = 18.5, P < 0.01). B: percent change from control. Values are means ± SE. **P < 0.01, significant differences from control; Delta P < 0.05, significant differences from palmitate-treated cells. F, fluorescence.

Palmitate induces apoptosis. To further examine the kind of cell death and to confirm that palmitate produces apoptosis, cellular DNA content was examined. Because apoptosis is associated with patterned DNA fragmentation, the loss of intact DNA content can be demonstrated by flow cytometry of permeabilized cardiomyocytes stained with PI, which intercalates nuclear DNA of the nuclei. Palmitate induced an increase in proportion of the population with low DNA content (Fig. 4). The proportion of the population with this characteristic increased significantly (P < 0.01) from 1,002 ± 70 (n = 28) in control to 1,505 ± 100 (n = 28) in palmitate-treated cells (per 10,000 cells). By comparing each experiment to its control, there was a significant (P < 0.01) and 1.6 ± 0.1-fold increase in nuclei with low DNA content (PI fluorescence <102). We (20) have shown by FACS analysis that apoptosis accounts for a small but important component of the overall amount of palmitate-induced cell death. The difference in the percent increase in cell death in MTT assay and the percent increase in apoptosis may be accounted for by the measurement of total cell death, i.e., apoptotic and oncotic cell death by MTT assay (20). There were no apparent changes in the population in DNA synthesis or growth/mitosis with palmitate treatment, which might indicate a change in the number of quiescent cells.


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  		Fig. 4.   Palmitate induces an increase in the population of cells with low DNA content. Top: histograms of 10,000 cells stained with propidium iodide (PI) and analyzed by flow cytometry. Resulting histograms are an indication of nuclear size and DNA content. G0/G1, quiescence/growth; S, synthesis; G2/M, mitosis; Apo, apoptotic DNA with low DNA content. Apoptotic cells with low DNA content are those with PI fluorescence <102. Palmitate-treated cells (n = 28) showed a significant (**P < 0.01) increase in the number of cells with nuclei with low DNA content compared with control (n = 28).

Carnitine enhances palmitate-induced apoptotic death. We then sought to determine whether carnitine enhanced palmitate-induced formation of apoptotic nuclei with low DNA content. The use of the racemic mixture of carnitine with palmitate (DL-carnitine; 10 mM) did not induce apoptosis whereas a high concentration (30 mM) induced a 4.1 ± 0.8-fold (n = 10) increase in apoptotic nuclei compared with control (Fig. 5). The higher concentrations of carnitine needed to induce apoptotic cell death compared with the previous data (Fig. 3) was likely due to the use of the racemic mixture of carnitine in these experiments plus the different end points: apoptotic cell death in FACS analysis compared with total cell death in the MTT assay (Fig. 3). DL-Carnitine (30 mM) co-incubation with palmitiate significantly (P < 0.01) increased the amount of cells exhibiting low-DNA content compared with that produced by palmitate or carnitine alone. The increase in apoptotic cells between palmitate and control (614 per 10,000 cells) was far less than the increase in apoptotic cells (2,623 per 10,000 cells) found when palmitate was combined with carnitine.


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  		Fig. 5.   Carnitine enhances palmitate-induced apoptotic cell death. Cardiomyocytes were treated with 30 mM carnitine (n = 10), 100 µM palmitate (n = 9), or the co-incubation of carnitine 30 mM + palmitate 100 µM: (n = 9) or diluent-control (10) for 24 h before fluorescent activated cell sorting (FACS) analysis of nuclei with low DNA content (as described in MATERIALS AND METHODS). A: change in apoptosis compared with control (divided by control). Data analysis shows a significant (F = 10.0, P < 0.001) difference in the means and each treatment significantly increased apoptosis compared with control (**P < 0.01). Carnitine + palmitate (Palm + Carn) significantly enhanced apoptosis compared with palmitate alone or carnitine alone (P < 0.01). B: data analysis used one-way analysis of variance (F = 16; P < 0.01) with Tukey's test for multiple group comparisons. **P < 0.05, each treatment significantly increased apoptosis compared with control. Delta Delta P < 0.01, carnitine + palmitate significantly enhanced nuclei with low DNA content (apoptosis) compared with palmitate alone or carnitine alone.

Oxfenicine blunts carnitine plus palmitate-induced formation of apoptotic nuclei. To further explore the interaction of palmitate and carnitine, we used oxfenicine, an inhibitor of CPT-1. Cardiomyocytes were treated with oxfenicine, either 1 or 10 mM, starting 30 min before DL-carnitine (30 mM) + palmitate (100 µM) stimulation, and were analyzed by flow cytometry (Fig. 6). Oxfenicine (10 mM) pretreatment yielded a significant (P < 0.05) decrease in the formation of apoptotic nuclei compared with carnitine (30 mM) + palmitate. The relative increase in apoptotic nuclei with oxfencine pretreatment was only 3.7 ± 0.6 greater than control, compared with the 8.0 ± 1.9-fold increase induced by DL-carnitine (30 mM) + palmitate (100 µM) in the absence of oxfenicine.


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  		Fig. 6.   Oxfenicine blunted the effect of carnitine and palmitate to induce apoptosis-low DNA content. Cardiomyocytes that had been grown in culture for 72 h were treated with either diluent (control) (n = 9), 100 µM palmitate (n = 8), 30 mM carnitine + 100 µM palmitate (n = 8), or 30 mM carnitine + 100 µM palmitate with oxfenicine pretreatment for 30 min, either 1 µm (n = 4) or 1 mM (n = 7). Permeabilized cells stained with PI were evaluated by flow cytometry. The data are presented as the means ± SE and were analyzed by one-way analysis of variance (F = 16.0; P < 0.01) with Tukey's test for multiple group comparisons that showed significant differences from control (**P < 0.01), or palmitate (Delta Delta P < 0.01) or carnitine + palmitate (+P < 0.05). P, 100 µM palmitate; C + P, 30 mM carnitine + 100 µM palmitate; Ox1 + C + P, 1 mM oxfenicine + 30 mM carnitine + 100 µM palmitate; Ox10 + C + P, 10 mM oxfenicine + 30 mM carnitine + 100 µM palmitate. Inset: relative change for each treatment group compared with control.

Fumonisin does not block palmitate-induced apoptotic cell death. To determine whether palmitate-induced apoptotic cell death might be mediated through an increased synthesis of ceramide, cardiomyocytes were treated with palmitate plus the ceramide synthetase inhibitor fumonisin B1 (32), beginning 30 min before and continuing with palmitate. Fumonisin (10 uM) did not alter palmitate-induced apoptotic cell death (low DNA content) regardless of whether cells were treated with palmitate for 24 or 48 h (Fig. 7).


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  		Fig. 7.   Fumonisin did not block palmitate-induced apoptosis. Cardiomyocytes, grown for 72 h, were treated with palmitate (100 µM) for 24 or 48 h (n = 3), its diluent (control) (n = 3), 10 µM fumonisin B1 (n = 3) or the combination of palmitate with fumonisin B (n = 3). Cells were stained with PI and analyzed by FACS. Values are means ± SE. **P < 0.01, significant differences from control.

Effect of glucose and insulin on palmitate-induced cell death. Because glucose and insulin enhance the glycolytic pathway and shift cardiac metabolism away from fatty acid oxidation, we sought to determine the effect of glucose and insulin on palmitate-induced cell death. Insulin concentrations above the 50% inhibitory concentration for effects on these cardiomyocytes were selected (54). Insulin (100 nM) plus additional glucose (2 g/l) maintained a similar A570 to control whereas glucose + insulin treatment, starting 30 min before, did not significantly affect palmitate-induced loss of A570 (Fig. 8). The percent loss of cell viability in palmitate-treated cells was no different from cells treated with glucose + insulin + palmitate.


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  		Fig. 8.   Palmitate induces a loss of cell viability that is unaffected by glucose + insulin. Cardiomyocytes, grown for 72 h, were treated with 100 µM palmitate (n = 7), with (n = 7) or without 100 nM insulin + 2 g/l glucose (n = 7) pretreatment, for 24 h and cell viability was assessed by the MTT assay. **P < 0.001, significant reduction in absorbance compared with control.

Insulin plus glucose pretreatment has no effect on palmitate-induced loss of DNA content. Cardiomyocytes were treated with palmitate (100 µM for 24 h), with or without glucose (2 g/l) + insulin (100 nM), and DNA content was examined by flow cytometry. There was no significant change in the number of cells exhibiting low DNA with insulin + glucose pretreatment compared with palmitate alone (Fig. 9). Indeed, the increase in nuclei with low DNA content produced by palmitate (100 µM) of 1.4 ± 0.1 was the same as that observed when insulin plus glucose pretreatment was combined with palmitate.


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  		Fig. 9.   Insulin + glucose pretreatment has no effect on palmitate-induced loss of DNA content. Cardiomyocytes treated with palmitate (100 µM for 24 h), with (n = 8) or without 2 g/l glucose + 100 nM insulin (n = 8) or diluent (control) (n = 8), were examined by flow cytometry using PI fluorescence in permeabilized cells. The proportion of the population with low DNA content (PI fluorescence <102) was determined. Values are means ± SE. *P < 0.05; **P < 0.01, significant differences compared with control.

Lactate does not affect palmitate-induced apoptosis or loss of DNA content. To further explore whether another product of glycolysis would alter palmitate-induced apoptosis, cardiomyocytes were treated with 1 mM lactate + 100 µM palmitate for 24 h before analysis by flow cytometry. Although palmitate induced a significant (P < 0.01) increase in the apoptotic populations, this was not altered by lactate (Fig. 10). Similarly, lactate had no effect on palmitate-induced formation of nuclei with low DNA content.


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  		Fig. 10.   Lactate has no effect on palmitate-induced loss of DNA content. Cardiomyocytes treated with 100 µM palmitate for 24 h (n = 6), with (n = 6) or without 1 mM lactate (n = 6), were examined by flow cytometry using PI fluorescence as a measure of nuclear size in permeabilized cardiomyocytes. **P < 0.01, significant differences of increase in proportion of the population with low DNA content compared with control.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study extends our knowledge of apoptosis in cardiomyocytes by delineating mechanisms that modulate palmitate-induced cell death. Despite the documentation of palmitate-induced cell death in various cell types (37, 44, 55), including cardiomyocytes (12, 20, 39), factors regulating this kind of cell death have been infrequently studied and are incompletely understood. The data in this study present novel findings on the effects of carnitine and oxfenicine and thus provide compelling new evidence to implicate carnitine palmitoyltransferase, which traverses the mitochondrial membrane, as a crucial step in the pathogenesis of palmitate-induced cardiomyocyte cell death. These data are briefly as follows. First, the combination of palmitate with L-carnitine, another substrate for CPT-1, enhanced palmitate-induced cell death. Second, the CPT-1 inhibitor oxfenicine blocked palmitate-induced cell death. Third, other potential pathways influencing palmitate metabolism, specifically those regulated by insulin or lactate, did not influence this kind of cell death.

To our knowledge, the ability of carnitine to accentuate palmitate-induced cell death has not been previously described. Exogenous carnitine is readily transported into the cell across the sarcolemmal membrane (50). The concentrations of carnitine used in this study increase intracellular carnitine concentration (6). We speculate that the mechanism of action of carnitine on palmitate-induced cell death is mediated through CPT-1 and palmitate metabolism because exogenous L-carnitine increases cardiac CPT-1 activity and palmitate oxidation (1). Increased exogenous palmitate concentrations also increase CPT-1 activity (46). Carnitine, however, can interact with palmitate in other metabolic pathways and transport processes. Carnitine facilitates the transfer of acetyl CoA, a product of both fatty acid and glycolytic oxidation, from inside the mitochondria to the cytosol via carnitine acetyltransferase (26). The cytosolic acetyl CoA is then converted into malonyl CoA, a potent intracellular inhibitor of CPT-1 (5). This is an unlikely mechanism of action for carnitine because one would expect the same results with oxfenicine as carnitine. Carnitine also blunts palmitate-induced reductions in glycolysis (5), but our data do not support a role for glycolysis to blunt palmitate-induced cell death.

This study is the first to describe the beneficial effects of oxfenicine on the prevention of cardiac cell death and specifically apoptotic cell death. It supports the beneficial effects of oxfenicine on cardiac contractility and enzyme release in the ischemic heart (8, 18, 21, 33, 51) and extends the observation to cell death. Other CPT-1 inhibitors such as etoxomir (25) and 2,5,4 chlorophenylpentyloxirane-2-carboxylate (56) confirm the beneficial role of inhibiting CPT-1 on cardiac contractile performance in the ischemic myocardium. Our data are at variance with that of Paumen et al. (37), who found in murine hematopoietic cell lines that etomoxir accentuated palmitate-induced apoptosis. The most likely explanation for the difference is the fact that the different cell types as the beneficial effect of CPT-1 inhibition has been established in the heart (8, 18, 21, 33, 51) and confirmed in proximal renal tubular cells subjected to hypoxia (38). Etomoxir differs from oxfenicine, however, in that etomoxir requires metabolism to its CoA ester to become active (46), whereas oxfenicine is transaminated in the heart by branched-chain amino acid aminotransferase to 4-hydroxyphenylglyoxyalte, which acts to directly inhibit CPT-1 (47). Oxfenicine does not, however, completely inhibit cardiac CPT-1 because the maximum CPT-1 inhibition produced by oxfenicine in isolated cardiac mitochondria was 70-80% compared with the 100% achievable by oxfenicine in liver mitochondria (47). This may explain why oxfenicine did not completely prevent the apoptosis induced by the combination of carnitine and palmitate.

Insulin and glucose shift cardiac metabolism away from fatty acid oxidation and shunt energy production of the embryonic cardiomyocyte primarily to glycolysis (11). Insulin also upregulates ACC activity, which, in turn, increases production of malonyl CoA, an intramitochondrial inhibitor of CPT-1 (23, 24, 48). We found that glucose and insulin pretreatment did not affect palmitate-induced cell death suggesting that the mechanism by which palmitate produces apoptosis in cardiomyocytes may not be dependent on energy production by glycolysis. It also suggests that the beneficial effect of oxfenicine is on fatty acid metabolism rather than an action to increase glucose oxidation (18). The absence of an effect of insulin and glucose on palmitate-induced cell death indicates that palmitate may override or bypass the malonyl CoA inhibition of fatty acid oxidation. These data suggest that palmitate may act via a mechanism operative through CPT-1 but not controlled by malonyl CoA or that the effects of glucose to inhibit fatty acid oxidation through malonyl CoA is less effective in the presence of palmitate or have resolved within the 24 h of palmitate treatment.

When exogenous lactate enters the cytosol, it undergoes a reversible oxidation to form pyruvate before entering the TCA cycle. Hence, lactic acid functions as an alternative source of pyruvate generation (11). Fetal and neonatal hearts utilize both lactate-based oxidation and glycolysis before fatty acid oxidation (15). The present study found that lactate did not affect palmitate-induced cell death suggesting that lactate oxidation is not involved in the action of palmitate to induce apoptotic cell death in cardiomyocytes or that the effects of lactate to inhibit fatty acid oxidation may have resolved within the time frame of palmitate treatment to induce cell death. The data with lactate are consonant with those of glucose and insulin.

Our study did not support the suggestion that palmitate-induced cell death is due to palmitate-induced enhancement of de novo synthesis of ceramide (37), an intracellular mediator of cell death apoptosis (32). In the present study, cardiomyocytes were treated with palmitate for sufficient time to permit ceramide synthesis (37). The use of fumonisin to inhibit de novo ceramide synthesis (34) was a crucial part of the evidence suggesting that palmitate-induced cell death was mediated by ceramide synthesis (37). We found that fumonisin at similar concentrations (37), did not alter palmitate-induced cell death or apoptosis in cardiomyocytes. The failure of fumonisin to influence apoptosis does not suggest that ceramide synthesis was not increased by palmitate. Rather, it suggests that ceramide is not playing a major role in palmitate-induced cell death because fumonisin is a potent inhibitor of ceramide formation in various cell types (32, 37) and is effective in antagonizing cardiomyocyte-induced cell death from other agents (S. W. Rabkin, unpublished observations). A likely explanation for the differences between this study and that of Paumen et al. (37) is the differences between cardiomyocytes and a hematopoietic cell line.

The failure of insulin to affect palmitate-induced cell death is not due to cell type. Chick embryonic cardiomyocytes are a suitable model for studying the effects of insulin because they possess insulin receptors and the effect of insulin in these cells has been recognized for a long time (14). These effects include the action of insulin on amino acid transport and free fatty acids metabolism (16). These cardiomyocytes readily metabolize palmitate, which is subject to regulatory control by carnitine and glucose (42, 53). Another potential explanation for our findings is the magnitude of the difference in this regulation. In these cardiomyocytes, 1 mM carnitine doubled palmitate oxidation, whereas 1 mM glucose decreased palmate metabolism by 50% (53).

The use of embryonic chick cardiomyocytes has limitations in the extent to which the data can be extrapolated to mammalian and adult heart. However, these cells have several advantages in the study of glucose and fatty acids because the embryonic heart utilizes glycolysis and lactate oxidation before fatty acid oxidation (15, 41). The switch in energy source to predominantly fatty acids may occur as the mitochondria mature structurally coinciding with increase in activities of enzymes involved in oxidative metabolism, such as reduced nicotinamide adenine dinucleotide oxidase, ATPase, and cytochrome c oxidase (2). However, embryonic chick cardiomyocytes have similar levels of CPT activity as mammalian (neonatal rat) heart (42). More importantly, these embryonic cardiomyocytes have components of the apoptotic pathway such as bcl-2 that have similarities to human cardiomyocytes (13). Whereas embryonic chick cardiomyocytes have high levels of CPT (42), the CPT-1 isoform distribution has not been studied in the developing avian heart. In newborn rat, the liver (L) L-CPT-1 Michaelis-Menten constant (Km; for carnitine of ~30 µM) is responsible for ~60% of total cardiac fatty acid oxidation but falls to ~4% in adult animals as the proportion of muscle isoforms (M)-CPT-1 (Km for carnitine of ~500 µM) increases (7). The extent to which CPT-1 isoform distribution impacts on palmitate-induced cell death, however, remains speculative. While the implications of our findings in the adult heart warrant investigation, the adverse effect of palmitate cannot simply be ascribed to the metabolism of the embryonic heart because palmitate-induced cell death in the heart has been demonstrated in the fetal, neonatal, and adult heart (12, 20, 39, 51).

In conclusion, cardiomyocytes are terminally differentiated cells whose inability to reproduce underscores the seriousness of the loss of even a small amount of cardiac muscle cells in myocardial infarction accompanied by high-circulating concentrations of palmitate. These data highlight the importance of the initial step in the transport of palmitate into the mitochondria and suggest that inhibition of CPT-1 can limit palmitate-induced cardiomyocyte cell death.


    ACKNOWLEDGEMENTS

This study was supported in part by a grant-in-aid from the Heart and Stroke Foundation of British Columbia and the Yukon.


    FOOTNOTES

Address for reprint requests and other correspondence: S. W. Rabkin, Univ. of British Columbia, D410 2733 Heather St., Vancouver, BC, Canada V5Z 3J5 (E-mail: rabkin{at}interchange.ubc.ca).

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.

10.1152/ajpheart.00257.2001

Received 28 March 2001; accepted in final form 18 October 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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