Preferential oxidation of triacylglyceride-derived fatty acids in heart is augmented by the nuclear receptor PPARalpha

Abstract

Rationale: Long chain fatty acids (LCFAs) are the preferred substrate for energy provision in hearts. However, the contribution of endogenous triacylglyceride (TAG) turnover to LCFA oxidation and the overall dependence of mitochondrial oxidation on endogenous lipid is largely unstudied.

Objective: We sought to determine the role of TAG turnover in supporting LCFA oxidation and the influence of the lipid-activated nuclear receptor, proliferator-activated receptor (PPAR)alpha, on this balance.

Methods and results: Palmitoyl turnover within TAG and palmitate oxidation rates were quantified in isolated hearts, from normal mice (nontransgenic) and mice with cardiac-specific overexpression of PPARalpha (MHC-PPARalpha). Turnover of palmitoyl units within TAG, and thus palmitoyl-coenzyme A recycling, in nontransgenic (4.5+/-2.3 micromol/min per gram dry weight) was 3.75-fold faster than palmitate oxidation (1.2+/-0.4). This high rate of palmitoyl unit turnover indicates preferential oxidation of palmitoyl units derived from TAG in normal hearts. PPARalpha overexpression augmented TAG turnover 3-fold over nontransgenic hearts, despite similar fractions of acetyl-coenzyme A synthesis from palmitate and oxygen use at the same workload. Palmitoyl turnover within TAG of MHC-PPARalpha hearts (16.2+/-2.9, P<0.05) was 12.5-fold faster than oxidation (1.3+/-0.2). Elevated TAG turnover in MHC-PPARalpha correlated with increased mRNA for enzymes involved in both TAG synthesis, Gpam (glycerol-3-phosphate acyltransferase, mitochondrial), Dgat1 (diacylglycerol acetyltransferase 1), and Agpat3 (1-acylglycerol-3-phospate O-acyltransferase 3), and lipolysis, Pnliprp1 (pancreatic lipase related protein 1).

Conclusions: The role of endogenous TAG in supporting beta-oxidation in the normal heart is much more dynamic than previously thought, and lipolysis provides the bulk of LCFA for oxidation. Accelerated palmitoyl turnover in TAG, attributable to chronic PPARalpha activation, results in near requisite oxidation of LCFAs from TAG.

Figure 1

Triacylglyceride content in hearts of MHC-PPARα low over-expressing mice and nontransgenic littermates on a regular chow diet or high fat diet (HFD) perfused with 0.4 mM palmitate or 1.2 mM palmtiate (high palmitate, HP). TAG content reported in nanomoles/mg protein. * P < 0.05, vs. NTG baseline; † P < 0.05, vs NTG HFD and MHC-PPARα HFD.

Figure 2

Representative ¹³C spectra from isolated mouse hearts displaying progressive ¹³C enrichment of TAG and glutamate over 120 minutes from A) MHC-PPARα and B) NTG hearts perfused with 0.4 mM [2,4,6,8,10,12,14,16 ¹³C₈] palmitate + 10 mM unlabeled glucose. The spectra signals from ¹³C enriched methylene carbons of TAG, at 30.5 ppm, due to ¹³C palmitate storage and the 4- and 3-carbons of glutamate produced by oxidation of ¹³C palmitate, at 34 and 28 ppm respectively (glu C-4 and glu C-3).

Figure 3

Triacylglyceride turnover in MHC-PPARα low over-expressing mice and NTG littermates. * P < 0.05, vs. NTG baseline TAG. † P < 0.05, vs. NTG + isoproterenol and PPARα baseline.

Figure 4

A. Acetyl CoA enrichment from ¹³C palmitate in MHC-PPARα low over-expressing mice (PPARα) and NTG littermates. B. Rates of palmitate oxidation and MHC-PPARα low over-expressing mice (PPARα) and NTG littermates (μmoles/min/gdw). C. Palmitate turnover of TAG units in NTG and MHC-PPARα low over-expressing mice (MHC-PPARα) (μmoles/min/gdw). D. Acetyl CoA enrichment from ¹³C palmitate in MHC-PPARα low over-expressing mice (PPARα) and NTG littermates fed a high fat diet (HFD) for 2 weeks and perfused with 1.2 mM Palmitate. E. Palmitate oxidation rates from MHC-PPARα low over-expressing mice (PPARα) and NTG littermates (μmoles/min/gdw) fed a HFD and perfused with 1.2 mM Palmitate. F. Palmitate turnover of TAG units in NTG and MHC-PPARα low over-expressing mice (MHC-PPARα) fed a HFD and perfused with 1.2 mM Palmitate (μmoles/min/gdw). * P<0.05 vs. NTG.

Figure 5

mRNA levels of genes encoding enzymes that regulate TAG synthesis and lipolysis in heart tissue from NTG and MHC-PPARα mice fed a regular chow diet (RCD) or a high fat diet (HFD). * P < 0.03, vs NTG. † Significantly different compared to NTG—RCD. ‡ P < 0.05, vs NTG—HFD. n.d., not detectable.

Figure 6

A. Fate of LCFA in the myocardium at baseline. Both MHC-PPARα and NTG hearts exhibit cytosolic mixing of endogenous LCFA (shown in the solid grey) with exogenous LCFA. B. Fate of LCFA during β-adrenergic stimulation. MHC-PPARα hearts exhibit increased mixing to meet energy demands, shown in darker shade. Thickness of arrow indicates pathway preference. Shaded grey areas represent mixing of exogenous and endogenous LCFA. Both figures show bidirectional pathway of LCFA entering and exiting the TAG pool.

Figure 7

Schematic of enzymatic pathways for the synthesis of triacylglycerol. Up arrow indicates an increased expression in the transgenic model. Down arrow indicates a decreased enzyme expression.