Substrate-enzyme competition attenuates upregulated anaplerotic flux through malic enzyme in hypertrophied rat heart and restores triacylglyceride content: attenuating upregulated anaplerosis in hypertrophy

Abstract

Recent work identifies the recruitment of alternate routes for carbohydrate oxidation, other than pyruvate dehydrogenase (PDH), in hypertrophied heart. Increased carboxylation of pyruvate via cytosolic malic enzyme (ME), producing malate, enables "anaplerotic" influx of carbon into the citric acid cycle. In addition to inefficient NADH production from pyruvate fueling this anaplerosis, ME also consumes NADPH necessary for lipogenesis. Thus, we tested the balance between PDH and ME fluxes in hypertrophied hearts and examined whether low triacylglyceride (TAG) was linked to ME-catalyzed anaplerosis. Sham-operated (SHAM) and aortic banded rat hearts (HYP) were perfused with buffer containing either 13C-palmitate plus glucose or (13)C glucose plus palmitate for 30 minutes. Hearts remained untreated or received dichloroacetate (DCA) to activate PDH and increase substrate competition with ME. HYP showed a 13% to 26% reduction in rate pressure product (RPP) and impaired dP/dt versus SHAM (P<0.05). DCA did not affect RPP but normalized dP/dt in HYP. HYP had elevated ME expression with a 90% elevation in anaplerosis over SHAM. Increasing competition from PDH reduced anaplerosis in HYP+DCA by 18%. Correspondingly, malate was 2.2-fold greater in HYP than SHAM but was lowered with PDH activation: HYP=1419+/-220 nmol/g dry weight; HYP+DCA=343+/-56 nmol/g dry weight. TAG content in HYP (9.7+/-0.7 micromol/g dry weight) was lower than SHAM (13.5+/-1.0 micromol/g dry weight). Interestingly, reduced anaplerosis in HYP+DCA corresponded with normalized TAG (14.9+/-0.6 micromol/g dry weight) and improved contractility. Thus, we have determined partial reversibility of increased anaplerosis in HYP. The findings suggest anaplerosis through NADPH-dependent, cytosolic ME limits TAG formation in hypertrophied hearts.

Figure 1

Route of pyruvate entry into competing reactions: oxidation via pyruvate dehydrogenase (PDH) versus anaplerotic carboxylation to malate via malic enzyme (ME). Bold arrows indicate the increased action of ME in the hypertrophied heart. Acetyl CoA is produced from long chain fatty acids via β-oxidation and from glycolytic pyruvate via PDH. Anaplerosis contributes malate and oxaloacetate in the second (5 carbon) span of the citric acid cycle and is fueled by both the amino acids (aspartate) and pyruvate. Enzymes catalyzing pyruvate entry into anaplerosis are pyruvate carboxylase (PC) and malic enzyme (ME). Propionyl CoA carboxylase also catalyzes anaplerosis, converting propionyl CoA into methylmalonyl CoA (not shown). Carboxylation of pyruvate, via ME, consumes NADPH necessary for triacylglyceride (TAG) synthesis and glutathione reduction. Aspartate fuels anaplerosis through aspartate aminotransferase (AAT), producing oxaloacetate. Glutamate is produced by transamination of 2-oxoglutarate that is transported out of mitochondria via the oxoglutarate-malate carrier (OMC).

Figure 2

Rate-pressure-product (RPP, mmHg × beats/min) in hypertrophied and sham hearts, with or without activation of PDH with DCA. Note reduced RPP in hypertrophy versus sham (*, p<0.05). Solid triangle, sham; open triangle, Sham + DCA; solid square, Hypertrophy; open square, Hypertrophy + DCA.

Figure 3

Expression of anaplerotic enzymes. Protein content of anaplerotic enzymes: A) Pyruvate Carboxylase (PC); B) Propionyl CoA Carboxylase (PCC); and C) Malic Enzyme (ME). Values are shown in arbitrary units (AU) relative to GAPDH content. Note increased ME in HYP and DCA HYP. *, P<0.002 vs. SHAM. †, P<0.002 vs. DCA SHAM.

Figure 4

Fractional contribution of anaplerotic flux to citric acid cycle flux. Note elevated anaplerosis in hypertrophied rat hearts (HYP). Activation of PDH to compete against increased malic enzyme in hypertrophied hearts (HYP+DCA) moderated anaplerosis, which was lower than in untreated HYP. Black bar, sham hearts; white bar, DCA-treated sham hearts; Striped bar, hypertrophied hearts; Gray bar, DCA-treated hypertrophied hearts. *, P<0.05 vs. other 3 groups. †, P<0.05 vs. HYP.