Glucose is preferentially utilized for biomass synthesis in pressure-overloaded hearts: evidence from fatty acid-binding protein-4 and -5 knockout mice

Abstract

Aims: The metabolism of the failing heart is characterized by an increase in glucose uptake with reduced fatty acid (FA) oxidation. We previously found that the genetic deletion of FA-binding protein-4 and -5 [double knockout (DKO)] induces an increased myocardial reliance on glucose with decreased FA uptake in mice. However, whether this fuel switch confers functional benefit during the hypertrophic response remains open to debate. To address this question, we investigated the contractile function and metabolic profile of DKO hearts subjected to pressure overload.

Methods and results: Transverse aortic constriction (TAC) significantly reduced cardiac contraction in DKO mice (DKO-TAC), although an increase in cardiac mass and interstitial fibrosis was comparable with wild-type TAC (WT-TAC). DKO-TAC hearts exhibited enhanced glucose uptake by 8-fold compared with WT-TAC. Metabolic profiling and isotopomer analysis revealed that the pool size in the TCA cycle and the level of phosphocreatine were significantly reduced in DKO-TAC hearts, despite a marked increase in glycolytic flux. The ingestion of a diet enriched in medium-chain FAs restored cardiac contractile dysfunction in DKO-TAC hearts. The de novo synthesis of amino acids as well as FA from glycolytic flux was unlikely to be suppressed, despite a reduction in each precursor. The pentose phosphate pathway was also facilitated, which led to the increased production of a coenzyme for lipogenesis and a precursor for nucleotide synthesis. These findings suggest that reduced FA utilization is not sufficiently compensated by a robust increase in glucose uptake when the energy demand is elevated. Glucose utilization for sustained biomass synthesis further enhances diminishment of the pool size in the TCA cycle.

Conclusions: Our data suggest that glucose is preferentially utilized for biomass synthesis rather than ATP production during pressure-overload-induced cardiac hypertrophy and that the efficient supplementation of energy substrates may restore cardiac dysfunction caused by energy insufficiency.

Figure 1

Cardiac contraction was reduced by TAC in DKO mice without difference of cardiac hypertrophy and fibrosis. (A) The survival curves of WT and DKO mice after TAC. The survival rate was not significantly different (n = 20) (P = 0.075). (B) Cardiac function was estimated by echocardiography before and 1, 4, and 8 weeks after TAC. Cardiac contraction was reduced in DKO mice with enlarged LV diameter after TAC. FS, fractional shortening; IVSd, thickness of interventricular septum in diastole; LVDd, diastolic diameter of left ventricle; LVDs, systolic diameter of left ventricle (n = 10–12). (C) Mice were sacrificed 1 week after TAC to isolate hearts after a 12 h fast. Heart weight was measured after removing atria. The heart weight/body weight ratio (HW/BW) was comparable between WT and DKO mice in the presence or absence of TAC (n = 6). (D) The capillary density estimated by isolectin B4 and wheat germ agglutinin staining was comparable (n = 6). (E), Fibrosis area estimated by Masson’s trichrome stain was comparable (n = 6). *P < 0.05, **P < 0.01, and ***P < 0.001. For Figure 1B, the data were analysed with an unpaired two-tailed Student’s t-test with Welch correction. For Figure 1C–E, an unpaired Student’s t-test was performed for each pair of four groups and subsequent multiple comparisons were made with use of the Bonferroni method.

Figure 2

Cardiac uptake and circulating levels of lipids and glucose in the absence or presence of pressure overload. Experiments were done 1 week after TAC. (A) The uptake of ¹²⁵I-BMIPP, an FA tracer, and ¹⁸F-FDG, a glucose tracer, by hearts in the absence or presence of TAC. Samples were collected after a 12 h fast. (B) Blood samples were collected 4 h after fasting (n = 5–7). *P < 0.05, **P < 0.01, and ***P < 0.001. An unpaired Student’s t-test was performed for each pair of four groups and subsequent multiple comparisons were made with use of the Bonferroni method.

Figure 3

The expression of genes associated with FA metabolism in hearts. Mice were sacrificed 1 week after TAC to isolate hearts with a prior 12 h fast. Data are normalized to WT control. Ppara, peroxisome proliferator-activated receptor a; Pgc1a/b, PPARG coactivator 1 alpha/beta; Fatp1, FA transport protein 1; Cpt1b/2, carnitine palmitoyltransferase 1B/2; Mcad, medium-chain acyl-CoA dehydrogenase; Lcad, long-chain acyl-CoA dehydrogenase; Atgl, adipose triglyceride lipase; Hsl, hormone sensitive lipase (n = 6). *P < 0.05, **P < 0.01, and ***P < 0.001. An unpaired Student’s t-test was performed for each pair of four groups and subsequent multiple comparisons were made with use of the Bonferroni method.

Figure 4

The expression of glucose transporters and metabolic profiling in glycolysis. Mice were sacrificed 1 week after TAC to isolate hearts with a prior 12 h fast. Data are normalized to WT control when unit is indicated by fold. (A) The expression of genes for glucose uptake by hearts with or without TAC. Glut1, glucose transporter 1; Glut4, glucose transporter 4 (n = 6). (B) The protein expression of GLUT4 by western blot analysis. Right panel: the protein expression of GLUT4 was normalized by that of GAPDH. Loading amount was also shown by silver staining. (C) The metabolic profiling in the glycolysis pathway. G1P, glucose-1-phosphate. G1P-pyruvate, total metabolites from G1P to pyruvate (n = 5–7). (D) A tracer study with C₆-glucose. After a 12 h fast, the hearts were isolated 10 min after an intraperitoneal injection of C₆-glucose (n = 5–7). *P < 0.05 and **P < 0.01. An unpaired Student’s t-test was performed for each pair of four groups and subsequent multiple comparisons were made with use of the Bonferroni method.

Figure 5

The pool size in the TCA cycle and PCr was significantly reduced in DKO-TAC hearts despite a marked increase in glycolytic flux. Mice were sacrificed 1 week after TAC to isolate hearts with a prior 12 h fast. (A) The metabolic profiling in the TCA cycle. Note that oxaloacetate (OAA) cannot be detected by this method. TCA cycle, tricarboxylic acid cycle; α-KG, α-ketoglutarate (n = 5–7). (B) A tracer study with C₆-glucose. After a 12 h fast, the hearts were isolated 10 min after an intraperitoneal injection of C₆-glucose (n = 5–7). Data are normalized to WT control. (C) The metabolic profiling in CP energy shuttle. ATP, adenosine triphosphate; PCr, phosphocreatine (n = 5-7). (D) DKO mice subjected to TAC were divided into two groups and fed standard chow (SC) and an MCFA-rich diet (MCFA). Feeding and fasting were carried out according to the depicted protocol. Cardiac function was evaluated by echocardiography at the indicated time points. *P < 0.05 and **P < 0.01. For Figure 5A–C, an unpaired Student’s t-test was performed for each pair of four groups and subsequent multiple comparisons were made with use of the Bonferroni method. For Figure 5D, the data were analysed with an unpaired two-tailed Student’s t-test with Welch correction.

Figure 6

The biosynthesis of non-essential amino acids seems to be enhanced in DKO-TAC hearts. Mice were sacrificed 1 week after TAC to isolate hearts with a prior 12 h fast. (A) The levels of non-essential amino acids in hearts. Glutamate (Glu), aspartate (Asp), glutamine (Gln), asparagine (Asn), and proline (Pro) can be generated from metabolites in the TCA cycle. Serine (Ser), glycine (Gly), and alanine (Ala) can be produced from glycolytic intermediates (n = 5-7). (B) Glu/α-KG, glutamate/α-ketoglutarate ratio; Ala/pyruvate, alanine/pyruvate ratio (n = 5-7). (C) A tracer study with C₆-glucose. After a 12 h fast, hearts were isolated 10 min after an intraperitoneal injection of C₆-glucose (n = 5–7). Data are normalized to WT control. *P < 0.05, **P < 0.01, and ***P < 0.001. An unpaired Student’s t-test was performed for each pair of four groups and subsequent multiple comparisons were made with use of the Bonferroni method.

Figure 7

The biosynthesis of nucleic acids and FAs seems to be enhanced in DKO-TAC hearts. Mice were sacrificed 1 week after TAC to isolate hearts with a prior 12 h fast. Data are normalized to WT control when unit is indicated by fold. A, The levels of nucleic acid precursors through the PPP. PRPP, 5-phosphoribosyl pyrophosphate; IMP, inosine monophosphate (n = 5–7). (B) A tracer study with C₆-glucose. After a 12 h fast, the hearts were isolated 10 min after an intraperitoneal injection of C₆-glucose. R5P, ribose 5-phosphate. (C) Levels of NADP⁺, NAPDH and NADP⁺/NADPH ratio (n = 5–7). (D) The levels of citrate-derived products through the TCA cycle (n = 5–7). (E) The expression levels of proteins associated with FA synthesis were elevated in both hearts after TAC. ACLY, ATP citrate lyase; FASN, FA synthase. The expression levels of ACLY and FASN were normalized by that of GAPDH (n = 5). Loading amount was also shown by silver staining. *P < 0.05 and **P < 0.01. An unpaired Student’s t-test was performed for each pair of four groups and subsequent multiple comparisons were made with use of the Bonferroni method.