One of the primary metabolic functions of a mature adipocyte is to supply energy via lipolysis, or the catabolism of stored lipids. Adipose triacylglycerol lipase (ATGL) and hormone-sensitive lipase ...(HSL) are critical lipolytic enzymes, and their phosphorylation generates phospho-binding sites for 14-3-3 proteins, a ubiquitously expressed family of molecular scaffolds. While we previously identified essential roles of the 14-3-3ζ isoform in murine adipogenesis, the presence of 14-3-3 protein binding sites on ATGL and HSL suggests that 14-3-3zcould also influence mature adipocyte processes like lipolysis. Herein, we demonstrate that 14-3-3ζ is necessary for lipolysis in male mice and fully differentiated 3T3-L1 adipocytes, as depletion of 14-3-3ζ significantly impaired glycerol and FFA release. Unexpectedly, reducing 14-3-3ζ expression was found to significantly impact adipocyte maturity, as observed by reduced abundance of PPARg2 protein and expression of mature adipocytes genes and those associated with de novo triglyceride synthesis and lipolysis. The impact of 14-3-3ζ depletion on adipocyte maturity was further examined with untargeted lipidomics, which revealed that reductions in 14-3-3ζ abundance promoted the acquisition of a lipidomic signature that resembled undifferentiated, pre-adipocytes. Collectively, these findings reveal a novel aspect of 14-3-3ζ in adipocytes, as reducing 14-3-3zwas found to have a negative effect on adipocyte maturity and adipocyte-specific processes like lipolysis.
δ-5 and δ-6 desaturases (D5D and D6D) catalyze the endogenous conversion of n–3 (ω-3) and n–6 (ω-6) polyunsaturated fatty acids (PUFAs). Their activities are negatively and positively associated with ...type 2 diabetes (T2D), respectively, by unclear mechanisms. Elevated plasma apoB-lipoproteins (measured as plasma apoB), which can be reduced by n–3 PUFA intake, promote T2D risk factors.
The aim of this study was to test the hypothesis that the association of D5D and D6D activities with T2D risk factors is dependent on plasma apoB.
This is a pooled analysis of 2 populations recruited for 2 different metabolic studies. It is a post hoc analysis of baseline data of these subjects n = 98; 60% women (postmenopausal); mean ± SD body mass index (in kg/m2): 32.8 ± 4.7; mean ± SD age: 57.6 ± 6.3 y. Glucose-induced insulin secretion (GIIS) and insulin sensitivity (IS) were measured using Botnia clamps. Plasma clearance of a high-fat meal (600 kcal/m2, 66% fat) and white adipose tissue (WAT) function (storage of 3H-triolein-labeled substrate) were assessed in a subpopulation (n = 47). Desaturase activities were estimated from plasma phospholipid fatty acids. Associations were examined using Pearson and partial correlations.
While both desaturase activities were positively associated with percentage of eicosapentaenoic acid, only D5D was negatively associated with plasma apoB (r = −0.30, P = 0.003). Association of D5D activity with second-phase GIIS (r = −0.23, P = 0.029), IS (r = 0.33, P = 0.015, in women) and 6-h area-under-the-curve (AUC6h) of plasma chylomicrons (apoB48, r = −0.47, P = 0.020, in women) was independent of age and adiposity, but was eliminated after adjustment for plasma apoB. D6D activity was associated in the opposite direction with GIIS (r = 0.24, P = 0.049), IS (r = −0.36, P = 0.004) and AUC6h chylomicrons (r = 0.52, P = 0.004), independent of plasma apoB. Both desaturases were associated with plasma interleukin-1-receptor antagonist (D5D: r = −0.45, P < 0.001 in women; D6D: r = −0.33, P = 0.007) and WAT function (trend for D5D: r = 0.30, P = 0.05; D6D: r = 0.39, P = 0.027) independent of any adjustment.
Association of D5D activity with IS, lower GIIS, and plasma chylomicron clearance is dependent on plasma apoB in overweight and obese adults.
There has been a resurgence of interest for the field of cardiac metabolism catalyzed by evidence demonstrating a role of metabolic dysregulation in the pathogenesis of heart disease as well as the ...increased need for new therapeutic targets for patients with these diseases. In this regard, measuring substrate fluxes is critical in providing insight into the dynamics of cellular metabolism and in delineating the regulation of metabolite production and utilization. This chapter provides a comprehensive description of concepts, guidelines, and tips to assess metabolic fluxes relevant to energy substrate metabolism using (13)C-labeled substrates and (13)C-isotopomer analysis by gas chromatography-mass spectrometry (GC-MS), and the ex vivo working heart as study model. The focus will be on the mouse and on flux parameters, which are commonly assessed in the field, namely, those relevant to substrate selection for energy metabolism, specifically the relative contribution of carbohydrate (glucose, lactate, and pyruvate) and fatty acid oxidation to acetyl-CoA formation for citrate synthesis, glycolysis, as well as anaplerosis. We provide detailed procedures for the heart isolation and perfusion in the working mode as well as for sample processing for metabolite extraction and analysis by GC-MS and subsequent data processing for calculation of metabolic flux parameters. Finally, we address practical considerations and discuss additional applications and future challenges.
In mice, genetic background is known to influence various parameters, including cardiac function. Its impact on cardiac energy substrate metabolism-a factor known to be closely related to function ...and contributes to disease development-is, however, unclear. This was examined in this study. In commonly used control mouse substrains SJL/JCrNTac, 129S6/SvEvTac, C57Bl/6J, and C57Bl/6NCrl, we assessed the functional and metabolic phenotypes of 3-mo-old working mouse hearts perfused ex vivo with physiological concentrations of (13)C-labeled carbohydrates (CHO) and a fatty acid (FA). Marked variations in various functional and metabolic flux parameters were observed among all mouse substrains, although the pattern observed differed for these parameters. For example, among all strains, C57Bl/6NCrl hearts had a greater cardiac output (+1.7-fold vs. SJL/JCrNTac and C57Bl/6J; P < 0.05), whereas at the metabolic level, 129S6/SvEvTac hearts stood out by displaying (vs. all 3 strains) a striking shift from exogenous FA (~-3.5-fold) to CHO oxidation as well as increased glycolysis (+1.7-fold) and FA incorporation into triglycerides (+2-fold). Correlation analyses revealed, however, specific linkages between 1) glycolysis, FA oxidation, and pyruvate metabolism and 2) cardiac work, oxygen consumption with heart rate, respectively. This implies that any genetically determined factors affecting a given metabolic flux parameter may impact on the associated functional parameters. Our results emphasize the importance of selecting the appropriate control strain for cardiac metabolic studies using transgenic mice, a factor that has often been neglected. Understanding the molecular mechanisms underlying the diversity of strain-specific cardiac metabolic and functional profiles, particularly the 129S6/SvEvTac, may ultimately disclose new specific metabolic targets for interventions in heart disease.
High plasma leucine levels strongly correlate with type 2 diabetes. Studies of muscle cells have suggested that leucine alters the insulin response for glucose transport by activating an ...insulin-negative feedback loop driven by the mammalian target of rapamycin/p70 ribosomal S6 kinase (mTOR/p70S6K) pathway. Here, we examined the molecular mechanism involved in leucine's action on cardiac glucose uptake. Leucine was indeed able to curb glucose uptake after insulin stimulation in both cultured cardiomyocytes and perfused hearts. Although leucine activated mTOR/p70S6K, the mTOR inhibitor rapamycin did not prevent leucine's inhibitory action on glucose uptake, ruling out the contribution of the insulin-negative feedback loop. α-Ketoisocaproate, the first metabolite of leucine catabolism, mimicked leucine's effect on glucose uptake. Incubation of cardiomyocytes with
Cleucine ascertained its metabolism to ketone bodies (KBs), which had a similar negative impact on insulin-stimulated glucose transport. Both leucine and KBs reduced glucose uptake by affecting translocation of glucose transporter 4 (GLUT4) to the plasma membrane. Finally, we found that leucine elevated the global protein acetylation level. Pharmacological inhibition of lysine acetyltransferases counteracted this increase in protein acetylation and prevented leucine's inhibitory action on both glucose uptake and GLUT4 translocation. Taken together, these results indicate that leucine metabolism into KBs contributes to inhibition of cardiac glucose uptake by hampering the translocation of GLUT4-containing vesicles via acetylation. They offer new insights into the establishment of insulin resistance in the heart.
Catabolism of the branched-chain amino acid leucine into ketone bodies efficiently inhibits cardiac glucose uptake through decreased translocation of glucose transporter 4 to the plasma membrane. Leucine increases protein acetylation. Pharmacological inhibition of acetylation reverses leucine's action, suggesting acetylation involvement in this phenomenon.Listen to this article's corresponding podcast at http://ajpheart.podbean.com/e/leucine-metabolism-inhibits-cardiac-glucose-uptake/.
There has been a resurgence of interest for the field of cardiac metabolism catalysed by the increased need for new therapeutic targets for patients with heart failure. The primary focus of research ...in this area to date has been on the impact of substrate selection for oxidative energy metabolism; however, anaplerotic metabolism also has significant interest for its potential cardioprotective role. Anaplerosis refers to metabolic pathways that replenish the citric acid cycle intermediates, which are essential to energy metabolism; however, our understanding of the role and regulation of this process in the heart, particularly under pathophysiological conditions, is very limited. Therefore, the goal of this article is to provide a foundation for future directions of research on cardiac anaplerosis and heart disease. We include an overview of anaplerotic metabolism, a critical evaluation of current methods available for its quantitation in the intact heart, and a discussion of its role and regulation both in health and disease as it is currently understood based mostly on animal studies. We also consider genetic diseases affecting anaplerotic pathways in humans and acute intervention studies with anaplerotic substrates in the clinics. Finally, as future perspectives, we will share our thoughts about potential benefits and practical considerations on modalities of interventions targeting anaplerosis in heart disease, including heart failure.
Background The right ventricle exposed to chronic pressure overload exhibits hypertrophy and decompensates when exposed to stress. We hypothesize that impaired ability to increase myocardial ...oxidative flux through pyruvate dehydrogenase leads to hypertrophied right ventricular (RV) dysfunction when exposed to hemodynamic stress, and pyruvate dehydrogenase stimulation can improve RV function. Methods and Results Infant male Yorkshire piglets (13.5±0.6 kg weight, n=19) were used to assess substrate fractional contribution to the citric acid cycle after sustained pulmonary artery banding (PAB). Carbon 13-labeled glucose, lactate, and leucine, oxidative substrate tracers for the citric acid cycle, were infused into the right coronary artery on 7 to 10 days after PAB. RV systolic pressure, RV free wall thickness, and individual cardiomyocyte cell size after PAB were significantly elevated compared with the sham group. Both fractional glucose and lactate oxidations in the PAB group were >2-fold higher than in the sham group. Pigs with overdrive atrial pacing (≈80% increase in heart rate) stress after PAB showed only a 22% increase in rate-pressure product from baseline before atrial pacing and limited carbohydrate oxidation rate in the right ventricle. Intracoronary infusion of dichloroacetate, a pyruvate dehydrogenase agonist, produced higher rate-pressure product (59% increase) in response to increased workload by atrial pacing in association with a marked increase in lactate oxidation. Conclusions The immature hypertrophied right ventricle shows limited ability to increase carbohydrate oxidation in response to tachycardia stress leading to energy supply/utilization imbalance and decreased systolic function. Enhanced pyruvate dehydrogenase activation by dichloroacetate increases energy supply and preserves hypertrophied RV contractile function during hemodynamic stress.
Very-long chain acyl-CoA dehydrogenase (VLCAD) catalyzes the initial step of mitochondrial long chain (LC) fatty acid β-oxidation (FAO). Inherited VLCAD deficiency (VLCADD) predisposes to neonatal ...arrhythmias whose pathophysiology is still not understood. We hypothesized that VLCADD results in global disruption of cardiac complex lipid homeostasis, which may set conditions predisposing to arrhythmia. To test this, we assessed the cardiac lipidome and related molecular markers in seven-month-old VLCAD
mice, which mimic to some extent the human cardiac phenotype. Mice were sacrificed in the fed or fasted state after receiving for two weeks a chow or a high-fat diet (HFD), the latter condition being known to worsen symptoms in human VLCADD. Compared to their littermate counterparts, HFD/fasted VLCAD
mouse hearts displayed the following lipid alterations: (1) Lower LC, but higher VLC-acylcarnitines accumulation, (2) higher levels of arachidonic acid (AA) and lower docosahexaenoic acid (DHA) contents in glycerophospholipids (GPLs), as well as (3) corresponding changes in pro-arrhythmogenic AA-derived isoprostanes and thromboxane B
(higher), and anti-arrythmogenic DHA-derived neuroprostanes (lower). These changes were associated with remodeling in the expression of gene or protein markers of (1) GPLs remodeling: higher calcium-dependent phospholipase A2 and lysophosphatidylcholine-acyltransferase 2, (2) calcium handling perturbations, and (3) endoplasmic reticulum stress. Altogether, these results highlight global lipid dyshomeostasis beyond FAO in VLCAD
mouse hearts, which may set conditions predisposing the hearts to calcium mishandling and endoplasmic reticulum stress and thereby may contribute to the pathogenesis of arrhythmias in VLCADD in mice as well as in humans.
Defects in fatty acid (FA) utilization have been well described in group 1 pulmonary hypertension (PH) and in heart failure (HF), yet poorly studied in group 2 PH. This study was to assess whether ...the metabolomic profile of patients with pulmonary hypertension (PH) due HF, classified as group 2 PH, differs from those without PH. We conducted a proof-of-principle cross-sectional analysis of 60 patients with chronic HF with reduced ejection fraction and 72 healthy controls in which the circulating level of 71 energy-related metabolites was measured using various methods. Echocardiography was used to classify HF patients as noPH-HF (
= 27; mean pulmonary artery pressure mPAP 21 mmHg) and PH-HF (
= 33; mPAP 35 mmHg). The profile of circulating metabolites among groups was compared using principal component analysis (PCA), analysis of covariance (ANCOVA), and Pearson's correlation tests. Patients with noPH-HF and PH-HF were aged 64 ± 11 and 68 ± 10 years, respectively, with baseline left ventricular ejection fractions of 27 ± 7% and 26 ± 7%. Principal component analysis segregated groups, more markedly for PH-HF, with long-chain acylcarnitines, acetylcarnitine, and monounsaturated FA carrying the highest loading scores. After adjustment for age, sex, kidney function, insulin resistance, and N-terminal pro-brain natriuretic peptide (NT-proBNP), 5/15 and 8/15 lipid-related metabolite levels were significantly different from controls in noPH-HF and PH-HF subjects, respectively. All metabolites for which circulating levels interacted between group and NT-proBNP significantly correlated with NT-proBNP in HF-PH, but none with HF-noPH. FA-related metabolites were differently affected in HF with or without PH, and may convey adverse outcomes given their distinct correlation with NT-proBNP in the setting of PH.
Abstract The cytoskeletal protein dystrophin has been implicated in hereditary and acquired forms of cardiomyopathy. However, much remains to be learned about the role of dystrophin in the heart. We ...hypothesized that the dystrophin-deficient heart displays early alterations in energy metabolism that precede overt cardiomyopathy. We evaluated the metabolic and functional phenotype of dystrophin-deficient mdx mouse hearts at 10–12 weeks, when no major histological or echocardiographic abnormalities are reported. Ex vivo working mdx heart perfusions with stable isotopes revealed a marked shift in substrate fuel selection from fatty acids to carbohydrates associated with enhanced oxygen consumption. They also unmasked in the mdx heart: (i) compromised cardiac contractile function and efficiency, (ii) reduced cellular integrity, and (iii) exacerbated alterations in mitochondrial citric acid cycle-related parameters and in nutrient signaling pathways related to Akt. The observed shift in substrate selection cannot be explained by metabolic gene remodeling. However, mdx mice hearts showed an increased expression of the atrial natriuretic factor ( anf ) gene, an activator of the nitric oxide (NO)/cGMP signaling pathway and marker of cardiac remodeling, and, only as the cardiomyopathy progresses (at 25 weeks of age), an increased expression of the α1 subunit of soluble guanylate cyclase, which is known to negatively correlate with the activity NO/cGMP pathway. Collectively, our results highlight early metabolic and signaling alterations in the dystrophin-deficient heart, which may predispose these hearts to contractile dysfunction and sarcolemmal fragility. They also suggest the presence of a “sub-clinical” defect in the NO/cGMP pathway, which in vivo , at an early age, may be compensated by enhanced anf gene expression.
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