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Intrauterine growth restriction elevates circulating acylcarnitines and suppresses fatty‐acid metabolism genes in the fetal sheep heart
Abstract
Key points:
The fetal heart relies on carbohydrates in utero and must be prepared to metabolize fatty acids after birth but the effects of compromised fetal growth on the maturation of this metabolic system are unknown. Plasma fatty-acylcarnitines are elevated in intrauterine growth restricted (IUGR) fetuses over control fetuses, indicative of impaired fatty acid metabolism in fetal organs. Fatty acid uptake and storage are not different in IUGR cardiomyocytes compared to controls. mRNA levels of genes regulating fatty acid transporter and metabolic enzymes are suppressed in the IUGR myocardium compared to controls, while protein levels remain unchanged. Mismatches in gene and protein expression, and increased circulating fatty acylcarnitines may have long term implications for offspring heart metabolism and adult health in IUGR individuals. This requires further investigation.
Abstract:
At birth, the mammalian myocardium switches from using carbohydrates as the primary energy substrate to free fatty acids as the primary fuel. Thus, a compromised switch could jeopardize normal heart function in the neonate. Placental embolization in sheep is a reliable model of intrauterine growth restriction (IUGR). It leads to suppression of both proliferation and terminal differentiation of cardiomyocytes. We hypothesized that the expression of genes regulating cardiac fatty acid metabolism would be similarly suppressed in IUGR, leading to compromised processing of lipids. Following 10 days of umbilicoplacental embolization in fetal sheep, IUGR fetuses had elevated circulating long chain fatty acylcarnitines compared to controls (C14: CTRL 0.012 ± 0.005 nmol/mL vs IUGR 0.018 ± 0.005 nmol/mL, p<0.05; C18: CTRL 0.027 ± 0.009 nmol/mol vs IUGR 0.043 ± 0.024 nmol/mol, p<0.05, n = 12 control, n = 12 IUGR) indicative of impaired fatty acid metabolism. Uptake studies using fluorescently tagged BODIPY-C12 saturated free fatty acid in live, isolated cardiomyocytes showed lipid droplet area and number were not different between control and IUGR cells. mRNA levels of sarcolemmal fatty acid transporters (CD36, FATP6), acylation enzymes (ACSL1, ACSL3), mitochondrial transporter (CPT1), ß-oxidation enzymes (LCAD, HADH, ACAT1), tricarboxylic acid cycle enzyme (IDH), esterification enzymes (PAP, DGAT) and regulator of lipid droplet formation (BSCL2) gene were all suppressed in IUGR myocardium (p<0.05). However, protein levels for these regulatory genes were not different between groups. This discordance between mRNA and protein levels in the stressed myocardium suggests an adaptive protection of key myocardial enzymes under conditions of placental insufficiency. Abstract figure. We investigated to degree to which the cardiac genes that regulate fatty acid metabolism were altered in growth restricted fetuses compared to those that grew normally. As shown by the red arrows, messenger RNA levels for the genes studied were suppressed compared to controls. However, the double headed arrows show that protein levels were unaffected. Circulating long chain fatty acylcarnitines in the fetus were elevated suggesting incomplete metabolism of free fatty acids. Whether these experimental conditions portend adverse outcomes in the postnatal period as the heart transitions to fatty acid metabolism requires further study. This article is protected by copyright. All rights reserved.
... Critically, pre-clinical studies in a variety of species demonstrate that FGR is also associated with effects on other organs such as the kidney, with reduced nephron endowment and associated reduced filtration capacity (Mitchell et al., 2004), and heart, with reduced cardiac myocyte endowment, impaired maturation, remodeling of cardiac vasculature and a switch in energy substrate use (Louey et al., 2007;Wang et al., 2013;Botting et al., 2018;Masoumy et al., 2018;Maréchal et al., 2021;Drake et al., 2022). Altered renal and cardiac development are strongly linked with later-life risks for cardiovascular disease (Masoumy et al., 2018;Fung and Zinkhan, 2021). ...
Fetal growth restriction (FGR) is a major cause of stillbirth, prematurity and impaired neurodevelopment. Its etiology is multifactorial, but many cases are related to impaired placental development and dysfunction, with reduced nutrient and oxygen supply. The fetus has a remarkable ability to respond to hypoxic challenges and mounts protective adaptations to match growth to reduced nutrient availability. However, with progressive placental dysfunction, chronic hypoxia may progress to a level where fetus can no longer adapt, or there may be superimposed acute hypoxic events. Improving detection and effective monitoring of progression is critical for the management of complicated pregnancies to balance the risk of worsening fetal oxygen deprivation in utero, against the consequences of iatrogenic preterm birth. Current surveillance modalities include frequent fetal Doppler ultrasound, and fetal heart rate monitoring. However, nearly half of FGR cases are not detected in utero, and conventional surveillance does not prevent a high proportion of stillbirths. We review diagnostic challenges and limitations in current screening and monitoring practices and discuss potential ways to better identify FGR, and, critically, to identify the “tipping point” when a chronically hypoxic fetus is at risk of progressive acidosis and stillbirth.
... Intriguingly, seipin expression levels are significantly increased in experimental Parkinson's disease models both in vitro and in vivo, and overexpression of seipin aggravates ER stress via the Grp94/Bip-ATF4-CHOP signalling pathway [166]. Seipin deficiency facilitated Aβ 25-35/1-42 -induced neuroinflammation via the PPARγ-GSK3β-TNF-α/IL-6 pathway in astrocytes, resulting in neurodegenerative disorders eventually [20,169]. In glioblastoma patients and glioblastoma cell lines, the expression of BSCL2 is increased [167]. ...
Seipin, a protein encoded by the Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2) gene, is famous for its key role in the biogenesis of lipid droplets and type 2 congenital generalised lipodystrophy (CGL2). BSCL2 gene mutations result in genetic diseases including CGL2, progressive encephalopathy with or without lipodystrophy (also called Celia’s encephalopathy), and BSCL2-associated motor neuron diseases. Abnormal expression of seipin has also been found in hepatic steatosis, neurodegenerative diseases, glioblastoma stroke, cardiac hypertrophy, and other diseases. In the current study, we comprehensively summarise phenotypes, underlying mechanisms, and treatment of human diseases caused by BSCL2 gene mutations, paralleled by animal studies including systemic or specific Bscl2 gene knockout, or Bscl2 gene overexpression. In various animal models representing diseases that are not related to Bscl2 mutations, differential expression patterns and functional roles of seipin are also described. Furthermore, we highlight the potential therapeutic approaches by targeting seipin or its upstream and downstream signalling pathways. Taken together, restoring adipose tissue function and targeting seipin-related pathways are effective strategies for CGL2 treatment. Meanwhile, seipin-related pathways are also considered to have potential therapeutic value in diseases that are not caused by BSCL2 gene mutations.
... This review focuses on a notable paper in the area by Drake et al. published recently in The Journal of Physiology. This is one of the first studies to perform an in-depth investigation into how metabolic pathways are affected in the heart of IUGR fetal sheep (Drake et al. 2022). They hypothesised that expression of genes regulating fatty acid metabolism in cardiomyocytes would be suppressed in IUGR fetal hearts. ...
Background:
Intrauterine growth restriction (IUGR) exerts a negative impact on developing cardiomyocytes and emerging evidence suggests activation of oxidative stress pathways plays a key role in this altered development. Here, we provided pregnant guinea pig sows with PQQ, an aromatic tricyclic o-quinone that functions as a redox cofactor antioxidant, during the last half of gestation as a potential antioxidant intervention for IUGR-associated cardiomyopathy.
Methods:
Pregnant guinea pig sows were randomly assigned to receive PQQ or placebo at mid gestation and fetuses were identified as spontaneous IUGR (spIUGR) or normal growth (NG) near term yielding four cohorts: NG ± PQQ and spIUGR ± PQQ. Cross sections of fetal left and right ventricles were prepared and cardiomyocyte number, collagen deposition, proliferation (Ki67) and apoptosis (TUNEL) were analyzed.
Results:
Cardiomyocyte endowment was reduced in spIUGR fetal hearts when compared to NG; however, PQQ exerted a positive effect on cardiomyocyte number in spIUGR hearts. Cardiomyocytes undergoing proliferation and apoptosis were more common in spIUGR ventricles when compared with NG animals, which was significantly reduced with PQQ supplementation. Similarly, collagen deposition was increased in spIUGR ventricles and was partially rescued in PQQ-treated spIUGR animals.
Conclusion:
The negative influence of spIUGR on cardiomyocyte number, apoptosis, and collagen deposition during parturition can be suppressed by antenatal administration of PQQ to pregnant sows. These data identify a novel therapeutic intervention for irreversible spIUGR-associated cardiomyopathy.
In clinical cohort studies, high expression of long-chain acyl-coenzyme A synthetases 1 (ACSL1 gene) in peripheral white blood cells of patients with acute myocardial infarction (AMI) has been utilized as molecular markers of myocardial infarction diagnosis. The plasma triglyceride level of AMI patients is significantly higher than that of healthy individuals. We hypothesized that the high expression of ACSL1 increases the level of triglyceride, which is one of the pathogenesis of AMI promoted by ACSL1. In this report, cell culture based methods were adopted to test the hypothesis and further investigate the effect and mechanism of ACSL1 on lipid metabolism. In this study, liver cells of healthy individuals were cultured, the overexpression and the knockdown vectors of ACSL1 were constructed and transfected into liver cells. The transfection was verified at the mRNA and protein level. Intracellular triglyceride content was quantitatively analyzed using ELISA. Changes of genes related to lipid metabolism were subsequently measured through PCR array. Overexpression of ACSL1 led to higher gene expression and protein levels compared to control and the triglyceride content was significantly increased in overexpressing cells. The expression level of fatty acid oxidation pathway PPARγ was significantly down-regulated compared with the control group, as were genes associated with fatty acid synthesis pathways: SREBP1, ACC, FAS, and SCD1. ACSL1 knockdown decreased the content of triglyceride whereas PPARγ was up-regulated and SREBP1, ACC, FAS, and SCD1 were down-regulated compared with the control group. In summary, high expression of ACSL1 reduced fatty acid β-oxidation through the PPARγ pathway, thereby increasing triglyceride levels.
Key points:
Skeletal muscle energy requirements increase at birth but little is known about the development of mitochondria that provide most of the cellular energy as ATP. Thyroid hormones are known regulators of adult metabolism and are important in driving several aspects of fetal development, including muscle fibre differentiation. Mitochondrial density and abundance of mitochondrial membrane proteins in skeletal muscle increased during late gestation. However, mitochondrial functional capacity, measured as oxygen consumption rate, increased primarily after birth. Fetal hypothyroidism resulted in significant reductions in mitochondrial function and density in skeletal muscle before birth. Mitochondrial function matures towards birth and is dependent on the presence of thyroid hormones with potential implications for the health of pre-term and hypothyroid infants.
Abstract:
Birth is a significant metabolic challenge with exposure to a pro-oxidant environment and the increased energy demands for neonatal survival. This study investigated the development of mitochondrial density and activity in ovine biceps femoris skeletal muscle during the perinatal period and examined the role of thyroid hormones in these processes. Muscle capacity for oxidative phosphorylation increased primarily after birth but was accompanied by prepartum increases in mitochondrial density and abundance of electron transfer system (ETS) complexes I-IV and ATP-synthase as well as by neonatal upregulation of uncoupling proteins. This temporal disparity between prepartum maturation and neonatal upregulation of mitochondrial oxidative capacity may protect against oxidative stress associated with birth while ensuring energy availability to the neonate. Fetal thyroid hormone deficiency reduced oxidative phosphorylation and prevented the prepartum upregulation of mitochondrial density and ETS proteins in fetal skeletal muscle. Overall, the data shows that mitochondrial function matures over the perinatal period and is dependent on thyroid hormones, with potential consequences for neonatal viability and adult metabolic health. This article is protected by copyright. All rights reserved.
Hypoxic exposure during development can have a profound influence on offspring physiology, including cardiac dysfunction, yet many reptile embryos naturally experience periods of hypoxia in buried nests. American alligators experimentally exposed to developmental hypoxia demonstrate morphological and functional changes to the heart that persist into later life stages; however, the molecular bases of these changes remain unknown. We tested if targeted and persistent changes in steady-state protein expression underlie this hypoxic heart phenotype, using isobaric tags for relative and absolute quantitation (iTRAQ) proteomics. Alligator eggs were reared under normoxia or 10% hypoxia, then either sampled (embryo) or returned to normoxia for 2 years (juvenile). Three salient findings emerge from the integrated analysis of the 145 differentially expressed proteins in hypoxia-reared animals: (1) significant protein-protein interaction networks were identified only in up-regulated proteins, indicating that the effects of developmental hypoxia are stimulatory and directed; (2) the up-regulated proteins substantially enriched processes related to protein turnover, cellular organization, and metabolic pathways, supporting increased resource allocation towards building and maintaining a higher functioning heart; and (3) the juvenile cardiac proteome retained many of the signature changes observed in embryonic hearts, supporting long-term reprogramming of cardiac myocytes induced by hypoxia during critical periods of development.
Oxygen deprivation or hypoxia characterizes a number of serious pathological conditions and elicits a number of adaptive changes that are mainly mediated at the transcriptional level by the family of hypoxia-inducible factors (HIFs). The HIF target gene repertoire includes genes responsible for the regulation of metabolism, oxygen delivery and cell survival. Although the involvement of HIFs in the regulation of carbohydrate metabolism and the switch to anaerobic glycolysis under hypoxia is well established, their role in the control of lipid anabolism and catabolism remains still relatively obscure. Recent evidence indicates that many aspects of lipid metabolism are modified during hypoxia or in tumor cells in a HIF-dependent manner, contributing significantly to the pathogenesis and/or progression of cancer and metabolic disorders. However, direct transcriptional regulation by HIFs has been only demonstrated in relatively few cases, leaving open the exact and isoform-specific mechanisms that underlie HIF-dependency. This review summarizes the evidence for both direct and indirect roles of HIFs in the regulation of genes involved in lipid metabolism as well as the involvement of HIFs in various diseases as demonstrated by studies with transgenic animal models.
Epidemiologic and clinical research has provided a large body of evidence supporting the developmental origins of health and disease (DOHaD), but there has been a relative dearth of mechanistic studies in humans due to the complexity of working with large, longitudinal cohorts. Nonetheless, animal models of undernutrition have provided substantial evidence for the potential epigenetic, metabolic, and endocrine mechanisms behind DOHaD. Furthermore, recent research has explored the interaction between the environment and the gastrointestinal system by investigating how the gut microbial ecology may impact the capacity for nutrient processing and absorption in a manner that may limit growth. This review presents a summary of current research that supports the concept of DOHaD, as well as potential mechanisms and interactions that explain how nutrition in utero and during early childhood influences lifelong health.
Abstract
Background Fetal growth restriction (FGR) is a condi- tion that a ects 5–10% of pregnancies and is the second most common cause of perinatal mortality. This review presents the most recent knowledge on FGR and focuses on the etiology, classi cation, prediction, diagnosis, and management of the condition, as well as on its neurological complications.
Methods The Pubmed, SCOPUS, and Embase databases were searched using the term “fetal growth restriction”. Results Fetal growth restriction (FGR) may be classi ed as early or late depending on the time of diagnosis. Early FGR (<32 weeks) is associated with substantial alterations in placental implantation with elevated hypoxia, which requires cardiovascular adaptation. Perinatal morbidity and mortality rates are high. Late FGR (≥32 weeks) presents with slight de ciencies in placentation, which leads to mild hypoxia and requires little cardiovascular adaptation. Peri- natal morbidity and mortality rates are lower. The diagno- sis of FGR may be clinical; however, an arterial and venous Doppler ultrasound examination is essential for diagnosis and follow-up. There are currently no treatments to control FGR; the time at which pregnancy is interrupted is of vital importance for protecting both the mother and fetus. Conclusion Early diagnosis of FGR is very important, because it enables the identi cation of the etiology of the condition and adequate monitoring of the fetal status, thereby minimizing risks of premature birth and intrauter- ine hypoxia.
Keywords Fetal growth restriction · Placental insu ciency · Prediction · Doppler · Management · Neurological development
Background: Intrauterine growth restriction (IUGR) is associated with short- and long-term metabolic consequences which are possibly dictated by in utero programming together with environmental and dietetic manipulation after birth. Early detection of metabolic derangements in these babies through metabolomics approach will help recognition of cases in need for further follow-up and can help future development of therapeutic and preventive strategies for the late consequences.
Objective: To compare amino acids and acyl carnitine levels in neonates with IUGR to normal birth weight controls; as a part of metabolic profiling.
Methods: Cord blood samples were collected at birth from 40 small-for-gestational-age (SGA) neonates and 20 normal birth weight gestational age-matched neonates, for quantification of amino acids and acylcarnitines using Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS).
Results: Significantly elevated acylcarnitine levels especially C18-OH and C16-OH were found in IUGR neonates vs. controls (p
While the human placenta must provide selected long-chain fatty acids to support the developing fetal brain, little is known about the mechanisms underlying the transport process. We tracked the movement of the fluorescently labeled long-chain fatty acid analogue, BODIPY-C12, across the cell layers of living explants of human term placenta. Although all layers took up the fatty acid, rapid esterification of long-chain fatty acids and incorporation into lipid droplets was exclusive to the inner layer cytotrophoblast cells rather than the expected outer syncytiotrophoblast layer. Cytotrophoblast is a progenitor cell layer previously relegated to a repair role. As isolated cytotrophoblasts differentiated into syncytialized cells in culture, they weakened their lipid processing capacity. Syncytializing cells suppress previously active genes that regulate fatty-acid uptake (SLC27A2/FATP2, FABP4, ACSL5) and lipid metabolism (GPAT3, LPCAT3). We speculate that cytotrophoblast performs a previously unrecognized role in regulating placental fatty acid uptake and metabolism.
How the fetus withstands an environment of reduced oxygenation during life in the womb has been a vibrant area of research since this field was introduced by Joseph Barcroft, a century ago. Studies spanning five decades have since used the chronically instrumented fetal sheep preparation to investigate the fetal compensatory responses to hypoxia. This defence is contingent on the fetal cardiovascular system, which in late gestation adopts strategies to decrease oxygen consumption and redistribute the cardiac output away from peripheral vascular beds and towards essential circulations, such as those perfusing the brain. The introduction of simultaneous measurement of blood flow in the fetal carotid and femoral circulations by ultrasonic transducers has permitted investigation of the dynamics of the fetal brain sparing response for the first time. Now we know that major components of fetal brain sparing during acute hypoxia are triggered exclusively by a carotid chemoreflex and that they are modified by endocrine agents and the recently discovered vascular oxidant tone. The latter is determined by the interaction between nitric oxide and reactive oxygen species. The fetal brain sparing response matures as the fetus approaches term, in association with the prepartum increase in fetal plasma cortisol and treatment of the preterm fetus with clinically-relevant doses of synthetic steroids mimics this maturation. Despite intense interest into how the fetal brain sparing response may be affected by adverse intrauterine conditions, this area of research has been comparatively scant but it is likely to take centre stage in the near future. This article is protected by copyright. All rights reserved.
Intrauterine growth restriction (IUGR) is associated with an increased risk of developing obesity, insulin resistance and cardiovascular disease. However, its effect on energetics in heart remains unknown. In the present study, we examined respiration in cardiac muscle and liver from adult mice that were undernourished in utero. We report that in utero undernutrition is associated with impaired cardiac muscle energetics, including decreased fatty acid oxidative capacity, decreased maximum oxidative phosphorylation rate and decreased proton leak respiration. No differences in oxidative characteristics were detected in liver. We also measured plasma acylcarnitine levels and found that short-chain acylcarnitines are increased with in utero undernutrition. Results reveal the negative impact of suboptimal maternal nutrition on adult offspring cardiac energy metabolism, which may have life-long implications for cardiovascular function and disease risk.
© 2015 Authors.
Hypoxia-inducible factor 1 (HIF-1) mediates a metabolic switch that blocks the conversion of pyruvate to acetyl-CoA in cancer cells. Here, we report that HIF-1α also inhibits fatty acid β-oxidation (FAO), another major source of acetyl-CoA. We identified a PGC-1β-mediated pathway by which HIF-1 inhibits the medium- and long-chain acyl-CoA dehydrogenases (MCAD and LCAD), resulting in decreased reactive oxygen species levels and enhanced proliferation. Surprisingly, we further uncovered that blocking LCAD, but not MCAD, blunts PTEN expression and dramatically affects tumor growth in vivo. Analysis of 158 liver cancer samples showed that decreased LCAD expression predicts patient mortality. Altogether, we have identified a previously unappreciated mechanism by which HIF-1 suppresses FAO to facilitate cancer progression.
Control of lipid droplet (LD) nucleation and copy number are critical, yet poorly understood, processes. We use model peptides that shift from the endoplasmic reticulum (ER) to LDs in response to fatty acids to characterize the initial steps of LD formation occurring in lipid-starved cells. Initially, arriving lipids are rapidly packed in LDs that are resistant to starvation (pre-LDs). Pre-LDs are restricted ER microdomains with a stable core of neutral lipids. Subsequently, a first round of "emerging" LDs is nucleated, providing additional lipid storage capacity. Finally, in proportion to lipid concentration, new rounds of LDs progressively assemble. Confocal microscopy and electron tomography suggest that emerging LDs are nucleated in a limited number of ER microdomains after a synchronized stepwise process of protein gathering, lipid packaging, and recognition by Plin3 and Plin2. A comparative analysis demonstrates that the acyl-CoA synthetase 3 is recruited early to the assembly sites, where it is required for efficient LD nucleation and lipid storage.
Heart failure is associated with impaired myocardial metabolism with a shift from fatty acids to glucose use for ATP generation. We hypothesized that cardiac accumulation of toxic lipid intermediates inhibits insulin signaling in advanced heart failure and that mechanical unloading of the failing myocardium corrects impaired cardiac metabolism.
We analyzed the myocardium and serum of 61 patients with heart failure (body mass index, 26.5±5.1 kg/m(2); age, 51±12 years) obtained during left ventricular assist device implantation and at explantation (mean duration, 185±156 days) and from 9 control subjects. Systemic insulin resistance in heart failure was accompanied by decreased myocardial triglyceride and overall fatty acid content but increased toxic lipid intermediates, diacylglycerol, and ceramide. Increased membrane localization of protein kinase C isoforms, inhibitors of insulin signaling, and decreased activity of insulin signaling molecules Akt and Foxo were detectable in heart failure compared with control subjects. Left ventricular assist device implantation improved whole-body insulin resistance (homeostatic model of analysis-insulin resistance, 4.5±0.6-3.2±0.5; P<0.05) and decreased myocardial levels of diacylglycerol and ceramide, whereas triglyceride and fatty acid content remained unchanged. Improved activation of the insulin/phosphatidylinositol-3 kinase/Akt signaling cascade after left ventricular assist device implantation was confirmed by increased phosphorylation of Akt and Foxo, which was accompanied by decreased membrane localization of protein kinase C isoforms after left ventricular assist device implantation.
Mechanical unloading after left ventricular assist device implantation corrects systemic and local metabolic derangements in advanced heart failure, leading to reduced myocardial levels of toxic lipid intermediates and improved cardiac insulin signaling.
Acylcarnitine profiling by electrospray ionization tandem mass spectrometry (ESI-MS/MS) is a potent tool for the diagnosis and screening of fatty acid oxidation and organic acid disorders. Few studies have analyzed free carnitine and acylcarnitines in dried blood spots (DBS) of umbilical cord blood (CB) and the postnatal changes in the concentrations of these analytes. We have investigated these metabolites in healthy exclusively breastfed neonates and examined possible effects of birth weight and gestational age. DBS of CB were collected from 162 adequate for gestational age neonates. Paired DBS of heel-prick blood were collected 4-8 days after birth from 106 of these neonates, the majority exclusively breastfed. Methanol extracts of DBS with deuterium-labeled internal standards were derivatized before analysis by ESI-MS/MS. Most of the analytes were measured using a full-scan method. The levels of the major long-chain acylcarnitines, palmitoylcarnitine, stearoylcarnitine, and oleoylcarnitine, increased by 27, 12, and 109%, respectively, in the first week of life. Free carnitine and acetylcarnitine had a modest increase: 8 and 11%, respectively. Propionylcarnitine presented a different behavior, decreasing 9% during the period. The correlations between birth weight or gestational age and the concentrations of the analytes in DBS were weak (r ≤ 0.20) or nonsignificant. Adaptation to breast milk as the sole source of nutrients can explain the increase of these metabolites along the early neonatal period. Acylcarnitine profiling in CB should have a role in the early detection of metabolic disorders in high-risk neonates.
Long-chain acyl coenzyme A (acyl-CoA) synthetase isoform 1 (ACSL1) catalyzes the synthesis of acyl-CoA from long-chain fatty
acids and contributes the majority of cardiac long-chain acyl-CoA synthetase activity. To understand its functional role in
the heart, we studied mice lacking ACSL1 globally (Acsl1T−/−) and mice lacking ACSL1 in heart ventricles (Acsl1H−/−) at different times. Compared to littermate controls, heart ventricular ACSL activity in Acsl1T−/− mice was reduced more than 90%, acyl-CoA content was 65% lower, and long-chain acyl-carnitine content was 80 to 90% lower.
The rate of [14C]palmitate oxidation in both heart homogenate and mitochondria was 90% lower than in the controls, and the maximal rates
of [14C]pyruvate and [14C]glucose oxidation were each 20% higher. The mitochondrial area was 54% greater than in the controls with twice as much mitochondrial
DNA, and the mRNA abundance of Pgc1α and Errα increased by 100% and 41%, respectively. Compared to the controls, Acsl1T−/− and Acsl1H−/− hearts were hypertrophied, and the phosphorylation of S6 kinase, a target of mammalian target of rapamycin (mTOR) kinase,
increased 5-fold. Our data suggest that ACSL1 is required to synthesize the acyl-CoAs that are oxidized by the heart, and
that without ACSL1, diminished fatty acid (FA) oxidation and compensatory catabolism of glucose and amino acids lead to mTOR
activation and cardiac hypertrophy without lipid accumulation or immediate cardiac dysfunction.
Low birth weight and low placental weight predict later coronary heart disease and hypertension. This has led to the hypothesis that these diseases are initiated by foetal programming, the process by which foetal malnutrition leads to permanent changes in the body in ways that lead to chronic disease in later life. Here we examine the association between body and placental size at birth and later chronic heart failure.
We identified 187 patients taking medications for chronic heart failure in a birth cohort of 13,345 people born in Helsinki, Finland during 1934-44. Chronic heart failure was associated with a small placental surface area. In people born with a placental area less than 225 cm(2), the odds ratio for chronic heart failure was 1.7 (1.1-2.5), compared with people born with a placental area greater than 295 cm(2). The risk of heart failure was further increased by rapid gain in body mass index after the age of 2 years, a path of growth known to be linked to insulin resistance in later life. In a simultaneous regression, low body mass index at 2 years and high body mass index at 11 years were each associated with chronic heart failure (P = 0.008 and 0.001, respectively).
Chronic heart failure in adult life may be initiated by impaired placental growth which adversely affects cardiac development. People born with a vulnerable heart are more likely to develop chronic heart failure if they become insulin resistant.
Lipid droplets (LDs) form from the endoplasmic reticulum (ER) and grow in size by obtaining triacylglycerols (TG). Triacylglycerol hydrolase (TGH), a lipase residing in the ER, is involved in the mobilization of TG stored in LDs for the secretion of very-low-density lipoproteins. In this study, we investigated TGH-mediated changes in cytosolic LD dynamics. We have found that TGH deficiency resulted in decreased size and increased number of LDs in hepatocytes. Using fluorescent fatty acid analogues to trace LD formation, we observed that TGH deficiency did not affect the formation of nascent LDs on the ER. However, the rate of lipid transfer into preformed LDs was significantly slower in the absence of TGH. Absence of TGH expression resulted in increased levels of membrane diacylglycerol and augmented phospholipid synthesis, which may be responsible for the delayed lipid transfer. Therefore, altered maturation (growth) rather than nascent formation (de novo synthesis) may be responsible for the observed morphological changes of LDs in TGH-deficient hepatocytes.
The two ventricles of the fetal sheep heart have anatomic and biochemical differences that account for their differing functional capabilities and blood flows. Coronary flows to both ventricles have been measured using radiolabeled microspheres [or left ventricular (LV) flow, by Doppler sensor on the circumflex coronary artery] during experiments of pressure loading and chronic and acute hypoxemia. Blood flow to the left ventricle with its lower wall tension is about two-thirds the flow per gram compared with the right ventricle (RV). Acute systolic pressure loading of the RV to its maximal work capability stimulates flow to double (from approximately 250 to 500 ml. min(-1). 100 g(-1)), but to a level less than stimulated by adenosine (750 ml. min(-1). 100 g(-1)). At all RV work loads, LV flow remains at two-thirds RV flow. Resting myocardial flow levels in fetuses that have been chronically hypoxemic are similar to maximal adenosine-stimulated flows of normal fetal sheep. This flow augmentation is evidently due to vascular remodeling because a normal "flow reserve" of approximately 500 ml. min(-1). 100 g(-1) during adenosine administration remains. Acute hypoxemia stimulates myocardial flow to extraordinary levels (>1.5 l. min(-1). 100 g(-1)), levels larger than can be obtained with chemical dilation alone. LV flows do not exceed adenosine-stimulated flows when nitric oxide synthase is antagonized. We conclude 1) fetal RV coronary flow increases with RV work but to levels less than during adenosine stimulation; 2) the fetal heart is designed to accommodate extremely high flows in response to acute hypoxemia, partially through large production of nitric oxide; and 3) the fetal coronary tree is dramatically remodeled in response to chronic hypoxemia.
Key points:
Glucocorticoids are steroid hormones that play a vital role in late pregnancy in maturing fetal organs, including the heart. In fetal cardiomyocytes in culture, glucocorticoids promote mitochondrial fatty acids oxidation, suggesting they facilitate the perinatal switch from carbohydrates to fatty acids as the predominant energy substrate. Administration of a synthetic glucocorticoid in late pregnancy in mice down-regulates the glucocorticoid receptor and interferes with the normal increase in genes involved in fatty acid metabolism in the heart. In a sheep model of preterm birth, antenatal corticosteroids (synthetic glucocorticoid) down-regulates glucocorticoid receptor and the gene encoding PGC-1α, a master regulator of energy metabolism. These experiments suggest that administration of antenatal corticosteroids in anticipation of preterm delivery may interfere with fetal heart maturation by down-regulating the ability to respond to glucocorticoids.
Abstract:
The late gestational rise in glucocorticoids contributes to the structural and functional maturation of the perinatal heart. Here, we hypothesised that glucocorticoid action contributes to the metabolic switch in perinatal cardiomyocytes from carbohydrate to fatty acid oxidation. In primary mouse fetal cardiomyocytes, dexamethasone treatment induced expression of genes involved in fatty acid oxidation and increased mitochondrial oxidation of palmitate, dependent upon glucocorticoid receptor (GR). Dexamethasone did not, however, induce mitophagy or alter the morphology of the mitochondrial network. In vivo, in neonatal mice, dexamethasone treatment induced cardiac expression of fatty acid oxidation genes. However, dexamethasone treatment of pregnant C57Bl/6 mice at embryonic day (E)13.5 or E16.5 failed to induce fatty acid oxidation genes in fetal hearts assessed 24 hours later. Instead, at E17.5, fatty acid oxidation genes were down-regulated by dexamethasone, as was GR itself. PGC-1α, required for glucocorticoid-induced maturation of primary mouse fetal cardiomyocytes in vitro, was also down-regulated in fetal hearts at E17.5, 24 hours after dexamethasone administration. Similarly, following a course of antenatal corticosteroids in a translational sheep model of preterm birth, both GR and PGC-1α were down-regulated in heart. These data suggest endogenous glucocorticoids support the perinatal switch to fatty acid oxidation in cardiomyocytes through changes in gene expression rather than gross changes in mitochondrial volume or mitochondrial turnover. Moreover, our data suggest that treatment with exogenous glucocorticoids may interfere with normal fetal heart maturation, possibly by down-regulating GR. This has implications for clinical use of antenatal corticosteroids when preterm birth is considered a possibility. This article is protected by copyright. All rights reserved.
Intrauterine growth restriction (IUGR) is a result of limited substrate supply to the developing fetus in utero, and can be caused by either placental, genetic or environmental factors. Babies born IUGR can have poor long-term health outcomes, including being at higher risk of developing cardiovascular disease. Limited substrate supply in the IUGR fetus not only changes the structure of the heart but may also affect metabolism and function of the developing heart. We have utilized two imaging modalities, two-photon microscopy and phase-contrast MRI (PC-MRI), to assess alterations in cardiac metabolism and function using a sheep model of IUGR. Two-photon imaging revealed that the left ventricle of IUGR fetuses (at 140-141 d GA) had a reduced optical redox ratio, suggesting a reliance on glycolysis for ATP production. Concurrently, the use of PC-MRI to measure fetal left ventricular cardiac output (LVCO) revealed a positive correlation between LVCO and redox ratio in IUGR, but not control fetuses. These data suggest that altered heart metabolism in IUGR fetuses is indicative of reduced cardiac output, which may contribute to poor cardiac outcomes in adulthood.
This article is protected by copyright. All rights reserved.
Background
Prostate cancer is characterized by aberrant lipid metabolism, including elevated fatty acid oxidation. Carnitine palmitoyltransferase 1B (CPT1B) catalyzes the rate‐limiting step of fatty acid oxidation. This study aimed to determine if CPT1B has a critical role in prostate cancer progression and to identify its regulatory mechanism.
Methods
CPT1B expression data from The Cancer Genome Atlas and Gene Expression Omnibus databases was compared with patient survival data. A tissue microarray was constructed with 60 samples of prostate cancer and immunohistochemically stained for CPT1B. Castration‐resistant prostate cancer (CRPC) cell lines 22RV1 and C4‐2 in which CPT1B expression had been stably knocked down were established; and cell proliferation, cell cycle distribution, and invasion were investigated by Cell Counting Kit‐8 (CCK‐8) and colony formation assays, flow cytometry, and Transwell assays, respectively. To examine the impact of androgen receptor (AR) inhibition on CPT1B expression, JASPAR CORE was searched to identify AR‐binding sites in CPT1B. Dual luciferase and ChIP assays were performed to confirm CPT1B activity and AR binding, respectively. Differentially expressed genes (DEGs) in prostate cancer underwent gene set enrichment analysis (GSEA). Enzalutamide‐resistant C4‐2 cells were generated and the mechanism of enzalutamide resistance and downstream signaling pathway changes of CPT1B to C4‐2 was explored through CCK‐8 test.
Results
CPT1B expression was upregulated in human prostate cancer compared with normal prostate tissue and was associated with poor disease‐free survival and overall survival. Silencing of CPT1B resulted in downregulated cell proliferation, reduced S‐phase distribution, and lower invasive ability, whereas the opposite was observed in CRPC cells overexpressing CPTB1. DEGS in prostate cancer were correlated with G‐protein–coupled receptor signaling, molecular transducer activity, and calcium ion binding. AR may regulate CPT1B expression and activity via specific binding sites, as confirmed by dual luciferase and ChIP assays. The CCK‐8 experiment demonstrated that CPT1B overexpression in C4‐2 cells did not significantly increase the ability of enzalutamide resistance. However, overexpression of CPT1B in C4‐2R cells significantly increased the enzalutamide resistance. Upregulation of CPT1B expression increased AKT expression and phosphorylation.
Conclusions
CPT1B is upregulated in prostate cancer and is correlated with poor prognosis, indicating its potential as a biomarker. AR inhibits the transcription of CPT1B. In the CRPC cell line, overexpression of CPT1B alone cannot promote enzalutamide resistance, but in the drug‐resistant line C4‐2R, overexpression of CPT1B can promote the resistance of C4‐2R to enzalutamide.
At birth, weight of the neonate is used as a marker of the 9-month journey as a fetus. Those neonates born less than the 10th centile for their gestational age are at risk of being intrauterine growth restricted. However, this depends on their genetic potential for growth and the intrauterine environment in which they grew. Alterations in the supply of oxygen and nutrients to the fetus will decrease fetal growth, but these alterations occur due to a range of causes that are maternal, placental or fetal in nature. Consequently, IUGR neonates are a heterogeneous population. For this reason, it is likely that these neonates will respond differently to interventions compared not only to normally grown fetuses, but also to other neonates that are IUGR but have travelled a different path to get there. Thus, a range of models of IUGR should be studied to determine the effects of IUGR on the development and function of the heart and lung and subsequently the impact of interventions to improve development of these organs. Here we focus on a range of models of IUGR caused by manipulation of the maternal, placental or fetal environment on cardiorespiratory outcomes.
The primary metabolic pathway required to produce ATP differs as a result of tissue type, developmental stage and substrate availability. We utilised molecular and histological techniques to define the metabolic status in fetal and adult, adipose and skeletal muscle tissues. Redox ratios of these tissues were also determined optically by two‐photon microscopy. Adult perirenal adipose tissue had a higher optical redox ratio than fetal perirenal adipose tissue, which aligned with glycolysis being used for ATP production; whereas adult skeletal muscle had a lower optical redox ratio than fetal skeletal muscle, which aligned with OXPHOS activity being utilised for ATP production. We have compared traditional molecular and microscopy techniques of metabolic tissue characterisation with optical redox ratios to provide a more comprehensive report on the dynamics of tissue metabolism. This article is protected by copyright. All rights reserved.
Fetal cardiomyocytes shift from glycolysis to oxidative phosphorylation around the time of birth. Myeloid ecotropic viral integration site 1 (MEIS1) is a transcription factor that promotes glycolysis in hematopoietic stem cells. We reasoned that MEIS1 could have a similar role in the developing heart. We hypothesized that suppression of MEIS1 expression in fetal sheep cardiomyocytes leads to a metabolic switch as found at birth. Expression of MEIS1 was assayed in left ventricular cardiac tissue and primary cultures of cardiomyocytes from fetal (100- and 135-d gestation, term = 145 d), neonatal, and adult sheep. Cultured cells were treated with short interfering RNA (siRNA) to suppress MEIS1. Oxygen consumption rate was assessed with the Seahorse metabolic flux analyzer, and mitochondrial activity was assessed by staining cells with MitoTracker Orange. Cardiomyocyte respiratory capacity increased with advancing age concurrently with decreased expression of MEIS1. MEIS1 suppression with siRNA increased maximal oxygen consumption in fetal cells but not in postnatal cells. Mitochondrial activity was increased and expression of glycolytic genes decreased when MEIS1 expression was suppressed. Thus, we conclude that MEIS1 is a key regulator of cardiomyocyte metabolism and that the normal down-regulation of MEIS1 with age underlies a gradual switch to oxidative metabolism.-Lindgren, I. M., Drake, R. R., Chattergoon, N. N., Thornburg, K. L. Down-regulation of MEIS1 promotes the maturation of oxidative phosphorylation in perinatal cardiomyocytes.
Experimental studies that are relevant to human pregnancy rely on the selection of appropriate animal models as an important element in experimental design. Consideration of the strengths and weaknesses of any animal model of human disease is fundamental to effective and meaningful translation of preclinical research. Studies in sheep have made significant contributions to our understanding of the normal and abnormal development of the fetus. As a model of human pregnancy, studies in sheep have enabled scientists and clinicians to answer questions about the etiology and treatment of poor maternal, placental and fetal health and to provide an evidence base for translation of interventions into the clinic. The aim of this review is to highlight the advances in perinatal human medicine that have been achieved following translation of research using the pregnant sheep and fetus.
Placental insufficiency causes intrauterine growth restriction (IUGR), a common complication of pregnancy. In skeletal muscle, IUGR reduces fetal myofibril size, reduces myoblast proliferation, and reduces expression of genes in cell cycle regulation clusters. The myocardium is striated like skeletal muscle, and IUGR also reduces cell cycle activity and maturation in cardiomyocytes, despite cardiac output preferentially directed to the coronary circulation. We hypothesized that cardiomyocyte growth restriction would be accompanied by similar changes in cell cycle regulation genes, and would reduce cardiomyocyte cell cycle activity, number, maturity and size. Pregnant ewes were housed in elevated ambient temperatures from ~40 to ~115 days of gestation (dGA) to produce placental insufficiency and IUGR; fetal hearts were studied at ~134 dGA. Hearts were biopsied for mRNA analysis and then dissociated into individual myocytes (Control n=8; IUGR n=15), or dissected (Control n=9; IUGR n=13). IUGR fetuses had low circulating insulin and insulin-like growth factor-1 (IGF-1), and high circulating cortisol. Bodies and hearts of IUGR fetuses were lighter than Controls. Cardiomyocytes of IUGR fetuses were smaller, less mature, less active in the cell cycle, and less numerous than Controls. Further, there was a pattern of down-regulation of cell cycle genes in IUGR ventricles. IUGR growth profiles in heart and skeletal muscle suggest similar regulation despite differences in blood and nutrient delivery prioritization. IGF-1 signaling is suggested as a mechanism regulating altered growth in IUGR striated muscle and a potential therapeutic candidate.
Intrauterine growth restriction (IUGR) increases the risk of ischaemic heart disease in adulthood. Studies in rats suggest cardiac vulnerability is more pronounced in males and in offspring that were exposed to hypoxia in utero. Therefore, we aimed to test the hypotheses that 1) IUGR adolescent males, but not females, have fewer cardiomyocytes and altered expression of cardiometabolic genes compared to Controls and 2) IUGR due to hypoxia has a greater effect on these parameters compared to IUGR due to nutrient restriction. IUGR was induced in guinea pigs by Maternal Hypoxia (MH, 10% O2, n=9), or Maternal Nutrient Restriction (MNR, ~30% reduction in food intake, n=9) in the second half of pregnancy and compared to Control (n=11). At 120 days of age, post-mortem was performed, and the left ventricle perfusion fixed for stereological determination of cardiomyocyte number or snap frozen to determine the abundance of cardiometabolic genes and proteins by qRT-PCR and Western Blotting, respectively. MH reduced the number of cardiomyocytes in female (P<0.05), but not male or MNR, adolescent offspring. Furthermore, IUGR males had decreased expression of genes responsible for fatty acid activation in the sarcoplasm (FACS) and transport into the mitochondria (AMPKa2 and ACC; P<0.05) and females exposed to MH had increased activation/phosphorylation of AMPK (P<0.05). We postulate that the changes in cardiomyocyte endowment and cardiac gene expression observed in the present study are a direct result of in utero programming as offspring at this age did not suffer from obesity, hypertension or left ventricular hypertrophy.
Significance
Engineered cardiac muscle can be used to promote the structural and functional maturation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). However, previous studies have not yet produced cardiac tissues with metabolic and proliferative maturation. Here, we develop a 96-well screening platform and screen for cardiac maturation conditions in engineered cardiac muscle. We found that simulating the postnatal switch in metabolic substrates from carbohydrates to fatty acids promoted a switch in metabolism, DNA damage response, and cell cycle arrest in hPSC-CM. Our study shows that this mechanism can be harnessed to enhance the maturation of human hPSC-CM and cardiac tissues, which has major implications for stem cell sciences, drug discovery, and regenerative medicine.
Poor maternal nutrition causes intrauterine growth restriction (IUGR); however, its effects on fetal cardiac development are unclear. We have developed a baboon model of moderate maternal undernutrition, leading to IUGR. We hypothesized that IUGR affects fetal cardiac structure and metabolism. Six control pregnant baboons ate ad-libitum (CTRL)) or 70% CTRL from 0.16 of gestation (G). Fetuses were euthanized at C-section at 0.9G under general anesthesia. Male but not female IUGR fetuses showed left ventricular fibrosis inversely correlated with birth weight. Expression of extracellular matrix protein TSP-1 was increased (p < 0.05) in male IUGR s. Expression of cardiac fibrotic markers TGFβ, SMAD3 and ALK-1 were downregulated in male IUGRs with no difference in females. Autophagy was present in male IUGR evidenced by upregulation of ATG7 expression and lipidation LC3B. Global miRNA expression profiling revealed 56 annotated and novel cardiac miRNAs exclusively dysregulated in female IUGR, and 38 cardiac miRNAs were exclusively dysregulated in males (p < 0.05). Fifteen (CTRL) and 23 (IUGR) miRNAs, were differentially expressed between males and. Females (p < 0.05) suggesting sexual dimorphism, which can be at least partially explained by differential expression of upstream transcription factors (e.g. HNF4α, and NFκB p50). Lipidomics analysis of fetal cardiac tissue exhibited a net increase in diacylglycerol and plasmalogens and a decrease in triglycerides and phosphatidylcholines. In summary, IUGR resulting from decreased maternal nutrition is associated with sex-dependent dysregulations in cardiac structure, miRNA expression, and lipid metabolism. If these changes persist postnatally, they may program offspring for higher later life cardiac risk.
Unlike other visceral organs, myocardial weight is maintained in relation to fetal body weight in intrauterine growth restriction (IUGR) fetal sheep despite hypoinsulinemia and global nutrient restriction. We designed experiments in fetal sheep with placental insufficiency and restricted growth to determine basal and insulin-stimulated myocardial glucose and oxygen metabolism and test the hypothesis that myocardial insulin sensitivity would be increased in the IUGR heart. IUGR was induced by maternal hyperthermia during gestation. Control (C) and IUGR fetal myocardial metabolism were measured at baseline and under acute hyperinsulinemic/euglycemic clamp conditions at 128-132 days gestation using fluorescent microspheres to determine myocardial blood flow. Fetal body and heart weights were reduced by 33% (P = 0.008) and 30% (P = 0.027), respectively. Heart weight to body weight ratios were not different. Basal left ventricular (LV) myocardial blood flow per gram of LV tissue was maintained in IUGR fetuses compared to controls. Insulin increased LV myocardial blood flow by ∼38% (P < 0.01), but insulin-stimulated LV myocardial blood flow in IUGR fetuses was 73% greater than controls. Similar to previous reports testing acute hypoxia, LV blood flow was inversely related to arterial oxygen concentration (r(2 )= 0.71) in both control and IUGR animals. Basal LV myocardial glucose delivery and uptake rates were not different between IUGR and control fetuses. Insulin increased LV myocardial glucose delivery (by 40%) and uptake (by 78%) (P < 0.01), but to a greater extent in the IUGR fetuses compared to controls. During basal and hyperinsulinemic-euglycemic clamp conditions LV myocardial oxygen delivery, oxygen uptake, and oxygen extraction efficiency were not different between groups. These novel results demonstrate that the fetal heart exposed to nutrient and oxygen deprivation from placental insufficiency appears to maintain myocardial energy supply in the IUGR condition via increased glucose uptake and metabolic response to insulin, which support myocardial function and growth.
Background:
Heart failure (HF) is characterized by perturbations in energy homeostasis and metabolism. The reversibility and prognostic value of circulating markers associated with these changes remain unclear.
Objectives:
This study sought to describe the metabolomic profiles of patients along the spectrum of systolic HF, determine their association with adverse outcomes in a clinical trial of HF, and evaluate whether identified metabolites change with treatment for end-stage systolic HF.
Methods:
To assess association of metabolites with clinical outcomes, we evaluated a population of 453 chronic systolic HF patients who had been randomized to exercise training versus usual care. To assess change in metabolites with mechanical circulatory support, 41 patients with end-stage HF who underwent left ventricular assist device (LVAD) placement were studied. Targeted, quantitative profiling of 60 metabolites using tandem flow injection mass spectrometry was performed on frozen plasma samples obtained prior to randomization, as well as prior to and ≥90 days post-placement in the LVAD group. Principal components analysis was used for data reduction.
Results:
Five principal components analysis-derived factors were significantly associated with peak Vo2 levels at baseline in fully adjusted models. Of these, factor 5 (composed of long-chain acylcarnitines) was associated with increased risk of all 3 pre-specified clinical trial outcomes: all-cause mortality/all-cause hospitalization, all cause-hospitalization, and cardiovascular death or cardiovascular hospitalization. Individual components of factor 5 were significantly higher in patients with end-stage HF prior to LVAD placement and decreased significantly post-implantation.
Conclusions:
In chronic HF patients, circulating long-chain acylcarnitine metabolite levels were independently associated with adverse clinical outcomes and decreased after long-term mechanical circulatory support. These metabolites may serve as potential targets for new diagnostics or therapeutic interventions. (Exercise Training Program to Improve Clinical Outcomes in Individuals With Congestive Heart Failure; NCT00047437).
Intrauterine growth restricted (IUGR) fetal sheep, produced by placental insufficiency, have lower oxygen concentrations, higher lactate concentrations, and increased hepatic glucose production that is resistant to suppression by insulin. We hypothesized that increased lactate production in the IUGR fetus results from reduced glucose oxidation, during basal and maximal insulin-stimulated conditions, and is used to support glucose production. To test this, studies were performed in late gestation control (CON) and IUGR fetal sheep under basal and hyperinsulinemic-clamp conditions. The basal glucose oxidation rate was similar and increased by 30-40% during insulin-clamp in CON and IUGR fetuses (P<0.005). However, the fraction of glucose oxidized was 15% lower in IUGR fetuses during basal and insulin-clamp periods (P=0.05). IUGR fetuses also had 4-fold higher lactate concentrations (P<0.001) and lower lactate uptake rates (P<0.05). In IUGR fetal muscle and liver, mRNA expression of pyruvate dehydrogenase kinase (PDK4), and inhibitor of glucose oxidation, was increased over 4-fold and protein expression was increased by 50% in muscle and by 2-fold in liver. In IUGR fetal liver, but not skeletal muscle, mRNA expression of lactate dehydrogenase (LDHA) was increased nearly 5-fold. Hepatic expression of the gluconeogenic genes, phosphoenolpyruvate carboxykinase (PCK)1 and PCK2, was correlated with expression of PDK4 and LDHA. Collectively, these in vivo and tissue data support limited capacity for glucose oxidation in the IUGR fetus via increased PDK4 in skeletal muscle and liver. We speculate that lactate production also is increased, which may supply carbon for glucose production in the IUGR fetal liver.
Copyright © 2015, American Journal of Physiology - Regulatory, Integrative and Comparative Physiology.
Studies in altricial rodents attribute dramatic changes in perinatal cardiomyocyte growth, maturation, and attrition to stimuli associated with birth. Our purpose was to determine whether birth is a critical trigger controlling perinatal cardiomyocyte growth, maturation and attrition in a precocial large mammal, sheep (Ovis aries). Hearts from 0-61 d postnatal lambs were dissected or enzymatically dissociated. Cardiomyocytes were measured by micromorphometry, cell cycle activity assessed by immunohistochemistry, and nuclear number counted after DNA staining. Integration of this new data with published fetal data from our laboratory demonstrate that a newly appreciated >30% decrease in myocyte number occurred in the last 10 d of gestation (P < 0.0005) concomitant with an increase in cleaved poly (ADP-ribose) polymerase 1 (P < 0.05), indicative of apoptosis. Bisegmental linear regressions show that most changes in myocyte growth kinetics occur before birth (median = 15.2 d; P < 0.05). Right ventricular but not left ventricular cell number increases in the neonate, by 68% between birth and 60 d postnatal (P = 0.028). We conclude that in sheep few developmental changes in cardiomyocytes result from birth, excepting the different postnatal degrees of free wall hypertrophy between the ventricles. Furthermore, myocyte number is reduced in both ventricles immediately before term, but proliferation increases myocyte number in the neonatal right ventricle.-Jonker, S. S., Louey, S., Giraud, G. D., Thornburg, K. L., Faber, J. J. Timing of cardiomyocyte growth, maturation, and attrition in perinatal sheep.
© FASEB.
Fatty acids (FAs) provide cellular energy under starvation, yet how they mobilize and move into mitochondria in starved cells, driving oxidative respiration, is unclear. Here, we clarify this process by visualizing FA trafficking with a fluorescent FA probe. The labeled FA accumulated in lipid droplets (LDs) in well-fed cells but moved from LDs into mitochondria when cells were starved. Autophagy in starved cells replenished LDs with FAs, increasing LD number over time. Cytoplasmic lipases removed FAs from LDs, enabling their transfer into mitochondria. This required mitochondria to be highly fused and localized near LDs. When mitochondrial fusion was prevented in starved cells, FAs neither homogeneously distributed within mitochondria nor became efficiently metabolized. Instead, FAs reassociated with LDs and fluxed into neighboring cells. Thus, FAs engage in complex trafficking itineraries regulated by cytoplasmic lipases, autophagy, and mitochondrial fusion dynamics, ensuring maximum oxidative metabolism and avoidance of FA toxicity in starved cells.
Copyright © 2015 Elsevier Inc. All rights reserved.
In the fetus, there is a redistribution of cardiac output in response to acute hypoxemia, to maintain perfusion of key organs including the brain, heart and adrenal glands. There may be a similar redistribution of cardiac output in the chronically hypoxemic, intrauterine growth restricted (IUGR) fetus. Surgical removal of uterine caruncles in non-pregnant ewe results in the restriction of placental growth (PR) and IUGR. Vascular catheters were implanted in 7 Control and 6 PR fetal sheep and blood flow to organs was determined using microspheres. Placental and fetal weight was significantly reduced in the PR group. Despite an increase in the relative brain weight in the PR group, there was no difference in blood flow to the brain between the groups, although PR fetuses had higher blood flow to the temporal lobe. Adrenal blood flow was significantly higher in PR fetuses and there was a direct relationship between mean gestational PaO2 and blood flow to the adrenal gland. There was no change in blood flow, but a decrease in oxygen and glucose delivery to the heart in the PR fetuses. In another group, there was a decrease in femoral artery blood flow in PR compared to Controls and this may support blood flow changes to the adrenal and temporal lobe. In contrast to the response to acute hypoxemia, these data show that there is a redistribution of blood flow to the adrenals and temporal lobe, but not the heart or whole brain, in chronically hypoxemic PR fetuses in late gestation.
Copyright © 2014, American Journal of Physiology - Regulatory, Integrative and Comparative Physiology.
Cellular energy homeostasis is a crucial function of oxidative tissues but becomes altered with obesity, a major health problem that is rising unabated and demands attention. Maintaining cardiac lipid homeostasis relies on complex processes and pathways that require concerted actions between lipid droplets (LDs) and mitochondria to prevent intracellular accumulation of bioactive or toxic lipids while providing an efficient supply of lipid for conversion into ATP. While cardiac mitochondria have been extensively studied, cardiac LDs and their role in heart function have not been fully characterized. The cardiac LD compartment is highly dynamic and individual LD is small, making their study challenging. Here, we describe a simple procedure to isolate cardiac LDs that provide sufficient amounts of highly enriched material to allow subsequent protein and lipid biochemical characterization. We also present a detailed protocol to image cardiac LDs by conventional transmission electronic microscopy to provide two-dimensional (2D) analyses of cardiac LDs and mitochondria. Finally, we discuss the potential advantages of dual ion beam and electron beam platform (FIB-SEM) technology to study the cardiac LDs and mitochondria by allowing 3D imaging analysis.
Intrauterine growth restriction that results in low birth weight (LBW) has been linked to the onset of pathological cardiac hypertrophy. An altered transition from a fetal to an adult energy metabolism phenotype, with increased reliance on glucose rather than fatty acids for energy production, could help explain this connection. We have therefore investigated cardiac metabolism in relation to left ventricular hypertrophy in LBW lambs, at 21days after birth.
The expression of regulatory molecules involved in cardiac glucose and fatty acid metabolism was measured using real-time PCR and Western blotting. A section of the left ventricle was fixed for Periodic Acid Schiff staining to determine tissue glycogen content.
There was increased abundance of insulin signalling pathway proteins (phospho-insulin receptor, insulin receptor and phospho-Akt) and the glucose transporter (GLUT)-1, but no change in GLUT-4 or glycogen content in the heart of LBW compared to ABW lambs. There was, however, increased abundance of cardiac pyruvate dehydrogenase kinase 4 (PDK-4) in LBW compared to ABW lambs. There were no significant changes in the mRNA expression of components of the peroxisome proliferator activated receptor regulatory complex or proteins involved in fatty acid metabolism.
We concluded that LBW induced left ventricular hypertrophy was associated with increased GLUT-1 and PDK-4, suggesting increased glucose uptake, but decreased efficacy for the conversion of glucose to ATP. A reduced capacity for energy conversion could have significant implications for vulnerability to cardiovascular disease in adults who are born LBW.
The endoplasmic reticulum (ER) is essential for lipid biosynthesis, and stress signals in this organelle are thought to alter lipid metabolism. Elucidating the mechanisms that underlie the dysregulation of lipid metabolism in hepatocytes may lead to novel therapeutic approaches for the treatment of lipid accumulation. We first tested the effects of several inhibitors on lipid dysregulation induced by tunicamycin, an ER stress inducer. Triacsin C, an inhibitor of long-chain acyl-CoA synthetase (ACSL) 1, 3, and 4, was the most potent among these inhibitors. We then analyzed the expression of the ACSL family during ER stress. The expression of ACSL3 was induced by ER stress in HuH-7 cells and in mice livers. ACSL3 shRNA, but not ACSL1 shRNA, inhibited the induction of lipid accumulation. GSK-3β inhibitors attenuated ACSL3 expression and the lipid accumulation induced by ER stress in HuH-7 cells. shRNA that target GSK-3β also inhibited the upregulation of ACSL3 and lipid accumulation in HuH-7 and HepG2 cells. The hepatitis B virus mutant large surface protein, which is known to induce ER stress, increased the lipid content of cells. Similarly, Triacsin C, and GSK-3β inhibitors abrogated the lipid dysregulation caused by the hepatitis B virus mutant large surface protein. Altogether, ACSL3 and GSK-3β represent novel therapeutic targets for lipid dysregulation by ER stress.
Dramatic maturational changes occur in cardiac energy metabolism during cardiac development, differentiation, and postnatal growth. These changes in energy metabolism have important impacts on the ability of the cardiomyocyte to proliferate during early cardiac development, as well as when cardiomyocytes terminally differentiate during later development. During early cardiac development, glycolysis is a major source of energy for proliferating cardiomyocytes. As cardiomyocytes mature and become terminally differentiated, mitochondrial oxidative capacity increases, with fatty acid beta-oxidation becoming a major source of energy for the heart. The increase in mitochondrial oxidative capacity seems to coincide with a decrease in the proliferative ability of the cardiomyocyte. The switch from glycolysis to mitochondrial oxidative metabolism during cardiac development includes both alterations in the transcriptional control and acute alterations in the control of each pathway. Interestingly, if a hypertrophic stress is placed on the adult heart, cardiac energy metabolism switches to a more fetal phenotype, which includes an increase in glycolysis and decrease in mitochondrial fatty acid beta-oxidation. In this article, we review the impact of alterations in energy substrate metabolism on cardiomyocyte proliferation, differentiation, and postnatal maturation.
Carnitine plays an essential role in fatty acid metabolism, as well as modulation of intracellular concentrations of free coenzyme A by esterification of acyl residues. Acylcarnitine analysis of various biological fluids is a sensitive method to detect >20 inborn errors of metabolism that result in abnormal accumulation of acylcarnitine species due to several organic acidemias and most fatty acid beta-oxidation disorders. In addition, acylcarnitine analysis may aid in monitoring treatment of known patients affected with these inborn errors of metabolism. This unit describes protocols that can be used to measure acylcarnitine species of various carbon chain lengths in several biological specimen types including plasma, dried blood and bile spots, and urine, by derivatization to butylesters and flow-injection electrospray ionization tandem mass spectrometry (ESI-MS/MS).
Chronic anaemia increases the workload of the growing fetal heart, leading to cardiac enlargement. To determine which cellular process increases cardiac mass, we measured cardiomyocyte sizes, binucleation as an index of terminal differentiation, and tissue volume fractions in hearts from control and anaemic fetal sheep. Fourteen chronically catheterized fetal sheep at 129 days gestation had blood withdrawn for 9 days to cause severe anaemia; 14 control fetuses were of similar age. At postmortem examination, hearts were either enzymatically dissociated or fixed for morphometric analysis. Daily isovolumetric haemorrhage reduced fetal haematocrit from a baseline value of 35% to 15% on the final day (P < 0.001). At the study conclusion, anaemic fetuses had lower arterial pressures than control fetuses (P < 0.05). Heart weights were increased by 39% in anaemic fetuses compared with control hearts (P < 0.0001), although the groups had similar body weights; the heart weight difference was not due to increased ventricular wall water content or disproportionate non-myocyte tissue expansion. Cardiomyocytes from anaemic fetuses tended to be larger than those of control fetuses. There were no statistically significant differences between groups in the cardiomyocyte cell cycle activity. The degree of terminal differentiation was greater in the right ventricle of anaemic compared with control fetuses by 8% (P < 0.05). Anaemia substantially increased heart weight in fetal sheep. The volume proportions of connective and vascular tissue were unchanged. Cardiomyocyte mass expanded by a balanced combination of cellular enlargement, increased terminal differentiation and accelerated proliferation.
To determine the capacity of the fetus to adapt to chronic O2 deficiency produced by decreased placental perfusion in the early development of growth retardation, we embolized the umbilical placental vascular bed of fetal sheep for a period of 9 days. Fetal umbilical placental embolization decreased arterial O2 content by 39%, decreased total placental blood flow by 33%, and produced a 20% reduction in mean fetal body weight. Neither the combined ventricular output nor the regional blood flow distribution was significantly different between the 8 growth-retarded and 7 normally grown fetuses despite the 39% decrease in fetal arterial O2 content. Thus a 33% reduction in total placental blood flow restricts normal fetal growth, but does not exceed the placental circulatory reserve capacity necessary to maintain normal basal metabolic oxygenation. Because the proportion of combined ventricular output to the placenta at rest is decreased in late IUGR fetuses but not in early IUGR fetuses, despite chronic oxygen deficiency, we conclude that the growth retarded fetus maintains a normal regional blood flow distribution until the placental circulatory reserve capacity is depleted.
Palmitate oxidation and the effect of palmitate on glucose and lactate utilization were investigated in isolated, perfused, fetal (0.9 gestation), and neonatal (2 day old) pig hearts. Hearts were perfused under working conditions, developing a mean aortic pressure of 50-55 mmHg, paced at 180 beats/min for 30 min, with Krebs-Henseleit buffer containing 3% albumin, glucose (5 mM), and insulin (100 microU/ml). Palmitate (1 mM) and lactate (5 mM), either individually or in combination, were added to the perfusion buffer. Palmitate oxidation was assessed from 14CO2 production from [U-14C]-palmitate, glucose uptake as 3H2O production from D-[2-3H]-glucose, and lactate metabolism from changes in buffer lactate content. After perfusion, ATP, creatine phosphate, triglycerides, and glycogen were measured. Substantial palmitate oxidation was observed at both ages but was greater in neonatal hearts. Nevertheless, palmitate inhibited lactate utilization and glucose uptake similarly in fetal and neonatal hearts. Lactate also reduced palmitate uptake and oxidation by 40-60% in both fetal and neonatal hearts. During perfusions with palmitate, tissue concentrations of triglycerides increased approximately threefold in fetal hearts and were unaffected by lactate. Thus both palmitate and lactate can act as major energy substrates for the immature heart. Both substrates significantly (P less than 0.01) suppress glucose utilization, and each has suppressive effects on the other's metabolism.
The role of lactate as an energy substrate in fetal (0.9 gestation) and newborn (2 day old) hearts was investigated in isolated, perfused hearts. Perfusions were performed with Krebs-Henseleit buffer supplemented with glucose (5 mM) in combination with varying concentrations of lactate. Isolated working heart perfusions, in which the heart ejects the buffer at controlled pressure, were carried out with glucose (5 mM) alone and with glucose (5 mM) and lactate (5 mM) combined. With glucose as sole substrate, lactate was produced by the heart and glucose uptake accounted for approximately two-thirds of oxygen consumption. When both glucose and lactate were provided, lactate accounted for more than 80% of oxygen consumption and profoundly suppressed glucose uptake. Further investigations using retrograde perfusion through the aorta demonstrated that lactate uptake was consistently observed when exogenous lactate concentrations exceeded 1.25 mM. Glucose uptake was suppressed with lactate concentrations as low as 0.5 mM and progressive suppression occurred with increasing lactate concentrations. Fetal and newborn pig hearts utilize lactate as a primary substrate for energy production when lactate concentrations are in the physiological range.
Circulatory responses to hypoxemia and acidemia were studied in 10 fetal lambs, in utero with gestational ages of 122 to 142 days. Vinyl catheters were placed in fetal and maternal vessels, and the fetuses were studied 2 to 5 days postoperatively. Fetal heart rate, arterial pressure, P(O2), P(CO2), and pH were measured during a control period, and while the standing ewe breathed 6% oxygen and 3% carbon dioxide, through a plastic bag over its head. Fetal cardiac output and distribution and absolute organ blood flows were calculated from injections of 15μ nuclide labeled microspheres, during the control and hypoxemic states. One group of 5 fetuses only became hypoxemic (mean P(O2) 12, and mean pH 7.36), but the other 5 fetuses also developed acidemia (mean P(O2) 12, and mean pH 7.28). Fetal arterial P(CO2) values were normal throughout. During hypoxemia fetal arterial pressure increased, and fetal heart rate decreased. Although cardiac output fell in all but one fetus, the decrease was significant only in the acidemic group. Blood flow to the fetal body decrease in all, but the change was significantly greater in the acidemic group. Umbilical blood flow was maintained in all fetuses during hypoxemia. The percentage distribution of cardiac output to the placenta rose from 41 to 48% and from 41 to 57% in the hypoxemic and acidemic groups, respectively. Blood flow to the brain, heart, and adrenals increased 2 to 3 fold in all fetuses during hypoxemia while pulmonary, renal, splenic, gut, and carcass flows decreased. The changes were of greater magnitude in fetuses with combined hypoxemia and acidemia. These studies quantitate the fetal circulatory changes that occur in unanesthetized fetal lambs in utero during maternal hypoxemia.
We measured myocardial oxygen, glucose, lactate, and pyruvate consumption in chronically instrumented fetal and adult sheep. Although ascending aortic blood concentration of oxygen was significantly lower in fetuses, myocardial consumption of oxygen was similar in the two groups. This was accomplished by a significantly greater myocardial blood flow in the fetuses. Although ascending aortic blood glucose concentration was significantly lower in fetuses, myocardial consumption of glucose was significantly greater in fetuses. Complete oxidative combustion of all glucose consumed by the fetal heart would supply only one-third of myocardial energy demands, as measured by oxygen consumption. Ascending aortic blood concentration of lactate was similar in fetuses and adults, but myocardial consumption of lactate was significantly greater in fetuses. Complete oxidative combustion of all lactate consumed by fetal hearts would supply almost 60% of myocardial energy demands. Small, but significant, amounts of pyruvate are consumed by both fetuses and adults. Our data indicate that fetal lamb myocardium requires substrates other than glucose alone. The large amount of lactate consumed indicates that there is oxidative metabolism in addition to glycolysis and that lactate is of equal, or perhaps greater, importance as a myocardial energy substrate.
Our goal was to determine the effect of chronic and acute umbilical-placental embolization on placental hemodynamic and fetal heart rate patterns in relation to fetal oxygenation in the near-term ovine fetus.
Daily fetal placental embolization was performed during 10 days in 9 sheep fetuses until fetal arterial oxygen content decreased by approximately 30%. Nine control fetuses received saline solution. Mean and pulsatile umbilical blood flow, perfusion pressure, placental vascular resistance, fundamental impedance, pressure pulsatility index, and umbilical artery resistance index corrected to a fetal heart rate of 160 beats/min were measured. On day 10 both groups were acutely embolized until fetal arterial pH decreased to approximately 7.00. Fetal heart rate was measured with the Sonicaid System 8000 (Oxford Sonicaid, Oxford, United Kingdom).
Chronic fetal placental embolization was associated with a progressive reduction in umbilical blood flow (p < 0.00001) and fetal arterial oxygen content (p < 0.001) whereas fetal heart rate patterns remained unaltered. A chronic increase in umbilical artery resistance index corrected to a fetal heart rate of 160 beats/min could be entirely explained only if the changes in umbilical artery pressure pulsatility index and the fundamental impedance were taken into account, in addition to the changes observed in placental vascular resistance. During acute embolization leading to a 50% reduction in umbilical blood flow (p < 0.0002) and a three times increase in placental vascular resistance (p < 0.0001), the most consistent change in fetal heart rate patterns related to progressive metabolic acidosis was an 84% decrease in absolute acceleration frequency (p < 0.0001) whereas short-term fetal heart rate variability remained unaltered.
Changes in umbilical artery resistance index induced by chronic umbilical-placental embolization resulting in fetal hypoxemia occurred before any changes in fetal heart rate patterns were detectable. A decrease in the absolute acceleration frequency was the only component of fetal heart rate patterns related to progressive metabolic acidosis in the near-term ovine fetus.
While intestinal transport systems for metabolites such as carbohydrates have been well characterized, the molecular mechanisms of fatty acid (FA) transport across the apical plasmalemma of enterocytes have remained largely unclear. Here, we show that FATP4, a member of a large family of FA transport proteins (FATPs), is expressed at high levels on the apical side of mature enterocytes in the small intestine. Further, overexpression of FATP4 in 293 cells facilitates uptake of long chain FAs with the same specificity as enterocytes, while reduction of FATP4 expression in primary enterocytes by antisense oligonucleotides inhibits FA uptake by 50%. This suggests that FATP4 is the principal fatty acid transporter in enterocytes and may constitute a novel target for antiobesity therapy.
Lactate accounts for a third of myocardial oxygen consumption before and in the first 2 weeks after birth. It is unknown how the remainder of myocardial oxygen is consumed. Glucose is thought to be important before birth, whereas long-chain fatty acids (LC-FA) are the prime substrate for the adult. However, the ability of the myocardium of the newborn to use LC-FA has been doubted.
We measured the myocardial metabolism of glucose and LC-FA with [U-(13)C]glucose and [1-(13)C]palmitate in chronically instrumented fetal and newborn lambs. In fetal lambs, myocardial oxidation of glucose was high and that of LC-FA was low. Glucose and LC-FA accounted for 48+/-4% and 2+/-2% of myocardial oxygen consumption, respectively. In newborn lambs, oxidation of glucose decreased, whereas oxidation of LC-FA increased. Glucose and LC-FA accounted for 12+/-3% and 83+/-19% of myocardial oxygen consumption. To test whether near-term fetal lambs could use LC-FA, we increased the supply of LC-FA with a fat infusion. In fetal lambs during fat infusion, the oxidation of LC-FA increased 15-fold. Although the oxidation of LC-FA was still lower than in newborn lambs, the contribution to myocardial oxygen consumption (70+/-13%) was the same as in newborn lambs.
These data show that glucose and lactate account for the majority of myocardial oxygen consumption in fetal lambs, whereas in newborn lambs, LC-FA and lactate account for the majority of myocardial oxygen consumption. Moreover, we showed that the fetal myocardium can use LC-FA as an energy substrate.