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Welcome to OXION, Universities of Oxford, Cambridge, London and MRC Harwell
Inhibition of sarcolemmal FAT/CD36 by sulfo-N-succinimidyl oleate rapidly corrects metabolism and restores function in the diabetic heart following hypoxia/reoxygenation.
Aims: The type 2 diabetic heart oxidizes more fat and less glucose, which can impair metabolic flexibility and function. Increased sarcolemmal fatty acid translocase (FAT/CD36) imports more fatty acid into the diabetic myocardium, feeding increased fatty acid oxidation and elevated lipid deposition. Unlike other metabolic modulators that target mitochondrial fatty acid oxidation, we proposed that pharmacologically inhibiting fatty acid uptake, as the primary step in the pathway, would provide an alternative mechanism to rebalance metabolism and prevent lipid accumulation following hypoxic stress. Methods and results: Hearts from type 2 diabetic and control male Wistar rats were perfused in normoxia, hypoxia and reoxygenation, with the FAT/CD36 inhibitor sulfo-N-succinimidyl oleate (SSO) infused 4 min before hypoxia. SSO infusion into diabetic hearts decreased the fatty acid oxidation rate by 29% and myocardial triglyceride concentration by 48% compared with untreated diabetic hearts, restoring fatty acid metabolism to control levels following hypoxia-reoxygenation. SSO infusion increased the glycolytic rate by 46% in diabetic hearts during hypoxia, increased pyruvate dehydrogenase activity by 53% and decreased lactate efflux rate by 56% compared with untreated diabetic hearts during reoxygenation. In addition, SSO treatment of diabetic hearts increased intermediates within the second span of the Krebs cycle, namely fumarate, oxaloacetate, and the FAD total pool. The cardiac dysfunction in diabetic hearts following decreased oxygen availability was prevented by SSO-infusion prior to the hypoxic stress. Infusing SSO into diabetic hearts increased rate pressure product by 60% during hypoxia and by 32% following reoxygenation, restoring function to control levels. Conclusions: Diabetic hearts have limited metabolic flexibility and cardiac dysfunction when stressed, which can be rapidly rectified by reducing fatty acid uptake with the FAT/CD36 inhibitor, SSO. This novel therapeutic approach not only reduces fat oxidation but also lipotoxicity, by targeting the primary step in the fatty acid metabolism pathway.
The 'Goldilocks zone' of fatty acid metabolism; to ensure that the relationship with cardiac function is just right.
Fatty acids (FA) are the main fuel used by the healthy heart to power contraction, supplying 60-70% of the ATP required. FA generate more ATP per carbon molecule than glucose, but require more oxygen to produce the ATP, making them a more energy dense but less oxygen efficient fuel compared with glucose. The pathways involved in myocardial FA metabolism are regulated at various subcellular levels, and can be divided into sarcolemmal FA uptake, cytosolic activation and storage, mitochondrial uptake and β-oxidation. An understanding of the critical involvement of each of these steps has been amassed from genetic mouse models, where forcing the heart to metabolize too much or too little fat was accompanied by cardiac contractile dysfunction and hypertrophy. In cardiac pathologies, such as heart disease and diabetes, aberrations in FA metabolism occur concomitantly with changes in cardiac function. In heart failure, FA oxidation is decreased, correlating with systolic dysfunction and hypertrophy. In contrast, in type 2 diabetes, FA oxidation and triglyceride storage are increased, and correlate with diastolic dysfunction and insulin resistance. Therefore, too much FA metabolism is as detrimental as too little FA metabolism in these settings. Therapeutic compounds that rebalance FA metabolism may provide a mechanism to improve cardiac function in disease. Just like Goldilocks and her porridge, the heart needs to maintain FA metabolism in a zone that is 'just right' to support contractile function.
Fatty Acids Prevent Hypoxia-Inducible Factor-1α Signaling Through Decreased Succinate in Diabetes.
Hypoxia-inducible factor (HIF)-1α is essential following a myocardial infarction (MI), and diabetic patients have poorer prognosis post-MI. Could HIF-1α activation be abnormal in the diabetic heart, and could metabolism be causing this? Diabetic hearts had decreased HIF-1α protein following ischemia, and insulin-resistant cardiomyocytes had decreased HIF-1α-mediated signaling and adaptation to hypoxia. This was due to elevated fatty acid (FA) metabolism preventing HIF-1α protein stabilization. FAs exerted their effect by decreasing succinate concentrations, a HIF-1α activator that inhibits the regulatory HIF hydroxylase enzymes. In vivo and in vitro pharmacological HIF hydroxylase inhibition restored HIF-1α accumulation and improved post-ischemic functional recovery in diabetes.
Dysregulation of hypoxia pathways in fumarate hydratase-deficient cells is independent of defective mitochondrial metabolism.
Mutations in the gene encoding the Krebs cycle enzyme fumarate hydratase (FH) predispose to hereditary leiomyomatosis and renal cell cancer in affected individuals. FH-associated neoplasia is characterized by defective mitochondrial function and by upregulation of transcriptional pathways mediated by hypoxia-inducible factor (HIF), although whether and by what means these processes are linked has been disputed. We analysed the HIF pathway in Fh1-/- mouse embryonic fibroblasts (MEFs), in FH-defective neoplastic tissues and in Fh1-/- MEFs re-expressing either wild-type or an extra-mitochondrial restricted form of FH. These experiments demonstrated that upregulation of HIF-1alpha occurs as a direct consequence of FH inactivation. Fh1-/- cells accumulated intracellular fumarate and manifested severe impairment of HIF prolyl but not asparaginyl hydroxylation which was corrected by provision of exogenous 2-oxoglutarate (2-OG). Re-expression of the extra-mitochondrial form of FH in Fh1-/- cells was sufficient to reduce intracellular fumarate and to correct dysregulation of the HIF pathway completely, even in cells that remained profoundly defective in mitochondrial energy metabolism. The findings indicate that upregulation of HIF-1alpha arises from competitive inhibition of the 2-OG-dependent HIF hydroxylases by fumarate and not from disruption of mitochondrial energy metabolism.
Critical role of complex III in the early metabolic changes following myocardial infarction.
AIMS: The chronically infarcted rat heart has multiple defects in metabolism, yet the location of the primary metabolic abnormality arising after myocardial infarction is unknown. Therefore, we investigated cardiac mitochondrial metabolism shortly after infarction. METHODS AND RESULTS: Myocardial infarctions (n = 11) and sham operations (n = 9) were performed on Wistar rats, at 2 weeks cardiac function was assessed using echocardiography, and rats were grouped into failing (ejection fraction < or =45%), moderately impaired (46-60%), and sham-operated (>60%). Respiration rates were decreased by 28% in both subsarcolemmal and interfibrillar mitochondria isolated from failing hearts, compared with sham-operated controls. However, respiration rates were not impaired in mitochondria from hearts with moderately impaired function. The mitochondrial defect in the failing hearts was located within the electron transport chain (ETC), as respiration rates were suppressed to the same extent when fatty acids, ketone bodies, or glutamate were used as substrates. Complex III protein levels were decreased by 46% and complex III activity was decreased by 26%, in mitochondria from failing hearts, but all other ETC complexes were unchanged. Decreased complex III activity was accompanied by a three-fold increase in complex III-derived H(2)O(2) production, decreased cardiolipin content, and a 60% decrease in mitochondrial cytochrome c levels from failing hearts. Respiration rates, complex III activity, cardiolipin content, and reactive oxygen species generation rates correlated with ejection fraction. CONCLUSION: In conclusion, a specific defect in complex III occurred acutely after myocardial infarction, which increased oxidative damage and impaired mitochondrial respiration. The extent of mitochondrial dysfunction in the failing heart was proportional to the degree of cardiac dysfunction induced by myocardial infarction.
In vivo alterations in cardiac metabolism and function in the spontaneously hypertensive rat heart.
AIMS: The aim of this work was to use hyperpolarized carbon-13 ((13)C) magnetic resonance (MR) spectroscopy and cine MR imaging (MRI) to assess in vivo cardiac metabolism and function in the 15-week-old spontaneously hypertensive rat (SHR) heart. At this time point, the SHR displays hypertension and concentric hypertrophy. One of the cellular adaptations to hypertrophy is a reduction in β-oxidation, and it has previously been shown that in response to hypertrophy the SHR heart switches to a glycolytic/glucose-oxidative phenotype. METHODS AND RESULTS: Cine-MRI (magnetic resonance imaging) was used to assess cardiac function and degree of cardiac hypertrophy. Wistar rats were used as controls. SHRs displayed functional changes in stroke volume, heart rate, and late peak-diastolic filling alongside significant hypertrophy (a 56% increase in left ventricular mass). Using hyperpolarized [1-(13)C] and [2-(13)C]pyruvate, an 85% increase in (13)C label flux through pyruvate dehydrogenase (PDH) was seen in the SHR heart and (13)C label incorporation into citrate, acetylcarnitine, and glutamate pools was elevated in proportion to the increase in PDH flux. These findings were confirmed using biochemical analysis of PDH activity and protein expression of PDH regulatory enzymes. CONCLUSIONS: Functional and structural alterations in the SHR heart are consistent with the hypertrophied phenotype. Our in vivo work indicates a preference for glucose metabolism in the SHR heart, a move away from predominantly fatty acid oxidative metabolism. Interestingly, (13)C label flux into lactate was unchanged, indicating no switch to an anaerobic glycolytic phenotype, but rather an increased reliance on glucose oxidation in the SHR heart.
Metabolic adaptation to chronic hypoxia in cardiac mitochondria.
Chronic hypoxia decreases cardiomyocyte respiration, yet the mitochondrial mechanisms remain largely unknown. We investigated the mitochondrial metabolic pathways and enzymes that were decreased following in vivo hypoxia, and questioned whether hypoxic adaptation was protective for the mitochondria. Wistar rats were housed in hypoxia (7 days acclimatisation and 14 days at 11% oxygen), while control rats were housed in normoxia. Chronic exposure to physiological hypoxia increased haematocrit and cardiac vascular endothelial growth factor, in the absence of weight loss and changes in cardiac mass. In both subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria isolated from hypoxic hearts, state 3 respiration rates with fatty acid were decreased by 17-18%, and with pyruvate were decreased by 29-15%, respectively. State 3 respiration rates with electron transport chain (ETC) substrates were decreased only in hypoxic SSM, not in hypoxic IFM. SSM from hypoxic hearts had decreased activities of ETC complexes I, II and IV, which were associated with decreased reactive oxygen species generation and protection against mitochondrial permeability transition pore (MPTP) opening. In contrast, IFM from hypoxic hearts had decreased activity of the Krebs cycle enzyme, aconitase, which did not modify ROS production or MPTP opening. In conclusion, cardiac mitochondrial respiration was decreased following chronic hypoxia, associated with downregulation of different pathways in the two mitochondrial populations, determined by their subcellular location. Hypoxic adaptation was not deleterious for the mitochondria, in fact, SSM acquired increased protection against oxidative damage under the oxygen-limited conditions.
Fatty acid transporter levels and palmitate oxidation rate correlate with ejection fraction in the infarcted rat heart.
OBJECTIVES: Cardiac fatty acid uptake occurs predominantly via sarcolemmal transporter proteins; fatty acid translocase (FAT/CD36), plasma membrane fatty acid binding protein (FABPpm) and fatty acid transporter proteins (FATP) 1 and 6. We hypothesised that levels of the fatty acid transporters would be reduced in the chronically infarcted rat heart, in parallel with reduced dependence on fatty acid utilisation. METHODS AND RESULTS: In vivo left ventricular ejection fractions, measured using echocardiography, were 36% lower in rats six months after coronary artery ligation than in sham-operated control rats. In isolated, perfused, infarcted hearts, 3H-palmitate oxidation was 30% lower, and correlated with in vivo ejection fractions. As myocardial lipid incorporation was also reduced by 25%, total palmitate utilisation was 29% lower in the infarcted rat heart. The protein levels of the cardiac fatty acid transporters were reduced in the infarcted rat heart; FAT/CD36 by 36%, FABPpm by 12%, FATP6 by 21% and FATP1 by 26%, and the cytosolic fatty acid binding protein (cFABP) was 47% lower than in sham-operated rat hearts. Fatty acid transporter levels correlated with both palmitate oxidation rates and cardiac ejection fractions. CONCLUSIONS: Reductions in fatty acid oxidation and lipid incorporation rates were accompanied by downregulation of the cardiac fatty acid transporters. The metabolic shift away from fatty acid utilisation was proportional to the degree of functional impairment in the chronically infarcted rat heart.
Fruitless isoforms and target genes specify the sexually dimorphic nervous system underlying Drosophila reproductive behavior.
Courtship is pivotal to successful reproduction throughout the animal kingdom. Sexual differences in the nervous system are thought to underlie courtship behavior. Male courtship behavior in Drosophila is in large part regulated by the gene fruitless (fru). fru has been reported to encode at least three putative BTB-zinc-finger transcription factors predicted to have different DNA-binding specificities. Although a large number of previous studies have demonstrated that fru plays essential roles in male courtship behavior, we know little about the function of Fru isoforms at the molecular level. Our recent study revealed that male-specific Fru isoforms are expressed in highly overlapping subsets of neurons in the male brain and ventral nerve cord. Fru isoforms play both distinct and redundant roles in male courtship behavior. Importantly, we have identified for the first time, by means of the DamID technique, direct Fru transcriptional target genes. Fru target genes overwhelmingly represent genes previously reported to be involved in the nervous system development, such as CadN, lola and pdm2. Our study provides important insight into how the sexually dimorphic neural circuits underlying reproductive behavior are established.
Molecular Mechanisms of Sexually Dimorphic Nervous System Patterning in Flies and Worms.
Male and female brains display anatomical and functional differences. Such differences are observed in species across the animal kingdom, including humans, but have been particularly well-studied in two classic animal model systems, the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans. Here we summarize recent advances in understanding how the worm and fly brain acquire sexually dimorphic features during development. We highlight the advantages of each system, illustrating how the precise anatomical delineation of sexual dimorphisms in worms has enabled recent analysis into how these dimorphisms become specified during development, and how focusing on sexually dimorphic neurons in the fly has enabled an increasingly detailed understanding of sex-specific behaviors.
Prolonged systemic hyperglycemia does not cause pericyte loss and permeability at the mouse blood-brain barrier.
Diabetes mellitus is associated with cognitive impairment and various central nervous system pathologies such as stroke, vascular dementia, or Alzheimer's disease. The exact pathophysiology of these conditions is poorly understood. Recent reports suggest that hyperglycemia causes cerebral microcirculation pathology and blood-brain barrier (BBB) dysfunction and leakage. The majority of these reports, however, are based on methods including in vitro BBB modeling or streptozotocin-induced diabetes in rodents, opening questions regarding the translation of the in vitro findings to the in vivo situation, and possible direct effects of streptozotocin on the brain vasculature. Here we used a genetic mouse model of hyperglycemia (Ins2AKITA) to address whether prolonged systemic hyperglycemia induces BBB dysfunction and leakage. We applied a variety of methodologies to carefully evaluate BBB function and cellular integrity in vivo, including the quantification and visualization of specific tracers and evaluation of transcriptional and morphological changes in the BBB and its supporting cellular components. These experiments did neither reveal altered BBB permeability nor morphological changes of the brain vasculature in hyperglycemic mice. We conclude that prolonged hyperglycemia does not lead to BBB dysfunction, and thus the cognitive impairment observed in diabetes may have other causes.
Adult-induced genetic ablation distinguishes PDGFB roles in blood-brain barrier maintenance and development
Platelet-derived growth factor B (PDGFB) released from endothelial cells is indispensable for pericyte recruitment during angiogenesis in embryonic and postnatal organ growth. Constitutive genetic loss-of-function of PDGFB leads to pericyte hypoplasia and the formation of a sparse, dilated and venous-shifted brain microvasculature with dysfunctional blood-brain barrier (BBB) in mice, as well as the formation of microvascular calcification in both mice and humans. Endothelial PDGFB is also expressed in the adult quiescent microvasculature, but here its importance is unknown. We show that deletion of Pdgfb in endothelial cells in 2-months-old mice causes a slowly progressing pericyte loss leading, at 12–18 months of age, to ≈50% decrease in endothelial:pericyte cell ratio, ≈60% decrease in pericyte longitudinal capillary coverage and >70% decrease in pericyte marker expression. Similar to constitutive loss of Pdgfb, this correlates with increased BBB permeability. However, in contrast to the constitutive loss of Pdgfb, adult-induced loss does not lead to vessel dilation, impaired arterio-venous zonation or the formation of microvascular calcifications. We conclude that PDFGB expression in quiescent adult microvascular brain endothelium is critical for the maintenance of pericyte coverage and normal BBB function, but that microvessel dilation, rarefaction, arterio-venous skewing and calcification reflect developmental roles of PDGFB.
Integration of spatial and single-cell transcriptomic data elucidates mouse organogenesis.
Molecular profiling of single cells has advanced our knowledge of the molecular basis of development. However, current approaches mostly rely on dissociating cells from tissues, thereby losing the crucial spatial context of regulatory processes. Here, we apply an image-based single-cell transcriptomics method, sequential fluorescence in situ hybridization (seqFISH), to detect mRNAs for 387 target genes in tissue sections of mouse embryos at the 8-12 somite stage. By integrating spatial context and multiplexed transcriptional measurements with two single-cell transcriptome atlases, we characterize cell types across the embryo and demonstrate that spatially resolved expression of genes not profiled by seqFISH can be imputed. We use this high-resolution spatial map to characterize fundamental steps in the patterning of the midbrain-hindbrain boundary (MHB) and the developing gut tube. We uncover axes of cell differentiation that are not apparent from single-cell RNA-sequencing (scRNA-seq) data, such as early dorsal-ventral separation of esophageal and tracheal progenitor populations in the gut tube. Our method provides an approach for studying cell fate decisions in complex tissues and development.
Lymphatic clearance of immune cells in cardiovascular disease
Recent advances in our understanding of the lymphatic system, its function, development, and role in pathophysiology have changed our views on its importance. Historically thought to be solely involved in the transport of tissue fluid, lipids, and immune cells, the lymphatic system displays great heterogeneity and plasticity and is actively involved in immune cell regulation. Interference in any of these processes can be deleterious, both at the developmental and adult level. Preclinical studies into the cardiac lymphatic system have shown that invoking lymphangiogenesis and enhancing immune cell trafficking in ischaemic hearts can reduce myocardial oedema, reduce inflammation, and improve cardiac outcome. Understanding how immune cells and the lymphatic endothelium interact is also vital to understanding how the lymphatic vascular network can be manipulated to improve immune cell clearance. In this Review, we examine the different types of immune cells involved in fibrotic repair following myocardial infarction. We also discuss the development and function of the cardiac lymphatic vasculature and how some immune cells interact with the lymphatic endothelium in the heart. Finally, we establish how promoting lymphangiogenesis is now a prime therapeutic target for reducing immune cell persistence, inflammation, and oedema to restore heart function in ischaemic heart disease.
