HIF-1α metabolically controls collagen synthesis and modification in chondrocytes.

Endochondral ossification, an important process in vertebrate bone formation, is highly dependent on correct functioning of growth plate chondrocytes1. Proliferation of these cells determines longitudinal bone growth and the matrix deposited provides a scaffold for future bone formation. However, these two energy-dependent anabolic processes occur in an avascular environment1,2. In addition, the centre of the expanding growth plate becomes hypoxic, and local activation of the hypoxia-inducible transcription factor HIF-1α is necessary for chondrocyte survival by unidentified cell-intrinsic mechanisms3-6. It is unknown whether there is a requirement for restriction of HIF-1α signalling in the other regions of the growth plate and whether chondrocyte metabolism controls cell function. Here we show that prolonged HIF-1α signalling in chondrocytes leads to skeletal dysplasia by interfering with cellular bioenergetics and biosynthesis. Decreased glucose oxidation results in an energy deficit, which limits proliferation, activates the unfolded protein response and reduces collagen synthesis. However, enhanced glutamine flux increases α-ketoglutarate levels, which in turn increases proline and lysine hydroxylation on collagen. This metabolically regulated collagen modification renders the cartilaginous matrix more resistant to protease-mediated degradation and thereby increases bone mass. Thus, inappropriate HIF-1α signalling results in skeletal dysplasia caused by collagen overmodification, an effect that may also contribute to other diseases involving the extracellular matrix such as cancer and fibrosis.

Prolyl hydroxylase-1 regulates hepatocyte apoptosis in an NF-κB-dependent manner.

Hepatocyte death is an important contributing factor in a number of diseases of the liver. PHD1 confers hypoxic sensitivity upon transcription factors including the hypoxia inducible factor (HIF) and nuclear factor-kappaB (NF-κB). Reduced PHD1 activity is linked to decreased apoptosis. Here, we investigated the underlying mechanism(s) in hepatocytes. Basal NF-κB activity was elevated in PHD1(-/-) hepatocytes compared to wild type controls. ChIP-seq analysis confirmed enhanced binding of NF-κB to chromatin in regions proximal to the promoters of genes involved in the regulation of apoptosis. Inhibition of NF-κB (but not knock-out of HIF-1 or HIF-2) reversed the anti-apoptotic effects of pharmacologic hydroxylase inhibition. We hypothesize that PHD1 inhibition leads to altered expression of NF-κB-dependent genes resulting in reduced apoptosis. This study provides new information relating to the possible mechanism of therapeutic action of hydroxylase inhibitors that has been reported in pre-clinical models of intestinal and hepatic disease.

Very long-chain acyl-CoA synthetase 3: overexpression and growth dependence in lung cancer.

Lung cancer is the leading cause of cancer deaths worldwide. In the United States, only one in six lung cancer patients survives five years after diagnosis. These statistics may improve if new therapeutic targets are identified. We previously reported that an enzyme of fatty acid metabolism, very long-chain acyl-CoA synthetase 3 (ACSVL3), is overexpressed in malignant glioma, and that depleting glioblastoma cells of ACSVL3 diminishes their malignant properties. To determine whether ACSVL3 expression was also increased in lung cancer, we studied tumor histologic sections and lung cancer cell lines. Immunohistochemical analysis of normal human lung showed moderate ACSVL3 expression only in bronchial epithelial cells. In contrast, all of 69 different lung tumors tested, including adeno-, squamous cell, large cell, and small cell carcinomas, had robustly elevated ACSVL3 levels. Western blot analysis of lung cancer cell lines derived from these tumor types also had significantly increased ACSVL3 protein compared to normal bronchial epithelial cells. Decreasing the growth rate of lung cancer cell lines did not change ACSVL3 expression. However, knocking down ACSVL3 expression by RNA interference reduced cell growth rates in culture by 65-76%, and the ability of tumor cells to form colonies in soft agar suspension by 65-80%. We also conducted studies to gain a better understanding of the biochemical properties of human ACSVL3. ACSVL3 mRNA was detected in many human tissues, but the expression pattern differed somewhat from that of the mouse. The enzyme activated long- and very long-chain saturated fatty acid substrates, as well as long-chain mono- and polyunsaturated fatty acids to their respective coenzyme A derivatives. Endogenous human ACSVL3 protein was found in a punctate subcellular compartment that partially colocalized with mitochondria as determined by immunofluorescence microscopy and subcellular fractionation. From these studies, we conclude that ACSVL3 is a promising new therapeutic target in lung cancer.

Role of PFKFB3-driven glycolysis in vessel sprouting.

Vessel sprouting by migrating tip and proliferating stalk endothelial cells (ECs) is controlled by genetic signals (such as Notch), but it is unknown whether metabolism also regulates this process. Here, we show that ECs relied on glycolysis rather than on oxidative phosphorylation for ATP production and that loss of the glycolytic activator PFKFB3 in ECs impaired vessel formation. Mechanistically, PFKFB3 not only regulated EC proliferation but also controlled the formation of filopodia/lamellipodia and directional migration, in part by compartmentalizing with F-actin in motile protrusions. Mosaic in vitro and in vivo sprouting assays further revealed that PFKFB3 overexpression overruled the pro-stalk activity of Notch, whereas PFKFB3 deficiency impaired tip cell formation upon Notch blockade, implying that glycolysis regulates vessel branching.

Crosstalk between oxygen- and nitric oxide-dependent signaling pathways in angiogenesis.

With every heart beat blood rushes through a complex network of tubes to deliver essential ingredients of life, oxygen and nutrients. Consequently, this network of blood vessels is an indispensable part of vertebrate physiology. Its organization and architecture is highly dynamic in its form and function. Understanding how blood vessels develop, a process referred to as angiogenesis, is equally important as to know how they function considering that failure or misalignment of this process results in disorder and disease, in many cases of which death is inevitable. Much has been learned about the angiogenic process and the critical contributors of blood vessel function. A central determinant is oxygen, an evident contributor given the fact that oxygen delivery is a primary feature of blood vessel function. Not only is oxygen however essential for mitochondrial energy production, it also serves as a key molecule in various biochemical reactions, such as the formation of nitric oxide (NO), on its part a critical regulator of vascular tone and vessel homeostasis. Hence, oxygen abundance relates to the production of NO, and NO in turn regulates oxygen delivery and consumption. Given the importance of the intrinsic link these two molecules exert on angiogenesis and vessel function; this review shall highlight our current understanding on how these two molecules cooperate to form blood vessels.

Impaired local production of proresolving lipid mediators in obesity and 17-HDHA as a potential treatment for obesity-associated inflammation.

Obesity-induced chronic low-grade inflammation originates from adipose tissue and is crucial for obesity-driven metabolic deterioration, including insulin resistance and type 2 diabetes. Chronic inflammation may be a consequence of a failure to actively resolve inflammation and could result from a lack of local specialized proresolving lipid mediators (SPMs), such as resolvins and protectins, which derive from the n-3 polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). We assessed obesity-induced changes of n-3-derived SPMs in adipose tissue and the effects of dietary EPA/DHA thereon. Moreover, we treated obese mice with SPM precursors and investigated the effects on inflammation and metabolic dysregulation. Obesity significantly decreased DHA-derived 17-hydroxydocosahexaenoic acid (17-HDHA, resolvin D1 precursor) and protectin D1 (PD1) levels in murine adipose tissue. Dietary EPA/DHA treatment restored endogenous biosynthesis of n-3-derived lipid mediators in obesity while attenuating adipose tissue inflammation and improving insulin sensitivity. Notably, 17-HDHA treatment reduced adipose tissue expression of inflammatory cytokines, increased adiponectin expression, and improved glucose tolerance parallel to insulin sensitivity in obese mice. These findings indicate that impaired biosynthesis of certain SPM and SPM precursors, including 17-HDHA and PD1, contributes to adipose tissue inflammation in obesity and suggest 17-HDHA as a novel treatment option for obesity-associated complications.

Carbohydrate metabolism is perturbed in peroxisome-deficient hepatocytes due to mitochondrial dysfunction, AMP-activated protein kinase (AMPK) activation, and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) suppression.

Hepatic peroxisomes are essential for lipid conversions that include the formation of mature conjugated bile acids, the degradation of branched chain fatty acids, and the synthesis of docosahexaenoic acid. Through unresolved mechanisms, deletion of functional peroxisomes from mouse hepatocytes (L-Pex5(-/-) mice) causes severe structural and functional abnormalities at the inner mitochondrial membrane. We now demonstrate that the peroxisomal and mitochondrial anomalies trigger energy deficits, as shown by increased AMP/ATP and decreased NAD(+)/NADH ratios. This causes suppression of gluconeogenesis and glycogen synthesis and up-regulation of glycolysis. As a consequence, L-Pex5(-/-) mice combust more carbohydrates resulting in lower body weights despite increased food intake. The perturbation of carbohydrate metabolism does not require a long term adaptation to the absence of functional peroxisomes as similar metabolic changes were also rapidly induced by acute elimination of Pex5 via adenoviral administration of Cre. Despite its marked activation, peroxisome proliferator-activated receptor α (PPARα) was not causally involved in these metabolic perturbations, because all abnormalities still manifested when peroxisomes were eliminated in a peroxisome proliferator-activated receptor α null background. Instead, AMP-activated kinase activation was responsible for the down-regulation of glycogen synthesis and induction of glycolysis. Remarkably, PGC-1α was suppressed despite AMP-activated kinase activation, a paradigm not previously reported, and they jointly contributed to impaired gluconeogenesis. In conclusion, lack of functional peroxisomes from hepatocytes results in marked disturbances of carbohydrate homeostasis, which are consistent with adaptations to an energy deficit. Because this is primarily due to impaired mitochondrial ATP production, these L-Pex5-deficient livers can also be considered as a model for secondary mitochondrial hepatopathies.

Loss or inhibition of stromal-derived PlGF prolongs survival of mice with imatinib-resistant Bcr-Abl1(+) leukemia.

Imatinib has revolutionized the treatment of Bcr-Abl1(+) chronic myeloid leukemia (CML), but, in most patients, some leukemia cells persist despite continued therapy, while others become resistant. Here, we report that PlGF levels are elevated in CML and that PlGF produced by bone marrow stromal cells (BMSCs) aggravates disease severity. CML cells foster a soil for their own growth by inducing BMSCs to upregulate PlGF, which not only stimulates BM angiogenesis, but also promotes CML proliferation and metabolism, in part independently of Bcr-Abl1 signaling. Anti-PlGF treatment prolongs survival of imatinib-sensitive and -resistant CML mice and adds to the anti-CML activity of imatinib. These results may warrant further investigation of the therapeutic potential of PlGF inhibition for (imatinib-resistant) CML.

Loss of prolyl hydroxylase-1 protects against colitis through reduced epithelial cell apoptosis and increased barrier function.

BACKGROUND & AIMS: Hypoxia inducible factor (HIF) prolyl hydroxylase inhibitors are protective in mouse models of inflammatory bowel disease (IBD). Here, we investigated the therapeutic target(s) and mechanism(s) involved. METHODS: The effect of genetic deletion of individual HIF-prolyl hydroxylase (PHD) enzymes on the development of dextran sulphate sodium (DSS)-induced colitis was examined in mice. RESULTS: PHD1(-/-), but not PHD2(+/-) or PHD3(-/-), mice were less susceptible to the development of colitis than wild-type controls as determined by weight loss, disease activity, colon histology, neutrophil infiltration, and cytokine expression. Reduced susceptibility of PHD1(-/-) mice to colitis was associated with increased density of colonic epithelial cells relative to wild-type controls, which was because of decreased levels of apoptosis that resulted in enhanced epithelial barrier function. Furthermore, with the use of cultured epithelial cells it was confirmed that hydroxylase inhibition reversed DSS-induced apoptosis and barrier dysfunction. Finally, PHD1 levels were increased with disease severity in intestinal tissue from patients with IBD and in colonic tissues from DSS-treated mice. CONCLUSIONS: These results imply a role for PHD1 as a positive regulator of intestinal epithelial cell apoptosis in the inflamed colon. Genetic loss of PHD1 is protective against colitis through decreased epithelial cell apoptosis and consequent enhancement of intestinal epithelial barrier function. Thus, targeted PHD1 inhibition may represent a new therapeutic approach in IBD.

Loss or silencing of the PHD1 prolyl hydroxylase protects livers of mice against ischemia/reperfusion injury.

BACKGROUND & AIMS: Liver ischemia/reperfusion (I/R) injury is a frequent cause of organ dysfunction. Loss of the oxygen sensor prolyl hydroxylase domain enzyme 1 (PHD1) causes tolerance of skeletal muscle to hypoxia. We assessed whether loss or short-term silencing of PHD1 could likewise induce hypoxia tolerance in hepatocytes and protect them against hepatic I/R damage. METHODS: Hepatic ischemia was induced in mice by clamping of the portal vessels of the left lateral liver lobe; 90 minutes later livers were reperfused for 8 hours for I/R experiments. Hepatocyte damage following ischemia or I/R was investigated in PHD1-deficient (PHD1(-/-)) and wild-type mice or following short hairpin RNA-mediated short-term inhibition of PHD1 in vivo. RESULTS: PHD1(-/-) livers were largely protected against acute ischemia or I/R injury. Among mice subjected to hepatic I/R followed by surgical resection of all nonischemic liver lobes, more than half of wild-type mice succumbed, whereas all PHD1(-/-) mice survived. Also, short-term inhibition of PHD1 through RNA interference-mediated silencing provided protection against I/R. Knockdown of PHD1 also induced hypoxia tolerance of hepatocytes in vitro. Mechanistically, loss of PHD1 decreased production of oxidative stress, which likely relates to a decrease in oxygen consumption as a result of a reprogramming of hepatocellular metabolism. CONCLUSIONS: Loss of PHD1 provided tolerance of hepatocytes to acute hypoxia and protected them against I/R-damage. Short-term inhibition of PHD1 is a novel therapeutic approach to reducing or preventing I/R-induced liver injury.

Regulation of angiogenesis by oxygen and metabolism.

Blood vessels form an important interface between the environment and the organism by carrying oxygen and nutrients to all cells and thus determining cellular metabolism. It is therefore not surprising that oxygen and metabolism influence the development of the vascular network. Here, we discuss recent insights regarding the emerging crosstalk between angiogenesis and metabolism. We will highlight advances in how oxygen and metabolism regulate angiogenesis as well as how angiogenic factors in turn also regulate metabolism.

Oxygen sensors at the crossroad of metabolism.

Aerobic organisms developed mechanisms to protect themselves against a shortage of oxygen (O(2)). Recent studies reveal that O(2) sensors, belonging to the novel class of 2-oxoglutarate dependent iron(ii)-dioxygenases, have more important roles in metabolism than anticipated. Here, we provide a "metabolo-centric" overview of the role of the PHD/FIH members of this family in metabolism, in particular on how they regulate O(2) supply and consumption, energy compensation and conservation, O(2) conformance and hypoxia tolerance, redox and pH homeostasis, and other vital metabolic processes with implications in health and disease. These insights may offer novel opportunities for the treatment of ischemic diseases.

Inhibition of oxygen sensors as a therapeutic strategy for ischaemic and inflammatory disease.

Cells in the human body need oxygen to function and survive, and severe deprivation of oxygen, as occurs in ischaemic heart disease and stroke, is a major cause of mortality. Nevertheless, other organisms, such as the fossorial mole rat or diving seals, have acquired the ability to survive in conditions of limited oxygen supply. Hypoxia tolerance also allows the heart to survive chronic oxygen shortage, and ischaemic preconditioning protects tissues against lethal hypoxia. The recent discovery of a new family of oxygen sensors--including prolyl hydroxylase domain-containing proteins 1-3 (PHD1-3)--has yielded exciting novel insights into how cells sense oxygen and keep oxygen supply and consumption in balance. Advances in understanding of the role of these oxygen sensors in hypoxia tolerance, ischaemic preconditioning and inflammation are creating new opportunities for pharmacological interventions for ischaemic and inflammatory diseases.

Fatty acid transport and activation and the expression patterns of genes involved in fatty acid trafficking.

These studies defined the expression patterns of genes involved in fatty acid transport, activation and trafficking using quantitative PCR (qPCR) and established the kinetic constants of fatty acid transport in an effort to define whether vectorial acylation represents a common mechanism in different cell types (3T3-L1 fibroblasts and adipocytes, Caco-2 and HepG2 cells and three endothelial cell lines (b-END3, HAEC, and HMEC)). As expected, fatty acid transport protein (FATP)1 and long-chain acyl CoA synthetase (Acsl)1 were the predominant isoforms expressed in adipocytes consistent with their roles in the transport and activation of exogenous fatty acids destined for storage in the form of triglycerides. In cells involved in fatty acid processing including Caco-2 (intestinal-like) and HepG2 (liver-like), FATP2 was the predominant isoform. The patterns of Acsl expression were distinct between these two cell types with Acsl3 and Acsl5 being predominant in Caco-2 cells and Acsl4 in HepG2 cells. In the endothelial lines, FATP1 and FATP4 were the most highly expressed isoforms; the expression patterns for the different Acsl isoforms were highly variable between the different endothelial cell lines. The transport of the fluorescent long-chain fatty acid C(1)-BODIPY-C(12) in 3T3-L1 fibroblasts and 3T3-L1 adipocytes followed typical Michaelis-Menten kinetics; the apparent efficiency (k(cat)/K(T)) of this process increases over 2-fold (2.1 x 10(6)-4.5 x 10(6)s(-1)M(-1)) upon adipocyte differentiation. The V(max) values for fatty acid transport in Caco-2 and HepG2 cells were essentially the same, yet the efficiency was 55% higher in Caco-2 cells (2.3 x 10(6)s(-1)M(-1) versus 1.5 x 10(6)s(-1)M(-1)). The kinetic parameters for fatty acid transport in three endothelial cell types demonstrated they were the least efficient cell types for this process giving V(max) values that were nearly 4-fold lower than those defined form 3T3-L1 adipocytes, Caco-2 cells and HepG2 cells. The same cells had reduced efficiency for fatty acid transport (ranging from 0.82 x 10(6)s(-1)M(-1) to 1.35 x 10(6)s(-1)M(-1)).

Hungry for blood vessels: linking metabolism and angiogenesis.

A new study by Arany et al. demonstrates that the metabolic master regulator peroxisome proliferator-activated receptor (PPARgamma) coactivator-1alpha (PGC-1alpha) can promote angiogenesis. These findings show that alterations in metabolic homeostasis are linked to angiogenesis, which raises exciting opportunities for novel proangiogenic and antiangiogenic therapies in various diseases.

Deficiency or inhibition of oxygen sensor Phd1 induces hypoxia tolerance by reprogramming basal metabolism.

HIF prolyl hydroxylases (PHD1-3) are oxygen sensors that regulate the stability of the hypoxia-inducible factors (HIFs) in an oxygen-dependent manner. Here, we show that loss of Phd1 lowers oxygen consumption in skeletal muscle by reprogramming glucose metabolism from oxidative to more anaerobic ATP production through activation of a Pparalpha pathway. This metabolic adaptation to oxygen conservation impairs oxidative muscle performance in healthy conditions, but it provides acute protection of myofibers against lethal ischemia. Hypoxia tolerance is not due to HIF-dependent angiogenesis, erythropoiesis or vasodilation, but rather to reduced generation of oxidative stress, which allows Phd1-deficient myofibers to preserve mitochondrial respiration. Hypoxia tolerance relies primarily on Hif-2alpha and was not observed in heterozygous Phd2-deficient or homozygous Phd3-deficient mice. Of medical importance, conditional knockdown of Phd1 also rapidly induces hypoxia tolerance. These findings delineate a new role of Phd1 in hypoxia tolerance and offer new treatment perspectives for disorders characterized by oxidative stress.

Topology of the yeast fatty acid transport protein Fat1p: mechanistic implications for functional domains on the cytosolic surface of the plasma membrane.

The fatty acid transport protein (FATP) Fat1p in the yeast Saccharomyces cerevisiae functions in concert with acyl-coenzyme A synthetase (ACSL; either Faa1p or Faa4p) in vectorial acylation, which couples the transport of exogenous fatty acids with activation to CoA thioesters. To further define the role of Fat1p in the transport of exogenous fatty acids, the topological orientation of two highly conserved motifs [ATP/AMP and FATP/very long chain acyl CoA synthetase (VLACS)], the carboxyl 124 amino acid residues, which bind the ACSL Faa1p, and the amino and carboxyl termini within the plasma membrane were defined. T7 or hemagglutinin epitope tags were engineered at both amino and carboxyl termini, as well as at multiple nonconserved, predicted random coil segments within the protein. Six different epitope-tagged chimeras of Fat1p were generated and expressed in yeast; the sidedness of the tags was tested using indirect immunofluorescence and protease protection by Western blotting. Plasma membrane localization of the tagged proteins was assessed by immunofluorescence. Fat1p appears to have at least two transmembrane domains resulting in a N(in)-C(in) topology. We propose that Fat1p has a third region, which binds to the membrane and separates the highly conserved residues comprising the two halves of the ATP/AMP motif. The N(in)-C(in) topology results in the placement of the ATP/AMP and FATP/VLACS domains of Fat1p on the inner face of the plasma membrane. The carboxyl-terminal region of Fat1p, which interacts with ACSL, is likewise positioned on the inner face of the plasma membrane. This topological orientation is consistent with the mechanistic roles of both Fat1p and Faa1p or Faa4p in the coupled transport/activation of exogenous fatty acids by vectorial acylation.

A novel mammalian bubblegum-related acyl-CoA synthetase restricted to testes and possibly involved in spermatogenesis.

We have characterized a new, membrane-associated acyl-CoA synthetase (ACS), termed bubblegum-related protein (BGR), which upon functional analysis demonstrated ACS activity capable of activating long- and very long-chain fatty acids. By multiple tissue RNA array and Northern blot analyses, human BGR mRNA was exclusively detected in testes. Murine Bgr mRNA was specifically expressed in pubertal and adult testes and was further demonstrated to be enriched in germ cells and Sertoli cells while present at a lower level in Leydig cells both by in situ hybridization and cell type fractionation. The complex 5'-end of the BGR mRNA appears to underlie translational control leading to differential utilization of alternative translation start sites. Thus, the BGR gene expands the bubblegum ACS family with a testes-specific, developmentally regulated member that may play a role in spermatogenesis.

Mouse very long-chain Acyl-CoA synthetase 3/fatty acid transport protein 3 catalyzes fatty acid activation but not fatty acid transport in MA-10 cells.

The family of proteins that includes very long-chain acyl-CoA synthetases (ACSVL) consists of six members. These enzymes have also been designated fatty acid transport proteins. We cloned full-length mouse Acsvl3 cDNA and characterized its protein product ACSVL3/fatty acid transport protein 3. The predicted amino acid sequence contains two highly conserved motifs characteristic of acyl-CoA synthetases. Northern blot analysis revealed that the mouse Acsvl3 mRNA is highly expressed in adrenal gland, testis, and ovary, with lower expression in the brain of adult mice. A developmental Northern blot revealed that Acsvl3 mRNA levels were significantly higher in embryonic mouse brain (embryonic days 12-14) than in newborn or adult mice, suggesting a possible role in nervous system development. Immunohistochemistry revealed high ACSVL3 expression in adrenal cortical cells, spermatocytes and interstitial cells of the testis, theca cells of the ovary, cerebral cortical neurons, and cerebellar Purkinje cells. Endogenous ACSVL3 was found primarily in mitochondria of MA-10 and Neuro2a cells by both Western blot analysis of subcellular fractions and immunofluorescence analysis. In MA-10 cells, loss-of-function studies using RNA interference confirmed that endogenous ACSVL3 is an acyl-CoA synthetase capable of activating both long-chain (C16:0) and very long-chain (C24:0) fatty acids. However, despite decreased acyl-CoA synthetase activity, initial rates of fatty acid uptake were unaffected by knockdown of Acsvl3 expression in MA-10 cells. These studies cast doubt on the designation of ACSVL3 as a fatty acid transport protein.