Elevated levels of the pro-inflammatory cytokine tumor necrosis factor-α (TNFα) inhibit erythropoiesis and cause anemia in patients with cancer and chronic inflammatory diseases. TNFα is also a ...potent activator of the sphingomyelinase (SMase)/ceramide pathway leading to ceramide synthesis and regulating cell differentiation, proliferation, apoptosis, senescence, and autophagy. Here we evaluated the implication of the TNFα/SMase/ceramide pathway on inhibition of erythropoiesis in human CD34
hematopoietic stem/progenitor cells (CD34/HSPCs) from healthy donors. Exogenous synthetic C2- and C6-ceramide as well as bacterial SMase inhibited erythroid differentiation in erythropoietin-induced (Epo)CD34/HSPCs shown by the analysis of various erythroid markers. The neutral SMase inhibitor GW4869 as well as the genetic inhibition of nSMase with small interfering RNA (siRNA) against sphingomyelin phosphodiesterase 3 (SMPD3) prevented the inhibition by TNFα, but not the acid SMase inhibitor desipramine. Moreover, sphingosine-1-phosphate (S1P), a ceramide metabolite, restored erythroid differentiation, whereas TNFα inhibited sphingosine kinase-1, required for S1P synthesis. Analysis of cell morphology and colony formation demonstrated that erythropoiesis impairment was concomitant with a granulomonocytic differentiation in TNFα- and ceramide-treated EpoCD34/HSPCs. Inhibition of erythropoiesis and induction of granulomonocytic differentiation were correlated to modulation of hematopoietic transcription factors (TFs) GATA-1, GATA-2, and PU.1. Moreover, the expression of microRNAs (miR)-144/451, miR-146a, miR-155, and miR-223 was also modulated by TNFα and ceramide treatments, in line with cellular observations. Autophagy plays an essential role during erythropoiesis and our results demonstrate that the TNFα/neutral SMase/ceramide pathway inhibits autophagy in EpoCD34/HSPCs. TNFα- and ceramide-induced phosphorylation of mTOR
and ULK1
, inhibited Atg13
phosphorylation, and blocked autophagosome formation as shown by transmission electron microscopy and GFP-LC3 punctae formation. Moreover, rapamycin prevented the inhibitory effect of TNFα and ceramides on erythropoiesis while inhibiting induction of myelopoiesis. In contrast, bafilomycin A1, but not siRNA against Atg5, induced myeloid differentiation, while both impaired erythropoiesis. We demonstrate here that the TNFα/neutral SMase/ceramide pathway inhibits erythropoiesis to induce myelopoiesis via modulation of a hematopoietic TF/miR network and inhibition of late steps of autophagy. Altogether, our results reveal an essential role of autophagy in erythroid vs. myeloid differentiation.
Abstract Purpose Recent studies have shown an increased incidence of congenital hypothyroidism over the past 2 or 3 decades. The etiology of this change is unknown, but it has been related by several ...authors to lowering of cutoffs. We sought to determine whether the incidence of congenital hypothyroidism (CH) in France has changed. Methods We analyzed data from the nationwide neonatal screening program for CH during the period 1982–2012. We included all children having thyroid-stimulating hormone values above the threshold and for whom diagnosis of CH confirmed by the pediatrician. We estimated multicentric temporal trends in the annual incidence rates adjusted for screening methods for thyroid dysgenesis and eutopic gland. Results We found 6622 cases of CH (28.0 per 100,000 newborns); 1895 had a eutopic gland, and 4727 had thyroid dysgenesis. The incidence of eutopic glands showed a significant annual average increase of (5.1%; 95% confidence interval: 4.3–5.9) regardless of the screening method or screening center. This increase was confirmed in severe cases (thyroid-stimulating hormone ≥50: 2.1%; 95% confidence interval, 1.4–2.9). The incidence of dysgenesis remained constant. Conclusions The incidence of eutopic glands increased in France, not only in mild forms but also in severe cases.
Creatine is physiologically provided equally by diet and by endogenous synthesis from arginine and glycine with successive involvements of arginine glycine amidinotransferase AGAT and ...guanidinoacetate methyl transferase GAMT. A specific plasma membrane transporter, creatine transporter CRTR (SLC6A8), further enables cells to incorporate creatine and through uptake of its precursor, guanidinoacetate, also directly contributes to creatine biosynthesis. Breakthrough in the role of creatine has arisen from studies on creatine deficiency disorders. Primary creatine disorders are inherited as autosomal recessive (mutations affecting GATM for glycine-amidinotransferase, mitochondrial) and GAMT genes) or X-linked (SLC6A8 gene) traits. They have highlighted the role of creatine in brain functions altered in patients (global developmental delay, intellectual disability, behavioral disorders). Creatine modulates GABAergic and glutamatergic cerebral pathways, presynaptic CRTR (SLC6A8) ensuring re-uptake of synaptic creatine. Secondary creatine disorders, addressing other genes, have stressed the extraordinary imbrication of creatine metabolism with many other cellular pathways. This high dependence on multiple pathways supports creatine as a cellular sensor, to cell methylation and energy status. Creatine biosynthesis consumes 40% of methyl groups produced as S-adenosylmethionine, and creatine uptake is controlled by AMP activated protein kinase, a ubiquitous sensor of energy depletion. Today, creatine is considered as a potential sensor of cell methylation and energy status, a neurotransmitter influencing key (GABAergic and glutamatergic) CNS neurotransmission, therapeutic agent with anaplerotic properties (towards creatine kinases creatine–creatine phosphate cycle and creatine neurotransmission), energetic and antioxidant compound (benefits in degenerative diseases through protection against energy depletion and oxidant species) with osmolyte behavior (retention of water by muscle). This review encompasses all these aspects by providing an illustrated metabolic account for brain and body creatine in health and disease, an algorithm to diagnose metabolic and gene bases of primary and secondary creatine deficiencies, and a metabolic exploration by 1H-MRS assessment of cerebral creatine levels and response to therapeutic measures.
•Primary creatine disorders highlight a role of creatine in brain physiology.•SLC6A8 takes part in creatine transport and re-uptake but also in biosynthesis.•Secondary creatine defects stress dependence of creatine metabolism on many pathways.•The high dependence on multiple metabolic pathways supports creatine as a cell sensor.•Creatine is a potential sensor of cell methylation and energy status.
Zellweger syndrome disorders (ZSD) is the principal group of peroxisomal disorders characterized by a defect of peroxisome biogenesis due to mutations in one of the 13 PEX genes. The clinical ...spectrum is very large with a continuum from antenatal forms to adult presentation. Whereas biochemical profile in body fluids is classically used for their diagnosis, the revolution of high-throughput sequencing has extended the knowledge about these disorders. The aim of this review is to offer a large panorama on molecular basis, clinical presentation and treatment of ZSD, and to update the diagnosis strategy of these disorders in the era of next-generation sequencing (NGS).
Dietary sphingomyelin (SM) has been reported to favorably modulate postprandial lipemia. Mechanisms underlying these beneficial effects on cardiovascular risk markers are not fully elucidated. Rodent ...studies showed that tritiated SM was hydrolyzed in the intestinal lumen into ceramides (Cer) and further to sphingosine (SPH) and fatty acids (FA) that were absorbed by the intestine. Our objective was to investigate the uptake and metabolism of SPH and/or tricosanoic acid (C23:0), the main FA of milk SM, as well as lipid secretion in Caco-2/TC7 cells cultured on semipermeable inserts. Mixed micelles (MM) consisting of different digested lipids and taurocholate were prepared without or with SPH, SPH and C23:0 (SPH+C23:0), or C23:0. Triglycerides (TG) were quantified in the basolateral medium, and sphingolipids were analyzed by tandem mass spectrometry. TG secretion increased 11-fold in all MM-incubated cells compared with lipid-free medium. Apical supply of SPH-enriched MM led to increased concentrations of total Cer in cells, and coaddition of C23:0 in SPH-enriched MM led to a preferential increase of C23:0 Cer and C23:0 SM. Complementary experiments using deuterated SPH demonstrated that SPH-d9 was partly converted to sphingosine-1-phosphate-d9, Cer-d9, and SM-d9 within cells incubated with SPH-enriched MM. A few Cer-d9 (2% of added SPH-d9) was recovered in the basolateral medium of (MM+SPH)-incubated cells, especially C23:0 Cer-d9 in (MM+SPH+C23:0)-enriched cells. In conclusion, present results indicate that MM enriched with (SPH+C23:0), such as found in postprandial micelles formed after milk SM ingestion, directly impacts sphingolipid endogenous metabolism in enterocytes, resulting in the secretion of TG-rich particles enriched with C23:0 Cer.
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The biological diagnosis of sphingolipidoses currently relies on the measurement of specific enzymatic activities and/or genetic studies. Lysosphingolipids have recently emerged as potential ...biomarkers of sphingolipidoses and Niemann-Pick type C in plasma.
We developed a sensitive and specific method enabling the simultaneous quantification of lysosphingolipids by LC-MS/MS: lysoglobotriaosylceramide for Fabry disease, lysohexosylceramide (i.e. lysoglucosylceramide and/or lysogalactosylceramide) for Gaucher and Krabbe diseases, lysosphingomyelin and its carboxylated analogue lysosphingomyelin-509 for Niemann-Pick type A or B, and C diseases, lysoGM1 ganglioside for GM1gangliosidosis and lysoGM2 ganglioside for GM2 gangliosidosis.
The diagnostic performances were validated in plasma samples analysing a large series of patients affected with sphingolipidoses and Niemann-Pick type C disease (n = 98), other inborn errors of metabolism (n = 23), and controls (n = 228). The multiplex measurement of lysosphingolipids allowed the screening of Fabry (including female patients and late-onset variants), Gaucher and infantile Krabbe, Niemann-Pick type A/B and C diseases with high sensitivity and specificity. LysoGM1 and LysoGM2 were elevated in most of the patients affected with GM1 and GM2 gangliosidosis respectively. In amniotic fluid supernatant from pregnancies presenting non-immune hydrops fetalis (n = 77, including previously diagnosed Gaucher (n = 5), GM1 gangliosidosis (n = 4) and galactosialidosis (n = 4) fetuses) and from normal pregnancies (n = 15), a specific and dramatic increase of lysohexosylceramide was observed only in the Gaucher amniotic fluid samples.
This multiplex assay which allows the simultaneous measurement of lysosphingolipids in plasma modifies the diagnostic strategy of sphingolipidoses and Niemann-Pick type C. Furthermore, in pregnancies presenting non-immune hydrops fetalis, lysohexosylceramide measurement in amniotic fluid offers a rapid screening of fetal Gaucher disease without waiting for glucocerebrosidase activity measurement in cultured amniocytes.
Severe progressive neurological paediatric disease mucopolysaccharidosis III type C is caused by mutations in the HGSNAT gene leading to deficiency of acetyl-CoA: α-glucosaminide N-acetyltransferase ...involved in the lysosomal catabolism of heparan sulphate. To understand the pathophysiology of the disease we generated a mouse model of mucopolysaccharidosis III type C by germline inactivation of the Hgsnat gene. At 6-8 months mice showed hyperactivity, and reduced anxiety. Cognitive memory decline was detected at 10 months and at 12-13 months mice showed signs of unbalanced hesitant walk and urinary retention. Lysosomal accumulation of heparan sulphate was observed in hepatocytes, splenic sinus endothelium, cerebral microglia, liver Kupffer cells, fibroblasts and pericytes. Starting from 5 months, brain neurons showed enlarged, structurally abnormal mitochondria, impaired mitochondrial energy metabolism, and storage of densely packed autofluorescent material, gangliosides, lysozyme, phosphorylated tau, and amyloid-β. Taken together, our data demonstrate for the first time that deficiency of acetyl-CoA: α-glucosaminide N-acetyltransferase causes lysosomal accumulation of heparan sulphate in microglial cells followed by their activation and cytokine release. They also show mitochondrial dysfunction in the neurons and neuronal loss explaining why mucopolysaccharidosis III type C manifests primarily as a neurodegenerative disease.
Sphingolipids are structural components of cell membranes and lipoproteins but also act as signaling molecules in many pathophysiological processes. Although sphingolipids comprise a small part of ...the plasma lipidome, some plasma sphingolipids are recognized as implicated in the development of metabolic diseases and cardiovascular diseases. Plasma sphingolipids are mostly carried out into lipoproteins and may modulate their functional properties. Lipids ingested from the diet contribute to the plasma lipid pool besides lipids produced by the liver and released from the adipose tissue. Depending on their source, quality and quantity, dietary lipids may modulate sphingolipids both in plasma and lipoproteins. A few human dietary intervention studies investigated the impact of dietary lipids on circulating sphingolipids and lipid-related cardiovascular risk markers. On the one hand, dietary saturated fatty acids, mainly palmitic acid, may increase ceramide concentrations in plasma, triglyceride-rich lipoproteins and HDL. On the other hand, milk polar lipids may decrease some molecular species of sphingomyelins and ceramides in plasma and intestine-derived chylomicrons. Altogether, different dietary fatty acids and lipid species can modulate circulating sphingolipids vehicled by postprandial lipoproteins, which should be part of future nutritional strategies for prevention of cardiovascular diseases.
Tandem mass spectrometry (MS/MS) is a highly sensitive and specific technique. Thanks to the development of triple quadrupole analyzers, it is becoming more widely used in laboratories working in the ...field of inborn errors of metabolism. We review here the state of the art of this technique applied to the diagnosis of lysosomal storage disorders (LSDs) and how MS/MS has changed the diagnostic rationale in recent years. This fine technology brings more sensitive, specific, and reliable methods than the previous biochemical ones for the analysis of urinary glycosaminoglycans, oligosaccharides, and sialic acid. In sphingolipidoses, the quantification of urinary sphingolipids (globotriaosylceramide, sulfatides) is possible. The measurement of new plasmatic biomarkers such as oxysterols, bile acids, and lysosphingolipids allows the screening of many sphingolipidoses and related disorders (Niemann–Pick type C), replacing tedious biochemical techniques. Applied to amniotic fluid, a more reliable prenatal diagnosis or screening of LSDs is now available for fetuses presenting with antenatal manifestations. Applied to enzyme measurements, it allows high throughput assays for the screening of large populations, even newborn screening. The advent of this new method can modify the diagnostic rationale behind LSDs.
Sphingolipids play a key structural role in cellular membranes and/or act as signaling molecules. Inherited defects of their catabolism lead to lysosomal storage diseases called sphingolipidoses. ...Although progress has been made toward a better understanding of their pathophysiology, several issues still remain unsolved. In particular, whether lysosphingolipids, the deacylated form of sphingolipids, both of which accumulate in these diseases, are simple biomarkers or play an instrumental role is unclear. In the meanwhile, evidence has been provided for a high risk of developing malignancies in patients affected with Gaucher disease, the most common sphingolipidosis. This article aims at analyzing the potential involvement of lysosphingolipids in cancer. Knowledge about lysosphingolipids in the context of lysosomal storage diseases is summarized. Available data on the nature and prevalence of cancers in patients affected with sphingolipidoses are also reviewed. Then, studies investigating the biological effects of lysosphingolipids toward pro or antitumor pathways are discussed. Finally, original findings exploring the role of glucosylsphingosine in the development of melanoma are presented. While this lysosphingolipid may behave like a protumorigenic agent, further investigations in appropriate models are needed to elucidate the role of these peculiar lipids, not only in sphingolipidoses but also in malignant diseases in general.
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