ABSTRACT
Adaptive thermogenesis has attracted much attention because of its ability to increase systemic energy expenditure and to counter obesity and diabetes1-3. Recent data have indicated that thermogenic fat cells use creatine to stimulate futile substrate cycling, dissipating chemical energy as heat4,5. This model was based on the super-stoichiometric relationship between the amount of creatine added to mitochondria and the quantity of oxygen consumed. Here we provide direct evidence for the molecular basis of this futile creatine cycling activity in mice. Thermogenic fat cells have robust phosphocreatine phosphatase activity, which is attributed to tissue-nonspecific alkaline phosphatase (TNAP). TNAP hydrolyses phosphocreatine to initiate a futile cycle of creatine dephosphorylation and phosphorylation. Unlike in other cells, TNAP in thermogenic fat cells is localized to the mitochondria, where futile creatine cycling occurs. TNAP expression is powerfully induced when mice are exposed to cold conditions, and its inhibition in isolated mitochondria leads to a loss of futile creatine cycling. In addition, genetic ablation of TNAP in adipocytes reduces whole-body energy expenditure and leads to rapid-onset obesity in mice, with no change in movement or feeding behaviour. These data illustrate the critical role of TNAP as a phosphocreatine phosphatase in the futile creatine cycle.
Subject(s)
Alkaline Phosphatase/metabolism , Mitochondria/enzymology , Phosphocreatine/metabolism , Thermogenesis , Adipocytes/metabolism , Adipose Tissue, Brown/cytology , Adipose Tissue, Brown/metabolism , Animals , Cold Temperature , Energy Metabolism , Hydrolysis , Male , Mice , Mice, Inbred C57BL , Microscopy, Electron, Transmission , Mitochondria/ultrastructure , Mitochondrial Proteins/metabolism , Obesity/metabolismABSTRACT
Tauopathies are a family of neurodegenerative diseases characterized by the presence of abnormally hyperphosphorylated Tau protein. Several studies have proposed that increased extracellular Tau (eTau) leads to the spread of cerebral tauopathy. However, the molecular mechanisms underlying eTau-induced neurotoxicity remain unclear. Previous in vitro studies reported that the ecto-enzyme tissue-nonspecific alkaline phosphatase (TNAP) dephosphorylate eTau at different sites increasing its neurotoxicity. Here, we confirm TNAP protein upregulation in the brains of Alzheimer's patients and found a similar TNAP increase in Pick's disease patients and P301S mice, a well-characterized mouse model of tauopathies. Interestingly, the conditional overexpression of TNAP causes intracellular Tau hyperphosphorylation and aggregation in cells neighbouring those overexpressing the ectoenzyme. Conversely, the genetic disruption of TNAP reduced the dephosphorylation of eTau and decreased neuronal hyperactivity, brain atrophy, and hippocampal neuronal death in P301S mice. TNAP haploinsufficiency in P301S mice prevents the decreased anxiety-like behaviour, motor deficiency, and increased memory capacity and life expectancy. Similar results were observed by the in vivo pharmacological blunting of TNAP activity. This study provides the first in vivo evidence demonstrating that raised TNAP activity is critical for Tau-induced neurotoxicity and suggest that TNAP blockade may be a novel and efficient therapy to treat tauopathies.
Subject(s)
Alkaline Phosphatase , Tauopathies , Alkaline Phosphatase/genetics , Alkaline Phosphatase/metabolism , Alkaline Phosphatase/therapeutic use , Animals , Brain/metabolism , Disease Models, Animal , Humans , Life Expectancy , Mice , Mice, Transgenic , Tauopathies/metabolism , Up-Regulation , tau Proteins/metabolismABSTRACT
Matrix vesicles (MVs) are a class of extracellular vesicles that initiate mineralization in cartilage, bone, and other vertebrate tissues by accumulating calcium ions (Ca2+) and inorganic phosphate (Pi) within their lumen and forming a nucleation core (NC). After further sequestration of Ca2+ and Pi, the NC transforms into crystalline complexes. Direct evidence of the existence of the NC and its maturation have been provided solely by analyses of dried samples. We isolated MVs from chicken embryo cartilage and used atomic force microscopy peak force quantitative nanomechanical property mapping (AFM-PFQNM) to measure the nanomechanical and morphological properties of individual MVs under both mineralizing (+Ca2+) and non-mineralizing (-Ca2+) fluid conditions. The elastic modulus of MVs significantly increased by 4-fold after incubation in mineralization buffer. From AFM mapping data, we inferred the morphological changes of MVs as mineralization progresses: prior to mineralization, a punctate feature, the NC, is present within MVs and this feature grows and stiffens during mineralization until it occupies most of the MV lumen. Dynamic light scattering showed a significant increase in hydrodynamic diameter and no change in the zeta potential of hydrated MVs after incubation with Ca2+. This validates that crystalline complexes, which are strongly negative relative to MVs, were forming within the lumen of MVs. These data were substantiated by transmission electron microscopy energy dispersive X-ray and Fourier transform infrared spectroscopic analyses of dried MVs, which provide evidence that the complexes increased in size, crystallinity, and Ca/P ratio within MVs during the mineralization process.
Subject(s)
Biomineralization/physiology , Extracellular Vesicles/chemistry , Extracellular Vesicles/metabolism , Microscopy, Atomic Force/methods , Animals , Biomechanical Phenomena , Cartilage/chemistry , Cartilage/metabolism , Cartilage/ultrastructure , Chick Embryo , Extracellular Vesicles/ultrastructure , Microscopy, Electron, Transmission , Spectroscopy, Fourier Transform InfraredABSTRACT
Arterial medial calcification (AMC) is thought to share some outward similarities to skeletal mineralization and has been associated with the transdifferentiation of vascular smooth muscle cells (VSMCs) to an osteoblast-like phenotype. ATP and UTP have previously been shown to inhibit bone mineralization. This investigation compared the effects of extracellular nucleotides on calcification in VSMCs with those seen in osteoblasts. ATP, UTP and the ubiquitous mineralization inhibitor, pyrophosphate (PPi ), dose dependently inhibited VSMC calcification by ≤85%. Culture of VSMCs in calcifying conditions was associated with an increase in apoptosis; treatment with ATP, UTP, and PPi reduced apoptosis to levels seen in non-calcifying cells. Extracellular nucleotides had no effect on osteoblast viability. Basal alkaline phosphatase (TNAP) activity was over 100-fold higher in osteoblasts than VSMCs. ATP and UTP reduced osteoblast TNAP activity (≤50%) but stimulated VSMC TNAP activity (≤88%). The effects of extracellular nucleotides on VSMC calcification, cell viability and TNAP activity were unchanged by deletion or inhibition of the P2Y2 receptor. Conversely, the actions of ATP/UTP on bone mineralization and TNAP activity were attenuated in osteoblasts lacking the P2Y2 receptor. Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) hydrolyses ATP and UTP to produce PPi . In both VSMCs and osteoblasts, deletion of NPP1 blunted the inhibitory effects of extracellular nucleotides suggesting involvement of P2 receptor independent pathways. Our results show that although the overall functional effect of extracellular nucleotides on AMC and bone mineralization is similar there are clear differences in the cellular mechanisms mediating these actions.
Subject(s)
Calcification, Physiologic , Extracellular Space/metabolism , Nucleotides/pharmacology , Tunica Media/pathology , Vascular Calcification/pathology , Adenosine Triphosphate/pharmacology , Alkaline Phosphatase/metabolism , Animals , Apoptosis/drug effects , Calcification, Physiologic/drug effects , Cell Survival/drug effects , Diphosphates/pharmacology , Mice , Models, Biological , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/enzymology , Muscle, Smooth, Vascular/pathology , Myocytes, Smooth Muscle/metabolism , Osteoblasts/drug effects , Osteoblasts/enzymology , Phosphoric Diester Hydrolases/deficiency , Phosphoric Diester Hydrolases/metabolism , Pyrophosphatases/deficiency , Pyrophosphatases/metabolism , Receptors, Purinergic P2/metabolism , Uridine Triphosphate/pharmacologyABSTRACT
Atomic force microscopy (AFM) is one of the most commonly used scanning probe microscopy techniques for nanoscale imaging and characterization of lipid-based particles. However, obtaining images of such particles using AFM is still a challenge. The present study extends the capabilities of AFM to the characterization of proteoliposomes, a special class of liposomes composed of lipids and proteins, mimicking matrix vesicles (MVs) involved in the biomineralization process. To this end, proteoliposomes were synthesized, composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phospho-l-serine (DPPS), with inserted tissue-nonspecific alkaline phosphatase (TNAP) and/or annexin V (AnxA5), both characteristic proteins of osteoblast-derived MVs. We then aimed to study how TNAP and AnxA5 insertion affects the proteoliposomes' membrane properties and, in turn, interactions with type II collagen, thus mimicking early MV activity during biomineralization. AFM images of these proteoliposomes, acquired in dynamic mode, revealed the presence of surface protrusions with distinct viscoelasticity, thus suggesting that the presence of the proteins induced local changes in membrane fluidity. Surface protrusions were measurable in TNAP-proteoliposomes but barely detectable in AnxA5-proteoliposomes. More complex surface structures were observed for proteoliposomes harboring both TNAP and AnxA5 concomitantly, resulting in a lower affinity for type II collagen fibers compared to proteoliposomes harboring AnxA5 alone. The present study achieved the topographic analysis of lipid vesicles by direct visualization of structural changes, resulting from protein incorporation, without the need for fluorescent probes.
Subject(s)
Alkaline Phosphatase/chemistry , Alkaline Phosphatase/metabolism , Annexin A5/chemistry , Annexin A5/metabolism , Proteolipids/chemistry , Proteolipids/metabolism , 1,2-Dipalmitoylphosphatidylcholine/analogs & derivatives , 1,2-Dipalmitoylphosphatidylcholine/chemistry , 1,2-Dipalmitoylphosphatidylcholine/metabolism , Animals , Biomimetics/methods , Calcification, Physiologic/physiology , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Collagen Type II/chemistry , Collagen Type II/metabolism , Liposomes/chemistry , Liposomes/metabolism , Membrane Fluidity/physiology , Membrane Lipids/chemistry , Membrane Lipids/metabolism , Microscopy, Atomic Force/methods , Rats , Serine/chemistry , Serine/metabolismABSTRACT
Agonists of mouse STING (TMEM173) shrink and even cure solid tumors by activating innate immunity; human STING (hSTING) agonists are needed to test this therapeutic hypothesis in humans. The endogenous STING agonist is 2'3'-cGAMP, a second messenger that signals the presence of cytosolic double-stranded DNA. We report activity-guided partial purification and identification of ecto-nucleotide pyrophosphatase/phosphodiesterase (ENPP1) to be the dominant 2'3'-cGAMP hydrolyzing activity in cultured cells. The hydrolysis activity of ENPP1 was confirmed using recombinant protein and was depleted in tissue extracts and plasma from Enpp1(-/-) mice. We synthesized a hydrolysis-resistant bisphosphothioate analog of 2'3'-cGAMP (2'3'-cG(s)A(s)MP) that has similar affinity for hSTING in vitro and is ten times more potent at inducing IFN-ß secretion from human THP1 monocytes. Studies in mouse Enpp1(-/-) lung fibroblasts indicate that resistance to hydrolysis contributes substantially to its higher potency. 2'3'-cG(s)A(s)MP is therefore improved over natural 2'3'-cGAMP as a model agonist and has potential as a vaccine adjuvant and cancer therapeutic.
Subject(s)
Antineoplastic Agents/pharmacology , Gene Expression Regulation, Neoplastic , Membrane Proteins/agonists , Nucleotides, Cyclic/metabolism , Organothiophosphorus Compounds/pharmacology , Pyrophosphatases/antagonists & inhibitors , Animals , Antineoplastic Agents/chemical synthesis , Cell Line, Tumor , Humans , Hydrolysis , Interferon-beta , Membrane Proteins/deficiency , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Monocytes/drug effects , Monocytes/metabolism , Monocytes/pathology , Nucleotides, Cyclic/chemistry , Nucleotides, Cyclic/pharmacology , Organothiophosphorus Compounds/chemical synthesis , Phosphoric Diester Hydrolases/genetics , Phosphoric Diester Hydrolases/metabolism , Pyrophosphatases/genetics , Pyrophosphatases/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Second Messenger Systems/genetics , Signal TransductionABSTRACT
Previous work has shown that acidosis prevents bone nodule formation by osteoblasts in vitro by inhibiting mineralisation of the collagenous matrix. The ratio of phosphate (Pi ) to pyrophosphate (PPi ) in the bone microenvironment is a fundamental regulator of bone mineralisation. Both Pi and PPi , a potent inhibitor of mineralisation, are generated from extracellular nucleotides by the actions of ecto-nucleotidases. This study investigated the expression and activity of ecto-nucleotidases by osteoblasts under normal and acid conditions. We found that osteoblasts express mRNA for a number of ecto-nucleotidases including NTPdase 1-6 (ecto-nucleoside triphosphate diphosphohydrolase) and NPP1-3 (ecto-nucleotide pyrophosphatase/phosphodiesterase). The rank order of mRNA expression in differentiating rat osteoblasts (day 7) was Enpp1 > NTPdase 4 > NTPdase 6 > NTPdase 5 > alkaline phosphatase > ecto-5-nucleotidase > Enpp3 > NTPdase 1 > NTPdase 3 > Enpp2 > NTPdase 2. Acidosis (pH 6.9) upregulated NPP1 mRNA (2.8-fold) and protein expression at all stages of osteoblast differentiation compared to physiological pH (pH 7.4); expression of other ecto-nucleotidases was unaffected. Furthermore, total NPP activity was increased up to 53% in osteoblasts cultured in acid conditions (P < 0.001). Release of ATP, one of the key substrates for NPP1, from osteoblasts, was unaffected by acidosis. Further studies showed that mineralised bone formation by osteoblasts cultured from NPP1 knockout mice was increased compared with wildtypes (2.5-fold, P < 0.001) and was partially resistant to the inhibitory effect of acidosis. These results indicate that increased NPP1 expression and activity might contribute to the decreased mineralisation observed when osteoblasts are exposed to acid conditions.
Subject(s)
Acidosis/metabolism , Osteoblasts/enzymology , Phosphoric Diester Hydrolases/metabolism , Pyrophosphatases/metabolism , Acidosis/genetics , Acidosis/pathology , Adenosine Triphosphate/metabolism , Animals , Animals, Newborn , Bone Density , Cells, Cultured , Gene Expression Regulation, Enzymologic , Hydrogen-Ion Concentration , Mice, 129 Strain , Mice, Knockout , Osteoblasts/pathology , Osteogenesis , Phosphoric Diester Hydrolases/deficiency , Phosphoric Diester Hydrolases/genetics , Pyrophosphatases/deficiency , Pyrophosphatases/genetics , RNA, Messenger/metabolism , Rats, Sprague-Dawley , Time Factors , Up-RegulationABSTRACT
Plasma levels of pyrophosphate, an endogenous inhibitor of vascular calcification, are reduced in end-stage renal disease and correlate inversely with arterial calcification. However, it is not known whether the low plasma levels are directly pathogenic or are merely a marker of reduced tissue levels. This was tested in an animal model in which aortas were transplanted between normal mice and Enpp1(-/-) mice lacking ectonucleotide pyrophosphatase phosphodiesterase, the enzyme that synthesizes extracellular pyrophosphate. Enpp1(-/-) mice had very low plasma pyrophosphate and developed aortic calcification by 2 months that was greatly accelerated with a high-phosphate diet. Aortas of Enpp1(-/-) mice showed no further calcification after transplantation into wild-type mice fed a high-phosphate diet. Aorta allografts of wild-type mice calcified in Enpp1(-/-) mice but less so than the adjacent recipient Enpp1(-/-) aorta. Donor and recipient aortic calcium contents did not differ in transplants between wild-type and Enpp1(-/-) mice, demonstrating that transplantation per se did not affect calcification. Histology revealed medial calcification with no signs of rejection. Thus, normal levels of extracellular pyrophosphate are sufficient to prevent vascular calcification, and systemic Enpp1 deficiency is sufficient to produce vascular calcification despite normal vascular extracellular pyrophosphate production. This establishes an important role for circulating extracellular pyrophosphate in preventing vascular calcification.
Subject(s)
Aorta/metabolism , Aortic Diseases/blood , Diphosphates/blood , Vascular Calcification/blood , Animals , Aorta/pathology , Aorta/transplantation , Aortic Diseases/genetics , Aortic Diseases/pathology , Aortic Diseases/prevention & control , Calcium/metabolism , Disease Models, Animal , Disease Progression , Mice, Inbred C57BL , Mice, Knockout , Phosphoric Diester Hydrolases/deficiency , Phosphoric Diester Hydrolases/genetics , Phosphorus, Dietary/adverse effects , Pyrophosphatases/deficiency , Pyrophosphatases/genetics , Time Factors , Vascular Calcification/genetics , Vascular Calcification/pathology , Vascular Calcification/prevention & controlABSTRACT
OBJECTIVE: To determine the role of intestinal alkaline phosphatase (IAP) in enteral starvation-induced gut barrier dysfunction and to study its therapeutic effect as a supplement to prevent gut-derived sepsis. BACKGROUND: Critically ill patients are at increased risk for systemic sepsis and, in some cases, multiorgan failure leading to death. Years ago, the gut was identified as a major source for this systemic sepsis syndrome. Previously, we have shown that IAP detoxifies bacterial toxins, prevents endotoxemia, and preserves intestinal microbiotal homeostasis. METHODS: WT and IAP-KO mice were used to examine gut barrier function and tight junction protein levels during 48-hour starvation and fed states. Human ileal fluid samples were collected from 20 patients postileostomy and IAP levels were compared between fasted and fed states. To study the effect of IAP supplementation on starvation-induced gut barrier dysfunction, WT mice were fasted for 48 hours +/- IAP supplementation in the drinking water. RESULTS: The loss of IAP expression is associated with decreased expression of intestinal junctional proteins and impaired barrier function. For the first time, we demonstrate that IAP expression is also decreased in humans who are deprived of enteral feeding. Finally, our data demonstrate that IAP supplementation reverses the gut barrier dysfunction and tight junction protein losses due to a lack of enteral feeding. CONCLUSIONS: IAP is a major regulator of gut mucosal permeability and is able to ameliorate starvation-induced gut barrier dysfunction. Enteral IAP supplementation may represent a novel approach to maintain bowel integrity in critically ill patients.
Subject(s)
Alkaline Phosphatase/administration & dosage , Alkaline Phosphatase/metabolism , Critical Illness , Dietary Supplements , Intestinal Mucosa/enzymology , Systemic Inflammatory Response Syndrome/prevention & control , Administration, Oral , Animals , Enteral Nutrition , Humans , Ileum/enzymology , Ileum/immunology , Inflammation/enzymology , Jejunum/enzymology , Jejunum/immunology , Mice , Permeability , Starvation , Tight Junction Proteins/metabolism , Up-RegulationABSTRACT
As broadly demonstrated for the formation of a functional skeleton, proper mineralization of periodontal alveolar bone and teeth - where calcium phosphate crystals are deposited and grow within an extracellular matrix - is essential for dental function. Mineralization defects in tooth dentin and cementum of the periodontium invariably lead to a weak (soft or brittle) dentition in which teeth become loose and prone to infection and are lost prematurely. Mineralization of the extremities of periodontal ligament fibers (Sharpey's fibers) where they insert into tooth cementum and alveolar bone is also essential for the function of the tooth-suspensory apparatus in occlusion and mastication. Molecular determinants of mineralization in these tissues include mineral ion concentrations (phosphate and calcium), pyrophosphate, small integrin-binding ligand N-linked glycoproteins and matrix vesicles. Amongst the enzymes important in regulating these mineralization determinants, two are discussed at length here, with clinical examples given, namely tissue-nonspecific alkaline phosphatase and phosphate-regulating gene with homologies to endopeptidases on the X chromosome. Inactivating mutations in these enzymes in humans and in mouse models lead to the soft bones and teeth characteristic of hypophosphatasia and X-linked hypophosphatemia, respectively, where the levels of local and systemic circulating mineralization determinants are perturbed. In X-linked hypophosphatemia, in addition to renal phosphate wasting causing low circulating phosphate levels, phosphorylated mineralization-regulating small integrin-binding ligand N-linked glycoproteins, such as matrix extracellular phosphoglycoprotein and osteopontin, and the phosphorylated peptides proteolytically released from them, such as the acidic serine- and aspartate-rich-motif peptide, may accumulate locally to impair mineralization in this disease.
Subject(s)
Alveolar Process/physiology , Calcification, Physiologic/physiology , Dental Enamel Proteins/physiology , Extracellular Matrix/physiology , Familial Hypophosphatemic Rickets/physiopathology , Hypophosphatasia/physiopathology , Periodontal Ligament/physiology , Alkaline Phosphatase/physiology , Alveolar Process/enzymology , Animals , Calcium Phosphates/metabolism , Diphosphates/metabolism , Disease Models, Animal , Endopeptidases/physiology , Extracellular Matrix/enzymology , Humans , Periodontal Ligament/enzymologyABSTRACT
Under conditions of starvation and disease, the gut barrier becomes impaired, and trophic feeding to prevent gut mucosal atrophy has become a standard treatment of critically ill patients. However, the mechanisms responsible for the beneficial effects of enteral nutrition have remained a mystery. Using in vitro and in vivo models, we demonstrate that the brush-border enzyme, intestinal alkaline phosphatase (IAP), has the ability to detoxify lipopolysaccharide and prevent bacterial invasion across the gut mucosal barrier. IAP expression and function are lost with starvation and maintained by enteral feeding. It is likely that the IAP silencing that occurs during starvation is a key component of the gut mucosal barrier dysfunction seen in critically ill patients.
Subject(s)
Antigens, Neoplasm/physiology , Enteral Nutrition , Intestinal Mucosa/immunology , Alkaline Phosphatase , Animals , Antigens, Neoplasm/immunology , Bacterial Translocation , Cell Line , Critical Care , Critical Illness , GPI-Linked Proteins , Humans , Immunity, Innate , Intestines/enzymology , Mice , Mice, Knockout , Microvilli/enzymology , Starvation/immunologyABSTRACT
OBJECTIVE: To study the association of polymorphisms in the genes encoding peroxisome proliferator-activated receptors (PPARs) with the polycystic ovary syndrome (PCOS). DESIGN: Case-control study and meta-analysis of published evidence. PATIENTS: One hundred and sixty-one polycystic ovary syndrome patients and 113 non-hyperandrogenic women. MEASUREMENTS: Genotyping for PPAR-gamma coactivator-1 gene (PPARGC1A) Gly482Ser, PPAR-alpha Leu162Val, PPAR-delta rs2267668A/G, PPAR-delta-87T/C, PPAR-gamma2 Pro12Ala and PPAR-gamma2 -681C/G variants and systematic review of the literature using the Entrez-PubMed search engine, followed by meta-analysis whenever possible. RESULTS: Polycystic ovary syndrome patients carried the Gly482Ser variant in PPARGC1A more frequently than controls (72%vs. 58%, chi(2 )=( )5.54 P = 0.019), whereas carriers of the PPAR-alpha Leu162Val, PPAR-delta rs2267668A/G, PPAR-delta-87T/C, PPAR-gamma2 Pro12Ala and PPAR-gamma2 -681C/G variants were distributed similarly among both groups. The interaction between the PPARGC1A Gly482Ser and PPAR-delta-87T/C variants was also associated with PCOS (OR = 1.24, 95% CI 1.05-1.50, P = 0.008). The systematic review identified 31 studies addressing associations between PPARs variants and PCOS; meta-analysis was possible for nine studies focusing on the PPAR-gamma2 Pro12Ala variant. Although the individual studies did not reveal any statistically significant association, meta-analysis uncovered that carrying the PPAR-gamma2 Pro12Ala variant was associated with a reduced probability of having PCOS (OR = 0.77, 95% CI 0.61-0.96, P = 0.025), and that this association may be mediated by an effect on insulin sensitivity. CONCLUSIONS: Common polymorphisms in the PPARGC1A, PPAR-delta and PPAR-gamma2 loci are associated with PCOS.
Subject(s)
Peroxisome Proliferator-Activated Receptors/genetics , Polycystic Ovary Syndrome/genetics , Adult , Case-Control Studies , Female , Genetic Variation , Humans , Insulin Resistance/genetics , Phenotype , Spain , Young AdultABSTRACT
Individuals who are at risk for autosomal dominant polycystic kidney disease are often screened by ultrasound using diagnostic criteria derived from individuals with mutations in PKD1. Families with mutations in PKD2 typically have less severe disease, suggesting a potential need for different diagnostic criteria. In this study, 577 and 371 at-risk individuals from 58 PKD1 and 39 PKD2 families, respectively, were assessed by renal ultrasound and molecular genotyping. Using sensitivity data derived from genetically affected individuals and specificity data derived from genetically unaffected individuals, various diagnostic criteria were compared. In addition, data sets were created to simulate the PKD1 and PKD2 case mix expected in practice to evaluate the performance of diagnostic criteria for families of unknown genotype. The diagnostic criteria currently in use performed suboptimally for individuals with mutations in PKD2 as a result of reduced test sensitivity. In families of unknown genotype, the presence of three or more (unilateral or bilateral) renal cysts is sufficient for establishing the diagnosis in individuals aged 15 to 39 y, two or more cysts in each kidney is sufficient for individuals aged 40 to 59 y, and four or more cysts in each kidney is required for individuals > or = 60 yr. Conversely, fewer than two renal cysts in at-risk individuals aged > or = 40 yr is sufficient to exclude the disease. These unified diagnostic criteria will be useful for testing individuals who are at risk for autosomal dominant polycystic kidney disease in the usual clinical setting in which molecular genotyping is seldom performed.
Subject(s)
Polycystic Kidney, Autosomal Dominant/diagnostic imaging , TRPP Cation Channels/genetics , Adolescent , Adult , Genotype , Humans , Middle Aged , Mutation , Polycystic Kidney, Autosomal Dominant/genetics , UltrasonographyABSTRACT
The genetic mechanisms underlying functional hyperandrogenism and the polycystic ovary syndrome (PCOS) remain largely unknown. Given the large number of genetic variants found in association with these disorders, the emerging picture is that of a complex multigenic trait in which environmental influences play an important role in the expression of the hyperandrogenic phenotype. Among others, genomic variants in genes related to the regulation of androgen biosynthesis and function, insulin resistance, and the metabolic syndrome, and proinflammatory genotypes may be involved in the genetic predisposition to functional hyperandrogenism and PCOS. The elucidation of the molecular genetic basis of these disorders has been burdened by the heterogeneity in the diagnostic criteria used to define PCOS, the limited sample size of the studies conducted to date, and the lack of precision in the identification of ethnic and environmental factors that trigger the development of hyperandrogenic disorders. Progress in this area requires adequately sized multicenter collaborative studies after standardization of the diagnostic criteria used to classify hyperandrogenic patients, in whom modifying environmental factors such as ethnicity, diet, and lifestyle are identified with precision. In addition to classic molecular genetic techniques such as linkage analysis in the form of a whole-genome scan and large case-control studies, promising genomic and proteomic approaches will be paramount to our understanding of the pathogenesis of functional hyperandrogenism and PCOS, allowing a more precise prevention, diagnosis, and treatment of these prevalent disorders.
Subject(s)
Hyperandrogenism/genetics , Polycystic Ovary Syndrome/genetics , Androgens/genetics , Female , Genetic Linkage/physiology , Humans , Hyperandrogenism/metabolism , Insulin Resistance/genetics , Insulin Resistance/physiology , Polycystic Ovary Syndrome/metabolismABSTRACT
CONTEXT: The diagnosis of the polycystic ovary syndrome requires the exclusion of nonclassical congenital adrenal hyperplasia (NCAH). OBJECTIVE: Our objective was to evaluate the actual prevalences of 21-hydroxylase and 11beta-hydroxylase deficiencies among women presenting with hyperandrogenic complaints. SETTINGS: This study was performed at an academic hospital. PATIENTS: A total of 270 consecutive unselected women presenting with hyperandrogenic symptoms were prospectively recruited. INTERVENTIONS: Basal and ACTH-stimulated 11-deoxycortisol and 17-hydroxyprogesterone concentrations were measured. MAIN OUTCOME MEASURES: The prevalences of 21-hydroxylase and 11beta-hydroxylase deficiencies were calculated, and the diagnostic performance of basal serum 17-hydroxyprogesterone levels for the screening of NCAH was evaluated by receiver operating characteristic curve analysis. RESULTS: Six of the 270 patients had 21-hydroxylase-deficient NCAH that was confirmed by CYP21 genotyping, whereas no patient was diagnosed with 11beta-hydroxylase deficiency, for an overall NCAH prevalence of 2.2% (95% confidence limits 0.5-3.9%). According to receiver operating characteristic analysis, a single basal serum 17-hydroxyprogesterone determination has a 0.97 (95% confidence interval: 0.934-1.008) chance of detecting NCAH in hyperandrogenic women. In our experience, the most appropriate cutoff value for the detection of NCAH is a 17-hydroxyprogesterone above 1.7 ng/ml, showing a 100% sensitivity and a 88.6% specificity. Five of the six 21-hydroxylase-deficient NCAH patients carried a severe CYP21 allele requiring genetic counseling and highlighting the importance of excluding this disorder among hyperandrogenic patients. CONCLUSIONS: The prevalence of NCAH among hyperandrogenic patients from Spain is 2.2%. Basal serum 17-hydroxyprogesterone measurements have an excellent diagnostic performance, yet the cutoff value should be established in each laboratory to avoid false-negative results.
Subject(s)
Adrenal Hyperplasia, Congenital/epidemiology , Hyperandrogenism/epidemiology , 17-alpha-Hydroxyprogesterone/blood , Adolescent , Adrenal Hyperplasia, Congenital/blood , Adrenal Hyperplasia, Congenital/diagnosis , Adrenal Hyperplasia, Congenital/enzymology , Adult , Cortodoxone/blood , Diagnosis, Differential , Female , Humans , Hyperandrogenism/blood , Hyperandrogenism/diagnosis , Hyperandrogenism/enzymology , Polycystic Ovary Syndrome/blood , Polycystic Ovary Syndrome/diagnosis , Polycystic Ovary Syndrome/enzymology , Polycystic Ovary Syndrome/epidemiology , Prevalence , Prospective Studies , ROC Curve , Sensitivity and Specificity , Spain/epidemiology , Steroid 11-beta-Hydroxylase/metabolism , Steroid 21-Hydroxylase/metabolismABSTRACT
BACKGROUND: Our aim was to study the protein expression profiles of omental adipose tissue biopsies obtained from morbidly obese women with or without polycystic ovary syndrome (PCOS) at the time of bariatric surgery to evaluate the possible involvement of visceral adiposity in the development of PCOS. METHODS: Ten PCOS patients and nine control samples were included. We used two-dimensional difference gel electrophoresis (2D-DIGE) followed by in-gel digestion, and mass spectrometry (MS) of selected protein spots. RESULTS: The 2D-DIGE technology allowed the analysis of approximately 1840 protein spots in the comparative study of control and patient proteomes, revealing 15 statistically significant spot changes (>2-fold, P < 0.05). Unambiguous protein identification was achieved for 9 of these 15 spots by MS. This preliminary study revealed differences in expression of proteins that may be involved in lipid and glucose metabolism, oxidative stress processes and adipocyte differentiation; they include proapolipoprotein Apo-A1, annexin V, glutathione S-transferase M3 (GSTM3), triosephosphate isomerase, peroxiredoxin 2 isoform a, actin and adipocyte plasma membrane-associated protein. The most relevant finding was an increase of GSTM3 in the omental fat of PCOS patients confirming previous studies conducted by our group. CONCLUSIONS: Proteomic analysis of omental fat reveals differential expression of several proteins in PCOS patients and non-hyperandrogenic women presenting with morbid obesity. The application of this novel methodology adds further evidence to support the role of visceral adiposity in the pathogenesis of PCOS.
Subject(s)
Abdominal Fat/metabolism , Omentum/metabolism , Polycystic Ovary Syndrome/physiopathology , Proteomics , Adult , Annexin A5/metabolism , Electrophoresis, Gel, Two-Dimensional/methods , Female , Gene Expression Profiling , Glutathione Transferase/metabolism , Humans , Hydrogen-Ion Concentration , Obesity, Morbid/physiopathology , Oxidative Stress , Peroxiredoxins/metabolism , Polycystic Ovary Syndrome/etiology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tandem Mass SpectrometryABSTRACT
BACKGROUND: Previous studies addressing the changes in serum visfatin levels after bariatric surgery yielded conflicting results. METHODS: We measured serum visfatin levels in 41 morbidly obese women before bariatric surgery and after losing at least 15% of the initial weight, and analyzed the results taking into account the type of surgery, reproductive and diabetic status, among others. Body mass index, waist circumference, lipid profile, and insulin resistance determined by homeostasis model assessment (HOMA-IR) were also measured. RESULTS: Patients lost 30.3 +/- 6.1% of the initial body weight, and serum visfatin levels increased from 22.2 +/- 20.9 to 32.2 +/- 27.6 ng/ml (P = 0.031). A multiple regression model (R (2) = 0.314, F = 3.555, P = 0.017) including the percentage of weight loss, changes in waist circumference, HOMA-IR, high-density lipoprotein-cholesterol, and triglycerides (also expressed as percentage from baseline), the surgical procedure, time elapsed since surgery, and previous diabetic status as independent variables showed that weight loss (beta = -0.670, P = 0.010), previous diabetic status (beta = -0.330, P = 0.036), and change in waist circumference (beta = 0.556, P = 0.031) were the main determinants of the percentual increase in serum visfatin levels observed after bariatric surgery. CONCLUSION: Serum visfatin increased after bariatric surgery in relation to the amount of weight lost and to the changes in waist circumference, and this increase was higher in diabetic patients.