Body size and early growth in appropriate- and large-for-gestational-age infants.

AIMS: To study weight, length, body composition, sleeping energy expenditure (SEE), and respiratory quotient (RQ) at birth and at 5 mo of age in both adequate-for-gestational-age (AGA) and large-for-gestational-age (LGA) subjects; to compare the changes in body weight and body composition adjusting for gender, age, SEE, RQ and several maternal factors; to investigate the contribution of initial SEE and RQ to changes in body weight and body composition. METHODS: Sixty-nine neonates were recruited among term infants in the University Hospital of Verona, Italy. Forty-nine subjects participated until follow-up. At birth and follow-up, weight and length were measured and arm-fat area and arm-muscle area were calculated from triceps and subscapular skinfolds. SEE and RQ were measured by indirect calorimetry. RESULTS: At birth, weight, length, arm-muscle and arm-fat areas were significantly higher in LGA subjects than in AGA subjects. Weight status, SEE and RQ at birth did not explain the relative weight change after adjusting for gestational weight, placental weight, age at follow-up and gender. Arm-fat area and weight/length ratio at birth were negatively associated with relative changes in body weight after adjusting for the above variables (p < 0.05). CONCLUSION: Early growth from birth to 5 mo of life is significantly affected by body size and adiposity at birth. Fatter newborns had a slower growth rate than thinner newborns.

A microarray-based system for the simultaneous analysis of single nucleotide polymorphisms in human genes involved in the metabolism of anti-malarial drugs

Background: In order to provide a cost-effective tool to analyse pharmacogenetic markers in malaria treatment, DNA microarray technology was compared with sequencing of polymerase chain reaction (PCR) fragments to detect single nucleotide polymorphisms (SNPs) in a larger number of samples. Methods: The microarray was developed to affordably generate SNP data of genes encoding the human cytochrome P450 enzyme family (CYP) and N-acetyltransferase-2 (NAT2) involved in antimalarial drug metabolisms and with known polymorphisms, i.e. CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP3A5, and NAT2. Results: For some SNPs, i.e. CYP2A6*2, CYP2B6*5, CYP2C8*3, CYP2C9*3/*5, CYP2C19*3, CYP2D6*4 and NAT2*6/*7/*14, agreement between both techniques ranged from substantial to almost perfect (kappa index between 0.61 and 1.00), whilst for other SNPs a large variability from slight to substantial agreement (kappa index between 0.39 and 1.00) was found, e. g. CYP2D6*17 (2850C>T), CYP3A4*1B and CYP3A5*3. Conclusion: The major limit of the microarray technology for this purpose was lack of robustness and with a large number of missing data or with incorrect specificity.

PVHL is a regulator of glucose metabolism and insulin secretion in pancreatic beta cells.

Insulin secretion from pancreatic beta cells is stimulated by glucose metabolism. However, the relative importance of metabolizing glucose via mitochondrial oxidative phosphorylation versus glycolysis for insulin secretion remains unclear. von Hippel-Lindau (VHL) tumor suppressor protein, pVHL, negatively regulates hypoxia-inducible factor HIF1alpha, a transcription factor implicated in promoting a glycolytic form of metabolism. Here we report a central role for the pVHL-HIF1alpha pathway in the control of beta-cell glucose utilization, insulin secretion, and glucose homeostasis. Conditional inactivation of Vhlh in beta cells promoted a diversion of glucose away from mitochondria into lactate production, causing cells to produce high levels of glycolytically derived ATP and to secrete elevated levels of insulin at low glucose concentrations. Vhlh-deficient mice exhibited diminished glucose-stimulated changes in cytoplasmic Ca(2+) concentration, electrical activity, and insulin secretion, which culminate in impaired systemic glucose tolerance. Importantly, combined deletion of Vhlh and Hif1alpha rescued these phenotypes, implying that they are the result of HIF1alpha activation. Together, these results identify pVHL and HIF1alpha as key regulators of insulin secretion from pancreatic beta cells. They further suggest that changes in the metabolic strategy of glucose metabolism in beta cells have profound effects on whole-body glucose homeostasis.

Altered high-energy phosphate metabolism predicts contractile dysfunction and subsequent ventricular remodeling in pressure-overload hypertrophy mice.

To study the role of early energetic abnormalities in the subsequent development of heart failure, we performed serial in vivo combined magnetic resonance imaging (MRI) and (31)P magnetic resonance spectroscopy (MRS) studies in mice that underwent pressure-overload following transverse aorta constriction (TAC). After 3 wk of TAC, a significant increase in left ventricular (LV) mass (74 +/- 4 vs. 140 +/- 26 mg, control vs. TAC, respectively; P < 0.000005), size [end-diastolic volume (EDV): 48 +/- 3 vs. 61 +/- 8 microl; P < 0.005], and contractile dysfunction [ejection fraction (EF): 62 +/- 4 vs. 38 +/- 10%; P < 0.000005] was observed, as well as depressed cardiac energetics (PCr/ATP: 2.0 +/- 0.1 vs. 1.3 +/- 0.4, P < 0.0005) measured by combined MRI/MRS. After an additional 3 wk, LV mass (140 +/- 26 vs. 167 +/- 36 mg; P < 0.01) and cavity size (EDV: 61 +/- 8 vs. 76 +/- 8 microl; P < 0.001) increased further, but there was no additional decline in PCr/ATP or EF. Cardiac PCr/ATP correlated inversely with end-systolic volume and directly with EF at 6 wk but not at 3 wk, suggesting a role of sustained energetic abnormalities in evolving chamber dysfunction and remodeling. Indeed, reduced cardiac PCr/ATP observed at 3 wk strongly correlated with changes in EDV that developed over the ensuing 3 wk. These data suggest that abnormal energetics due to pressure overload predict subsequent LV remodeling and dysfunction.

Thermogenic effect of the new beta-adrenoreceptor agonist Ro 16-8714 in healthy male volunteers.

Previous investigations in experimental animals have shown that a new type of beta-adrenoceptor agonist (Ro 16-8714) possesses both thermogenic and antihyperglycemic properties. The aim of the study was to assess the thermogenic capacity of the compound in man after acute administration. Following an overnight fast three different doses (5, 10 and 20 mg) and a placebo were given per os to six normal-weight young men. The rate of energy expenditure (EE) and substrate utilization were determined by indirect calorimetry (hood system) before and for 6 h following the drug administration. Heart rate and blood pressure as well as plasma glucose, insulin and free fatty acid (FFA) concentrations were also measured at regular intervals throughout the study. The increment relative to base-line (mean +/- s.e.m.) in EE with placebo, 5, 10 and 20 mg was 4 +/- 3, 10 +/- 2, 11 +/- 2 and 21 +/- 2 percent respectively whereas heart rate was enhanced by 2 +/- 2, 8 +/- 3, 22 +/- 2, and 49 +/- 8 percent. Systolic blood pressure increased less (1 +/- 2, 8 +/- 1, 11 +/- 1 and 13 +/- 2 percent), and diastolic blood pressure did not change significantly. Simultaneously we observed a slight and transient increase in blood glucose, insulin and FFA concentrations. It is concluded that in lean individuals Ro 16-8714 is a potent thermogenic agent; however, new beta-adrenoceptor agonists should be developed in order to avoid the tachycardia associated with the thermogenic effect.

Circadian clock orchestration of signaling pathways influences mouse metabolism

Circadian clocks, present in organisms leaving in a rhythmic environment, constitute the mechanisms allowing anticipation and adaptation of behavior and physiology in response to these environmental variations. As a consequence, most aspects of metabolism and behavior are under the control of this circadian clock. At a molecular level, in all the studied species, the rhythmic expression of the genes involved are generated by interconnected transcriptional and translational feedback loops. In mammals, the heterodimer composed of BMAL1 and its partners CLOCK or NPAS2 constitutes a transcriptional activator regulating transcription of Per and Cry genes. These genes encode for repressors of the activity of BMAL1:CLOCK or BMAL1: NPAS2 heterodimers, thus closing a negative feedback loop that generates rhythms of approximately 24 hours. The aim of my doctoral work consisted in the investigation of the role of circadian clock in the regulation of different aspects of mouse metabolism through the rhythmic activation of signaling pathways. First, we showed that one way how the circadian clock exerts its function as an oscillator is through the regulation of mRNA translation. Indeed, we present evidence showing that circadian clock influences the temporal translation of a subset of mRNAs involved in ribosome biogenesis by controlling the transcription of translation initiation factors as well as the clock-dependent rhythmic activation of signaling pathways involved in their regulation. Moreover, the circadian oscillator regulates the transcription of ribosomal protein mRNAs and ribosomal RNAs. Thus the circadian clock exerts a major role in coordinating transcription and translation steps underlying ribosome biogenesis. In the second part, we showed the involvement of the circadian clock in lipid metabolism. Indeed, the three PAR bZip transcription factors DBP, TEF and HLF, are regulated by the molecular clock and play key roles in the control of lipid metabolism. Here we present evidence concerning the circadian expression and activity of PPARα via the circadian transcription of genes involved in the release of fatty acids, natural ligands of PPARα. It leads to the rhythmic activation of PPARα itself which could then play its role in the transcription of genes encoding proteins involved in lipid, cholesterol and glucose metabolism. In addition, we considered the possible role of lipid transporters, here SCP2, in the modulation of circadian activation of signaling pathways such as TORC1, PPARα and SREBP, linked to metabolism, and its feedback on the circadian clock. In the last part of this work, we studied the effects of these circadian clock-orchestrated pathways in physiology, as clock disruptions have been shown to be linked to metabolic disorders. We performed in vivo experiments on genetically and high-fat induced obese mice devoid of functional circadian clock. The results obtained showed that clock disruption leads to impaired triglycerides and glucose homeostasis in addition to insulin secretion and sensitivity. -- Les rythmes circadiens, présents chez tout organisme vivant dans un environnement rythmique, constituent l'ensemble de mécanismes permettant des réponses comportementales et physiologiques anticipées et adaptées aux variations environnementales. De ce fait, la plupart des aspects liés au métabolisme et au comportement de ces organismes apparaissent être sous le contrôle de l'horloge circadienne contrôlant ces rythmes. Au niveau moléculaire, dans toutes les espèces étudiées, l'expression rythmique de gènes impliqués sont générés par l'interconnexion de boucles de contrôle transcriptionnelles et traductionnelles. Chez les mammifères, l'hétérodimère composé de BMAL1 et de ses partenaires CLOCK ou NPAS2 constitue un activateur transcriptionnel régulant la transcription des gènes Per et Cry. Ces gènes codent pour des répresseurs de l'activité des hétérodimères BMAL1:CLOCK ou BMAL1:NPAS2. Cela a pour effet de fermer la boucle négative, générant ainsi des rythmes d'environ 24 heures. Le but de mon travail de thèse a consisté en l'investigation du rôle de l'horloge circadienne dans la régulation de certains aspects du métabolisme chez la souris via la régulation de l'activation rythmique des voies de signalisation. Nous avons tout d'abord montré que l'horloge circadienne exerce sa fonction d'oscillateur notamment au niveau de la régulation de la traduction des ARNm. En effet, nous présentons des preuves montrant que l'horloge circadienne influence la traduction temporelle d'un groupe d'ARNm impliqués dans la biogénèse des ribosomes en contrôlant la transcription de facteurs d'initiation de la traduction ainsi que l'activation rythmique des voies de signalisation qui sont impliquées dans leur régulation. De plus, l'oscillateur circadien régule la transcription d'ARNm codant pour les protéines ribosomales et d'ARN ribosomaux. De cette façon, l'horloge circadienne exerce un rôle majeur dans la coordination des étapes de transcription et traduction permettant la biogénèse des ribosomes. Dans la deuxième partie, nous montrons les implications de l'horloge circadienne dans le métabolisme des lipides. En effet, DBP, TEF et HLF, trois facteurs de transcription de la famille des PAR bZip qui sont régulés par l'horloge circadienne, jouent un rôle clé dans le contrôle du métabolisme des lipides par l'horloge circadienne. Nous apportons ici des preuves concernant l'expression et l'activité rythmiques de PPARα via la transcription circadienne de gènes impliqués dans le relargage d'acides gras, ligands naturels de PPARα, conduisant à l'activation circadienne de PPARα lui-même, pouvant ainsi jouer son rôle de facteur de transcription de gènes codant pour des protéines impliquées dans le métabolisme des lipides, du cholestérol et du glucose. De plus, nous nous sommes penchés sur le rôle possible de transporteurs de lipides, ici SCP2, dans la modulation de l'activation circadienne de voies de signalisation, telles que TORC1, PPARα et SREBP, qui sont liées au métabolisme, ainsi que son impact sur l'horloge elle-même. Dans la dernière partie de ce travail, nous avons étudié les effets de l'activation de ces voies de signalisation régulées par l'horloge circadienne dans le contexte physiologique puisqu'il a été montré que la perturbation de l'horloge pouvait être associée à des désordres métaboliques. Pour ce faire, nous avons fait des expériences in vivo sur des souris déficientes pour l'horloge moléculaire pour lesquelles l'obésité est induite génétiquement ou induite par la nourriture riche en lipides. Les résultats que nous obtenons montrent des dérèglements au niveau de l'homéostasie des triglycérides et du glucose ainsi que sur l'expression et la réponse à l'insuline.

Mutations in NDUFB11, Encoding a Complex I Component of the Mitochondrial Respiratory Chain, Cause Microphthalmia with Linear Skin Defects Syndrome.

Microphthalmia with linear skin defects (MLS) syndrome is an X-linked male-lethal disorder also known as MIDAS (microphthalmia, dermal aplasia, and sclerocornea). Additional clinical features include neurological and cardiac abnormalities. MLS syndrome is genetically heterogeneous given that heterozygous mutations in HCCS or COX7B have been identified in MLS-affected females. Both genes encode proteins involved in the structure and function of complexes III and IV, which form the terminal segment of the mitochondrial respiratory chain (MRC). However, not all individuals with MLS syndrome carry a mutation in either HCCS or COX7B. The majority of MLS-affected females have severe skewing of X chromosome inactivation, suggesting that mutations in HCCS, COX7B, and other as-yet-unidentified X-linked gene(s) cause selective loss of cells in which the mutated X chromosome is active. By applying whole-exome sequencing and filtering for X-chromosomal variants, we identified a de novo nonsense mutation in NDUFB11 (Xp11.23) in one female individual and a heterozygous 1-bp deletion in a second individual, her asymptomatic mother, and an affected aborted fetus of the subject's mother. NDUFB11 encodes one of 30 poorly characterized supernumerary subunits of NADH:ubiquinone oxidoreductase, known as complex I (cI), the first and largest enzyme of the MRC. By shRNA-mediated NDUFB11 knockdown in HeLa cells, we demonstrate that NDUFB11 is essential for cI assembly and activity as well as cell growth and survival. These results demonstrate that X-linked genetic defects leading to the complete inactivation of complex I, III, or IV underlie MLS syndrome. Our data reveal an unexpected role of cI dysfunction in a developmental phenotype, further underscoring the existence of a group of mitochondrial diseases associated with neurocutaneous manifestations.

A bovine model of a respiratory Parachlamydia acanthamoebae infection.

The aim of this study was to evaluate the pathogenicity of Parachlamydia (P.) acanthamoebae as a potential agent of lower respiratory tract disease in a bovine model of induced lung infection. Intrabronchial inoculation with P. acanthamoebae was performed in healthy calves aged 2-3 months using two challenge doses: 10(8) and 10(10) bacteria per animal. Controls received 10(8) heat-inactivated bacteria. Challenge with 10(8) viable Parachlamydia resulted in a mild degree of general indisposition, whereas 10(10) bacteria induced a more severe respiratory illness becoming apparent 1-2 days post inoculation (dpi), affecting 9/9 (100%) animals and lasting for 6 days. The extent of macroscopic pulmonary lesions was as high as 6.6 (6.0)% [median (range)] of lung tissue at 2-4 dpi and correlated with parachlamydial genomic copy numbers detected by PCR, and with bacterial load estimated by immunohistochemistry in lung tissue. Clinical outcome, acute phase reactants, pathological findings and bacterial load exhibited an initial dose-dependent effect on severity. Animals fully recovered from clinical signs of respiratory disease within 5 days. The bovine lung was shown to be moderately susceptible to P. acanthamoebae, exhibiting a transient pneumonic inflammation after intrabronchial challenge. Further studies are warranted to determine the precise pathophysiologic pathways of host-pathogen interaction.

Reduction of respiratory motion artifacts for free-breathing whole-heart coronary MRA by weighted iterative reconstruction.

PURPOSE: To combine weighted iterative reconstruction with self-navigated free-breathing coronary magnetic resonance angiography for retrospective reduction of respiratory motion artifacts. METHODS: One-dimensional self-navigation was improved for robust respiratory motion detection and the consistency of the acquired data was estimated on the detected motion. Based on the data consistency, the data fidelity term of iterative reconstruction was weighted to reduce the effects of respiratory motion. In vivo experiments were performed in 14 healthy volunteers and the resulting image quality of the proposed method was compared to a navigator-gated reference in terms of acquisition time, vessel length, and sharpness. RESULT: Although the sampling pattern of the proposed method contained 60% more samples with respect to the reference, the scan efficiency was improved from 39.5 ± 10.1% to 55.1 ± 9.1%. The improved self-navigation showed a high correlation to the standard navigator signal and the described weighting efficiently reduced respiratory motion artifacts. Overall, the average image quality of the proposed method was comparable to the navigator-gated reference. CONCLUSION: Self-navigated coronary magnetic resonance angiography was successfully combined with weighted iterative reconstruction to reduce the total acquisition time and efficiently suppress respiratory motion artifacts. The simplicity of the experimental setup and the promising image quality are encouraging toward future clinical evaluation. Magn Reson Med 73:1885-1895, 2015. © 2014 Wiley Periodicals, Inc.

Role of rhinovirus load in the upper respiratory tract and severity of symptoms in lung transplant recipients.

BACKGROUND: Rhinovirus is the most common cause of respiratory viral infections and leads to frequent respiratory symptoms in lung transplant recipients. However, it remains unknown whether the rhinovirus load correlates with the severity of symptoms. OBJECTIVES: This study aimed to better characterize the pathogenesis of rhinoviral infection and the way in which viral load correlates with symptoms. STUDY DESIGN: We assessed rhinovirus load in positive upper respiratory specimens of patients enrolled prospectively in a cohort of 116 lung transplant recipients. Rhinovirus load was quantified according to a validated in-house, real-time, reverse transcription polymerase chain reaction in pooled nasopharyngeal and pharyngeal swabs. Symptoms were recorded in a standardised case report form completed at each screening/routine follow-up visit, or during any emergency visit occurring during the 3-year study. RESULTS: Rhinovirus infections were very frequent, including in asymptomatic patients not seeking a specific medical consultation. Rhinovirus load ranged between 4.1 and 8.3 log copies/ml according to the type of visit and clinical presentation. Patients with highest symptom scores tended to have higher viral loads, particularly those presenting systemic symptoms. When considering symptoms individually, rhinovirus load was significantly higher in the presence of symptoms such as sore throat, fever, sputum production, cough, and fatigue. There was no association between tacrolimus levels and rhinovirus load. CONCLUSIONS: Rhinovirus infections are very frequent in lung transplant recipients and rhinoviral load in the upper respiratory tract is relatively high even in asymptomatic patients. Patients with the highest symptom scores tend to have a higher rhinovirus load.

Brain energy metabolism measured by (13) C magnetic resonance spectroscopy in vivo upon infusion of [3-(13) C]lactate.

The brain uses lactate produced by glycolysis as an energy source. How lactate originated from the blood stream is used to fuel brain metabolism is not clear. The current study measures brain metabolic fluxes and estimates the amount of pyruvate that becomes labeled in glial and neuronal compartments upon infusion of [3-(13) C]lactate. For that, labeling incorporation into carbons of glutamate and glutamine was measured by (13) C magnetic resonance spectroscopy at 14.1 T and analyzed with a two-compartment model of brain metabolism to estimate rates of mitochondrial oxidation, glial pyruvate carboxylation, and the glutamate-glutamine cycle as well as pyruvate fractional enrichments. Extracerebral lactate at supraphysiological levels contributes at least two-fold more to replenish the neuronal than the glial pyruvate pools. The rates of mitochondrial oxidation in neurons and glia, pyruvate carboxylase, and glutamate-glutamine cycles were similar to those estimated by administration of (13) C-enriched glucose, the main fuel of brain energy metabolism. These results are in agreement with primary utilization of exogenous lactate in neurons rather than astrocytes. © 2014 Wiley Periodicals, Inc.

A Cellular Perspective on Brain Energy Metabolism and Functional Imaging.

The energy demands of the brain are high: they account for at least 20% of the body's energy consumption. Evolutionary studies indicate that the emergence of higher cognitive functions in humans is associated with an increased glucose utilization and expression of energy metabolism genes. Functional brain imaging techniques such as fMRI and PET, which are widely used in human neuroscience studies, detect signals that monitor energy delivery and use in register with neuronal activity. Recent technological advances in metabolic studies with cellular resolution have afforded decisive insights into the understanding of the cellular and molecular bases of the coupling between neuronal activity and energy metabolism and point at a key role of neuron-astrocyte metabolic interactions. This article reviews some of the most salient features emerging from recent studies and aims at providing an integration of brain energy metabolism across resolution scales.