Multimeric complexes of the PML-retinoic acid receptor alpha fusion protein in acute promyelocytic leukemia cells and interference with retinoid and peroxisome-proliferator signaling pathways.

The t(15;17) chromosomal translocation, specific for acute promyelocytic leukemia (APL), fuses the PML gene to the retinoic acid receptor alpha (RAR alpha) gene, resulting in expression of a PML-RAR alpha hybrid protein. In this report, we analyzed the nature of PML-RAR alpha-containing complexes in nuclear protein extracts of t(15;17)-positive cells. We show that endogenous PML-RAR alpha can bind to DNA as a homodimer, in contrast to RAR alpha that requires the retinoid X receptor (RXR) dimerization partner. In addition, these cells contain oligomeric complexes of PML-RAR alpha and endogenous RXR. Treatment with retinoic acid results in a decrease of PML-RAR alpha protein levels and, as a consequence, of DNA binding by the different complexes. Using responsive elements from various hormone signaling pathways, we show that PML-RAR alpha homodimers have altered DNA-binding characteristics when compared to RAR alpha-RXR alpha heterodimers. In transfected Drosophila SL-3 cells that are devoid of endogenous retinoid receptors PML-RAR alpha inhibits transactivation by RAR alpha-RXR alpha heterodimers in a dominant fashion. In addition, we show that both normal retinoid receptors and the PML-RAR alpha hybrid bind and activate the peroxisome proliferator-activated receptor responsive element from the Acyl-CoA oxidase gene, indicating that retinoids and peroxisome proliferator receptors may share common target genes. These properties of PML-RAR alpha may contribute to the transformed phenotype of APL cells.

The protein phosphatase 7 regulates phytochrome signaling in Arabidopsis.

The psi2 mutant of Arabidopsis displays amplification of the responses controlled by the red/far red light photoreceptors phytochrome A (phyA) and phytochrome B (phyB) but no apparent defect in blue light perception. We found that loss-of-function alleles of the protein phosphatase 7 (AtPP7) are responsible for the light hypersensitivity in psi2 demonstrating that AtPP7 controls the levels of phytochrome signaling. Plants expressing reduced levels of AtPP7 mRNA display reduced blue-light induced cryptochrome signaling but no noticeable deficiency in phytochrome signaling. Our genetic analysis suggests that phytochrome signaling is enhanced in the AtPP7 loss of function alleles, including in blue light, which masks the reduced cryptochrome signaling. AtPP7 has been found to interact both in yeast and in planta assays with nucleotide-diphosphate kinase 2 (NDPK2), a positive regulator of phytochrome signals. Analysis of ndpk2-psi2 double mutants suggests that NDPK2 plays a critical role in the AtPP7 regulation of the phytochrome pathway and identifies NDPK2 as an upstream element involved in the modulation of the salicylic acid (SA)-dependent defense pathway by light. Thus, cryptochrome- and phytochrome-specific light signals synchronously control their relative contribution to the regulation of plant development. Interestingly, PP7 and NDPK are also components of animal light signaling systems.

Is the formula of Traub still up to date in antemortem blood glucose level estimation?

According to the hypothesis of Traub, also known as the 'formula of Traub', postmortem values of glucose and lactate found in the cerebrospinal fluid or vitreous humor are considered indicators of antemortem blood glucose levels. However, because the lactate concentration increases in the vitreous and cerebrospinal fluid after death, some authors postulated that using the sum value to estimate antemortem blood glucose levels could lead to an overestimation of the cases of glucose metabolic disorders with fatal outcomes, such as diabetic ketoacidosis. The aim of our study, performed on 470 consecutive forensic cases, was to ascertain the advantages of the sum value to estimate antemortem blood glucose concentrations and, consequently, to rule out fatal diabetic ketoacidosis as the cause of death. Other biochemical parameters, such as blood 3-beta-hydroxybutyrate, acetoacetate, acetone, glycated haemoglobin and urine glucose levels, were also determined. In addition, postmortem native CT scan, autopsy, histology, neuropathology and toxicology were performed to confirm diabetic ketoacidosis as the cause of death. According to our results, the sum value does not add any further information for the estimation of antemortem blood glucose concentration. The vitreous glucose concentration appears to be the most reliable marker to estimate antemortem hyperglycaemia and, along with the determination of other biochemical markers (such as blood acetone and 3-beta-hydroxybutyrate, urine glucose and glycated haemoglobin), to confirm diabetic ketoacidosis as the cause of death.

Association of N-acetylaspartate and cerebrospinal fluid Aβ42 in dementia.

The interplay of amyloid and mitochondrial function is considered crucial in the pathophysiology of Alzheimer's disease (AD). We tested the association of the putative marker of mitochondrial function N-acetylaspartate (NAA) as measured by proton magnetic resonance spectroscopy within the medial temporal lobe and cerebrospinal fluid amyoid-β42 (Aβ42), total Tau and pTau181. 109 patients were recruited in a multicenter study (40 mild AD patients, 14 non-AD dementia patients, 29 mild cognitive impairment (MCI) AD-type patients, 26 MCI of non-AD type patients). NAA correlated with Aβ42 within the AD group. Since the NAA concentration is coupled to neuronal mitochondrial function, the correlation between NAA and Aβ42 may reflect the interaction between disrupted mitochondrial pathways and amyloid production.

Oligodendroglia metabolically support axons and contribute to neurodegeneration.

Oligodendroglia support axon survival and function through mechanisms independent of myelination, and their dysfunction leads to axon degeneration in several diseases. The cause of this degeneration has not been determined, but lack of energy metabolites such as glucose or lactate has been proposed. Lactate is transported exclusively by monocarboxylate transporters, and changes to these transporters alter lactate production and use. Here we show that the most abundant lactate transporter in the central nervous system, monocarboxylate transporter 1 (MCT1, also known as SLC16A1), is highly enriched within oligodendroglia and that disruption of this transporter produces axon damage and neuron loss in animal and cell culture models. In addition, this same transporter is reduced in patients with, and in mouse models of, amyotrophic lateral sclerosis, suggesting a role for oligodendroglial MCT1 in pathogenesis. The role of oligodendroglia in axon function and neuron survival has been elusive; this study defines a new fundamental mechanism by which oligodendroglia support neurons and axons.

Sweet sixteen for ANLS.

Since its introduction 16 years ago, the astrocyte-neuron lactate shuttle (ANLS) model has profoundly modified our understanding of neuroenergetics by bringing a cellular and molecular resolution. Praised or disputed, the concept has never ceased to attract attention, leading to critical advances and unexpected insights. Here, we summarize recent experimental evidence further supporting the main tenets of the model. Thus, evidence for distinct metabolic phenotypes between neurons (mainly oxidative) and astrocytes (mainly glycolytic) have been provided by genomics and classical metabolic approaches. Moreover, it has become clear that astrocytes act as a syncytium to distribute energy substrates such as lactate to active neurones. Glycogen, the main energy reserve located in astrocytes, is used as a lactate source to sustain glutamatergic neurotransmission and synaptic plasticity. Lactate is also emerging as a neuroprotective agent as well as a key signal to regulate blood flow. Characterization of monocarboxylate transporter regulation indicates a possible involvement in synaptic plasticity and memory. Finally, several modeling studies captured the implications of such findings for many brain functions. The ANLS model now represents a useful, experimentally based framework to better understand the coupling between neuronal activity and energetics as it relates to neuronal plasticity, neurodegeneration, and functional brain imaging.

Uric acid is a danger signal activating NALP3 inflammasome in lung injury inflammation and fibrosis.

RATIONALE: Lung injury leads to pulmonary inflammation and fibrosis through myeloid differentiation primary response gene 88 (MyD88) and the IL-1 receptor 1 (IL-1R1) signaling pathway. The molecular mechanisms by which lung injury triggers IL-1beta production, inflammation, and fibrosis remain poorly understood. OBJECTIVES: To determine if lung injury depends on the NALP3 inflammasome and if bleomycin (BLM)-induced lung injury triggers local production of uric acid, thereby activating the NALP3 inflammasome in the lung. Methods: Inflammation upon BLM administration was evaluated in vivo in inflammasome-deficient mice. Pulmonary uric acid accumulation, inflammation, and fibrosis were analyzed in mice treated with the inhibitor of uric acid synthesis or with uricase, which degrades uric acid. MEASUREMENTS AND MAIN RESULTS: Lung injury depends on the NALP3 inflammasome, which is triggered by uric acid locally produced in the lung upon BLM-induced DNA damage and degradation. Reduction of uric acid levels using the inhibitor of uric acid synthesis allopurinol or uricase leads to a decrease in BLM-induced IL-1beta production, lung inflammation, repair, and fibrosis. Local administration of exogenous uric acid crystals recapitulates lung inflammation and repair, which depend on the NALP3 inflammasome, MyD88, and IL-1R1 pathways and Toll-like receptor (TLR)2 and TLR4 for optimal inflammation but are independent of the IL-18 receptor. CONCLUSIONS: Uric acid released from injured cells constitutes a major endogenous danger signal that activates the NALP3 inflammasome, leading to IL-1beta production. Reducing uric acid tissue levels represents a novel therapeutic approach to control IL-1beta production and chronic inflammatory lung pathology.

Angiotensin II favors progression to heart failure in diabetic hearts: role of myocardial fatty acid oxidation downregulation

Purpose: Diabetic myocardium is particularly vulnerable to develop heart failure in response to chronic stress conditions including hypertension or myocardial infarction. We have recently observed that angiotensin II (Ang II)-mediated downregulation of the fatty acid oxidation pathway favors occurrence of heart failure by myocardial accumulation of lipids (lipotoxicity). Because diabetic heart is exposed to high levels of circulating fatty acid, we determined whether insulin resistance favors development of heart failure in mice with Ang II-mediated myocardial remodeling.Methods: To study the combined effect of diabetes and Ang II-induced heart remodeling, we generated leptin-deficient/insulin resistant (Lepob/ob) mice with cardiac targeted overexpression of angiotensinogen (TGAOGN). Left ventricular (LV) failure was indicated by pulmonary congestion (lung weight/tibial length>+2SD of wild-type mice). Myocardial metabolism and function were assessed during in vitro isolated working heart perfusion.Results: Forty-eight percent of TGAOGN mice without insulin resistance exhibited pulmonary congestion at the age of 6 months associated with increased myocardial BNP expression (+375% compared with WT) and reduced LV power (developed pressure x cardiac output; -15%). The proportion of mice presenting heart failure was markedly increased to 71% in TGAOGN mice with insulin resistance (TGAOGN/Lepob/ob). TGAOGN/Lepob/ob mice with heart failure exhibited further increase of BNP compared with failing non-diabetic TGAOGN mice (+146%) and further reduction of cardiac power (-59%). Mice with insulin resistance alone (Lepob/ob) did not exhibit signs of heart failure or LV dysfunction. Myocardial fatty acid oxidation measured during in vitro perfusion was markedly increased in non-failing hearts from Lepob/ob mice (+380% compared with WT) and glucose oxidation decreased (-72%). In contrast, fatty acid and glucose oxidation did not differ from Lepob/ob mice in hearts from TGAOGN/Lepob/ob mice without heart failure. However, both fatty acid and glucose oxidation were markedly decreased (-47% and -48%, respectively, compared with WT/Lepob/+) in failing hearts from TGAOGN/Lepob/ob mice. Reduction of fatty acid oxidation was associated with marked reduction of protein expression of a number of regulatory enzymes implied in fatty acid oxidation.Conclusions: Insulin resistance favors the progression to heart failure during chronic exposure of the myocardium to Ang II. Our results are compatible with a role of Ang II-mediated downregulation of fatty acid oxidation, potentially promoting lipotoxicity.

Enteral versus parenteral nutrition: comparison of energy metabolism in healthy subjects.

Continuous respiratory exchange measurements were performed on 10 healthy young women for 1 h before, 3 h during, and 3 h after either parenteral (iv) or intragastric (ig) administration of a nutrient mixture (52% glucose, 18% amino acid, and 30% lipid energy) infused at twice the postabsorptive resting energy expenditure (REE). REE rose from 0.98 +/- 0.02 (iv) and 0.99 +/- 0.02 kcal/min (ig) postabsorptively to 1.13 +/- 0.03 (iv) and 1.13 +/- 0.02 kcal/min (ig), resulting in nutrient-induced thermogenesis of 10 +/- 0.6 and 9.3 +/- 0.9%, respectively, when related to the metabolizable energy. The respiratory quotient rose from preinfusion values of 0.81 +/- 0.02 (iv) and 0.80 +/- 0.01 (ig) to 0.86 +/- 0.01 (iv) and 0.85 +/- 0.01 (ig). After nutrient administration the respiratory quotient fell significantly to below the preinfusion values. Plasma glucose and insulin concentrations rose during nutrient administration but were higher during the intravenous route. It is concluded that, although the response time to intragastric administration was delayed, the thermic effects and overall substrate oxidations were comparable during intravenous or intragastric administration, albeit, at lower plasma glucose and insulin concentrations via the intragastric route.

NEUROPATHOPHYSIOLOGICAL MECHANISMS IN GLUTARIC ACIDURIA TYPE I: 3-HYDROXYGLUTARIC ACID LEADS TO AMMONIA INCREASE AND NON-APOPTOTIC CELL DEATH

A 3D in vitro model of rat organotypic brain cell cultures in aggregates was used to investigate neurotoxicity mechanisms in glutaric aciduria type I (GA-I). 1 mM glutarate (GA) or 3-hydroxyglutarate (3OHGA) were repeatedly added to the culture media at two different time points. In cultures treated with 3OHGA, we observed an increase in lactate in the medium, pointing to a possible inhibition of Krebs cycle and respiratory chain. We further observed that 3OHGA and to a lesser extend GA induced an increase in ammonia production with concomitant decrease of glutamine concentrations, which may suggest an inhibition of the astrocytic enzyme glutamine synthetase. These previously unreported findings may uncover a pathogenic mechanism in this disease which has deleterious effects on early stages of brain development. By immunohistochemistry we showed that 3OHGA increased non-apoptotic cell death. On the cellular level, 3OHGA and to a lesser extend GA led to cell swelling and loss of astrocytic fibers whereas a loss of oligodendrocytes was only observed for 3OHGA. We conclude that 3OHGAwas the most toxic metabolite in our model for GA-I. 3OHGA induced deleterious effects on glial cells, an increase of ammonia production, and resulted in accentuated cell death of non-apoptotic origin.

Compartmentalized Cerebral Metabolism of [1,6-(13)C]Glucose Determined by in vivo (13)C NMR Spectroscopy at 14.1 T.

Cerebral metabolism is compartmentalized between neurons and glia. Although glial glycolysis is thought to largely sustain the energetic requirements of neurotransmission while oxidative metabolism takes place mainly in neurons, this hypothesis is matter of debate. The compartmentalization of cerebral metabolic fluxes can be determined by (13)C nuclear magnetic resonance (NMR) spectroscopy upon infusion of (13)C-enriched compounds, especially glucose. Rats under light α-chloralose anesthesia were infused with [1,6-(13)C]glucose and (13)C enrichment in the brain metabolites was measured by (13)C NMR spectroscopy with high sensitivity and spectral resolution at 14.1 T. This allowed determining (13)C enrichment curves of amino acid carbons with high reproducibility and to reliably estimate cerebral metabolic fluxes (mean error of 8%). We further found that TCA cycle intermediates are not required for flux determination in mathematical models of brain metabolism. Neuronal tricarboxylic acid cycle rate (V(TCA)) and neurotransmission rate (V(NT)) were 0.45 ± 0.01 and 0.11 ± 0.01 μmol/g/min, respectively. Glial V(TCA) was found to be 38 ± 3% of total cerebral oxidative metabolism, accounting for more than half of neuronal oxidative metabolism. Furthermore, glial anaplerotic pyruvate carboxylation rate (V(PC)) was 0.069 ± 0.004 μmol/g/min, i.e., 25 ± 1% of the glial TCA cycle rate. These results support a role of glial cells as active partners of neurons during synaptic transmission beyond glycolytic metabolism.

Effect of carbohydrate overfeeding on whole body macronutrient metabolism and expression of lipogenic enzymes in adipose tissue of lean and overweight humans

OBJECTIVE: Lipids stored in adipose tissue can originate from dietary lipids or from de novo lipogenesis (DNL) from carbohydrates. Whether DNL is abnormal in adipose tissue of overweight individuals remains unknown. The present study was undertaken to assess the effect of carbohydrate overfeeding on glucose-induced whole body DNL and adipose tissue lipogenic gene expression in lean and overweight humans. DESIGN: Prospective, cross-over study. SUBJECTS AND METHODS: A total of 11 lean (five male, six female, mean BMI 21.0+/-0.5 kg/m(2)) and eight overweight (four males, four females, mean BMI 30.1+/-0.6 kg/m(2)) volunteers were studied on two occasions. On one occasion, they received an isoenergetic diet containing 50% carbohydrate for 4 days prior to testing; on the other, they received a hyperenergetic diet (175% energy requirements) containing 71% carbohydrates. After each period of 4 days of controlled diet, they were studied over 6 h after having received 3.25 g glucose/kg fat free mass. Whole body glucose oxidation and net DNL were monitored by means of indirect calorimetry. An adipose tissue biopsy was obtained at the end of this 6-h period and the levels of SREBP-1c, acetyl CoA carboxylase, and fatty acid synthase mRNA were measured by real-time PCR. RESULTS: After isocaloric feeding, whole body net DNL amounted to 35+/-9 mg/kg fat free mass/5 h in lean subjects and to 49+/-3 mg/kg fat free mass/5 h in overweight subjects over the 5 h following glucose ingestion. These figures increased (P<0.001) to 156+/-21 mg/kg fat free mass/5 h in lean and 64+/-11 mg/kg fat free mass/5 h (P<0.05 vs lean) in overweight subjects after carbohydrate overfeeding. Whole body DNL after overfeeding was lower (P<0.001) and glycogen synthesis was higher (P<0.001) in overweight than in normal subjects. Adipose tissue SREBP-1c mRNA increased by 25% in overweight and by 43% in lean subjects (P<0.05) after carbohydrate overfeeding, whereas fatty acid synthase mRNA increased by 66 and 84% (P<0.05). CONCLUSION: Whole body net DNL is not increased during carbohydrate overfeeding in overweight individuals. Stimulation of adipose lipogenic enzymes is also not higher in overweight subjects. Carbohydrate overfeeding does not stimulate whole body net DNL nor expression of lipogenic enzymes in adipose tissue to a larger extent in overweight than lean subjects.

Analysis of the contribution of the β-oxidation auxiliary enzymes in the degradation of the dietary conjugated linoleic acid 9-cis-11-trans-octadecadienoic acid in the peroxisomes of Saccharomyces cerevisiae

Beta-oxidation of the conjugated linoleic acid 9-cis,11-trans-octadecadienoic acid (rumenic acid) was analyzed in vivo in Saccharomyces cerevisiae by monitoring polyhydroxyalkanoate production in the peroxisome. Polyhydroxyalkanoate is synthesized by the polymerization of the beta-oxidation intermediates 3-hydroxyacyl-CoAs via a bacterial polyhydroxyalkanoate synthase targeted to the peroxisome. The amount of polyhydroxyalkanaote synthesized from the degradation of rumenic acid was found to be similar to the amount synthesized from the degradation of 10-trans,12-cis-octadecadienoic acid, oleic acid or 10-cis-heptadecenoic acid. Furthermore, the degradation of 10-cis-heptadecenoic acid was found to be unaffected by the presence of rumenic acid in the media. Efficient degradation of rumenic acid was found to be independent of the Delta(3,5),Delta(2,4)-dienoyl-CoA isomerase but instead relied on the presence of Delta(3),Delta(2)-enoyl-CoA isomerase activity. The presence of the unsaturated monomer 3-hydroxydodecenoic acid in polyhydroxyalkanoate derived from rumenic acid degradation was found to be dependent on the presence of a Delta(3),Delta(2)-enoyl-CoA isomerase activity. Together, these data indicate that rumenic acid is mainly degraded in vivo in S. cerevisiae through a pathway requiring only the participation of the auxiliary enzymes Delta(3),Delta(2)-enoyl-CoA isomerase, along with the enzyme of the core beta-oxidation cycle.

Liver-specific loss of lipin-1-mediated phosphatidic acid phosphatase activity does not mitigate intrahepatic TG accumulation in mice.

Lipin proteins (lipin 1, 2, and 3) regulate glycerolipid homeostasis by acting as phosphatidic acid phosphohydrolase (PAP) enzymes in the TG synthesis pathway and by regulating DNA-bound transcription factors to control gene transcription. Hepatic PAP activity could contribute to hepatic fat accumulation in response to physiological and pathophysiological stimuli. To examine the role of lipin 1 in regulating hepatic lipid metabolism, we generated mice that are deficient in lipin-1-encoded PAP activity in a liver-specific manner (Alb-Lpin1(-/-) mice). This allele of lipin 1 was still able to transcriptionally regulate the expression of its target genes encoding fatty acid oxidation enzymes, and the expression of these genes was not affected in Alb-Lpin1(-/-) mouse liver. Hepatic PAP activity was significantly reduced in mice with liver-specific lipin 1 deficiency. However, hepatocytes from Alb-Lpin1(-/-) mice had normal rates of TG synthesis, and steady-state hepatic TG levels were unaffected under fed and fasted conditions. Furthermore, Alb-Lpin1(-/-) mice were not protected from intrahepatic accumulation of diacylglyerol and TG after chronic feeding of a diet rich in fat and fructose. Collectively, these data demonstrate that marked deficits in hepatic PAP activity do not impair TG synthesis and accumulation under acute or chronic conditions of lipid overload.