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.

Transient stimulation of glucose metabolism by insulin in the 1-day chick embryo.

Effects of insulin upon glucose metabolism were investigated in chick embryos explanted in vitro during the first 30 h of incubation. Insulin stimulated the glucose consumption of the chick gastrula (18 h) and neurula (24 h), but had no effect on the late blastula (0 h:laying) and on the stage of six to eight somites (30 h). The increase in glucose consumption concerned both the embryonic area pellucida (AP) and extraembryonic area opaca (AO). AP responded to a greater extent (50%) and at a lower range of concentrations (0.1-1.0 ng/ml) than AO (30%; 1-100 ng/ml). Insulin had no effect on the oxygen consumption of blastoderms, whereas it stimulated the aerobic lactate production (approximately 70% of the additional glucose consumption was converted to lactate). The nanomolar range of stimulating concentrations suggests that insulin has a specific effect in the chick embryo, and that it could modulate glucose metabolism in ovo as well. The transient sensitivity of the embryo to insulin is discussed in relation to behavior of mesodermal cells.

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.

Ataxia telangiectasia mutated (ATM) inhibition transforms human mammary gland epithelial cells.

Carriers of mutations in the cell cycle checkpoint protein kinase ataxia telangiectasia mutated (ATM), which represent 1-2% of the general population, have an increased risk of breast cancer. However, experimental evidence that ATM deficiency contributes to human breast carcinogenesis is lacking. We report here that in MCF-10A and MCF-12A cells, which are well established normal human mammary gland epithelial cell models, partial or almost complete stable ATM silencing or pharmacological inhibition resulted in cellular transformation, genomic instability, and formation of dysplastic lesions in NOD/SCID mice. These effects did not require the activity of exogenous DNA-damaging agents and were preceded by an unsuspected and striking increase in cell proliferation also observed in primary human mammary gland epithelial cells. Increased proliferation correlated with a dramatic, transient, and proteasome-dependent reduction of p21(WAF1/CIP1) and p27(KIP1) protein levels, whereas little or no effect was observed on p21(WAF1/CIP1) or p27(KIP1) mRNAs. p21(WAF1/CIP1) silencing also increased MCF-10A cell proliferation, thus identifying p21(WAF1/CIP1) down-regulation as a mediator of the proliferative effect of ATM inhibition. Our findings provide the first experimental evidence that ATM is a human breast tumor suppressor. In addition, they mirror the sensitivity of ATM tumor suppressor function and unveil a new mechanism by which ATM might prevent human breast tumorigenesis, namely a direct inhibitory effect on the basal proliferation of normal mammary epithelial cells.

Skin permeation and metabolism of di(2-ethylhexyl) phthalate (DEHP).

Phthalates are suspected to be endocrine disruptors. Di(2-ethylhexyl) phthalate (DEHP) is assumed to have low dermal absorption; however, previous in vitro skin permeation studies have shown large permeation differences. Our aims were to determine DEHP permeation parameters and assess extent of skin DEHP metabolism among workers highly exposed to these lipophilic, low volatile substances. Surgically removed skin from patients undergoing abdominoplasty was immediately dermatomed (800 μm) and mounted on flow-through diffusion cells (1.77 cm(2)) operating at 32°C with cell culture media (aqueous solution) as the reservoir liquid. The cells were dosed either with neat DEHP or emulsified in aqueous solution (166 μg/ml). Samples were analysed by HPLC-MS/MS. DEHP permeated human viable skin only as the metabolite MEHP (100%) after 8h of exposure. Human skin was able to further oxidize MEHP to 5-oxo-MEHP. Neat DEHP applied to the skin hardly permeated skin while the aqueous solution readily permeated skin measured in both cases as concentration of MEHP in the receptor liquid. DEHP pass through human skin, detected as MEHP only when emulsified in aqueous solution, and to a far lesser degree when applied neat to the skin. Using results from older in vitro skin permeation studies with non-viable skin may underestimate skin exposures. Our results are in overall agreement with newer phthalate skin permeation studies.

Alteration of glucose metabolism in cultured astrocytes after AQP9-small interference RNA application.

Aquaglyceroporin-9 (AQP9) facilitates diffusion of water and energy substrates such as glycerol and monocarboxylates. AQP9 is present in plasma membrane and mitochondria of astrocytes and catecholaminergic neurons, suggesting that it plays a role in the energetic status of these cells. Using specific small interference RNA directed against AQP9 in astrocyte cultures, we showed that glycerol uptake is decreased which is associated with an increase in glucose uptake and oxidative metabolism. Our results not only confirm the presence of AQP9 in astrocytes but also suggest that changes in AQP9 expression alter glial energy metabolism.

ENaC-mediated alveolar fluid clearance and lung fluid balance depend on the channel-activating protease 1.

Sodium transport via epithelial sodium channels (ENaC) expressed in alveolar epithelial cells (AEC) provides the driving force for removal of fluid from the alveolar space. The membrane-bound channel-activating protease 1 (CAP1/Prss8) activates ENaC in vitro in various expression systems. To study the role of CAP1/Prss8 in alveolar sodium transport and lung fluid balance in vivo, we generated mice lacking CAP1/Prss8 in the alveolar epithelium using conditional Cre-loxP-mediated recombination. Deficiency of CAP1/Prss8 in AEC induced in vitro a 40% decrease in ENaC-mediated sodium currents. Sodium-driven alveolar fluid clearance (AFC) was reduced in CAP1/Prss8-deficient mice, due to a 48% decrease in amiloride-sensitive clearance, and was less sensitive to beta(2)-agonist treatment. Intra-alveolar treatment with neutrophil elastase, a soluble serine protease activating ENaC at the cell surface, fully restored basal AFC and the stimulation by beta(2)-agonists. Finally, acute volume-overload increased alveolar lining fluid volume in CAP1/Prss8-deficient mice. This study reveals that CAP1 plays a crucial role in the regulation of ENaC-mediated alveolar sodium and water transport and in mouse lung fluid balance.

Systems Genetics of Metabolism: The Use of the BXD Murine Reference Panel for Multiscalar Integration of Traits.

Metabolic homeostasis is achieved by complex molecular and cellular networks that differ significantly among individuals and are difficult to model with genetically engineered lines of mice optimized to study single gene function. Here, we systematically acquired metabolic phenotypes by using the EUMODIC EMPReSS protocols across a large panel of isogenic but diverse strains of mice (BXD type) to study the genetic control of metabolism. We generated and analyzed 140 classical phenotypes and deposited these in an open-access web service for systems genetics (www.genenetwork.org). Heritability, influence of sex, and genetic modifiers of traits were examined singly and jointly by using quantitative-trait locus (QTL) and expression QTL-mapping methods. Traits and networks were linked to loci encompassing both known variants and novel candidate genes, including alkaline phosphatase (ALPL), here linked to hypophosphatasia. The assembled and curated phenotypes provide key resources and exemplars that can be used to dissect complex metabolic traits and disorders.