Novel mechanisms involved in leptin sensitization in obesity.

Leptin is a hormone that is secreted by adipocytes in proportion to adipose tissue size, and that informs the brain about the energy status of the body. Leptin acts through its receptor LepRb, expressed mainly in the hypothalamus, and induces a negative energy balance by potent inhibition of feeding and activation of energy expenditure. These actions have led to huge expectations for the development of therapeutic targets for metabolic complications based on leptin-derived compounds. However, the majority of patients with obesity presents elevated leptin production, suggesting that in this setting leptin is ineffective in the regulation of energy balance. This resistance to the action of leptin in obesity has led to the development of "leptin sensitizers," which have been tested in preclinical studies. Much research has focused on generating combined treatments that act on multiple levels of the gastrointestinal-brain axis. The gastrointestinal-brain axis secretes a variety of different anorexigenic signals, such as uroguanylin, glucagon-like peptide-1, amylin, or cholecystokinin, which can alleviate the resistance to leptin action. Moreover, alternative mechanism such as pharmacokinetics, proteostasis, the role of specific kinases, chaperones, ER stress and neonatal feeding modifications are also implicated in leptin resistance. This review will cover the current knowledge regarding the interaction of leptin with different endocrine factors from the gastrointestinal-brain axis and other novel mechanisms that improve leptin sensitivity in obesity.

Evaluation of Essential, Toxic and Potentially Toxic Elements in Leafy Vegetables Grown in the Canary Islands.

Forty-seven elements in leafy green vegetables were studied to estimate the daily intakes from this food category in different scenarios (average and high consumers) and age groups of the Canary Islands population. The contribution of the consumption of each type of vegetable to the reference intakes of essential, toxic and potentially toxic elements was assessed and the risk-benefit ratio was evaluated. The leafy vegetables that provide the highest levels of elements are spinach, arugula, watercress and chard. While spinach, chard, arugula, lettuce sprouts and watercress were the leafy vegetables with the highest concentrations of essential elements (38,743 ng/g of Fe in spinach, 3733 ng/g of Zn in watercress), the high levels of Mn in chard, spinach and watercress are noteworthy. Among the toxic elements, Cd is the element with the highest concentration, followed by As and Pb. The vegetable with the highest concentration of potentially toxic elements (Al, Ag, Be, Cr, Ni, Sr and V) is spinach. In average adult consumers, while the greatest contribution of essential elements comes from arugula, spinach and watercress, insignificant dietary intakes of potentially toxic metals are observed. Toxic metal intakes from the consumption of leafy vegetables in the Canary Islands do not show significant values, so the consumption of these foods does not pose a health risk. In conclusion, the consumption of leafy vegetables provides significant levels of some essential elements (Fe, Mn, Mo, Co and Se), but also of some potentially toxic elements (Al, Cr and Tl). A high consumer of leafy vegetables would see their daily nutritional needs regarding Fe, Mn, Mo, and Co covered, although they are also exposed to moderately worrying levels of Tl. To monitor the safety of dietary exposure to these metals, total diet studies on those elements with dietary exposures above the reference values derived from the consumption of this food category, mainly Tl, are recommended.

Prolonged breastfeeding protects from obesity by hypothalamic action of hepatic FGF21.

Early-life determinants are thought to be a major factor in the rapid increase of obesity. However, while maternal nutrition has been extensively studied, the effects of breastfeeding by the infant on the reprogramming of energy balance in childhood and throughout adulthood remain largely unknown. Here we show that delayed weaning in rat pups protects them against diet-induced obesity in adulthood, through enhanced brown adipose tissue thermogenesis and energy expenditure. In-depth metabolic phenotyping in this rat model as well as in transgenic mice reveals that the effects of prolonged suckling are mediated by increased hepatic fibroblast growth factor 21 (FGF21) production and tanycyte-controlled access to the hypothalamus in adulthood. Specifically, FGF21 activates GABA-containing neurons expressing dopamine receptor 2 in the lateral hypothalamic area and zona incerta. Prolonged breastfeeding thus constitutes a protective mechanism against obesity by affecting long-lasting physiological changes in liver-to-hypothalamus communication and hypothalamic metabolic regulation.

Crosstalk between Melanin Concentrating Hormone and Endocrine Factors: Implications for Obesity.

Melanin-concentrating hormone (MCH) is a 19aa cyclic peptide exclusively expressed in the lateral hypothalamic area, which is an area of the brain involved in a large number of physiological functions and vital processes such as nutrient sensing, food intake, sleep-wake arousal, memory formation, and reproduction. However, the role of the lateral hypothalamic area in metabolic regulation stands out as the most relevant function. MCH regulates energy balance and glucose homeostasis by controlling food intake and peripheral lipid metabolism, energy expenditure, locomotor activity and brown adipose tissue thermogenesis. However, the MCH control of energy balance is a complex mechanism that involves the interaction of several neuroendocrine systems. The aim of the present work is to describe the current knowledge of the crosstalk of MCH with different endocrine factors. We also provide our view about the possible use of melanin-concentrating hormone receptor antagonists for the treatment of metabolic complications. In light of the data provided here and based on its actions and function, we believe that the MCH system emerges as an important target for the treatment of obesity and its comorbidities.

Salt-Sensitive Hypertension in GR+/- Rats Is Accompanied with Dysregulation in Adrenal Soluble Epoxide Hydrolase and Polyunsaturated Fatty Acid Pathways.

Mutations within the glucocorticoid receptor (GR) gene locus lead to glucocorticoid resistance which is characterized by several clinical symptoms such as adrenal gland hyperplasia and salt-sensitive hypertension, although the underlying mechanisms are still unknown. We studied GR haploinsufficient (GR+/-) Sprague Dawley rats which, on a standard diet, showed significantly increased plasma aldosterone and corticosterone levels and an adrenocortex hyperplasia accompanied by a normal systolic blood pressure. Following a high salt diet, these rats developed salt-sensitive hypertension and maintained elevated enzyme-soluble epoxide hydrolase (sEH) in adrenal glands, while sEH was significantly decreased in wild-type rats. Furthermore, GR+/- rats showed dysregulation of the equilibrated linoleic and arachidonic acid pathways, with a significant increase of less active metabolites such as 8,9-DiHETrE. In Sprague Dawley rats, GR haploinsufficiency induced steroid disturbances, which provoked hypertension only in combination with high salt intake, which was accompanied by disturbances in sEH and fatty acid metabolism. Our results suggest that sEH inhibition could be a potential target to treat hypertension in patients with GR haploinsufficiency.

Evaluation of nitrate contents in regulated and non-regulated leafy vegetables of high consumption in the Canary Islands, Spain: Risk assessment.

The nitrate content of the most consumed green leafy vegetables in the European Region of the Canary Islands was determined. The sampling included chard and watercress, which are not regulated but highly consumed in this region. The levels of nitrates in organic vegetables were significantly higher than those of conventional cultivation. However, no seasonal differences were observed, and overall nitrate levels were lower than those reported in other studies. Median nitrate levels in the analyzed vegetables were: lettuce (3 varieties) = 573.7 mg/kg; ready-to-eat salad mixes = 595.0 mg/kg; spinach = 1044.2 mg/kg; arugula = 3144.2 mg/kg; watercress = 450.5 mg/kg; and chard = 1788.4 mg/kg. In general, the nitrate levels of watercress and chard were significantly higher than those of regulated vegetables with similar culinary uses. The average per capita daily intake of nitrates through regulated vegetables was 17.5-32.5% of acceptable daily intake (ADI). On the contrary, the consumption of unregulated vegetables in this archipelago represents a similar, or even higher, percentage of ADI (23.6-44.3%). We, therefore, consider that the establishment of maximum limits of nitrate by the EU regulatory authorities would be appropriate for chard and watercress and similar to those set for spinach.

mTOR Pathway is Involved in Energy Homeostasis Regulation as a Part of the Gut-Brain Axis.

Mammalian, or mechanic, target of rapamycin (mTOR) signaling is a crucial factor in the regulation of the energy balance that functions as an energy sensor in the body. The present review explores how the mTOR/S6k intracellular pathway is involved in modulating the production of different signals such as ghrelin and nesfatin-1 in the gastrointestinal tract to regulate food intake and body weight. The role of gastric mTOR signaling in different physiological processes was studied in depth through different genetic models that allow the modulation of mTOR signaling in the stomach and specifically in gastric X/A type cells. It has been described that mTOR signaling in X/A-like gastric cells has a relevant role in the regulation of glucose and lipid homeostasis due to its interaction with different organs such as liver and adipose tissue. These findings highlight possible therapeutic strategies, with the gut-brain axis being one of the most promising targets in the treatment of obesity.

Oral Pharmacological Activation of Hypothalamic Guanylate Cyclase 2C Receptor Stimulates Brown Fat Thermogenesis to Reduce Body Weight.

Linaclotide is a synthetic peptide approved by the FDA for the treatment of constipation-predominant irritable bowel syndrome and chronic constipation. Linaclotide binds and activates the transmembrane receptor guanylate cyclase 2C (Gucy2c). Uroguanylin (UGN) is a 16 amino acid peptide that is mainly secreted by enterochromaffin cells in the duodenum and proximal small intestine. UGN is the endogenous ligand of Gucy2c and decreases body weight in diet-induced obese (DIO) mice via the activation of the thermogenic program in brown adipose tissue. Therefore, we wanted to evaluate whether oral linaclotide could also improve DIO mice metabolic phenotype. In this study, we have demonstrated that DIO mice orally treated with linaclotide exhibited a significant reduction of body weight without modifying food intake. Linaclotide exerts its actions through the central nervous system, and more specifically, via Gucy2c receptors located in the mediobasal hypothalamus, leading to the activation of the sympathetic nervous system to trigger the thermogenic activity of brown fat stimulating energy expenditure. These findings indicate for first time that, in addition to its effects at intestinal level to treat irritable bowel syndrome with constipation and chronic constipation, linaclotide also exerts a beneficial effect in whole body metabolism.

Graphene-based materials do not impair physiology, gene expression and growth dynamics of the aeroterrestrial microalga Trebouxia gelatinosa.

The effects of two graphene-based materials (GBMs), few-layers graphene (FLG) and graphene oxide (GO), were studied in the aeroterrestrial green microalga Trebouxia gelatinosa. Algae were subjected to short- and long-term exposure to GBMs at 0.01, 1 and 50 µg mL - 1. GBMs internalization after short-term exposures was investigated with confocal microscopy, Raman spectroscopy and TEM. Potential negative effects of GBMs, compared to the oxidative stress induced by H2O2, were verified by analyzing chlorophyl a fluorescence (ChlaF), expression of stress-related genes and membrane integrity. Effects of up to 4-week-long exposures were assessed analyzing growth dynamics, ChlaF and photosynthetic pigments. GBMs were not observed in cells but FLG was detected at the interface between the cell wall and plasma membrane, whereas GO was observed adherent to the external wall surface. FLG caused the down-regulation of the HSP70-1 gene, with the protein levels remaining stable, whereas GO had no effect. In comparison, H2O2 produced dose- and time-dependent effects on ChlaF, gene expression and HSP70 protein level. Long-term exposures to GBMs did not affect growth dynamics, ChlaF or photosynthetic pigment contents, indicating that the few observed short-term effects were not dangerous on the long-term. Results suggest that interactions between FLG and plasma membrane were harmless, activating a down-regulation of the HSP70-1 gene similar to that induced by H2O2. Our work shows that studying GBMs effects on non-model organisms is important since the results of model green microalgae are not representative of the whole taxonomic group.

ANGPTL-4 is Associated with Obesity and Lipid Profile in Children and Adolescents.

Angiopoietin-like protein 4 (ANGPTL-4) regulates lipidic metabolism and affects energy homeostasis. However, its function in children with obesity remains unknown. We investigated plasma ANGPTL-4 levels in children and its relationship with body mass index (BMI) and different lipidic parameters such as free fatty acids (FFA). Plasma ANGPTL-4 levels were analyzed in two different cohorts. In the first cohort (n = 150, age 3-17 years), which included children with normal weight or obesity, we performed a cross-sectional study. In the second cohort, which included only children with obesity (n = 20, age 5-18 years) followed up for two years after an intervention for weight loss, in which we performed a longitudinal study measuring ANGPTL-4 before and after BMI-loss. In the cross-sectional study, circulating ANGPTL-4 levels were lower in children with obesity than in those with normal weight. Moreover, ANGPTL-4 presented a negative correlation with BMI, waist circumference, weight, insulin, homeostasis model assessment of insulin resistance index (HOMA index), triglycerides, and leptin, and a positive correlation with FFA and vitamin-D. In the longitudinal study, the percent change in plasma ANGPTL-4 was correlated with the percent change in FFA, total-cholesterol and high-density lipoprotein cholesterol. This study reveals a significant association of ANGPTL-4 with pediatric obesity and plasma lipid profile.

The E6/E7 oncogenes of human papilloma virus and estradiol regulate hedgehog signaling activity in a murine model of cervical cancer.

Human papilloma virus oncogenes and estradiol are major etiologic factors associated with cervical cancer. In order to understand the mechanism by which these two factors promote carcinogenesis, the role of the Hedgehog (Hh) signaling pathway was evaluated during the normal growth of cervical epithelium and in the presence of E6/E7 oncogenes and exogenous estradiol. Hh signaling activity was determined in live animals (i.e., Gli-Luc reporter levels) during the estrous cycle and was found to be higher in the cervical area during the major growth phases, proestrus-estrus, in comparison to the diestrus phase. The same pattern was observed in transgenic mice expressing the E6/E7 oncogenes, though with notably higher levels than in control mice. Adding estradiol also markedly increased Gli activity in the cervix and the skin. In agreement with the correlation between high bioluminescence and tissue growth in different context, cervical cell proliferation was reduced upon Hh signaling inhibition in mice. Treatment with itraconazole, a putative novel Hh inhibitor, at an early stage of cervical carcinogenesis, did not decrease Hh signaling but it did reduce growth. Therefore, Hh signaling likely contributes to cervical carcinogenesis and itraconazole is effective to reduce growth but by a mechanism involving additional signaling pathways.

Hedgehog signaling dynamics in mouse embryos determined by a bioluminiscent reporter.

Determination of cellular signaling in live embryos is key to understand the molecular processes that drive development. Here, we show that a transgenic mouse line carrying a luciferase-based gene reporter of Gli-mediated transcriptional activation (Gli-Luc) displays sonic hedgehog (Shh) signaling in discrete developmental processes during short-term cultures of whole embryos or embryo explants. The bioluminescence in E9.5 embryos was detected in regions in which Shh activity has been demonstrated. Later, in E10.5 embryos, bioluminescence intensity markedly increased, mostly corresponding to the high Shh activity of the developing midbrain and limb. Notably, the dynamic range of the Gli-Luc reporter in the developing limb revealed the progressive emergence of bioluminescence in the zone of polarizing activity, where reporter activity locally increased and spatially spread in agreement with the signaling gradient expected for Shh. In the midbrain of E9.5 mouse embryos, bioluminescence was not detected along the ventral region as expected but, instead, Shh-dependent anterior and posterior bioluminescence foci emerged by E10.5 indicating that the Gli-Luc reporter can only respond transcriptionally to relatively high levels of GliA and/or without the interaction with other transcription factors. The present work supports the use of bioluminescence to identify and study the dynamics of centers of morphogen signaling during mouse embryogenesis.

Circulating Pro-Uroguanylin Levels In Children And Their Relation To Obesity, Sex And Puberty.

Uroguanylin is a 16 amino acid peptide that constitutes a key component of the gut- brain axis with special relevance in body weight regulation. In childhood and adolescence, periods of life with notable metabolic changes; limited data exist, with measurements of pro-uroguanylin in adolescence but not in prepubertal children. This study investigates pro-uroguanylin circulating levels in children with obesity and its relationship with obesity, sex and pubertal development. We analyzed circulating prouroguanylin levels in 117 children (62) and adolescents (55), including 73 with obesity and 44 with normal weight. The pro-uroguanylin concentration is higher in lean girls during pre-puberty versus lean boys (1111 vs 635, p < 0.001). During puberty, pro-uroguanylin levels are higher in lean males with respect to lean females (1060 vs 698, p < 0.01). In girls, a negative correlation exists between pro-uroguanylin and age, Tanner stage, weight, height, BMI (body mass index), waist circumference and plasma levels of leptin and testosterone; a positive correlation was found between pro-uroguanylin and free triiodothyronine. In boys, a positive correlation was found between pro-uroguanylin and BMI and waist circumference and a negative correlation was found with high density lipoprotein-cholesterol. We conclude that a sexual dimorphism exists in circulating pro-uroguanylin levels with respect to BMI. Uroguanylin presents also an opposed circulating pattern during puberty in both sexes.

Production of ready-to-use few-layer graphene in aqueous suspensions.

Graphene has promising physical and chemical properties such as high strength and flexibility, coupled with high electrical and thermal conductivities. It is therefore being incorporated into polymer-based composites for use in electronics and photonics applications. A main constraint related to the graphene development is that, being of a strongly hydrophobic nature, almost all dispersions (usually required for its handling and processing toward the desired application) are prepared in poisonous organic solvents such as N-methyl pyrrolidone or N,N-dimethyl formamide. Here, we describe how to prepare exfoliated graphite using a ball mill. The graphene produced is three to four layers thick and ∼500 nm in diameter on average, as measured by electron microscopy and Raman spectroscopy; can be stored in the form of light solid; and is easily dispersed in aqueous media. Our methodology consists of four main steps: (i) the mechanochemical intercalation of organic molecules (melamine) into graphite, followed by suspension in water; (ii) the washing of suspended graphene to eliminate most of the melamine; (iii) the isolation of stable graphene sheets; and (iv) freeze-drying to obtain graphene powder. This process takes 6-7 or 9-10 d for aqueous suspensions and dry powders, respectively. The product has well-defined properties and can be used for many science and technology applications, including toxicology impact assessment and the production of innovative medical devices.

Pharmacological inhibition of cannabinoid receptor 1 stimulates gastric release of nesfatin-1 via the mTOR pathway.

AIM: To determine whether Nucb2/nesfatin1 production is regulated by the cannabinoid system through the intracellular mTOR pathway in the stomach. METHODS: Sprague Dawley rats were treated with vehicle, rimonabant, rapamycin or rapamycin+rimonabant. Gastric tissue obtained from the animals was used for biochemical assays: Nucb2 mRNA measurement by real time PCR, gastric Nucb2/nesfatin protein content by western blot, and gastric explants to obtain gastric secretomes. Nucb2/nesfatin levels were measured in gastric secretomes and plasma using enzyme-linked immunosorbent assay. RESULTS: The inhibition of cannabinoid receptor 1 (CB1) by the peripheral injection of an inverse agonist, namely rimonabant, decreases food intake and increases the gastric secretion and circulating levels of Nucb2/nesfatin-1. In addition, rimonabant treatment activates mTOR pathway in the stomach as showed by the increase in pmTOR/mTOR expression in gastric tissue obtained from rimonabant treated animals. These effects were confirmed by the use of a CB1 antagonist, AM281. When the intracellular pathway mTOR/S6k was inactivated by chronic treatment with rapamycin, rimonabant treatment was no longer able to stimulate the gastric secretion of Nucb2/nesfatin-1. CONCLUSION: The peripheral cannabinoid system regulates food intake through a mechanism that implies gastric production and release of Nucb2/Nesfatin-1, which is mediated by the mTOR/S6k pathway.

Few-Layer Graphene Kills Selectively Tumor Cells from Myelomonocytic Leukemia Patients.

In the cure of cancer, a major cause of today's mortality, chemotherapy is the most common treatment, though serious frequent challenges are encountered by current anticancer drugs. We discovered that few-layer graphene (FLG) dispersions have a specific killer action on monocytes, showing neither toxic nor activation effects on other immune cells. We confirmed the therapeutic application of graphene on an aggressive type of cancer that is myelomonocytic leukemia, where the monocytes are in their malignant form. We demonstrated that graphene has the unique ability to target and boost specifically the necrosis of monocytic cancer cells. Moreover, the comparison between FLG and a common chemotherapeutic drug, etoposide, confirmed the higher specificity and toxicity of FLG. Since current chemotherapy treatments of leukemia still cause serious problems, these findings open the way to new and safer therapeutic approaches.

Differential cytotoxic effects of graphene and graphene oxide on skin keratinocytes.

Impressive properties make graphene-based materials (GBMs) promising tools for nanoelectronics and biomedicine. However, safety concerns need to be cleared before mass production of GBMs starts. As skin, together with lungs, displays the highest exposure to GBMs, it is of fundamental importance to understand what happens when GBMs get in contact with skin cells. The present study was carried out on HaCaT keratinocytes, an in vitro model of skin toxicity, on which the effects of four GBMs were evaluated: a few layer graphene, prepared by ball-milling treatment (FLG), and three samples of graphene oxide (GOs, a research-grade GO1, and two commercial GOs, GO2 and GO3). Even though no significant effects were observed after 24 h, after 72 h the less oxidized compound (FLG) was the less cytotoxic, inducing mitochondrial and plasma-membrane damages with EC50s of 62.8 µg/mL (WST-8 assay) and 45.5 µg/mL (propidium iodide uptake), respectively. By contrast, the largest and most oxidized compound, GO3, was the most cytotoxic, inducing mitochondrial and plasma-membrane damages with EC50s of 5.4 and 2.9 µg/mL, respectively. These results suggest that only high concentrations and long exposure times to FLG and GOs could impair mitochondrial activity associated with plasma membrane damage, suggesting low cytotoxic effects at the skin level.

Graphene Oxide Nanosheets Reshape Synaptic Function in Cultured Brain Networks.

Graphene offers promising advantages for biomedical applications. However, adoption of graphene technology in biomedicine also poses important challenges in terms of understanding cell responses, cellular uptake, or the intracellular fate of soluble graphene derivatives. In the biological microenvironment, graphene nanosheets might interact with exposed cellular and subcellular structures, resulting in unexpected regulation of sophisticated biological signaling. More broadly, biomedical devices based on the design of these 2D planar nanostructures for interventions in the central nervous system require an accurate understanding of their interactions with the neuronal milieu. Here, we describe the ability of graphene oxide nanosheets to down-regulate neuronal signaling without affecting cell viability.

Surface Area of Carbon Nanoparticles: A Dose Metric for a More Realistic Ecotoxicological Assessment.

Engineered nanoparticles such as graphenes, nanodiamonds, and carbon nanotubes correspond to different allotropes of carbon and are among the best candidates for applications in fast-growing nanotechnology. It is thus likely that they may get into the environment at each step of their life cycle: production, use, and disposal. The aquatic compartment concentrates pollutants and is expected to be especially impacted. The toxicity of a compound is conventionally evaluated using mass concentration as a quantitative measure of exposure. However, several studies have highlighted that such a metric is not the best descriptor at the nanoscale. Here we compare the inhibition of Xenopus laevis larvae growth after in vivo exposure to different carbon nanoparticles for 12 days using different dose metrics and clearly show that surface area is the most relevant descriptor of toxicity for different types of carbon allotropes.

Graphene-Based Interfaces Do Not Alter Target Nerve Cells.

Neural-interfaces rely on the ability of electrodes to transduce stimuli into electrical patterns delivered to the brain. In addition to sensitivity to the stimuli, stability in the operating conditions and efficient charge transfer to neurons, the electrodes should not alter the physiological properties of the target tissue. Graphene is emerging as a promising material for neuro-interfacing applications, given its outstanding physico-chemical properties. Here, we use graphene-based substrates (GBSs) to interface neuronal growth. We test our GBSs on brain cell cultures by measuring functional and synaptic integrity of the emerging neuronal networks. We show that GBSs are permissive interfaces, even when uncoated by cell adhesion layers, retaining unaltered neuronal signaling properties, thus being suitable for carbon-based neural prosthetic devices.