Hypothalamic Inhibition Of Acetyl-coa Carboxylase Stimulates Hepatic Counter-regulatory Response Independent Of Ampk Activation In Rats.

Hypothalamic AMPK acts as a cell energy sensor and can modulate food intake, glucose homeostasis, and fatty acid biosynthesis. Intrahypothalamic fatty acid injection is known to suppress liver glucose production, mainly by activation of hypothalamic ATP-sensitive potassium (K(ATP)) channels. Since all models employed seem to involve malonyl-CoA biosynthesis, we hypothesized that acetyl-CoA carboxylase can modulate the counter-regulatory response independent of nutrient availability. In this study employing immunoblot, real-time PCR, ELISA, and biochemical measurements, we showed that reduction of the hypothalamic expression of acetyl-CoA carboxylase by antisense oligonucleotide after intraventricular injection increased food intake and NPY mRNA, and diminished the expression of CART, CRH, and TRH mRNA. Additionally, as in fasted rats, in antisense oligonucleotide-treated rats, serum glucagon and ketone bodies increased, while the levels of serum insulin and hepatic glycogen diminished. The reduction of hypothalamic acetyl-CoA carboxylase also increased PEPCK expression, AMPK phosphorylation, and glucose production in the liver. Interestingly, these effects were observed without modification of hypothalamic AMPK phosphorylation. Hypothalamic ACC inhibition can activate hepatic counter-regulatory response independent of hypothalamic AMPK activation.8e6266

Nanoparticles In Treatment Of Thermal Injured Rats: Is It Safe?

The aim of this study was to assess whether thermal trauma induced oxidative stress altered the balance between oxidant and antioxidant systems in the blood of burn wound rats in the absence and presence of silver nanoparticles and S-nitrosoglutathione, GSNO. Free silver nanoparticles, free GSNO and silver nanoparticles + GSNO had no cytotoxic effects. Under anesthesia, the shaved dorsum of the rats was exposed to 90°C (burn group) water bath. Studied compounds were administered topically immediately and at 28 days after the burn injury, four times a day. Silver nanoparticles and silver nanoparticles + GSNO were no toxic in vitro and in vivo. There were no significant differences in the levels of urea, creatinine, aminotransferases and hematological parameters, in control-burn groups (free silver nanoparticles) and treated-burn groups (free GSNO or silver nanoparticles + GSNO). There were no differences in lipid peroxidation and in the levels of protein carbonyls and glutathione, used as oxidative stress markers. A little inflammatory cell response, papillary dermis vascularization, fibroblasts differentiated into contractile myofibroblasts and the presence of a large amount of extracellular matrix were evidenced in treated groups following skin injury. These results indicate that silver nanoparticles and GSNO may provide an effective action on wound healing.3041Tian, J., Wong, K.K.Y., Ho, C.M., Lok, C.N., Yu, W.Y., Che, C.M., Chiu, J.F., Tam, P.K.H., (2007) J. Chem. Med. Chem., 2, p. 129Teli, M.K., Mutalik, S., Rajanikant, G.K., (2010) Cur. Pharm. Design., 16, p. 1882Schaller, M., Laude, J., Bodewaldt, H., Hamm, G., Korting, H.C., (2004) Skin Pharmacol. Physiol., 17, p. 31Seabra, A.B., Da Silva, R., De Souza, G.F.P., De Oliveira, M.G., (2008) Artif. Organs, 32, p. 262Seabra, A.B., Pankotai, E., Fehér, M., Somlai, A., Kiss, L., Bíró, L., Szabó, C., Lacza, Z., (2007) Br. J. Dermatol., 156, p. 814Seabra, A.B., Martins, D., Simes, M.M.S.G., Da Silva, R., Brocchi, M., De Oliveira, M.G., (2010) Artif. Organs, 34, p. 204Durán, N., Marcato, P.D., Alves, O.L., De Souza, G.I.H., Esposito, E., (2005) J. Nanobiotechnol., 3, p. 1Durán, N., Marcato, P.D., De Souza, G.I.H., Alves, O.L., Esposito, E., (2007) J. Biomed. Nanotechnol., 3, p. 203Amadeu, T.P., Seabra, A.B., De Oliveira, M.G., Costa, A.M.A., (2007) J. Eur. Acad. Dermatol. Venereol., 21, p. 629Amadeu, T.P., Seabra, A.B., De Oliveira, M.G., Costa, A.M.A., (2008) J. Surg Res., 149, p. 84Correa, D.H.A., Melo, P.S., De Carvalho, C.A.A., De Azevedo, M.B.M., Durán, N., Haun, M., (2005) Eur. J. Pharmacol., 510, p. 17De Conti, R., Oliveira, D.A., Fernandes, A.M.A.P., Melo, P.S., Rodriguez, J.A., Haun, M., Castro, S.L., Durán, N., (1998) Vitro Mol. Toxicol, 11, p. 153Borefreund, E., Puerner, J.A., (1984) J. Tissue Cult. Methods, 9, p. 7Denizot, F., Lang, R., (1986) J. Immunol. Methods, 89, p. 271Michailidis, Y., Jamurta, A.Z., Nikolaidis, M.G., Fatouros, I.G., Koutedakis, Y., Papassotiriou, I., (2007) Med. Sci. Sport Exerc., 39, p. 1107Davis, T.A., Amare, M., Naik, S., Kovalchuk, A.L., Tadaki, D., (2007) Wound Repair Regen., 15, p. 57

Differential Regulation of Adhesion Complex Turnover by ROCK1 and ROCK2

ROCK1 and ROCK2 are serine/threonine kinases that function downstream of the small GTP-binding protein RhoA. Rho signalling via ROCK regulates a number of cellular functions including organisation of the actin cytoskeleton, cell adhesion and cell migration.In this study we use RNAi to specifically knockdown ROCK1 and ROCK2 and analyse their role in assembly of adhesion complexes in human epidermal keratinocytes. We observe that loss of ROCK1 inhibits signalling via focal adhesion kinase resulting in a failure of immature adhesion complexes to form mature stable focal adhesions. In contrast, loss of ROCK2 expression results in a significant reduction in adhesion complex turnover leading to formation of large, stable focal adhesions. Interestingly, loss of either ROCK1 or ROCK2 expression significantly impairs cell migration indicating both ROCK isoforms are required for normal keratinocyte migration.ROCK1 and ROCK2 have distinct and separate roles in adhesion complex assembly and turnover in human epidermal keratinocytes

The Nutritional Induction of COUP-TFII Gene Expression in Ventromedial Hypothalamic Neurons Is Mediated by the Melanocortin Pathway

BACKGROUND: The nuclear receptor chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) is an important coordinator of glucose homeostasis. We report, for the first time, a unique differential regulation of its expression by the nutritional status in the mouse hypothalamus compared to peripheral tissues. METHODOLOGY/PRINCIPAL FINDINGS: Using hyperinsulinemic-euglycemic clamps and insulinopenic mice, we show that insulin upregulates its expression in the hypothalamus. Immunofluorescence studies demonstrate that COUP-TFII gene expression is restricted to a subpopulation of ventromedial hypothalamic neurons expressing the melanocortin receptor. In GT1-7 hypothalamic cells, the MC4-R agonist MTII leads to a dose dependant increase of COUP-TFII gene expression secondarily to a local increase in cAMP concentrations. Transfection experiments, using a COUP-TFII promoter containing a functional cAMP responsive element, suggest a direct transcriptional activation by cAMP. Finally, we show that the fed state or intracerebroventricular injections of MTII in mice induce an increased hypothalamic COUP-TFII expression associated with a decreased hepatic and pancreatic COUP-TFII expression. CONCLUSIONS/SIGNIFICANCE: These observations strongly suggest that hypothalamic COUP-TFII gene expression could be a central integrator of insulin and melanocortin signaling pathway within the ventromedial hypothalamus. COUP-TFII could play a crucial role in brain integration of circulating signal of hunger and satiety involved in energy balance regulation

DAF-16 and Δ9 Desaturase Genes Promote Cold Tolerance in Long-Lived Caenorhabditis elegans age-1 Mutants

In Caenorhabditis elegans, mutants of the conserved insulin/IGF-1 signalling (IIS) pathway are long-lived and stress resistant due to the altered expression of DAF-16 target genes such as those involved in cellular defence and metabolism. The three Δ9 desaturase genes, fat-5, fat-6 and fat-7, are included amongst these DAF-16 targets, and it is well established that Δ9 desaturase enzymes play an important role in survival at low temperatures. However, no assessment of cold tolerance has previously been reported for IIS mutants. We demonstrate that long-lived age-1(hx546) mutants are remarkably resilient to low temperature stress relative to wild type worms, and that this is dependent upon daf-16. We also show that cold tolerance following direct transfer to low temperatures is increased in wild type worms during the facultative, daf-16 dependent, dauer stage. Although the cold tolerant phenotype of age-1(hx546) mutants is predominantly due to the Δ9 desaturase genes, additional transcriptional targets of DAF-16 are also involved. Surprisingly, survival of wild type adults following a rapid temperature decline is not dependent upon functional daf-16, and cellular distributions of a DAF-16::GFP fusion protein indicate that DAF-16 is not activated during low temperature stress. This suggests that cold-induced physiological defences are not specifically regulated by the IIS pathway and DAF-16, but expression of DAF-16 target genes in IIS mutants and dauers is sufficient to promote cross tolerance to low temperatures in addition to other forms of stress

Involvement Of Available Sh Groups In The Heterogeneity Of Hemoglobin From The Tortoise Geochelone Carbonaria.

Geochelone carbonaria hemoglobin (Hb) was analyzed by polyacrylamide gel electrophoresis (PAGE) and purified by ion exchange chromatography on CM-cellulose. Seven fractions were obtained using fresh Hb preparations. CM-cellulose chromatography of Hb reacted with iodoacetamide, showed one minor (HbI) and one major band (HbII). Analysis of the molecular masses of recently collected Hb and of aged solutions determined by gel filtration showed that polymerization increased with the duration of storage. The reaction with oxidized glutathione changed the electrophoretic pattern of Hb, and highlighted the bands corresponding to glutathionyl-Hb. The presence of these bands in fresh Hb solutions and in alkylated preparations suggests that they may occur in vivo. PAGE under dissociating conditions showed that the hemolysate contained 3 different polypeptide chains (G1, G2 and G3). Both Hb components shared the G1 globin chain with HbI containing G1 and G2 and HbII, G1 and G3 chains.44851-6

TNFα-Induced Oxidative Stress and Mitochondrial Dysfunction Alter Hypothalamic Neurogenesis and Promote Appetite Versus Satiety Neuropeptide Expression in Mice

Maternal obesity results in programmed offspring hyperphagia and obesity. The increased offspring food intake is due in part to the preferential differentiation of hypothalamic neuroprogenitor cells (NPCs) to orexigenic (AgRP) vs. anorexigenic (POMC) neurons. The altered neurogenesis may involve hypothalamic bHLH (basic helix–loop–helix) neuroregulatory factors (Hes1, Mash1, and Ngn3). Whilst the underlying mechanism remains unclear, it is known that mitochondrial function is critical for neurogenesis and is impacted by proinflammatory cytokines such as TNFα. Obesity is associated with the activation of inflammation and oxidative stress pathways. In obese pregnancies, increased levels of TNFα are seen in maternal and cord blood, indicating increased fetal exposure. As TNFα influences neurogenesis and mitochondrial function, we tested the effects of TNFα and reactive oxidative species (ROS) hydrogen peroxide (H2O2) on hypothalamic NPC cultures from newborn mice. TNFα treatment impaired NPC mitochondrial function, increased ROS production and NPC proliferation, and decreased the protein expression of proneurogenic Mash1/Ngn3. Consistent with this, AgRP protein expression was increased and POMC was decreased. Notably, treatment with H2O2 produced similar effects as TNFα and also reduced the protein expression of antioxidant SIRT1. The inhibition of STAT3/NFκB prevented the effects of TNFα, suggesting that TNFα mediates its effects on NPCs via mitochondrial-induced oxidative stress that involves both signaling pathways

Functional behavior of tortoise hemoglobin Geochelone denticulata

The hemolysate from Geochelone denticulata contains two main hemoglobin components, as shown by ion exchange chromatography and polyacrylamide gel electrophoresis (PAGE). Electrophoresis under dissociating conditions showed three types of globin chains. The apparent molecular mass, as determined by gel filtration on Sephadex G-200, was compatible with tetrameric Hb, which was unable to polymerize. The G. denticulata Hb has a P50 value of 9.56 mm Hg at pH 7.4. The Hb oxygenation appears to be under the control of organic phosphates and hydrogen ion since it is strongly affected by those species. In the presence ATP or IHP the P50 values increased to 29.51 mm Hg and 54.95 mm Hg, respectively, at pH 7.4. The n50 was generally lower than 1.5 in stripped Hb, suggesting a dissociation of tetramers. In the presence of organic phosphates n50 values increased to approximately 2.5. The Bohr effect was evident in oxygen equilibrium experiments. The hematocrit (32%) and Hb concentration (5.7 mM as heme) of G. denticulata blood were substantially larger than those of G. carbonaria, but the methemoglobin levels were similar in both species, approximately 1%. Thus, the oxygen capacity of blood appears to be higher in G. denticulata than in G. carbonaria, particularly considering the functional properties of their Hbs, which would guarantee the survival of animals