Antonio Díaz-Cruz

Regulation of glycogen metabolism in hepatocytes through adenosine receptors. Role of Ca2+ and cAMP.

The objective of this work is to identify the adenosine receptor subtype and the triggered events involved in the regulation of hepatic glycogen metabolism. Glycogenolysis, gluconeogenesis, cAMP, and cytosolic Ca2+ ([Ca2+](cyt)) were measured in isolated hepatocytes challenged with adenosine A1, A2A, and A3 receptor-selective agonists. Stimulation of adenosine receptor subtypes with selective agonists in Ca2+ media produced a dose-dependent increase in [Ca2+]cyt with A1>A2=A3, cAMP with A2A, glycogenolysis with A1>A2A>A3, and gluconeogenesis with A2A>A1>A3, in addition, a decrease in cAMP was observed with A1=A3. Comparatively, in Ca2+-free media or with a cell membrane-permeant Ca2+ chelator, activation of these adenosine receptors with the same selective agonists produced a smaller and transient rise in [Ca2+]cyt with A1=A3>A2, no rise in glycogenolysis and gluconeogenesis with A3>A1, but a full rise with A2A. Thus, in isolated rat hepatocytes activation of the adenosine A1 receptor triggered Ca2+-mediated glycogenolysis, activation of the adenosine A2A receptor stimulated cAMP-mediated gluconeogenesis, and activation of the adenosine A3 receptor increased [Ca2+]cyt and decreased cAMP with minor changes in glycogen metabolism.

Adrenaline stimulates H2O2 generation in liver via NADPH oxidase

It is known that adrenaline promotes hydroxyl radical generation in isolated rat hepatocytes. The aim of this work was to investigate a potential role of NADPH oxidase (Nox) isoforms for an oxidative stress signal in response to adrenaline in hepatocytes. Enriched plasma membranes from isolated rat liver cells were prepared for this purpose. These membranes showed catalytic activity of Nox isoforms, probably Nox 2 based on its complete inhibition with specific antibodies. NADPH was oxidized to convert O2 into superoxide radical, later transformed into H2O2. This enzymatic activity requires previous activation with either 3 mM Mn2+ or guanosine 5′-0-(3-thiotriphosphate) (GTPγS) plus adrenaline. Experimental conditions for activation and catalytic steps were set up: ATP was not required; S0.5 for NADPH was 44 μM; S0.5 for FAD was 8 μM; NADH up to 1 mM was not substrate, and diphenyleneiodonium was inhibitory. Activation with GTPγS plus adrenaline was dose- and Ca2+-dependent and proceeded through α1-adrenergic receptors (AR), whereas β-AR stimulation resulted in inhibition of Nox activity. These results lead us to propose H2O2 as additional transduction signal for adrenaline response in hepatic cells.

Prophylactic action of lipoic acid on oxidative stress and growth performance in broilers at risk of developing ascites syndrome

The objective of this study was to assess the effects of dietary supplementation with lipoic acid (LA) on broilers maintained at 2235 m above sea level with high risk to develop ascites syndrome (AS). A total of 2040 chicks were fed under commercial conditions with water and specific diets ad libitum during 7 weeks in two consecutive experiments. Mortality and indicators of performance and oxidative stress were compared weekly in broilers fed a basal diet plus 0, 10, 20, or 40 parts/106 LA. The effects of LA at 40 parts/106 were also studied during the initial 3 weeks or the last 4 weeks of the production cycle. Diets supplemented with 40 parts/106 of LA during 7 weeks significantly improved feed conversion, decreased general mortality and mortality attributable to AS, and lowered thiobarbituric acid reactive substances and hydroxyl radicals in liver, and increased total glutathione pool. Smaller doses or shorter periods of exposure to LA were partially effective. In conclusion, LA under our experimental conditions has a prophylactic action in broilers with high risk to develop AS due to oxygen availability limitation.

Newly synthesized cAMP is integrated at a membrane protein complex signalosome to ensure receptor response specificity.

Spatiotemporal regulation of cAMP within the cell is required to achieve receptor‐specific responses. The mechanism through which the cell selects a specific response to newly synthesized cAMP is not fully understood. In hepatocyte plasma membranes, we identified two functional and independent cAMP‐responsive signaling protein macrocomplexes that produce, use, degrade, and regulate their own nondiffusible (sequestered) cAMP pool to achieve their specific responses. Each complex responds to the stimulation of an adenosine G protein‐coupled receptor (Ado‐GPCR), bound to either A2A or A2B, but not simultaneously to both. Each isoprotein involved in each signaling cascade was identified by measuring changes in cAMP levels after receptor activation, and its participation was confirmed by antibody‐mediated inactivation. A2A‐Ado‐GPCR selective stimulation activates adenylyl cyclase 6 (AC6), which is bound to AKAP79/150, to synthesize cAMP which is used by two other AKAP79/150‐tethered proteins: protein kinase A (PKA) and phosphodiesterase 3A (PDE3A). In contrast, A2B‐Ado‐GPCR stimulation activates D‐AKAP2‐attached AC5 to generate cAMP, which is channeled to two other D‐AKAP2‐tethered proteins: guanine‐nucleotide exchange factor 2 (Epac2) and PDE3B. In both cases, prior activation of PKA or Epac2 with selective cAMP analogs prevents de novo cAMP synthesis. In addition, we show that cAMP does not diffuse between these protein macrocomplexes or ‘signalosomes’. Evidence of coimmunoprecipitation and colocalization of some proteins belonging to each signalosome is presented. Each signalosome constitutes a minimal functional signaling unit with its own machinery to synthesize and regulate a sequestered cAMP pool. Thus, each signalosome is devoted to ensure the transmission of a unique and unequivocal message through the cell.

NOX2 activated by α1-adrenoceptors modulates hepatic metabolic routes stimulated by β-adrenoceptors

Abstract The NADPH oxidase (NOX) family of enzymes oxidase catalyzes the transport of electrons from NADPH to molecular oxygen and generates O2•−, which is rapidly converted into H2O2. We aimed to identify in hepatocytes the protein NOX complex responsible for H2O2 synthesis after α1-adrenoceptor (α1-AR) stimulation, its activation mechanism, and to explore H2O2 as a potential modulator of hepatic metabolic routes, gluconeogenesis, and ureagenesis, stimulated by the ARs. The dormant NOX2 complex present in hepatocyte plasma membrane (HPM) contains gp91phox, p22phox, p40phox, p47phox, p67phox and Rac 1 proteins. In HPM incubated with NADPH and guanosine triphosphate (GTP), α1-AR-mediated H2O2 synthesis required all of these proteins except for p40phox. A functional link between α1-AR and NOX was identified as the Gα13 protein. Alpha1-AR stimulation in hepatocytes promotes Rac1-GTP generation, a necessary step for H2O2 synthesis. Negative cross talk between α1-/β-ARs for H2O2 synthesis was observed in HPM. In addition, negative cross talk of α1-AR via H2O2 to β-AR-mediated stimulation was recorded in hepatocyte gluconeogenesis and ureagenesis, probably involving aquaporine activity. Based on previous work we suggest that H2O2, generated after NOX2 activation by α1-AR lightening in hepatocytes, reacts with cAMP-dependent protein kinase A (PKA) subunits to form an oxidized PKA, insensitive to cAMP activation that prevented any rise in the rate of gluconeogenesis and ureagenesis.

In Vivo activation by diets and in vitro activation by hormones are additive for urea synthesis in the rat liver

Abstract The basal rate of urea or glucose synthesis, set in vivo by different diets, was challenged by addition of hormones to isolated hepatocytes. Basal rate of urea synthesis ranged from 7.1 to 23.5 and 27.8 nmol/mg wet weight cells/hr in hepatocytes prepared from rats with a protein free diet or with two distinct but complete protein diets. Maximum difference in the basal rate of glucose synthesis from lactate was only 26.7% for the same diets. Depending on the basal rate of urea synthesis fixed in vivo by the diets, significant in vivo activations superimposed on these, either low or high, basal rates with a remarkable constancy: 2.4-fold with glucagon, 1.9-fold with epinephrine and 1.4-to 1.8-fold with adenosine. Likewise, the basal rate of glucose synthesis from lactate, established by these diets, superimposed 3.7-fold with glucagon, 2.4-fold with epinephrine, 2.1-to 2.4-fold with adenosine and 2.0-to 2.5-fold with inosine. The stimulation of hepatocytes in vivo showed a direct relationship in the increased rates of urea and glucose synthesis. The short-term activation in urea and glucose synthesis superimposed in proportion to the long-term status of urea or glucose formation set in the liver by the diet.