Person:
Blázquez Fernández, Enrique

First Name

Enrique

Last Name

Blázquez Fernández

URI

https://hdl.handle.net/20.500.14352/82765

Affiliation

Universidad Complutense de Madrid

Faculty / Institute

Medicina

Area

Bioquímica y Biología Molecular

Identifiers

Full item page

Search Results

Now showing 1 - 10 of 14

Functional Glucokinase Isoforms Are Expressed in Rat Brain
(Journal of Neurochemistry, 2000) Roncero Rincón, Isabel; Álvarez García, Elvira; Vázquez Pérez, Patricia; Blázquez Fernández, Enrique; Wiley
Recently, the description of glucokinase mRNA in certain neuroendocrine cells has opened new ways to characterize this enzyme in the rat brain. In this study, we found glucokinase mRNA and a similar RNA splicing pattern of the glucokinase gene product in rat hypothalamus and pancreatic islets; the mRNA that codes for B1 isoform was the most abundant, with minor amounts of those coding for the B2, P1, P2, P1/B2, and P2/B2 isoforms. Glucokinase gene expression in rat brain gave rise to a protein of 52 kDa with a high apparent Km for glucose and no product inhibition by glucose 6-phosphate, with a contribution to the total glucose phosphorylating activity of between 40 and 14%; the hypothalamus and cerebral cortex were the regions of maximal activity. Low and high Km hexokinases were characterized by several criteria. Also, using RT-PCR analysis we found a glucokinase regulatory protein mRNA similar to that previously reported in liver. These findings indicate that the glucokinase present in rat brain should facilitate the adaptation of this organ to fluctuations in blood glucose concentrations, and the expression of glucokinase and GLUT-2 in the same hypothalamic neurons suggests a role in glucose sensing.
Aplicaciones terapúticas de la utililzación de células madre humanas presentes en tejidos adultos.
(Fundamentos Moleculares de la Medicina II, 2007) Blázquez Fernández, Enrique; Sanz Miguel, María Del Carmen; Vázquez Pérez, Patricia; Barrio, Pedro; Alvárez, Mar; Roncero Rincón, Isabel; Álvarez García, Elvira
The cytoplasmic domain close to the transmembrane region of the glucagon-like peptide-1 receptor contains sequence elements that regulate agonist-dependent internalisation
(Journal of Endocrinology, 2005) Vázquez Pérez, Patricia; Roncero Rincón, Isabel; Blázquez Fernández, Enrique; Álvarez García, Elvira
In order to gain better insight into the molecular events involved in the signal transduction generated through glucagon-like peptide-1 (GLP-1) receptors, we tested the effect of deletions and point mutations within the cytoplasmic tail of this receptor with a view to establishing relationships between signal transduction desensitisation and receptor internalisation. Wild-type and truncated (deletion of the last 27 amino acids (GLPR 435R) and deletion of 44 amino acids (GLPR 418R)) GLP-1 receptors bound the agonist with similar affinity. Deletion of the last 27 amino acids decreased the internalisation rate by 78%, while deletion of 44 amino acids containing all the phosphorylation sites hitherto described in this receptor decreased the internalisation rate by only 47%. Binding of the ligand to both receptors stimulated adenylyl cyclase. In contrast, deletion of the region containing amino acids 419 to 435 (GLPR 419delta435) increased the internalisation rate by 268%, and the replacement of EVQ(408-410) by alanine (GLPR A(408-410)) increased this process to 296%. In both receptors, the efficacy in stimulating adenylate cyclase was decreased. All the receptors studied were internalised by coated pits, except for the receptor with a deletion of the last 44 amino acids, which also had a faster resensitisation rate. Our findings indicate that the neighbouring trans-membrane domain of the carboxyl-terminal tail of the GLP-1 receptor contains sequence elements that regulate agonist-dependent internalisation and transmembrane signalling.
Expression of glucose transporter isoform GLUT‐2 and glucokinase genes in human brain
(Journal of Neurochemistry, 2004) Chowen, Julie A.; Rábano, Alberto; Vázquez Pérz, Patricia; Roncero Rincón, Isabel; Álvarez García, Elvira; Sanz Miguel, María Del Carmen; Blázquez Fernández, Enrique; Wiley
The glucose transporter isoform-2 (GLUT-2) and glucokinase are considered to be components of a glucose sensor system controlling several key processes, and hence may modulate feeding behaviour. We have found GLUT-2 and glucokinase mRNAs in several brain regions, including the ventromedial and arcuate nuclei of the hypothalamus. GLUT-2, glucokinase and glucokinase regulatory protein mRNAs and proteins were present in these areas as determined by biochemical approaches. In addition, glucose-phosphorylating activity with a high apparent Km for glucose that displayed no product inhibition by glucose-6-phosphate was observed. Increased glycaemia after meals may be recognized by specific hypothalamic neurones due to the high Km of GLUT-2 and glucokinase. This enzyme is considered to be the true glucose sensor because it catalyses the rate-limiting step of glucose catabolism its activity being regulated by interaction with glucokinase regulatory protein, that functions as a metabolic sensor
Substitution of the cysteine 438 residue in the cytoplasmic tail of the glucagon-like peptide-1 receptor alters signal transduction activity
(Journal of Endocrinology, 2005) Vázquez Pérez, Patricia; Roncero Rincón, Isabel; Blázquez Fernández, Enrique; Álvarez García, Elvira; Bioscientifica
Several G-protein-coupled receptors contain cysteine residues in the C-terminal tail that may modulate receptor function. In this work we analysed the substitution of Cys438 by alanine in the glucagon-like peptide-1 (GLP-1) receptor (GLPR), which led to a threefold decrease in cAMP production, although endocytosis and cellular redistribution of GLP-1 receptor agonist-induced processes were unaffected. Additionally, cysteine residues in the C-terminal tail of several G-protein-coupled receptors were found to act as substrates for palmitoylation, which might modify the access of protein kinases to this region. His-tagged GLP-1 receptors incorporated 3H-palmitate. Nevertheless, substitution of Cys438 prevented the incorporation of palmitate. Accordingly, we also investigated the effect of substitution of the consensus sequence by protein kinase C (PKC) Ser431/432 in both wild-type and Ala438 GLP-1 receptors. Substitution of Ser431/432 by alanine did not modify the ability of wild-type receptors to stimulate adenylate cyclase or endocytosis and recycling processes. By contrast, the substitution of Ser431/432 by alanine in the receptor containing Ala438 increased the ability to stimulate adenylate cyclase. All types of receptors were mainly internalised through coated pits. Thus, cysteine 438 in the cytoplasmic tail of the GLP-1 receptor would regulate its interaction with G-proteins and the stimulation of adenylyl cyclase. Palmitoylation of this residue might control the access of PKC to Ser431/432.
Glucagon-Like Peptide 1 (GLP-1) Can Reverse AMP-Activated Protein Kinase (AMPK) and S6 Kinase (P70S6K) Activities Induced by Fluctuations in Glucose Levels in Hypothalamic Areas Involved in Feeding Behaviour
(Molecular Neurobiology, 2012) Hurtado Carneiro, Verónica; Sanz Miguel, María Del Carmen; Roncero Rincón, Isabel; Vázquez Pérez, Patricia; Blázquez Fernández, Enrique; Álvarez García, Elvira; Rancán, Lisa; Bazán, Nicolas G.
The anorexigenic peptide, glucagon-like peptide-1 (GLP-1), reduces glucose metabolism in the human hypothalamus and brain stem. The brain activity of metabolic sensors such as AMP-activated protein kinase (AMPK) responds to changes in glucose levels. The mammalian target of rapamycin (mTOR) and its downstream target, p70S6 kinase (p70S6K), integrate nutrient and hormonal signals. The hypothalamic mTOR/p70S6K pathway has been implicated in the control of feeding and the regulation of energy balances. Therefore, we investigated the coordinated effects of glucose and GLP-1 on the expression and activity of AMPK and p70S6K in the areas involved in the control of feeding. The effect of GLP-1 on the expression and activities of AMPK and p70S6K was studied in hypothalamic slice explants exposed to low- and high-glucose concentrations by quantitative real-time RT-PCR and by the quantification of active-phosphorylated protein levels by immunoblot. In vivo, the effects of exendin-4 on hypothalamic AMPK and p70S6K activation were analysed in male obese Zucker and lean controls 1 h after exendin-4 injection to rats fasted for 48 h or after re-feeding for 2–4 h. High-glucose levels decreased the expression of Ampk in the lateral hypothalamus and treatment with GLP-1 reversed this effect. GLP-1 treatment inhibited the activities of AMPK and p70S6K when the activation of these protein kinases was maximum in both the ventromedial and lateral hypothalamic areas. Furthermore, in vivo s.c. administration of exendin-4 modulated AMPK and p70S6K activities in those areas, in both fasted and re-fed obese Zucker and lean control rats.
PAS Kinase Is a Nutrient and Energy Sensor in Hypothalamic Areas Required for the Normal Function of AMPK and mTOR/S6K1
(Molecular Neurobiology, 2014) Hurtado Carneiro, Verónica; Roncero Rincón, Isabel; Egger, Sascha S.; Wenger, Roland H.; Blázquez Fernández, Enrique; Sanz Miguel, María Del Carmen; Álvarez García, Elvira; Bazán, Nicolas G.
The complications caused by overweight, obesity and type 2 diabetes are one of the main problems that increase morbidity and mortality in developed countries. Hypothalamic metabolic sensors play an important role in the control of feeding and energy homeostasis. PAS kinase (PASK) is a nutrient sensor proposed as a regulator of glucose metabolism and cellular energy. The role of PASK might be similar to other known metabolic sensors, such as AMP-activated protein kinase (AMPK) and the mammalian target of rapamycin (mTOR). PASK-deficient mice resist diet-induced obesity. We have recently reported that AMPK and mTOR/S6K1 pathways are regulated in the ventromedial and lateral hypothalamus in response to nutritional states, being modulated by anorexigenic glucagon-like peptide-1 (GLP-1)/exendin-4 in lean and obese rats. We identified PASK in hypothalamic areas, and its expression was regulated under fasting/re-feeding conditions and modulated by exendin-4. Furthermore, PASK-deficient mice have an impaired activation response of AMPK and mTOR/S6K1 pathways. Thus, hypothalamic AMPK and S6K1 were highly activated under fasted/re-fed conditions. Additionally, in this study, we have observed that the exendin-4 regulatory effect in the activity of metabolic sensors was lost in PASK-deficient mice, and the anorexigenic properties of exendin-4 were significantly reduced, suggesting that PASK could be a mediator in the GLP-1 signalling pathway. Our data indicated that the PASK function could be critical for preserving the nutrient effect on AMPK and mTOR/S6K1 pathways and maintain the regulatory role of exendin-4 in food intake. Some of the antidiabetogenic effects of exendin-4 might be modulated through these processes.
Glucagon-like peptide-1 and its implications in obesity.
(Hot Topics in Endocrine and Endocrine-Related Diseases, 2013) Hurtado Carneiro, Verónica; Roncero Rincón, Isabel; Blázquez Fernández, Enrique; Álvarez García, Elvira; Sanz Miguel, María Del Carmen; Fedele, Monica
Evidence that glucokinase regulatory protein is expressed  and interacts with glucokinase in rat brain
(Journal of Neurochemistry, 2001) Chowen, Julie A.; Vázquez Pérez, Patricia; Álvarez García, Elvira; Roncero Rincón, Isabel; Blázquez Fernández, Enrique; Wiley
Our previous description of functional glucokinase isoforms in the rat brain has opened new questions concerning the presence of glucokinase regulatory protein in the brain and the functional role of its interactions with glucokinase. In this study, we found glucokinase regulatory protein mRNA in rat brain, pancreatic islets and liver. In addition, we found two other variant splicing isoforms, both identified in hypothalamus, pancreatic islets and liver. In situ hybridization studies revealed the presence of glucokinase regulatory protein mRNA, the highest number of positive cells being found in the paraventricular nucleus of the hypothalamus. Glucokinase regulatory protein gene expression gave rise to a protein of 69 kDa mainly in nuclear and soluble cell fractions. Glutathione S-transferase protein fused either to rat liver or human pancreatic islet glucokinase were able to precipitate glucokinase regulatory protein from liver or hypothalamic extracts in the presence of fructose-6-phosphate, the amount of protein co-precipitated being decreased with fructose-1-phosphate. These findings suggest that the presence of glucokinase and glucokinase regulatory protein in the rat brain would facilitate the adaptation of this organ to fluctuations in blood glucose concentrations, and both proteins may participate in glucose-sensing and metabolic regulation in the central nervous system.
Insulin-Receptor Substrate-2 (IRS-2) Is Required for Maintaining Glucokinase and Glucokinase Regulatory Protein Expression in Mouse Liver
(Plos One, 2013) Roncero Rincón, Isabel; Álvarez García, Elvira; Acosta, Carlos; Sanz Miguel, María Del Carmen; Barrio, Pedro; Hurtado Carneiro, Verónica; Burks, Deborah; Blázquez Fernández, Enrique; Hennige, Anita Magdalena
Insulin receptor substrate (IRS) proteins play important roles in hepatic nutrient homeostasis. Since glucokinase (GK) and glucokinase regulatory protein (GKRP) function as key glucose sensors, we have investigated the expression of GK and GKRP in liver of Irs-2 deficient mice and Irs2(-/-) mice where Irs2 was reintroduced specifically into pancreatic β-cells [RIP-Irs-2/IRS-2(-/-)]. We observed that liver GK activity was significantly lower (p<0.0001) in IRS-2(-/-) mice. However, in RIP-Irs-2/IRS-2(-/-) mice, GK activity was similar to the values observed in wild-type animals. GK activity in hypothalamus was not altered in IRS-2(-/-) mice. GK and GKRP mRNA levels in liver of IRS-2(-/-) were significantly lower, whereas in RIP-Irs-2/IRS-2(-/-) mice, both GK and GKRP mRNAs levels were comparable to wild-type animals. At the protein level, the liver content of GK was reduced in IRS-2(-/-) mice as compared with controls, although GKRP levels were similar between these experimental models. Both GK and GKRP levels were lower in RIP-Irs-2/IRS-2(-/-) mice. These results suggest that IRS-2 signalling is important for maintaining the activity of liver GK. Moreover, the differences between liver and brain GK may be explained by the fact that expression of hepatic, but not brain, GK is controlled by insulin. GK activity was restored by the β-cell compensation in the RIP-Irs-2/IRS-2 mice. Interestingly, GK and GKRP protein expression remained low in RIP-Irs-2/IRS-2(-/-) mice, perhaps reflecting different mRNA half-lives or alterations in the process of translation and post-translational regulation.