Carlos de Miguel

Caveolin expression and activation in retroperitoneal and subcutaneous adipocytes: influence of a high-fat diet.

The effect of a high‐fat diet on the expression of the three main isoforms of caveolins in adipocytes isolated from rat retroperitoneal and subcutaneous white adipose tissue was investigated. Two distinct phases can be distinguished on a time‐dependent response in adipocytes from both locations. The early stage affects only to retroperitoneal adipocytes and implies caveolin‐1 activation and caveolin‐2 inactivation, together with increased expression of insulin signaling intermediaries. This initial response would be aimed to counterbalance the energy overload. Continued exposure to the high‐fat diet produces an increase in circulating glucose and insulin levels, inducing a late stage in which adipocytes from both locations are affected. This late stage is characterized by general increased caveolin‐1 and caveolin‐2 expression; while on the other hand, the insulin signaling intermediaries are downregulated, with the noticeable exception of GLUT‐4, whose expression remains high. Therefore, it seems that at this stage caveolins and GLUT‐4 are regulated independently of the insulin pathway, through a mechanism that could be mediated by inflammation and oxidative stress associated with obesity. Although this GLUT‐4 upregulation suggests a response against the raise in circulating glucose, this might not be the case, since the developing insulin resistance at this stage indicates a prediabetic state. We have also found that the high‐fat diet is able to induce the expression of muscle‐specific caveolin‐3 in retroperitoneal adipocytes since the initial phase. This observation is similar to what we reported previously in skeletal muscle (Gómez‐Ruiz et al., 2009, FEBS Lett 583:3259–3264), suggesting a similar regulatory mechanism for this isoform. J. Cell. Physiol. 225: 206–213, 2010.

Methodological approaches to assess body-weight regulation and aetiology of obesity.

Obesity, which is becoming one of the major health hazards in developed and developing societies, results from a long-term positive energy balance. Body-weight regulation and stability depend on an axis with three interrelated components: food intake, energy expenditure and adipogenesis, although there are still many unknown features concerning fuel homeostasis and energy balance. Biochemical processes are interconnected, and a separate consideration of each component is often useful for methodological purposes and to achieve a better understanding of the whole system. Thus, many different experimental approaches can be applied by using laboratory animals, cell culture or human subjects to unravel the molecular mechanisms which participate in body-weight regulation. Thus, both in vitro (cellular and subcellular models) and in vivo methods have dramatically increased our knowledge of weight control. Several strategies in obesity research are reported here, exploiting the opportunities of the molecular era as well as novel whole-body approaches, which will impact on the development of new targets for obesity management and prevention.

Expression of Caveolin 1 Is Enhanced by DNA Demethylation during Adipocyte Differentiation. Status of Insulin Signaling

Caveolin 1 (Cav-1) is an essential constituent of adipocyte caveolae which binds the beta subunit of the insulin receptor (IR) and is implicated in the regulation of insulin signaling. We have found that, during adipocyte differentiation of 3T3-L1 cells the promoter, exon 1 and first intron of the Cav-1 gene undergo a demethylation process that is accompanied by a strong induction of Cav-1 expression, indicating that epigenetic mechanisms must have a pivotal role in this differentiation process. Furthermore, IR, PKB-Akt and Glut-4 expression are also increased during the differentiation process suggesting a coordinated regulation with Cav-1. Activation of Cav-1 protein by phosphorylation arises during the differentiation process, yet in fully mature adipocytes insulin is no longer able to significantly increase Cav-1 phosphorylation. However, these long-term differentiated cells are still able to respond adequately to insulin, increasing IR and PKB-Akt phosphorylation and glucose uptake. The activation of Cav-1 during the adipocyte differentiation process could facilitate the maintenance of insulin sensitivity by these fully mature adipocytes isolated from additional external stimuli. However, under the influence of physiological conditions associated to obesity, such as chronic inflammation and hypoxia, insulin sensitivity would finally be compromised.

Time-dependent regulation of muscle caveolin activation and insulin signalling in response to high-fat diet

We studied the effect of high‐fat diet on the expression and activation of the three caveolins in rat skeletal muscle and their association with the insulin signalling cascade. Initial response was characterized by increased signalling through Cav‐1 and Cav‐3 phosphorylation, suggesting that both participate in an initial acute response to the calorie surplus. Afterwards, Cav‐1 signalling was slightly reduced, whereas Cav‐3 remained active. Late chronic phase signalling through both proteins was impaired inducing a prediabetic state. Summarizing, caveolins seem to mediate a time‐dependent regulation of insulin cascade in response to high‐fat diet in muscle.

PROTEOLYSIS OF ALZHEIMER'S DISEASE BETA -AMYLOID PRECURSOR PROTEIN BY FACTOR XA

Amyloid beta-protein is a 4-kDa peptide which originates from proteolysis of a larger protein precursor (APP) and accumulates in senile plaques in brains of Alzheimers disease (AD) patients. Since secreted APP inhibits factors IXa, Xa and XIa, and thrombin appears to play a role in APP secretion and proteolysis, a relationship between hemostasis system and APP metabolism seems to exist. In this work we investigate the susceptibility to proteolytic cleavage by factor Xa of a fusion construct containing full-length APP prepared in bacteria, and demonstrate that both APP695 and APP770 are substrates for this protease. Factor Xa was found to cleave APP after arginines 102, 268, 510, 573 and 601 (APP695 numeration); most of these sites appear to be common for different coagulation factors. In addition, APP incubation with factor Xa generates an array of six potentially amyloidogenic fragments. Comparative kinetic analysis of APP695 and APP770 cleavage by factor Xa suggests that Kunitz-type inhibitor-containing isoforms exert an inhibitory effect on the protease. However, this inhibition is far from complete even at a 5-fold molar excess of inhibitor. Our results raise the possibility that proteases from the coagulation cascade may contribute to APP proteolysis, and support the notion that these proteases play a role in AD pathogenesis.

Effect of TNF-Alpha on Caveolin-1 Expression and Insulin Signaling During Adipocyte Differentiation and in Mature Adipocytes

Background/Aims: Tumor necrosis factor-α (TNF-α)-mediated chronic low-grade inflammation of adipose tissue is associated with obesity and insulin resistance. Caveolin-1 (Cav-1) is the central component of adipocyte caveolae and has an essential role in the regulation of insulin signaling. The effects of TNF-α on Cav-1 expression and insulin signaling during adipocyte differentiation and in mature adipocytes were studied. Methods: 3T3-L1 cells were differentiated (21 days) in the presence TNF-α (10 ng/mL) and mature adipocytes were also treated with TNF-α for 48 hours. Cav-1 and insulin receptor (IR) gene methylation were determined as well as Cav-1, IR, PKB/AKT-2 and Glut-4 expression and activation by real time RT-PCR and western blot. Baseline and insulin-induced glucose uptake was measured by the 2-[C14]-deoxyglucose uptake assay. Results: TNF-α slowed down the differentiation program, hindering the expression of some insulin signaling intermediates without fully eliminating insulin-mediated glucose uptake. In mature adipocytes, TNF-α did not compromise lipid-storage capacity, but downregulated the expression of the insulin signaling intermediates, totally blocking insulin-mediated glucose uptake. Insulin sensitivity correlated with the level of activated phospho-Cav-1 in both situations, strongly suggesting the direct contribution of Cav-1 to the maintenance of this physiological response. Conclusion: Cav-1 activation by phosphorylation seems to be essential for the maintenance of an active and insulin-sensitive glucose uptake.

High-fat diet feeding alters metabolic response to fasting/non fasting conditions. Effect on caveolin expression and insulin signalling

BackgroundThe effect of food intake on caveolin expression in relation to insulin signalling was studied in skeletal muscle and adipocytes from retroperitoneal (RP) and subcutaneous (SC) adipose tissue, comparing fasted (F) to not fasted (NF) rats that had been fed a control or high-fat (HF) diet for 72 days.MethodsSerum glucose was analysed enzymatically and insulin and leptin by ELISA. Caveolins and insulin signalling intermediaries (IR, IRS-1 and 2 and GLUT4) were determined by RT-PCR and western blotting. Caveolin and IR phosphorylation was measured by immunoprecipitation. Data were analysed with Mann-Whitney U test.ResultsHigh-fat fed animals showed metabolic alterations and developed obesity and insulin resistance. In skeletal muscle, food intake (NF) induced activation of IR and increased expression of IRS-2 in control animals with normal metabolic response. HF animals became overweight, hyperglycaemic, hyperinsulinemic, hyperleptinemic and showed insulin resistance. In skeletal muscle of these animals, food intake (NF) also induced IRS-2 expression together with IR, although this was not active. Caveolin 3 expression in this tissue was increased by food intake (NF) in animals fed either diet. In RP adipocytes of control animals, food intake (NF) decreased IR and IRS-2 expression but increased that of GLUT4. A similar but less intense response was found in SC adipocytes. Food intake (NF) did not change caveolin expression in RP adipocytes with either diet, but in SC adipocytes of HF animals a reduction was observed. Food intake (NF) decreased caveolin-1 phosphorylation in RP but increased it in SC adipocytes of control animals, whereas it increased caveolin-2 phosphorylation in both types of adipocytes independently of the diet.ConclusionsAnimals fed a control-diet show a normal response to food intake (NF), with activation of the insulin signalling pathway but without appreciable changes in caveolin expression, except a small increase of caveolin-3 in muscle. Animals fed a high-fat diet develop metabolic changes that result in insulin signalling impairment. In these animals, caveolin expression in muscle and adipocytes seems to be regulated independently of insulin signalling.

All‐trans‐retinoic acid inhibits collapsin response mediator protein‐2 transcriptional activity during SH‐SY5Y neuroblastoma cell differentiation

Neurons are highly polarized cells composed of two structurally and functionally distinct parts, the axon and the dendrite. The establishment of this asymmetric structure is a tightly regulated process. In fact, alterations in the proteins involved in the configuration of the microtubule lattice are frequent in neuro‐oncologic diseases. One of these cytoplasmic mediators is the protein known as collapsin response mediator protein‐2, which interacts with and promotes tubulin polymerization. In this study, we investigated collapsin response mediator protein‐2 transcriptional regulation during all‐trans‐retinoic acid‐induced differentiation of SH‐SY5Y neuroblastoma cells. All‐trans‐retinoic acid is considered to be a potential preventive and therapeutic agent, and has been extensively used to differentiate neuroblastoma cells in vitro. Therefore, we first demonstrated that collapsin response mediator protein‐2 mRNA levels are downregulated during the differentiation process. After completion of deletion construct analysis and mutagenesis and mobility shift assays, we concluded that collapsin response mediator protein‐2 basal promoter activity is regulated by the transcription factors AP‐2 and Pax‐3, whereas E2F, Sp1 and NeuroD1 seem not to participate in its regulation. Furthermore, we finally established that reduced expression of collapsin response mediator protein‐2 after all‐trans‐retinoic acid exposure is associated with impaired Pax‐3 and AP‐2 binding to their consensus sequences in the collapsin response mediator protein‐2 promoter. Decreased attachment of AP‐2 is a consequence of its accumulation in the cytoplasm. On the other hand, Pax‐3 shows lower binding due to all‐trans‐retinoic acid‐mediated transcriptional repression. Unraveling the molecular mechanisms behind the action of all‐trans‐retinoic acid on neuroblastoma cells may well offer new perspectives for its clinical application.

Interaction of f1-atpase and its inhibitor peptide effect of pH

1. The inhibition of F1-ATPase by its natural peptide inhibitor is mixed non-competitive with two pH optimum values (5.5 and 8.2). 2. A two-step model for the interaction is suggested in which two enzyme conformations would exhibit different affinities for the peptide. 3. At low pH, interaction would be favoured. At high pH, a conformation (not susceptible to inhibition) changes into another (susceptible to inhibition) through the hydrolytic reaction stimulation, due to high pH.

Interaction of F1-ATPase and its inhibitor peptide. Effect of dinitrophenol, nucleotides and anions.

1. ATPase natural inhibitor interacted in a mixed non-competitive manner with compounds affecting hydrolytic activity. 2. Kas for DNP, HCO3- and free ATP, and Kis for SCN- and ADP became smaller as inhibitor peptide concentration increased, reflecting an increase in affinity of F1-ATPase for these compounds induced by the peptide. 3. Activators increased the peptide inhibitory effect, whereas inhibitors decreased it. 4. A two-step model for the peptide-enzyme interaction is suggested in which ATP hydrolysis is a key factor.