Daniel Da Silva

Microcapsules of alginate/chitosan containing magnetic nanoparticles for controlled release of insulin

The challenge of this work was to investigate the potential of alginate/chitosan beads containing magnetite nanoparticles as a drug delivery system. The insulin beads were prepared by dripping a solution of sodium alginate containing insulin into a CaCl(2) solution. Magnetite nanoparticles of 5 nm mean size were synthesized inside the alginate egg-box structure by co-precipitation of Fe(III) and Fe(II) in the presence of NH(4)OH. Quantitative analysis revealed that insulin encapsulation depends on the initial protein content and 35% of insulin was entrapped by alginate beads for a protein concentration of 10 wt%. It was verified that approximately 50% of the insulin was released to Milli-Q water in 800 h release experiments. The application of oscillating magnetic field increased three fold the insulin release. The results suggest that the alginate/chitosan system containing magnetite nanoparticles is a promising system for clinical applications of controlled release of insulin in the presence of an oscillating magnetic field in a subcutaneous implant approach.

Lactate favours the dissociation of skeletal muscle 6-phosphofructo-1- kinase tetramers down-regulating the enzyme and muscle glycolysis

For a long period lactate was considered as a dead-end product of glycolysis in many cells and its accumulation correlated with acidosis and cellular and tissue damage. At present, the role of lactate in several physiological processes has been investigated based on its properties as an energy source, a signalling molecule and as essential for tissue repair. It is noteworthy that lactate accumulation alters glycolytic flux independently from medium acidification, thereby this compound can regulate glucose metabolism within cells. PFK (6-phosphofructo-1-kinase) is the key regulatory glycolytic enzyme which is regulated by diverse molecules and signals. PFK activity is directly correlated with cellular glucose consumption. The present study shows the property of lactate to down-regulate PFK activity in a specific manner which is not dependent on acidification of the medium. Lactate reduces the affinity of the enzyme for its substrates, ATP and fructose 6-phosphate, as well as reducing the affinity for ATP at its allosteric inhibitory site at the enzyme. Moreover, we demonstrated that lactate inhibits PFK favouring the dissociation of enzyme active tetramers into less active dimers. This effect can be prevented by tetramer-stabilizing conditions such as the presence of fructose 2,6-bisphosphate, the binding of PFK to f-actin and phosphorylation of the enzyme by protein kinase A. In conclusion, our results support evidence that lactate regulates the glycolytic flux through modulating PFK due to its effects on the enzyme quaternary structure.

Lactate downregulates the glycolytic enzymes hexokinase and phosphofructokinase in diverse tissues from mice

We examined the effects of lactate on the enzymatic activity of hexokinase (HK), phosphofructokinase (PFK) and pyruvate kinase (PK) in various mouse tissues. Our results showed that lactate inhibited PFK activity in all the analyzed tissues. This inhibitory effect was observed in skeletal muscle even in the presence of insulin. Lactate directly inhibited the phosphorylation of PFK tyrosine residues in skeletal muscle, an important mechanism of the enzyme activation. Moreover, lactate indirectly inhibited HK activity, which resulted from its cellular redistribution, here attributed to alterations of HK structure. PK activity was not affected by lactate. The activity of HK and PFK is directly related to glucose metabolism. Thus, it is conceivable that lactate exposure can induce inhibition of glucose consumption in tissues.

Regulation of mammalian muscle type 6‐phosphofructo‐1‐kinase and its implication for the control of the metabolism

Phosphofructokinase (PFK) is a major regulatory glycolytic enzyme and is considered to be the pacemaker of glycolysis. This enzyme presents a puzzling regulatory mechanism that is modulated by a large variety of metabolites, drugs, and intracellular proteins. To date, the mammalian enzyme structure has not yet been resolved. However, it is known that PFK undergoes an intricate oligomerization process, shifting among monomers, dimers, tetramers, and more complex oligomeric structures. The equilibrium between PFK dimers and tetramers is directly correlated with the enzyme regulation, because the dimer exhibits very low catalytic activity, whereas the tetramer is fully active. Several PFK ligands modulate the enzyme, favoring the formation of its dimers or tetramers. The present review integrates recent findings regarding the regulatory aspects of muscle type PFK and discusses their relation to the control of metabolism.

Differential expression of phosphofructokinase-1 isoforms correlates with the glycolytic efficiency of breast cancer cells

Cancer cells are characterized by increased aerobic glycolysis, which correlates with a negative prognosis. Although this correlation is well known, the mechanism of the elevated rate of glycolysis in cancer and the role of glycolytic enzymes have yet to be determined. The present work aims to evaluate the activity of the major enzymes that regulate glycolysis in breast cancer cell lines of varying aggressiveness. MCF10A, MCF-7 and MDA-mb-231 are human breast-derived cell lines with non-tumorigenic, tumorigenic and metastatic profiles, respectively. These cell lines have increasing degrees of glycolytic efficiency, i.e., lactate produced per glucose consumed, corresponding to their metastatic potential. Although, there are no differences in phosphofructokinase (PFK) or pyruvate kinase (PK) activities, the activity of hexokinase (HK) activity is higher in both tumorigenic cell lines compared to MCF10A cells. No difference in HK activity is observed between MCF-7 and MDA-mb-231 cells, suggesting that the difference in their glycolytic efficiency could not be attributed to this enzyme. However, we find that expression of the PFK-L isoform directly and strongly correlates with aggressiveness and glycolytic efficiency in these cell lines. Thus, we conclude that glycolytic efficiency, which is important for the survival of cancer cells, depends primarily on the preferential expression of PFK-L over the M and P isoforms.

Resveratrol decreases breast cancer cell viability and glucose metabolism by inhibiting 6-phosphofructo-1-kinase

Cancer cells are highly dependent on glycolysis to supply the energy and intermediates required for cell growth and proliferation. The enzyme 6-phosphofructo-1-kinase (PFK) is critical for glycolysis, and its activity is directly correlated with cellular glucose consumption. Resveratrol is a potential anti-tumoral drug that decreases glucose metabolism and viability in cancer cells. However, the mechanism involved in resveratrol-mediated anti-tumor activity is not entirely clear. In this work, it is demonstrated that resveratrol decreases viability, glucose consumption and ATP content in the human breast cancer cell line MCF-7. These effects are directly correlated with PFK inhibition by resveratrol in these cells. Moreover, resveratrol directly inhibits purified PFK, promoting the dissociation of the enzyme from fully active tetramers into less active dimers. This effect is exacerbated by known negative regulators of the enzyme, such as ATP and citrate. On the other hand, positive modulators that stabilize the tetrameric form of the enzyme, such as fructose-2,6-bisphosphate and ADP, prevent the inhibition of PFK activity by resveratrol, an effect not observed with increased pH. In summary, our results provide evidence that resveratrol directly inhibits PFK activity, therefore disrupting glucose metabolism and reducing viability in cancer cells.

Filamentous actin and its associated binding proteins are the stimulatory site for 6-phosphofructo-1-kinase association within the membrane of human erythrocytes.

Glycolytic enzymes reversibly associate with the human erythrocyte membrane (EM) as part of their regulatory mechanism. The site for this association has been described as the amino terminus of band 3, a transmembrane anion transporter. Binding of glycolytic enzymes to this site is recognized to inhibit glycolysis, since binding inhibits the catalytic activity of these enzymes, including the rate-limiting enzyme 6-phosphofructo-1-kinase (PFK). However, the existence of a putative stimulatory site for glycolytic enzymes within the EM has been proposed. PFK has been described as able to reversibly associate with other proteins, such as microtubules, which inhibit the enzyme, and filamentous actin, which activates the enzyme. Here, it is demonstrated that PFK also binds to actin filaments and its associated binding proteins in the protein meshwork that forms the erythrocyte cytoskeleton. Through fluorescence resonance energy transfer experiments using either confocal microscopy or fluorescence spectroscopy, we show that, within the EM, PFK and actin filaments containing its associated binding proteins are located close enough to propose binding between them. Moreover, specifically blocking PFK binding to band 3 results in an association of the enzyme with the EM that increases the enzymes catalytic activity. Conversely, disruption of the association between PFK and actin filaments containing its associated binding proteins potentiates the inhibitory action of the EM on the enzyme. Furthermore, it is shown that insulin signaling increases the association of PFK to actin filaments and its associated binding proteins, revealing that this event may play a role on the stimulatory effects of insulin on erythrocyte glycolysis. In summary, the present work presents evidence that filamentous actin and its associated binding proteins are the stimulatory site for PFK within the EM.

Metformin reverses hexokinase and 6-phosphofructo-1-kinase inhibition in skeletal muscle, liver and adipose tissues from streptozotocin-induced diabetic mouse.

The present work describes the effects of metformin on hexokinase (HK) and phosphofructokinase (PFK) activities and localization in different tissues from streptozotocin-induced diabetic mice. Diabetic mice present lower HK and PFK activities (50%) in skeletal muscle, liver and adipose tissue, as compared with control (P<0.05). Treatment with 250 mg/kg metformin reverses this pattern of enzyme inhibition with concomitant reversal of hyperglycemia and hypolactacidemia. Furthermore, the treatment increases the cytoskeleton-associated PFK activity in skeletal muscle; this activity has been described as an important mechanism for the enzyme activation. This effect might be due to the increased phosphorylation of serine residues in the enzyme, a modification which has been described to increase the interaction of PFK with f-actin. The current work supports the hypothesis that metformin hypoglycemic effects involve the activation of glycolysis through its regulatory enzymes, which may be potential targets for the development of new hypoglycemic drugs.

Polymeric particles for the controlled release of human amylin

Since its discovery the therapeutic use of the pancreatic hormone amylin has been limited due to its poor water solubility and propensity for amyloid aggregation. We have entrapped the human amylin protein in polymeric nanoparticles, using a single emulsion-solvent evaporation method and investigated its effectiveness in the controlled release of the peptide. Typical preparations composed of poly-ε-caprolactone had a mean particle size of approximately 200 nm, low polydispersity index, high protein entrapment efficiency (80%) and process yield (90%), and spherical and smooth surfaces. These nanoparticles presented a controlled release in vitro for approximately 240 h. Pharmacological evaluation in vivo by subcutaneous administration in fasting mice demonstrated the bioactivity and effectiveness of the released human amylin, resulting in reduced glycemia lasting for at least 36 h. These features indicate the potential for the use of a confined particulate system in the therapeutic controlled and sustained release of human amylin.

Serotonin modulates hepatic 6-phosphofructo-1-kinase in an insulin synergistic manner

Human and rat hepatic tissue express many serotonin (5-HT) receptor subtypes, such as 5-HT(1B), 5-HT(2A), 5-HT(2B) and 5-HT(7) receptors, which mediate diverse effects. 5-HT is known to regulate several key aspects of liver biology including hepatic blood flow, innervations and wound healing. 5-HT is also known to enhance net glucose uptake during glucose infusion in fasted dogs, but little is known about the ability of 5-HT to control hepatic glucose metabolism, especially glycolysis. This study addresses the potential of 5-HT to regulate PFK activity and the mechanisms related to the enzyme activity. Based on our results, we are the first to provide evidence that 5-HT up-regulates PFK in mouse hepatic tissue. Activation of the enzyme occurs through the 5-HT(2A) receptor and phospholipase C (PLC), resulting in PFK intracellular redistribution and favoring PFK association to the cytoskeletal f-actin-enriched fractions. Interestingly, 5-HT and insulin act in a synergistic manner, likely because of the ability of insulin to increase fructose-2,6-bisphosphate because the presence of this PFK allosteric regulator enhances the 5-HT effect on the enzyme activity. Together, these data demonstrate the ability of 5-HT to control hepatic glycolysis and present clues about the mechanisms involved in these processes, which may be important in understanding the action of 5-HT during the hepatic wound healing process.