Biomedical subjects

Enrique Roche

Publications and source records attributed to Enrique Roche.

10 recordsLinked to original sources

PubMed2006

Response of antioxidant defences to oxidative stress induced by prolonged exercise: antioxidant enzyme gene expression in lymphocytes.

The response of lymphocyte and plasma antioxidant defences to a prolonged exercise as a cycling stage in a professional race was analysed. Antioxidant enzyme activities and gene expression, carbonyl derivative and MDA levels were determined in lymphocytes. Plasma levels of vitamin E, carotenes, protein carbonyl derivatives and the test d-Roms were measured. Significant increases in plasmatic carbonyls and in the test d-Roms were observed after the cycling stage. No significant differences were found in the lymphocyte MDA and carbonyl derivative levels. A significant increase was found in plasma vitamin E concentration after the cycling stage; however, the lymphocyte vitamin E concentration did not change. Significant increases were observed in lymphocyte total superoxide dismutase (SOD) activity and in the levels of CuZn-SOD and Mn-SOD isoenzymes. The moderate levels of oxidative stress in the lymphocyte induced a cellular adaptation to exercise enough to counteract the negative effects of oxidative stress.

Antioxidants↗

PubMed2006

Role of small bioorganic molecules in stem cell differentiation to insulin-producing cells.

The use of small specific molecules has been instrumental in the modulation of stem cell proliferation and differentiation to obtain insulin-containing cells. Examples include nutrients (glucose, nicotinamide and retinoic acid), acids (butyrate), alkaloids (cyclopamine and conophylline) and pharmacological agents (LY294002 and wortmannin). These molecules, alone or in combination with specific growth factors and hormones, will likely provide key information to design specific culture media in order to obtain customized cells for implantation in diabetes. In addition, the study of such molecules will help to understand the mechanisms involved in stem cell biology as well as contribute to the design of specific drugs for islet repair and regeneration in diabetes.

Alkaloids↗

PubMed2005

Insulin-secreting cells derived from stem cells: clinical perspectives, hypes and hopes.

Diabetes is a degenerative disease that results from the selective destruction of pancreatic beta-cells. These cells are responsible for insulin production and secretion in response to increases in circulating concentrations of nutrients, such as glucose, fatty acids and amino acids. This degenerative disease can be treated by the transplantation of differentiated islets obtained from cadaveric donors, according to a new surgical intervention developed as Edmonton protocol. Compared to the classical double transplant kidney-pancreas, this new protocol presents several advantages, concerning to the nature of the implant, immunosuppressive drug regime and the surgical procedure itself. However, the main problem to face in any islet transplantation program is the scarcity of donor pancreases and the low yield of islets isolated (very often around 50%) from each pancreas. Nevertheless, transplanted patients presented no adverse effects and no progression of diabetic complications. In the search of new cell sources for replacement trials, stem cells from embryonic and adult origins represent a key alternative. In order to become a realistic clinical issue transplantation of insulin-producing cells derived from stem cells, it needs to overcome multiple experimental obstacles. The first one is to develop a protocol that may allow obtaining a pure population of functional insulin-secreting cells as close as possible to the pancreatic beta-cell. The second problem should concern to the transplantation itself, considering issues related to immune rejection, tumour formation, site for implant, implant survival, and biosafety mechanisms. Although transplantation of bioengineered cells is still far in time, experience accumulated in islet transplantation protocols and in experiments with appropriate animal models will give more likely the clues to address this question in the future.

Animals↗

PubMed2005

Ectodermal commitment of insulin-producing cells derived from mouse embryonic stem cells.

Embryonic stem cells possess the ability to differentiate in vitro into a variety of cell lineages, including insulin-producing cells. Pancreatic beta-cells derive from foregut endoderm during embryonic development. However, previous reports using transgenic mice strongly indicate that insulin-positive cells may be generated also through the neuroectoderm pathway. To analyze this point, a culture system was performed in which only ectoderm committed cells were present. Based on published work, we achieved this by maintaining transfected clonal R1 mouse embryonic stem cells in monolayer in the absence of LIF. Contrary to differentiation protocols via embryoid body formation, monolayer cultured cells displayed ectodermal fates according to the marker gene expression pattern. Under these particular conditions, neomycin was added in order to select insulin-expressing cells. The cell lineage obtained expressed Pdx1, Pax6, Isl1, AChE, MBP, TH, and GS genes, confirming ectodermal commitment, even though some of these factors are also expressed in endoderm. In addition these cells displayed excitatory properties similar to astrocytes. Co-expression of insulin II and nestin was observed in monolayer culture and in the presence of specific conditioned media. No expression of early endodermal markers was detected along monolayer cultures. Altogether, these observations suggest that cells with ectoderm fates could participate in vitro in the derivation of insulin-producing cells. These results have implications for insulin gene regulation and hormone secretion in order to generate insulin-producing cells for replacement protocols in the treatment of diabetes.

Animals↗

PubMed2005

Generation of insulin-producing cells from stem cells.

Islet transplantation as a potential treatment for diabetes will always be limited mainly because of the difficulty in obtaining sufficiently large numbers of purified islets from cadaveric donors. One alternative to organ or tissue transplantation is the use of a renewable source of cells. Stem cells are clonogenic cells capable of both self-renewal and multilineage differentiation. Therefore, these cells have the potential to proliferate and differentiate into any type of cell and to be genetically modified in vitro, thus providing cells which can be isolated and used for transplantation. Moreover, these derived cells have proven to be useful in different animal models. In this regard, insulin-secreting cells derived from mouse embryonic stem cells normalize blood glucose when transplanted into streptozotocin-induced diabetic animals. Using a combination of several differentiation methods and a 'cell trapping' system, we have obtained insulin-secreting cells from undifferentiated embryonic stem cells. The construct used allows the expression of a neomycin selection system under the control of the regulatory regions of insulin gene and other beta cell genes, such as Nkx6.1. Transplanted animals correct hyperglycaemia within 1 week and restore body weight in four weeks. Graft removal rescued the diabetic condition. Glucose tolerance test (IPGTT) and blood glucose normalization after a challenge meal was similar in control and in transplanted animals. This approach opens new possibilities for tissue transplantation in the treatment of type 1 and 2 diabetes.

Animals↗

PubMed2004

Generation of new islets from stem cells.

Spain ranks number one in organ donors (35 per million per yr). Although the prevalence of diabetes is low (100,000 type 1 diabetic patients and 2 million type 2 diabetic patients), the expected number of patients receiving islet transplants should be estimated at 200 per year. Islet replacement represents a promising cure for diabetes and has been successfully applied in a limited number of type 1 diabetic patients, resulting in insulin independence for periods longer than 3 yr. However, it has been difficult to obtain sufficient numbers of islets from cadaveric donors. Interesting alternatives include acquiring renewable sources of cells using either embryonic or adult stem cells to overcome the islet scarcity problem. Stem cells are capable of extensive proliferation rates and are capable of differentiating into other cell types of the body. In particular, totipotent stem cells are capable of differentiating into all cell types in the body, whereas pluripotent stem cells are limited to the development of a certain number of differentiated cell types. Insulin-producing cells have been obtained from both embryonic and adult stem cells using several approaches. In animal models of diabetes, the therapeutic application of bioengineered insulin-secreting cells derived from stem cells has delivered promising results. This review will summarize the different approaches that have been used to obtain insulin-producing cells from embryonic and adult stem cells and highlights the key points that will allow in vitro differentiation and subsequent transplantation in the future.

Animals↗

PubMed2004

Nutrients induce different Ca(2+) signals in cytosol and nucleus in pancreatic beta-cells.

Specific activation of Ca(2+)-dependent functions is achieved by the particular dynamics and local restriction of Ca(2+) signals. It has been shown that changes in amplitude, duration, or frequency of Ca(2+) signals modulate gene transcription. Thus, Ca(2+) variations should be finely controlled within the nucleus. Although a variety of mechanisms in the nuclear membrane have been demonstrated to regulate nuclear Ca(2+), the existence of an autonomous Ca(2+) homeostasis within the nucleus is still questioned. In the pancreatic beta-cell, besides their effect on insulin secretion, Ca(2+) messages generated by nutrients also exert their action on gene expression. However, the dynamics of these Ca(2+) signals in relation to nuclear function have been explored little in islet cells. In the current study, Ca(2+) changes both in the nucleoplasm and in the cytosol of INS-1 and pancreatic beta-cells were monitored using spot confocal microscopy. We show that nutrients trigger Ca(2+) signals of higher amplitude in the nucleus than in the cytosol. These amplitude-modulated Ca(2+) signals transmitted to the nucleus might play an important role in the control of gene expression in the pancreatic beta-cell.

ATP-Binding Cassette Transporters↗

PubMed2003

Mitochondrial dysfunction is involved in apoptosis induced by serum withdrawal and fatty acids in the beta-cell line INS-1.

The potential toxic effects of high extracellular concentrations of fatty acids were tested in beta(INS-1)-cells cultured in the absence of serum, a condition known to alter cell survival in various systems. This may in part mimic the situation in type 1 or 2 diabetes where beta-cells are already insulted by various stressful conditions, such as cytokines and oxidative stress. Serum removal caused, over a 36-h period, oxidative stress and an early impairment of mitochondrial function, as revealed by increased superoxide production and markedly reduced mitochondrial membrane potential, but a lack of cytochrome c and apoptosis-inducing factor release in the cytosol. The fatty acids palmitate and oleate considerably accelerated the apoptosis process in serum-starved cells, as revealed by fluorescence-activated cell sorting analysis, morphological changes, chromatin condensation, DNA laddering, poly(ADP-ribose) polymerase cleavage, cytochrome c and apoptosis-inducing factor release, and increased levels of Bax and cytosolic caspase-2. The fatty acids also increased nitric oxide production, apparently independently of inducible nitric oxide synthase induction. Under the same experimental conditions, elevated glucose alone had only a marginal effect on beta-cell apoptosis. Together the results indicate that elevated concentrations of fatty acids are particularly efficient in accelerating the rate of apoptosis of already stressed beta(INS-1)-cells displaying altered mitochondrial function, and that the mitochondrial arm of the apoptosis process is involved in beta-cell lipotoxicity.

Apoptosis↗

PubMed2002

Nuclear KATP channels trigger nuclear Ca(2+) transients that modulate nuclear function.

Glucose, the principal regulator of endocrine pancreas, has several effects on pancreatic beta cells, including the regulation of insulin release, cell proliferation, apoptosis, differentiation, and gene expression. Although the sequence of events linking glycemia with insulin release is well described, the mechanism whereby glucose regulates nuclear function is still largely unknown. Here, we have shown that an ATP-sensitive K(+) channel (K(ATP)) with similar properties to that found on the plasma membrane is also present on the nuclear envelope of pancreatic beta cells. In isolated nuclei, blockade of the K(ATP) channel with tolbutamide or diadenosine polyphosphates triggers nuclear Ca(2+) transients and induces phosphorylation of the transcription factor cAMP response element binding protein. In whole cells, fluorescence in situ hybridization revealed that these Ca(2+) signals may trigger c-myc expression. These results demonstrate a functional K(ATP) channel in nuclei linking glucose metabolism, nuclear Ca(2+) signals, and nuclear function.

Adenosine Triphosphate↗

PubMedDate not supplied

Isolation and characterization of residual undifferentiated mouse embryonic stem cells from embryoid body cultures by fluorescence tracking.

The differentiation of mouse embryonic stem (ES) cells can be induced in vitro after leukemia inhibitory factor (LIF) withdrawal and further enhanced by the formation of "embryoid body" (EB) aggregates. This strategy is being used in order to optimize differentiation protocols that would result in functional cells for experimental cell replacement therapies. However, this study presents the possibility for residual undifferentiated cells to survive after standard in vitro procedures. Mouse ES cells were stably transfected with the enhanced green fluorescent protein (EGFP), under the control of the Oct4 promoter, a transcription factor that is expressed in undifferentiated ES cells but down-regulated on differentiation. Residual fluorescent cells were isolated from EBs that were cultured in standard conditions in absence of LIF. These residual cells displayed recurrent gain of chromosomes 8 and 9. Residual fluorescent cells, further expanded in absence of LIF and cultured as EBs, still displayed a significant Oct4 expression in comparison with parental transfected ES cells. Consequently, these residual cells have an intrinsic resistance to differentiate. The behavior of these cells, observed in vitro, can be overcome in vivo, as they were able to induce teratomas in subcutaneously injected nude mice. Residual undifferentiated cells displayed slight levels of VASA and DAZL expression. These results demonstrate that mouse ES cells cultured in vitro, in standard conditions, can spontaneously acquire recurrent karyotypical changes that may promote an undifferentiated stage, being selected in standard culture conditions in vitro.

Animals↗