Biomedical subjects

C Fillat

Publications and source records attributed to C Fillat.

29 records · Page 2Linked to original sources

PubMed1996

Tissue-specific expression and dietary regulation of chimeric mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A synthase/human growth hormone gene in transgenic mice.

We have studied the role of the mitochondrial 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) synthase gene in regulating ketogenesis. The gene exhibits expression in various tissues and it is regulated in a tissue-specific manner. To investigate the underlying mechanisms of this expression, we linked a 1148-base-pair portion of the mitochondrial HMG-CoA synthase promoter to the human growth hormone (hGH) gene and analyzed the expression of the hGH reporter gene in transgenic mice. mRNA levels of hGH were observed in liver, testis, ovary, stomach, colon, cecum, brown adipose tissue, spleen, adrenal glands, and mammary glands from adult mice, and also in liver and stomach, duodenum, jejunum, brown adipose tissue, and heart of suckling mice. There was no expression either in kidney or in any other nonketogenic tissue. The comparison between these data and those of the endogenous mitochondrial HMG-CoA synthase gene suggests that the 1148 base pairs of the promoter contain the elements necessary for expression in liver and testis, but an enhancer is necessary for full expression in intestine of suckling animals and that a silencer prevents expression in stomach, brown adipose tissue, spleen, adrenal glands, and mammary glands in wild type adult mice. In starvation, transgenic mice showed higher expression in liver than did wild type. Both refeeding and insulin injection reduced the expression. Fat diets, composed in each case of different fatty acids, produced similar expression levels, respectively, to those found in wild type animals, suggesting that long-, medium-, and short-chain fatty acids may exert a positive influence on the transcription rate in this 1148-base-pair portion of the promoter. The ketogenic capacity of liver and the blood ketone body levels were equal in transgenic mice and in nontransgenic mice.

Animals↗

PubMed1995

Fluorescence-based selection of retrovirally transduced cells in the absence of a marker gene: direct selection of transduced type B Niemann-Pick disease cells and evidence for bystander correction.

Types A and B Niemann-Pick disease (NPD) are lysosomal storage disorders resulting from the deficient activity of acid sphingomyelinase (ASM). Type A NPD is characterized by the absence of residual ASM activity, massive accumulation of sphingomyelin and cholesterol within lysosomes, and a rapid, neurodegenerative course that leads to death by 3 years of age. In contrast, type B NPD patients have low, but detectable, levels of residual ASM activity and little or no neurologic disease. Thus, individuals with type B NPD may survive into late adolescence or adulthood and are considered excellent candidates for somatic cell gene therapy. To facilitate the development of gene therapy for this disorder, a novel procedure was devised to isolate metabolically corrected type B NPD cells in the absence of marker gene expression. Type B NPD cells were transduced with retroviral vectors expressing ASM, labeled with lissamine rhodamine sphingomyelin (LR-SPM), and subjected to preparative fluorescence-activated cell sorting (FACS). Two non-overlapping cell populations were isolated, corresponding to enzymatically corrected (i.e., low fluorescence) and noncorrected (i.e., high fluorescence) cells. Quantitative PCR analysis demonstrated that the enzymatically corrected cells were enriched for vector sequences. Moreover, the corrected cells could be regrown and continued to express high levels of ASM activity after numerous passages, consistent with the fact that they were stably transduced. Notably, coculture of FACS-sorted, overexpressing cells with untreated type B NPD fibroblasts resulted in a homogeneous cell population with low fluorescence whose FACS distribution overlapped that of the corrected cells. Computerized fluorescence microscopy confirmed that nearly all of these cocultured cells expressed ASM activity and could hydrolyze LR-SPM.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1994

Regulated expression of human insulin in the liver of transgenic mice corrects diabetic alterations.

Transgenic mice expressing the P-enolpyruvate carboxykinase (PEPCK)/human insulin chimeric gene have been obtained as a model to study the feasibility of gene therapy for diabetes. These transgenic animals were healthy and normoglycemic and expressed human insulin in a physiologically regulated manner, mainly in the liver. Streptozotocin-treated transgenic mice had high levels of human insulin immunoreactivity in serum and showed a significant decrease (up to 40%) in glycemia compared with streptozotocin-treated control mice. The expression of genes involved in liver glucose metabolism, such as glucokinase, pyruvate kinase, and PEPCK, which is markedly altered by diabetes, was significantly recovered in transgenic mice treated with streptozotocin. In addition, the activity of both glucokinase and glycogen synthase, and the content of glucose 6-phosphate and glycogen, were normal in the liver, even when transgenic animals were treated with diabetogenic doses of streptozotocin. These results constitute an indication in vivo that diabetes gene therapy is possible, by means of the production of insulin in extrapancreatic tissues.

Animals↗

PubMed1994

Overexpression of mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase in transgenic mice causes hepatic hyperketogenesis.

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase) is a key enzyme in the ketone body pathway. To determine its role in the regulation of liver ketogenesis, transgenic mice expressing a P-enolpyruvate carboxykinase/HMG-CoA synthase chimeric gene have been obtained. An increase in the concentration of mitochondrial HMG-CoA synthase mRNA was detected in these mice, which was associated with a 3-fold increase in HMG-CoA synthase activity in liver mitochondrial extracts. Transgenic mice were normoglycemic and had normal levels of plasma triglycerides and lower free fatty acids. However, the plasma concentration of ketone bodies was about three times higher in transgenic mice than in control animals. Hepatocytes in primary culture from transgenic mice expressed the chimeric gene in a regulated manner and showed a 3-fold increase in beta-hydroxybutyrate and acetoacetate concentrations in the medium. This animal model thus shows that the overexpression of mitochondrial HMG-CoA synthase causes ketone body overproduction, suggesting that this enzyme may be a regulatory step in liver ketogenesis.

3-Hydroxybutyric Acid↗

PubMed1993

Epidermal growth factor inhibits phosphoenolpyruvate carboxykinase gene expression in rat hepatocytes in primary culture.

Epidermal growth factor (EGF) decreased the basal, and blocked the dibutyryl cyclic AMP (Bt2cAMP)-induced, expression of P-enolpyruvate carboxykinase (GTP) (PEPCK) and tyrosine aminotransferase (TAT) genes in both rat hepatocytes in primary culture and the FTO-2B hepatoma cell line. Treatment of hepatocytes with EGF in combination with phorbol ester (TPA) resulted in an additive decrease of PEPCK mRNA levels. Overnight pretreatment of hepatocytes with TPA, which is known to downregulate protein kinase C, abolished the TPA and reduced the EGF-mediated inhibition of PEPCK gene expression. These results suggested that EGF caused its effect, at least in part, through protein kinase C.

Animals↗

PubMed1993

Stimulation of glucose utilization by fructose in isolated rat hepatocytes.

In rat hepatocytes fructose at low concentrations (below 1 mM) stimulated the glycolytic flux as measured by the release of 3H2O from [3-3H]glucose and increased fructose 2,6-bisphosphate [Fru(2,6)P2] levels, without modifying the activity of 6-phosphofructo-2-kinase. Maximal stimulation of the glycolytic pathway by 0.1 mM fructose was observed when hepatocytes were incubated in the presence of physiological concentrations of glucose (8 mM). The rise in Fru(2,6)P2 levels was probably due to an increase in glucose 6-phosphate, which in turn resulted from the stimulation of glucose phosphorylation as measured by the formation of 3H2O from [2-3H]glucose. Furthermore, no effects of low doses of fructose on the glycolytic flux or on glucose phosphorylation were observed in hepatocytes from streptozocin-diabetic rats in which glucokinase is almost absent, or in hepatocytes incubated in the presence of mannoheptulose where glucokinase is inhibited. These results suggest that fructose at low concentrations increases the glycolytic flux by raising Fru(2,6)P2 levels solely as a consequence of the stimulation of glucose phosphorylation.

Animals↗

PubMed1992

Molybdate and tungstate act like vanadate on glucose metabolism in isolated hepatocytes.

In rat hepatocytes, molybdate and tungstate inactivate glycogen synthase by a mechanism independent of Ca2+ and activate glycogen phosphorylase by a Ca(2+)-dependent mechanism. On the other hand, both molybdate and tungstate increase fructose 2,6-bisphosphate levels and counteract the decrease in this metabolite induced by glucagon. These effectors do not directly modify 6-phosphofructo-2-kinase activity, even though they partially counteract the inactivation of this enzyme induced by glucagon. These effects are related to an increase on the glycolytic flux, as indicated by the increase in L-lactate and CO2 production and the decrease in glucose 6-phosphate levels in the presence of glucose. All these effects are similar to those previously reported for vanadate, although molybdate and tungstate are less effective than vanadate. These results could indicate that molybdate, tungstate and vanadate act on glucose metabolism in isolated hepatocytes by a similar mechanism of action.

Animals↗

PubMed1991

Activation by vanadate of glycolysis in hepatocytes from diabetic rats.

In hepatocytes from starved streptozocin-induced diabetic rats, vanadate increases the glycolytic flux because it raises the levels of fructose-2,6-bisphosphate (Fru-2,6-P2), the main regulatory metabolite of this pathway. This effect of vanadate on Fru-2,6-P2 levels is time and dose dependent, and it remains in cells incubated in a calcium-depleted medium. Vanadate is also able to counteract the decrease on Fru-2,6-P2 levels produced by glucagon, colforsin, or exogenous cAMP. However, vanadate does not modify 6-phosphofructo-2-kinase and pyruvate kinase activities, but it does counteract the inactivation of these enzymes induced by glucagon. Likewise, Fru-2,6-P2ase activity is also not affected by vanadate. In addition, vanadate is able to increase the production of both lactate and CO2 in hepatocytes from streptozocin-induced diabetic rats incubated in the presence of glucose in the medium. Vanadate behaves as a glycolytic effector in these cells, and this effect may be related to its ability to normalize blood glucose levels in diabetic animals.

Animals↗

PubMed1988

Vanadate raises fructose 2,6-bisphosphate concentrations and activates glycolysis in rat hepatocytes.

In rat hepatocytes, vanadate increases fructose 2,6-bisphosphate (Fru-2,6-P2) in a time- and dose-dependent manner, and counteracts the decrease in this metabolite caused by glucagon, forskolin or exogenous cyclic AMP. Vanadate does not directly modify the activity of 6-phosphofructo-2-kinase, even though it can counteract the inactivation of this enzyme caused by glucagon. Furthermore, vanadate raises the yield of 3H2O from [3-3H]glucose, indicating that it increases the flux through 6-phosphofructo-1-kinase. Moreover, vanadate in hepatocytes incubated in the presence of glucose increases the production of both lactate and CO2. Therefore vanadate has insulin-like effects on the glycolytic pathway in rat hepatocytes. These results clearly contrast with our previous observation that vanadate exerts glycogenolytic non-insulin-like effects on glycogen synthase and phosphorylase.

Animals↗

PubMed1988

Determination of glucose-6-phosphatase activity using the glucose dehydrogenase-coupled reaction.

We present a method to determine glucose 6-phosphate activity. This assay measures the rate of glucose released in the glucose-6-phosphatase reaction. The glucose is oxidized to beta-D-gluconolactone by glucose dehydrogenase in a coupled reaction that uses NAD(P)+. The determination is rapid, reproducible, and does not require withdrawal, precipitation, centrifugation, or neutralization steps. This method provides a simple resolution to the problem of the nonspecific appearance of Pi, which is especially important in studies of regulation of glucose-6-phosphatase performed in the presence of ATP.

Animals↗

PubMedDate not supplied

Genetic background determines the response to adenovirus-mediated wild-type p53 expression in pancreatic tumor cells.

The development of new therapies is particularly urgent with regard to pancreatic tumors. Gene therapy approaches involving p53 replacement are promising due to the central role of p53 in the cellular response to DNA damage and the high incidence of p53 mutations in pancreatic tumors. Adenoviruses containing wild-type (wt) p53 cDNA (Ad5CMV-p53) were introduced into four human pancreatic cell lines to examine the impact caused by exogenous wt p53 on these cells. Introduction of wt p53 in mutant p53 cells (NP-9, NP-18, and NP-31) caused marked falls in cell proliferation and rises in the level of apoptosis. In contrast, overexpression of p53 did not induce apoptosis in NP-29 (wt p53). The presence of p16 contributes to the induction of apoptosis, as demonstrated by introduction of the wt p16 gene (Ad5RSV-p16). Analysis of cell cycle and apoptosis in etoposide-treated cells corroborated the inability of NP-29 to die by apoptosis, suggesting that this wt p53 cell line lacks p53 downstream functions in the apoptosis pathway. Taken together, our results indicate that the effects elicited by exogenous p53 protein depend upon the molecular alterations related to p53 actions on cell cycle and apoptosis. Therefore, knowledge of the genetic background of tumor cells is crucial to the development of efficient therapies based on the introduction of tumor suppressor genes.

Adenocarcinoma↗