RESUMEN
The combined effects of carbohydrates and glutamine were investigated in diploid strains of normal human skin fibroblasts cultured for 21 days under eight different culture conditions: hexose-free medium or medium containing D-glucose, D-galactose, or D-fructose, with or without added glutamine. Cell growth, hexose consumption, lactate production, intracellular glycogen content and extracellular amino acid levels were measured every third to fourth day. In the presence of glutamine, cells reached a higher saturation density in fructose medium than in glucose or galactose medium but per cell consumption of fructose and galactose was much less than that of glucose. Consumption of all three carbohydrates per unit cell growth exhibited three distinct phases: Days 1-3, 3-10, and 10-20, respectively. In the absence of glutamine the rate of cell growth was not altered in glucose or galactose medium, but slowed down considerably in fructose medium. Glutamine deprivation also led to changes in hexose consumption. In hexose-free media the cell growth rate at first was very slow, but rose after 2 or 3 weeks of culture. The levels of extracellular nonessential amino acids varied according to medium and growth phase. One of the most exciting findings was that human fibroblasts are able to maintain a slight excess of glutamine in all media not supplemented with glutamine and, more surprisingly, to synthesize it in a medium containing galactose and glutamine.
Asunto(s)
Glutamina/metabolismo , Hexoquinasa/metabolismo , Piel/citología , Aminoácidos/metabolismo , División Celular , Células Cultivadas , Niño , Fibroblastos/citología , Fibroblastos/metabolismo , Fructosa/metabolismo , Galactosa/metabolismo , Glucosa/metabolismo , Humanos , Lactatos/metabolismo , Piel/metabolismoRESUMEN
UNLABELLED: Many publications indicate the beneficial effect of n-6 polyunsaturated fatty acids (n-6 PUFAs) in the control of coronary heart disease and diabetes, although the mechanism is not clear. Some of our previous results suggest that, in contrast to other lipids, n-6 PUFAs could have a permissive effect on carbohydrate oxidation. To check this hypothesis, we determined pyruvate dehydrogenase (PDH, decarboxylase: EC 1.2.4.1) activity in infant skin fibroblasts (ISF) incubated 6 hours in the presence of 0.25 mM linoleic (LI) or arachidonic (AR) acid, compared to oleic acid (OL) and control ISF incubated without addition of fatty acids. The four groups of cells were preincubated 36 hours either in the presence of fetal bovine serum (FBS), or in the presence of lipoprotein-deprived serum (LPDS). RESULTS: (1) When the ISF were maintained in the medium containing FBS, the two PUFAs had little inhibitory effect on PDH activity, in contrast with the effect of OL. (2) When the ISF were kept in the lipoprotein-deficient medium, PDH activity was low in controls and in the OL cells, but the addition of LI or AR increased the activity. This suggests the role of n-6 PUFAs in enhancing carbohydrate oxidation, under certain conditions.
Asunto(s)
Metabolismo de los Hidratos de Carbono , Enfermedad Coronaria/prevención & control , Diabetes Mellitus/prevención & control , Ácidos Grasos Insaturados/farmacología , Piel/metabolismo , Ácidos Araquidónicos/farmacología , Células Cultivadas , Fibroblastos/metabolismo , Humanos , Ácidos Linoleicos/farmacología , Ácidos Oléicos/farmacología , Oxidación-Reducción , Fosfolípidos/análisis , Complejo Piruvato Deshidrogenasa/análisisRESUMEN
Growth of human skin fibroblasts was dramatically enhanced when serum in the culture medium was replaced by Ultroser G. Compared to the same cells cultured in the presence of serum, alterations in glucose and lipid metabolism and an increase in the activity of prolidase (EC 3.4.13.9) and prolinase (EC 3.4.13.8) were also observed. Consequently, we advise extreme caution in the use of Ultroser G in metabolic studies, especially for periods of culture exceeding 10 days. However, Ultroser G can help to produce a large number of cells and so facilitate purification of the proteins produced during the stationary growth phase.
Asunto(s)
Sustitutos Sanguíneos , Técnicas Citológicas , Piel/citología , Ácido Ascórbico/farmacología , Sangre , Sustitutos Sanguíneos/farmacología , División Celular/efectos de los fármacos , Células Cultivadas , Dipeptidasas/metabolismo , Fibroblastos/citología , Fibroblastos/enzimología , Glucosa/metabolismo , Humanos , Lactatos/biosíntesis , Ácido Láctico , Compuestos Orgánicos , Piel/enzimologíaRESUMEN
The growth rate of human skin fibroblasts was evaluated when glucose was replaced by fructose in the culture medium. Four mediums containing respectively 5.5 mmol/l glucose (G1), 27.5 mmol/l glucose (G5), 5.5 mmol/l fructose (F1), and 27.5 mmol/fructose (F5) were used. Skin fibroblasts from fourteen subjects were continuously cultured for 20 days and the number of cells was counted at days 1, 3, 7, 10, 15 and 20 after plating. The morphological patterns were observed and compared, the pH values of the medium were calculated, as were hexose consumption and lactate production. The results established clear differences in cell growth, pH and morphology: up to day 7, the growth rate was lower in fructose than in glucose medium, and the pH values were higher. In addition, marked steatosis appeared, with increased pyruvate dehydrogenase (PDH) activity. After day 10, the mean values gave a significant increase in the number of cells grown in fructose mediums, even if variations occurred between different cell strains. This increase was accompanied by loss of density-dependent growth inhibition and a reduction in the quantity and size of the vacuoles caused by steatosis. These findings were also established for other cell types, like aponeurosis fibroblasts. In addition, the longevity of the strains increased. These observations indicate that intermediary metabolism is considerably influenced by the carbohydrate present in the cell culture medium and that there are also repercussions on the growth rate. Under our experimental conditions, metabolism pathways seemed to differ on day 7 and on day 20. The various metabolic events suggested by the differences in the pH values are now being studied in our laboratory.
Asunto(s)
División Celular/efectos de los fármacos , Fibroblastos/citología , Fructosa/farmacología , Glucosa/farmacología , Línea Celular , Membrana Celular/ultraestructura , Supervivencia Celular/efectos de los fármacos , Medios de Cultivo , Fibroblastos/metabolismo , Fructosa/metabolismo , Glucosa/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Complejo Piruvato Deshidrogenasa/metabolismo , Piel , Coloración y EtiquetadoRESUMEN
In order to determine the incorporation of C1-14C derived from mono- and poly-unsaturated fatty acids into cholesterol of human cells cultured in exponential phase, infant skin fibroblasts (SF) were used at the 5th passage. On Day 6, the SF were preincubated 36 h in a medium containing 5 per cent lipoprotein-deficient serum, and thereafter [1-14C] oleic, -linoleic or -arachidonic acid-without (OL1, LI1 and AR1 group SF), or with the addition of 0.25 mM cold fatty acids (OL2), LI2 and AR2 group SF). Cholesterol specific radioactivity (SRA) peaked 1 h after, and leveled off afterwards in the OL1, LI1 and AR1 groups. Cholesterol-SRA was relatively low in the other groups, but increased progressively, giving a biphasic response: C1-14C derived from from linoleic and arachidonic acids was actively incorporated into cholesterol during the first hours, as compared to C1-14C derived from oleic acid, but stabilized between 6 and 12 h for the LI2 and AR2 group SF incubation. This result appears to be due to the stimulation of pyruvate decarboxylation, observed elsewhere, and consequently to the dilution of the radioactive units in a large pool of non-labeled acetyl-CoA units derived from glucose, when these SF were incubated with 0.25 mM polyunsaturated fatty acids.
Asunto(s)
Colesterol/biosíntesis , Ácidos Grasos/metabolismo , Piel/metabolismo , Ácido Araquidónico , Ácidos Araquidónicos/metabolismo , Adhesión Celular , Medios de Cultivo , Fibroblastos/metabolismo , Humanos , Lactante , Ácidos Linoleicos/metabolismo , Masculino , Mitosis , Ácidos Oléicos/metabolismoRESUMEN
Explants for long term monolayer liver cell cultures were taken from individuals having PiM or PiZ phenotypes. Radioactive labelling, using I125 and an original method, permitted the quantitative measurement of AAT in vitro and more important, allowed Pi phenotype determination.