Potential of short chain fatty acids to modulate the induction of DNA damage and changes in the intracellular calcium concentration by oxidative stress in isolated rat distal colon cells.
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Short chain fatty acids (SCFA) are considered to be beneficial fermentation products in the gut by exerting trophic effects in non-transformed colon cells and by slowing proliferation and enhancing differentiation in colonic tumour cells. We have studied the further effects of SCFA on cellular events of early carcinogenesis, genotoxicity and cytotoxicity in rat distal colon cells. Cytotoxicity was assessed by measuring trypan blue exclusion and by determining the H2O2-induced changes in intracellular calcium concentration ([Ca2+]i) using a fluorospectrophotometer and the calcium-sensitive fluorescent dye Fura-2. The microgel electrophoresis technique (COMET assay) was used to assess oxidative DNA damage. Individual SCFA and physiological SCFA mixtures were investigated for their potential to prevent DNA and cell damage induced by H2O2. For this, freshly isolated colon cells were treated with H2O2 (100-500 microM) and 6.25 mM SCFA. We have found 100-500 microM H2O2 to cause a fast initial increase in [Ca2+]i, whereafter the levels gradually further increased. Addition of SCFA did not affect [Ca2+]i nor did it reduce the H2O2-induced increase in [Ca2+]i. Butyrate and acetate were able to reduce the induction of DNA damage by 100, 200 and 500 microM H2O2, respectively. In contrast, i-butyrate and propionate were ineffective. The degree of reduction of DNA damage for the two protective SCFA was similar. Physiological mixtures containing acetate, propionate and butyrate in ratios of 41:21:38 or 75:15:10 that are expected to arise in the colon after fermentation of resistant starches and pectin, respectively, did not show significant antigenotoxic effects. The major difference between butyrate and acetate, on one hand, and i-butyrate and propionate, on the other hand, is that the former compounds are utilized best as energy sources by the colon cells. Therefore, our results on antigenotoxicity coupled with the findings on [Ca2+]i homeostasis indicate that molecular effects on the energy system render these non-transformed, freshly isolated colon cells to be less susceptible to H2O2. (+info)
Localization of adipocyte long-chain fatty acyl-CoA synthetase at the plasma membrane.
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Long-chain fatty acyl-CoA synthetase (FACS) catalyzes esterification of long-chain fatty acids (LCFAs) with coenzyme A (CoA), the first step in fatty acid metabolism. FACS has been shown to play a role in LCFA import into bacteria and implicated to function in mammalian cell LCFA import. In the present study, we demonstrate that FACS overexpression in fibroblasts increases LCFA uptake, and overexpression of both FACS and the fatty acid transport protein (FATP) have synergistic effects on LCFA uptake. To explore how FACS contributes to LCFA import, we examined the subcellular location of this enzyme in 3T3-L1 adipocytes which natively express this protein and which efficiently take up LCFAs. We demonstrate for the first time that FACS is an integral membrane protein. Subcellular fractionation of adipocytes by differential density centrifugation reveals immunoreactive and enzymatically active FACS in several membrane fractions, including the plasma membrane. Immunofluorescence studies on adipocyte plasma membrane lawns confirm that FACS resides at the plasma membrane of adipocytes, where it co-distributes with FATP. Taken together, our data support a model in which imported LCFAs are immediately esterified at the plasma membrane upon uptake, and in which FATP and FACS function coordinately to facilitate LCFA movement across the plasma membrane of mammalian cells. (+info)
Developmental changes in rat brain membrane lipids and fatty acids. The preferential prenatal accumulation of docosahexaenoic acid.
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Information on the prenatal accumulation of rat brain membrane lipids is scarce. In this study we investigated in detail the fatty acid (FA) composition of the rat brain, on each day from embryonic day 12 (E12) up to birth, and on 8 time points during the first 16 days of postnatal life, and correlated the FA changes with well-described events of neurogenesis and synaptogenesis. Between E14 and E17, there was a steep increase in the concentration of all the FAs: 16:0 increased by 136%, 18:0 by 139%, 18:1 by 92%, 20:4n-6 by 98%, 22:4n-6 by 116%, 22:5n-6 by 220%, and 22:6n-3 by 98%. After this period and up to birth, the concentration of the FAs plateaued, except that of 22:6n-3, which accumulated further, reaching an additional increase of 75%. After birth, except 22:5n-6, all FAs steadily increased at various rates. Estimation of the FA/PL molar ratios showed that prenatally the ratios of all the FAs either decreased or remained constant, but that of 22:6n-3 increased more than 2-fold; postnatally the ratios remained constant, with the exception of 22:4n-6 and 22:5n-6, which decreased. In conclusion, prenatal accumulation of brain fatty acids parallels important events in neurogenesis. 22:6n-3 is exceptional inasmuch in its steep accumulation occurs just prior to synaptogenesis. (+info)
Targeted disruption of the adipocyte lipid-binding protein (aP2 protein) gene impairs fat cell lipolysis and increases cellular fatty acid levels.
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The availability of mice containing an adipocyte lipid-binding protein (ALBP/aP2) gene disruption allowed for a direct examination of the presumed role of lipid-binding proteins in the mobilization and trafficking of intracellular fatty acids. Total body and epididymal fat pad weights, as well as adipose cell morphology, were unaltered in male ALBP/aP2 disrupted mice when compared to their wild-type littermates. Analysis of adipocytes isolated from wild-type and ALBP/aP2 null mice revealed that a selective 40- and 13-fold increase in the level of the keratinocyte lipid-binding protein (KLBP) mRNA and protein, respectively, accompanied the ALBP/aP2 gene disruption. Although KLBP protein was significantly up-regulated, the total lipid-binding protein level decreased 8 -fold as a consequence of the disruption. There was no appreciable difference in the rate of fatty acid influx or esterification in adipocytes of wild-type and ALBP/aP2 null animals. To the contrary, basal lipolysis decreased approximately 40% in ALBP/aP2 nulls as compared to wild-type littermates. The glycerol release from isproterenol-stimulated ALBP/aP2 null fat cells was similarly reduced by approximately 35%. Consistent with a decrease in basal efflux, the non-esterified fatty acid (NEFA) level was nearly 3-fold greater in adipocytes from ALBP/aP2 nulls as compared to wild-type animals. The significant decrease in both basal and isoproterenol-stimulated lipolysis in adipose tissue of ALBP/aP2 null mice supports the model whereby intracellular lipid-binding proteins function as lipid chaperones, facilitating the movement of fatty acids out of the fat cell. (+info)
Requirement for the heart-type fatty acid binding protein in cardiac fatty acid utilization.
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Nonenzymatic cytosolic fatty acid binding proteins (FABPs) are abundantly expressed in many animal tissues with high rates of fatty acid metabolism. No physiological role has been demonstrated for any FABP, although these proteins have been implicated in transport of free long-chain fatty acids (LCFAs) and protection against LCFA toxicity. We report here that mice lacking heart-type FABP (H-FABP) exhibit a severe defect of peripheral (nonhepatic, non-fat) LCFA utilization. In these mice, the heart is unable to efficiently take up plasma LCFAs, which are normally its main fuel, and switches to glucose usage. Altered plasma levels of LCFAs, glucose, lactate and beta-hydroxybutyrate are consistent with depressed peripheral LCFA utilization, intensified carbohydrate usage, and increased hepatic LCFA oxidation; these changes are most pronounced under conditions favoring LCFA oxidation. H-FABP deficiency is only incompletely compensated, however, causing acute exercise intolerance and, at old age, a localized cardiac hypertrophy. These data establish a requirement for H-FABP in cardiac intracellular lipid transport and fuel selection and a major role in metabolic homeostasis. This new animal model should be particularly useful for investigating the significance of peripheral LCFA utilization for heart function, insulin sensitivity, and blood pressure. (+info)
Regulatory role of cAMP in transport of Na+, Cl- and short-chain fatty acids across sheep ruminal epithelium.
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Sodium is absorbed in considerable amounts across the ruminal epithelium, whilst its transport is strongly interrelated with the permeation of chloride and short-chain fatty acids (SCFAs). However, regulation of ruminal Na+, Cl-, and SCFA absorption is hardly understood. The present study was therefore performed to characterize the influence of cAMP on sodium and sodium-coupled transport mechanisms in short-circuited, stripped ruminal epithelia of sheep. Elevation of intracellular cAMP concentrations by theophylline (10 mM) or theophylline in combination with forskolin (0.1 mM) significantly reduced mucosal-to-serosal sodium transport, leading to a reduction of net transport. The theophylline- or theophylline-forskolin-induced reduction of sodium transport was accompanied by a decrease in chloride net transport but revealed no effect on propionate flux. Short-chain fatty acids stimulated Na+ transport but their stimulatory effect was almost completely blocked by theophylline-forskolin. In solutions with and without SCFAs, the inhibitory effect of 1 mM amiloride on sodium transport was strongly reduced after theophylline-forskolin pretreatment of the tissues. Blocking the production of endogenous prostaglandins by addition of indomethacin (10 microM) led to a theophylline-sensitive stimulation of unidirectional and net fluxes of sodium. The findings indicate that apical, amiloride-sensitive Na+-H+ exchange and/or basolateral Na+-K+-ATPase can effectively be blocked by cAMP, leading to a decrease in sodium and chloride transport. In the ruminal epithelium, cAMP is a second messenger of prostaglandins, which are released spontaneously under in vitro conditions. (+info)
Breast growth and the urinary excretion of lactose during human pregnancy and early lactation: endocrine relationships.
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Breast volume and morphology of eight subjects were measured before conception and at intervals throughout pregnancy until 1 month of lactation. Breast volume before conception ranged from 293 to 964 ml. At the end of pregnancy the volume of breast tissue had increased by 145+/-19 ml (mean+/-S.E.M., n = 13 breasts, range 12-227 ml) with a further increase to 211+/-16 ml (n = 12 breasts, range 129-320 ml) by 1 month of lactation. Urinary excretion of lactose increased at 22 weeks of pregnancy, signalling the capacity of the breast to synthesize lactose at this time. During pregnancy, both the change in breast volume and the change in cross-sectional area of the areola were related to the concentration of human placental lactogen in the plasma. The growth of the nipple and the rate of excretion of lactose were related to the concentration of prolactin in the plasma. During the first 3 days after birth, the rate of excretion of lactose was related to the rate of excretion of progesterone. There was no relationship between the growth of the breast during pregnancy and the amount of milk produced at 1 month of lactation. (+info)
Breast volume and milk production during extended lactation in women.
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Quantitative measurements were made of relative breast volume and milk production from 1 month of lactation until 3 months after weaning, and the storage capacity of the breasts was calculated. The increase in breast tissue volume from before conception until 1 month of lactation was maintained for the first 6 months of lactation (means+/-S.E.M.) (190.3+/-13.1 ml, number of breasts, nb = 46). During this period of exclusive breast-feeding, 24 h milk production from each breast remained relatively constant (453.6+/-201 g, nb = 48), and storage capacity was 209.9+/-11.0 ml (nb = 46). After 6 months, breast volume, milk production and storage capacity all decreased. There was a relationship between 24 h milk production and the storage capacity of the breasts, and these both appeared to be responding to infant demand for milk. At 15 months of lactation, the 24 h milk production of each breast was substantial (208.0+/-56.7 g, nb = 6), even though the breasts had returned to preconception size. This was associated with an apparent increased efficiency of the breast (milk production per unit breast tissue) after 6 months, which may have been due to redistribution of tissues within the breast. The possible causes of the decrease in breast volume are discussed. (+info)