Biomedical subjects

S E Fleming

Publications and source records attributed to S E Fleming.

At least 19 recordsLinked to original sources

PubMed2002

Absorption and metabolism of octanoate by the rat colon in vivo: concentration dependency and influence of alternative fuels.

BACKGROUND: Compared with short and long chain fatty acids, medium chain fatty acids (MCFAs) have been shown to provide the highest colonic absorption of substrate carbon. Moreover, colonic epithelial cells fulfil their basic energy requirements as easily from MCFAs as from short chain fatty acids. AIMS: To further characterise octanoate as a colonic luminal substrate, we determined in vivo the influence of (i) substrate concentration and (ii) alternative luminal fuels, on rat colonic transport and metabolism. METHODS: Segments of rat proximal colon (8 cm) were cannulated and perfused for 100 min with (14)C labelled octanoate. The right colic vein was also cannulated and venous blood analysed for total (14)C, (14)CO(2), and metabolites by scintillation counting and high performance liquid chromatography. RESULTS: Tracer appearance in mesenteric blood stabilised after 20-40 minutes of perfusion. Increasing luminal octanoate concentrations from 2 to 40 mM resulted in linear increases in total carbon absorption. Maximum CO(2) production was reached near 10 mM. A substantial proportion of octanoate was absorbed without being metabolised (59-94%). The luminal presence of a mixture of alternative fuels had no influence on either octanoate transport or metabolism. CONCLUSIONS: This study demonstrated substantial concentration dependent colonic absorption of octanoate, rendering this MCFA a potential and much needed high energy substrate for patients with compromised small bowel function. Moreover, octanoate meets the basic energy requirements of colonic epithelial cells in vivo as well as butyrate. This study also demonstrates the divergence of in vitro and in vivo data regarding fatty acid absorption and metabolism in the colonic epithelium.

Animals↗

PubMed2001

In vivo absorption of medium-chain fatty acids by the rat colon exceeds that of short-chain fatty acids.

BACKGROUND AND AIMS: Short-chain fatty acids (SCFAs) are main fuels of the colonic epithelium, and are avidly absorbed by the colon of animal and man. The current knowledge on colonic metabolism and absorption of medium-chain fatty acids (MCFAs) is limited. In some clinical situations, colonic absorption of high-energy substances could compensate for reduced absorptive capacity because of a shortened or malfunctioning small bowel. We evaluated and compared colonic absorption and metabolism of MCFAs (octanoate, decanoate, and dodecanoate), SCFAs (acetate and butyrate), and long-chain fatty acids (LCFAs) (oleate). METHODS: Rats were surgically operated on to cannulate a 7-cm segment of proximal colon, isolate the vasculature, and cannulate the right colic vein draining this segment. The lumen was perfused with (14)C-labeled substrates for 100 minutes. Right colic vein blood was analyzed for total (14)C, (14)CO(2), and metabolites by scintillation counting and high-performance liquid chromatography. RESULTS: The transport from the colonic lumen to mesenteric blood of substrate carbon from MCFAs exceeded by 2-13-fold that of SCFAs and LCFAs. The CO(2) production from the oxidation of MCFAs was as high as or higher than that from SCFAs. CO(2) produced from the LCFA, oleate, was lower than from SCFAs or MCFAs. In addition to CO(2), ketone bodies were major metabolites of SCFAs and MCFAs. Ketogenesis from butyrate and the MCFAs was significantly higher than from acetate and oleate. A substantial proportion (50%-90%) of all substrates was absorbed without being metabolized. CONCLUSIONS: The colonic epithelium serves to absorb and partially metabolize MCFAs. For patients with a compromised small-bowel function, colonic absorption of MCFAs could represent an important way of receiving calories.

Animals↗

PubMed1999

Metabolism of short-chain fatty acids by rat colonic mucosa in vivo.

To determine the influence of substrate concentration and substrate interactions on short-chain fatty acid metabolism in vivo, a surgical procedure was established. Rats were surgically operated to cannulate a 5-cm segment of proximal colon, isolate the vasculature, and cannulate the right colic vein draining this segment. Thus metabolism was restricted to the defined colonic segment. The appearance of total (14)C and (14)CO(2) in the mesenteric blood stabilized after 30 min of perfusion. Increasing luminal concentrations of butyrate from 2 to 40 mmol/l resulted in linear increases in total (14)C, but (14)CO(2) production from [(14)C]butyrate increased as a function of concentration only up to 10 mmol/l and was stable at higher butyrate concentrations. In addition to CO(2), 3-hydroxybutyrate and lactate were major metabolites of acetate and butyrate in vivo. The presence of a mixture of alternative substrates in the lumen had no influence on the metabolism of butyrate to CO(2) but significantly reduced the metabolism of acetate to CO(2). When compared with young (4 mo old) animals, transport of butyrate was significantly lower for aged (48 mo old) animals, as evidenced by the rate of appearance in blood of total (14)C (P = 0.04) and (14)C in butyrate (P = 0.03), but metabolism was similar, since differences were not significant for (14)C in the major metabolites 3-hydroxybutyrate (P = 0.06) and CO(2) (P = 0.17). These results show that important aspects of short-chain fatty acid transport and metabolism are not predicted from data using isolated colonocytes but require study using an in vivo model.

Acetates↗

PubMed1998

Rate at which glutamine enters TCA cycle influences carbon atom fate in intestinal epithelial cells.

Glutamine carbon entry into the tricarboxylic acid (TCA) cycle was assessed in small intestinal epithelial cells by measuring CO2 production from [1-14C]glutamine, and these data together with [U-14C]glutamine data were used to calculate fractional oxidation rates for glutamine. CO2 production from either [1-14C]glutamine or [U-14C]glutamine showed saturation kinetics, and the concentration needed to achieve the half-maximal rate of CO2 production was 0.7 and 0.4 mmol/l, respectively. Maximal rate for [1-14C]glutamine was twice that for [U-14C]glutamine. Increasing glutamine concentration did not cause proportional increases in glutamine entry into the TCA cycle and glutamine oxidation. Consequently, fractional oxidation of glutamine decreased with increasing glutamine concentration. Fractional oxidation could be predicted from the rate at which glutamine carbon entered the TCA cycle. (Aminooxy)acetic acid, an aminotransferase inhibitor, reduced entry of glutamine into the TCA cycle and increased fractional oxidation of glutamine. Glutamate carbon entered the TCA cycle at about one-half the rate of glutamine-derived glutamate carbon and had a higher fractional oxidation rate when provided at equivalent concentrations to glutamine. These differences in the rate of entry predictably account for the differences in the metabolic fate of glutamine vs. glutamate carbon.

Animals↗

PubMed1997

Aging stimulates fatty acid oxidation in rat colonocytes but does not influence the response to dietary fiber.

Metabolism was studied in colonocytes isolated from young (4 mo) and aged (24 mo) Fischer 344 rats. Animals were fed fiber-free, low-fiber (5% cellulose), or high-fiber (oat bran or NIH 31 stock) diets. Colonocytes isolated from aged animals oxidized both short- and long-chain fatty acids at significantly higher rates than did colonocytes isolated from young animals. No differences between the young and aged were noted for the oxidation to CO2 of glucose and glutamine or for flux of glucose through glycolysis. Net adenosine triphosphate (ATP) production by colonocytes was calculated to be 20% higher for the aged than for the young, although the relative contribution of substrates to net ATP production from exogenous substrates was similar for the young and aged (45-50% from butyrate, 20-25% from glucose, and 30% from other substrates including acetate, propionate, palmitate, and glutamine). Substrate oxidation was generally higher in colonocytes from the oat bran (17% total dietary fiber, highly soluble fiber) versus fiber-free diet.

Aging↗

PubMed1997

Glycolysis is a source of pyruvate for transamination of glutamine amino nitrogen in jejunal epithelial cells.

Previous research has shown that glucose increases transamination of glutamine amino nitrogen with pyruvate. It is unclear whether glucose or glutamine provides the pyruvate used for transamination. In the current study, it was hypothesized that glucose provides pyruvate for transamination of glutamine amino nitrogen. This hypothesis was tested by tracing the metabolism of [2-(13)C]glucose in these cells incubated in the presence of [2-(13)C] glucose or [2-(13)C] glucose and glutamine using (13)C nuclear magnetic resonance. Glutamine supplementation increased alanine production but did not affect lactate production. The 1-(13)C, 2-(13)C, 3-(13)C, 1,2-(13)C, and 2,3-(13)C isotopomers of alanine and lactate were produced when glutamine was supplemented. Glutamine supplementation increased production of 2-(13)C, 1,2-(13)C, and 2,3-(13)C isotopomers of alanine but did not affect the production of isotopomers of lactate. The ratio of production of [2-(13)C]alanine to [3-(13)C]alanine was 37:1 when glutamine was present. The predominance of production of [2-(13)C]alanine vs. all other isotopomers demonstrates that a large proportion of the pyruvate used for transamination of glutamine amino nitrogen was derived from glycolysis.

Animals↗

PubMed1997

Glucose and glutamine provide similar proportions of energy to mucosal cells of rat small intestine.

The objectives of this study were to establish a reliable method for quantifying glycolytic flux in intestinal epithelial cells, to determine the proportion of energy provided to small intestine epithelial cells by glucose vs. glutamine, and to determine whether there was an energetic advantage to having both substrates present simultaneously. There was substantial retention of 3H in alanine and lactate when [2-(3)H]glucose was used as tracer for quantifying glycolysis, and the magnitude of the 3H retention was influenced by the presence of other substrates and metabolites. Detritiation was at least 99% complete, however, when [3-(3)H]glucose was used as tracer in this system and the tritium was recovered as 3H2O. Glycolytic flux was six- to sevenfold higher in cells of the proximal than distal small intestine but was not significantly different for young adult (4 mo) vs. aged adult (24 mo) rats. Net ATP production from exogenous substrates was higher when both glucose and glutamine were present simultaneously than when either substrate was present alone, and glucose was calculated to provide 50-60% of the net ATP produced from these two substrates. Most of the energy produced from glucose was produced via the anaerobic metabolic pathways (78% for glucose alone, 95% with glucose and glutamine). Net energy production was calculated to be 10% lower in cells from aged animals than in those from young animals, since CO2 production from these major substrates was lower in cells from aged animals.

Aging↗

PubMed1995

Oxidation of glucose carbon entering the TCA cycle is reduced by glutamine in small intestine epithelial cells.

The influence of glutamine on glucose oxidation was assessed in epithelial cells isolated from the mucosa of the proximal, mid-, and distal small intestine of young, fed, male rats. Glucose oxidation declined along the length of the small intestine, with values from the mid- and distal segments representing approximately 55% and 40%, respectively, of the value from the proximal segment. A gradient along the small intestine was noted also in the influence of glutamine on glucose oxidation: glutamine suppressed glucose oxidation approximately 60% in the proximal small intestine, 39% in the mid-intestine, and 31% in the distal small intestine. Glutamine suppressed the oxidation of glucose carbon that entered the tricarboxylic acid (TCA) cycle; this was determined using CO2 ratios derived from acetate and glucose isotopes. In cells from the proximal segment, the probability that carbon entering the cycle would complete one full turn was reduced by glutamine from 0.77 to 0.28. The entry of glucose-derived pyruvate into the TCA cycle did not appear to be influenced by the presence of glutamine, however. Glutamine had no influence on the proportion of glucose metabolism that occurred via the pentose phosphate pathway (which averaged 5% or less), but reduced flux of carbon through pyruvate carboxylase relative to flux through pyruvate dehydrogenase from 40% to 9% in cells from the proximal segment. These data suggest that, in the presence of glutamine, the fate of pyruvate carbon (derived from glucose or elsewhere) entering the TCA cycle is altered from that of oxidation to anaplerosis and subsequent efflux of TCA cycle intermediates into newly synthesized compounds.

Animals↗

PubMed1994

Intestinal cell proliferation is influenced by intakes of protein and energy, aflatoxin, and whole-body radiation.

Intestinal epithelial cell proliferation in young male F344 rats was measured in response to dietary protein content (5%, 10%, and 20% casein diets), energy restriction (energy intake was 60% of ad libitum energy intakes of animals consuming the 20% casein diet), total diet restriction (dietary intake was 60% of the ad libitum intake of 20% casein diet group), aflatoxin administration, and whole body irradiation. Cellular proliferation was measured in sections of jejunum, ileum, proximal colon, and distal colon with the [3H]thymidine technique. Restricting energy or total diet intakes by 40% from ad libitum levels reduced proliferation in epithelial cells throughout the intestine. In comparison to the 5% casein diet, the 20% casein diet resulted in modestly lower cellular proliferation in all intestinal segments. Radiation induced a decrease in cellular proliferation in the jejunum and ileum; this decrease was prevented by a 20% casein diet. Pretreatment with aflatoxin B1 decreased intestinal cell proliferation throughout the intestine, and this decrease was not influenced by the protein content of the diet.

Aflatoxins↗

PubMed1994

Intestinal cell respiration is influenced by animal age, strain, and feeding status.

The objectives of this study were to evaluate the influence of aging and the fasted vs fed state on substrate oxidation by jejunal and colonic cells in vitro, and to determine whether the effects of these factors would be influenced by rat strain. Young (4 mo) and aged (24 mo) male rats of the Fischer 344 (F344) and Fischer x Brown Norway (F x BN) strains were used either following a 48-hr fast or in the ad libitum fed state. On the morning of experimentation, cells were removed from segments of the jejunum and colon and aliquots of these suspensions were incubated in 5 mM concentrations of substrates containing trace quantities of 14C-labeled isotopes. Following 60 min of incubation, 14CO2 was collected and quantified to determine substrate oxidation. The oxidation of glucose, glutamine, and 3-hydroxybutyrate was studied in jejunal and colonic cells, and the oxidation of acetate and butyrate was studied in colonic cells only. Glucose oxidation by jejunal cells was lower when cells were taken from 48-hr fasted animals than from fed animals, but the feeding status of the animal did not significantly influence oxidation of other substrates by jejunal or colonic cells. Substrate oxidation was not different for the F344 vs F x BN strains when jejunal and colonic cells were taken from young animals. Differences due to rat strain became apparent in the aged animals, however, with oxidation of several substrates being higher for the aged F344 than for the aged F x BN animals.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Hydroxybutyric Acid↗

PubMed1994

The TCA cycle as an oxidative and synthetic pathway is suppressed with aging in jejunal epithelial cells.

The influence of aging on glucose and glutamine metabolism by isolated jejunal cells was studied using young (4 months) and aged (24 months) Fischer 344 male rats when fed ad libitum or fasted 48 h. Concentration-dependent oxidation of glucose ([14C(U)]glucose) followed Michaelis-Menten kinetics. Neither K(ox) nor Vmax was influenced by animal age or feeding status, but at 1 mM, glucose oxidation was significantly higher for aged than young fed animals. In all animal groups, glutamine reduced glucose oxidation by ca. 60%, glucose stimulated glutamine oxidation by ca. 25%, and succinate CO2 ratios ranged from 1.37 for 20 mM glucose to 5.46 for 20 mM glucose+glutamine. The probability that a substrate that enters the TCA cycle will either remain in the cycle for one complete turn or leave and reenter as acetyl-CoA averaged 0.85 for glucose, 0.36 for glutamine, and 0.31 for glucose+glutamine. In comparison with the young fed animals, cells from fed aged animals showed lower oxygen uptake in the absence and presence of exogenous substrate, lower glucose oxidation, lower entry of glucose and glutamine into the TCA cycle, and lower contribution of glucose and glutamine carbon to anaplerosis and subsequent synthetic compounds. Differences between the young and aged animals were more pronounced in cells from fed animals than from fasted animals.

Aging↗

PubMed1993

Nutrient oxidation by rat intestinal epithelial cells is concentration dependent.

The kinetics of oxidative metabolism of glucose, glutamine, acetate and butyrate were determined in cells isolated from the jejunum and colon of young, fed rats. Jejunal and colonic cells were isolated from the same animal and incubated with a single radiolabeled substrate at concentrations ranging from 0.005 to 25 mmol/L, and 14CO2 production was determined. Carbon dioxide production was concentration dependent and saturable for all substrates. Data points within the range of the apparent half-maximal oxidation rate were transformed by use of a Lineweaver-Burk plot to calculate K(ox), the concentration at which there was half-maximal oxidation, and Vmax, the calculated maximal rate of oxidation. In jejunal cells, the K(ox) for glucose and glutamine were 0.40 and 0.45 mmol/L, respectively. The K(ox) for glucose, glutamine and acetate ranged from 0.80 to 0.88 mmol/L in colonocytes, whereas the K(ox) for butyrate was 0.33 mmol/L. Except for butyrate in colonocytes, the observed maximal rate of oxidation was comparable to the calculated maximal rate. The substrate concentration required to ensure that substrate was not limiting for its oxidation was estimated at 5 mmol/L for glucose and glutamine in enterocytes and colonocytes, 5 mmol/L for acetate in colonocytes, and 0.50 mmol/L for butyrate in colonocytes. This study showed that attention needs to be given to the concentration of substrate used for in vitro studies.

Acetates↗

PubMed1992

Wheat bran and corn oil do not influence proliferation in the colon of healthy rats when energy intakes are equivalent.

Dietary intakes of fiber (wheat bran) and fat (corn oil) by rats were quantitatively varied for 6 wk while intakes of energy and essential nutrients were constant among the diets. The influence of wheat bran and corn oil levels on colonic epithelial cell proliferation was assessed using [3H]thymidine incorporation assays. Cellular proliferation in the cecum, proximal colon and distal colon was not significantly higher when healthy rats consumed low fiber in comparison to high fiber diets, nor when healthy rats consumed high fat diets in comparison to low fat diets. Additionally, there were no significant interactions between these variables. In comparison to the low fiber diets, diets high in fiber caused a significantly lower pH of luminal contents of the cecum and distal colon, but there were no significant differences in the total short-chain fatty acid concentrations of cecal digesta. These results suggest that when energy intakes are equivalent among diets, low levels of fiber or high levels of fat in the diet do not cause biological changes in colonic mucosa that may be associated with increased risk of colonic cancer.

Animals↗

PubMed1992

The influence of dietary fiber on proliferation of intestinal mucosal cells in miniature swine may not be mediated primarily by fermentation.

Cannulated miniature pigs were used as a model for evaluating the effects of fiber-containing diets on proliferation of colonic epithelial cells. Biopsy specimens taken from the mucosa of the cecum and distal colon were incubated in [3H]thymidine and processed using autoradiography. Digesta from the cecum was analyzed for concentrations of short-chain fatty acids (SCFA), pH and volume. Cellular proliferation at both intestinal sites was influenced by the source or quantity of dietary fiber. These dietary variables also influenced SCFA concentrations and pH of cecal digesta and the volume of contents in the fermentation chamber. Cellular proliferation was not significantly correlated, however, with the quantity of water-soluble dietary fiber consumed, with SCFA concentrations or pH of the cecal digesta, or with the volume of the fermentation chamber. We suggest that the influence of dietary fiber on intestinal cell proliferation cannot be predicted from markers of the fermentation capacity of specific diets, because fiber likely influences intestinal proliferation through several interactive mechanisms, some of which include luminal factors.

Animals↗

PubMed1991

Absorption of short-chain fatty acids from the rat cecum in vivo.

Absorption of the short-chain fatty acids (SCFA), acetate, propionate and butyrate, was studied in vivo by measuring their disappearance from the washed cecum of anesthetized rats. The SCFA seemed to be absorbed predominantly by simple passive diffusion, because there was linear absorption over a wide concentration range, and no evidence of competition or enhancement of absorption when more than one SCFA was present in the lumen. Absorption of these three SCFA was independent of chain length. Their absorption was clearly limited by the lipid membrane, because permeability through the unstirred water layers was much greater than through the lipid membrane. Absorption was unaffected by altering the pH of the luminal infusate between 5.4 and 7.4. Based on our observation that, during absorption, there were increases in pH, total CO2 and HCO3- but no change in pCO2, we suggest that SCFA are absorbed primarily via diffusion involving anionic exchange with HCO3-.

Acetates↗

PubMed1991

Nutrient utilization by cells isolated from rat jejunum, cecum and colon.

Cells isolated from the jejunum, cecum and colon of rats were used to study the oxidation of nutrients by quantifying the production of 14CO2 from 5 mmols/L 14C-labeled exogenous substrate. In colonic cells, the decreasing order of oxidation was as follows: butyrate greater than acetate greater than propionate, glucose and glutamine. Acetate and butyrate significantly suppressed oxidation of both glucose and glutamine. In cells taken from the cecum, butyrate was oxidized at a greater rate than all other substrates. Cells taken from the jejunum produced CO2 from exogenous substrates in decreasing order as follows: glutamine greater than glucose much greater than acetate, propionate and butyrate. Butyrate oxidation was significantly reduced in colonic cells by 3-hydroxybutyrate, and it was reduced in cecal cells by glucose. Comparisons among the three gut segments showed no differences in glutamine oxidation. Glucose oxidation was greater in cells taken from the colon than from the cecum or jejunum, which were similar. Butyrate and acetate were oxidized at higher rates in cells taken from the cecum and colon than in cells taken from the jejunum, and propionate was oxidized at a greater rate in cells taken from the colon than from the jejunum. These studies demonstrate that relative rates of substrate oxidation differ along the intestinal tract of rats.

Acetates↗

PubMed1989

Abnormal fatty acid composition of biotin-responsive multiple carboxylase deficiency fibroblasts.

Clinical and biochemical correlations in the biotin-responsive multiple carboxylase deficiencies have suggested that disordered lipogenesis plays a role in the pathogenesis of the disease. In particular, the activity of biotin-dependent acetyl CoA carboxylase and the de novo synthesis of fatty acids are reduced in mutant fibroblasts. In the present work, we examine the biochemical consequences of these deficiencies, and document and characterize an abnormal fatty acid composition in holocarboxylase synthetase deficiency fibroblasts. Following growth in biotin-restricted medium, the total fatty acid content of mutant cells is reduced. There were significant reductions in the percentage as 16:0, 18:0 and 20:3N9 fatty acids, with the proportion of longer-chain fatty acids either increased or maintained at control levels. The cellular content of 16:0, 16:1, 18:0, 18:1 and 20:3N9 fatty acids was reduced, while that of the longer-chain fatty acids was preserved at control levels in mutant cells deprived of biotin. We speculate that the components of the altered fatty acid pools may be disproportionately incorporated into complex lipids in mutant cells, with pathologic effects on the multiple carboxylase deficiency phenotype.

Biotin↗

PubMed1989

High-fiber diets: influence on characteristics of cecal digesta including short-chain fatty acid concentrations and pH.

The effects of two sources of dietary fiber on the characteristics of cecal contents were assessed directly with miniature swine cannulated to facilitate frequent collections of cecal digesta. The short-chain fatty acid (SCFA) concentrations increased and the pH decreased at the same time that meal constituents entered the cecum; PEG was used as a dietary marker. The bean diet resulted in higher concentrations of acetate and total SCFA in cecal digesta, lower concentrations of butyrate, a larger SCFA pool size, and a more acidic pH than did the bran diet. Thus, we conclude that SCFA concentrations and acidity of the digesta are directly related and that dietary fibers can affect simultaneously several variables that are implicated as factors influencing colonic health.

Acetates↗